Effect of exosomes derived from Echinococcus multilocularis on macrophage polarization: A preliminary study / 临床肝胆病杂志

Objective To investigate the effect of exosomes derived from Echinococcus multilocularis on macrophage polarization after treatment for different durations and concentrations. Methods A total of 60 BALB/c mice were used for modeling, among which 4 mice were selected to observe the growth of abdominal lesions on 7.0T MRI. The mice for modeling were dissected, and the protoscoleces was taken from the abdominal lesion and cultured in vitro ; ultracentrifugation was used to extract the exosomes from the supernatant, and transmission electron microscopy and Western blotting were used for the characterization of exosomes. The macrophages without exosome treatment were established as control group, and the macrophages co-cultured with different concentrations of exosomes derived from Echinococcus multilocularis were established as experimental group (10 μg/mL group and 50 μg/mL group) and were cultured for 48 and 72 hours. The morphological changes of macrophages were observed under a microscope, and flow cytometry and ELISA were used to observe polarization state. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results The results of 7.0T MRI showed the formation of diffuse lesions with different sizes in the abdominal cavity of mice, and the exosomes derived from Echinococcus multilocularis were approximately 100 nm in diameter and were cup-shaped or saucer-shaped, with the positive expression of the surface markers CD9, TSG101, and CD63. After co-culture, most of the cells in the experimental group were elongated with an irregular and polygonal shape. Flow cytometry showed that after 48 hours of co-culture, the positive rates of CD16/32, CD206, and CD369 in the control group were 99.53%±0.06%, 90.27%±0.21%, and 2.40%±0.20%, respectively; compared with the control group, except that the 10 μg/mL exosome group had a significant reduction in the positive rate of CD369 (0.80%±0.00%) ( P < 0.05), all the other groups had a significant increase in the positive rates of CD16/32, CD206, and CD369 (all P < 0.000 1); after 72 hours of co-culture, the positive rates of CD16/32, CD206, and CD369 in the control group were 99.67%±0.06%, 85.47%±0.55%, and 6.60%±0.20%, respectively, and compared with the control group, the experimental group had significant increases in the positive rates of CD16/32, CD206, and CD369 (all P < 0.05). ELISA showed that after 48 hours of co-culture, the levels of IL-6 and TNFα in the control group were 58.53±15.52 pg/mL and 320.70±5.30 pg/mL, respectively, and when the exosome concentration was 50 μg/mL, the level of IL-6 in the experimental group was 98.81±15.55 pg/mL, which was higher than that in the control group ( P < 0.05); after 72 hours of co-culture, the levels of IL-6 and TNFα in the control group were 76.22±9.68 pg/mL and 323.90±87.37 pg/mL, respectively, and when the exosome concentration was 10 μg/mL, the level of TNFα was 164.20±14.17 pg/mL, which was significantly lower than that in the control group ( P < 0.05); when the exosome concentration was 50 μg/mL, the level of IL-6 was 99.52±8.35 pg/mL, which was significantly higher than that in the control group ( P < 0.05). Conclusion Exosomes derived from Echinococcus multilocularis can regulate macrophage polarization and induce M2-like polarization of macrophages after co-culture at a concentration of 10 μg /mL for 72 hours, and further studies are needed to clarify the specific method.

Role of D-bifunctional protein in promoting the growth of hepatocellular carcinoma in rats and nude mice / 临床肝胆病杂志

ObjectiveTo investigate the expression of D-bifunctional protein (DBP) in hepatocellular carcinoma (HCC) tissue in rats and the growth of DBP-induced HCC in nude mice. MethodsA total of 22 male Sprague-Dawley rats were randomly divided into normal control group with 8 rats and model group with 14 rats treated with intraperitoneally injected diethylnitrosamine to induce HCC, and Western blotting, immunohistochemistry, and RT-PCR were used to measure the expression of DBP. A total of 14 specific pathogen-free male BALB/c-nu mice were randomly divided into two groups. HepG2 cells were transfected with empty plasmid or DBP overexpression plasmid and were then injected subcutaneously into nude mice. There were 8 mice in the empty plasmid control group and 6 mice in the DBP high-expression plasmid group, and tumor size was measured for both groups. The t-test was used for comparison of continuous data between groups. ResultsThe rats with HCC had significantly higher protein and mRNA expression of DBP in liver tissue than normal rats (protein: 1.10±0.35 vs 0.67±0.12, t=-7.48, P＜0.05; mRNA: 3.70±0.85 vs 1.17±0.72, t=-20.46, P＜0.05). The DBP high-expression plasmid group had a significantly higher tumor volume than the empty plasmid group ［(7590.50±1867.97)mm3 vs (1663.78±420.24)mm3, t=-39.78, P＜0.01］. ConclusionHighly expressed DBP can promote the progression of HCC in rats and thus provides a new target for the treatment of HCC and the research and development of inhibitory drugs.

Clinical effects of bone marrow stem cell transplantation through different approaches in mice with acute liver injury / 临床肝胆病杂志

ObjectiveTo investigate the migration of bone marrow stem cells (BMSCs) to the liver and liver repair in mice with acute liver injury treated with BMSC transplantation through four approaches. MethodsMale BALB/c mice were divided into groups A, B, C, D, E, and F, with 10 mice in each group. Groups A, B, C, and D were treated by transplantation, group E was used as the donor of BMSCs, and group F was used as the model of acute liver injury. CCL4/2-AFF was used to establish the model of acute liver injury. Mouse BMSCs were isolated, labeled with the red fluorescent dye PKH26, and then transplanted into the mice with acute liver injury through the portal vein (group A), the tail vein (group B), the abdominal cavity (group C), and the spleen (group D). The mice were sacrificed 2 weeks later. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (Alb) were measured. The pathology of liver tissue was observed to evaluate the migration of BMSCs to the liver and the degree of liver repair. The mice in group F were sacrificed on day 8 to measure the levels of ALT, AST, and Alb. The t test was used for comparison of continuous data between two groups, and one-way analysis of variance was used for comparison of continuous data between multiple groups. ResultsIn groups A, B, C, and D, transplanted BMSCs migrated to the liver under a microscope, and newly formed hepatocytes were observed on pathological images. There were significant differences in the levels of ALT, AST, and Alb between groups A, B, C, and D and group F (ALT: t=2.372, 2.473, 2.354, and 2.383, all P＜0.05; AST: t=2.534, 2.423, 2.437, and 2.643, all P＜0.05; Alb: t=2.336, 2.243, 2.373, and 2.352, all P＜0.05). ConclusionBMSCs can promote repair of the liver in mice with acute liver injury, and the degree of liver repair is not related to the transplantation approach.

The protective effect of Xuebijing on mouse model of immunosuppressed sepsis / 天津医药

@#Objective To observe the effect of Xuebijing on immunosuppressed sepsis of model mice. Methods The 152 mice were randomly divided into control group (Control), immunosuppression group (IM), immunosuppressed sepsis model group (ISM) and Xuebijing treatment group (XT). There were thirty – eight mice for each group. For group IM, cyclosporine A was injected intraperitoneally along the median line of the lower abdomen for immunosuppression, 25 mg/kg， 1 time every other day for a total of 3 times. For group ISM, after immunosuppression, 300 μL escherichia coli 44102 with a concentration of 1×109 CFU/mL was injected intraperitoneally along the midline of the lower abdomen. The 30 minutes after the establishment of the immunosuppressive sepsis model, Xuebijing 4 mL/kg was intraperitoneally injected along the midline of the lower abdomen in the ISM group. After 30 minutes, the same dose of Xuebijing injection was repeated once. Control group was injected intraperitoneally with the same amount of normal saline. (1) After 8 h, 4 mice in each group were taken for blood bacterial culture. (2) After 12 h, 10 mice in each group were taken for detecting CD3+CD4+ and CD3+CD8+ in peripheral blood using flow cytometry. (3) After 12 h, 10 mice in each group were taken for detecting blood levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA). (4) After 12 h, 4 mice in each group were taken for detecting high-mobility group protein (HMGB1) by Western blot assay. (5) After 12 h, 10 mice in each group were taken for detecting alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and serum creatinine (CR) levels by automatic biochemical analyzer. Results Compared with the ISM group, the ratio of CD3+CD4+/CD3+CD8+ was significantly increased, the number of blood bacteria culture decreased obviously in the XT group. Liver and kidney function indicators ALT, AST, CR, BUN and inflammatory factor indicators TNF-α, IL-6 and HMGB1 were also significantly decreased in the XT group. Conclusion Xuebijing can obviously modulate the immunosuppression, against bacteria, inhibit the inflammatory reaction, and protect the vital organs.

Relationship between the miR-96 expression in the cochlea and ABR threshold and cochlear morphology of BALB/c mice / 中国耳鼻咽喉头颈外科

OBJECTIVE Through researching the ABR threshold, the cochlear morphology and miR-96 expression in the cochlear of BALB/c mice at different month's age, to find out if the miR-96 can regulate the age related hearing loss of BALB/c mice.METHODS ABR testing, AO/PI staining and scanning electron microscope were used to observe the ABR threshold and cochlear morphology of the BALB/c mice at the ages of 3 months, 6 months, 12 months and 18 months. Real-time PCR was used to detect the expression of miR-96 in the cochlea of BALB/c mice at the ages of 3 months, 6 months, 12 months and 18 months.RESULTS The ABR thresholds of BALB/c mice were (18.5±8.3), (45.8±7.8), and (85.6±15.6) dB SPL separately at the age of 3, 6 and 12 months. At the age of 18 months, no response was observed in the ABR testing with 120 dB SPL acoustic stimulation. In the AO/PI staining, we found that the outer hair cells was apparently lost since the age of 6 months and the loss of hair cells aggravated as the month's age increased. At the age of 12 months, no outer hair cells was left, inner hair cells was lost apparently too. With the scanning electron microscope, we found the changes of deficiency, lodging, fusion, shortening and inversion in the hair cell cilia. And these changes were aggravated as the month's ages increased. At the age of 3 months, the relative expression of miR-96 (2-△CT) was 0.0225±0.0073. The relative expression of miR-96 (2-△CT) in the cochlea were 0.0162±0.0048, 0.0116±0.0048, and 0.0050±0.0014 at the age of 6 months, 12 months and 18 months separately, comparing with the relative expression of miR-96 at the age of 3 months, the differences were significant(P<0.05).CONCLUSION The hearing loss, hair cells loss, and cilia damage aggravated as the month's age increased, but the miR-96 expression in the cochlea decreased. Which suggest that miR-96 might play an important role in the age related hearing loss.

Overexpression of amphiregulin promotes the growth of mouse colon carcinoma CT26 cells in vivo / 肿瘤

Objective: To investigate the effect of epidermal growth factoramphiregulin (AREG) on the growth of mouse colon carcinoma CT26cells and its related mechanisms.Methods: The protein expression level of AREG in different mouse cancer cells was detected by ELISA. Mouse colon carcinoma CT26 cells, melanoma B16 cellsand hepatocellular carcinoma LPC-Akt cells were transfected with the recombinant lentiviralplasmid carrying AREG gene, while the ones transfected with empty plasmid were used asthe negative controls. After AREG overexpression, the cell proliferation, colony-formingabilities and cell cycle progression in vitro were detected by MTT, colony-forming assay andFCM, respectively. After the homograft mouse model of CT26 cells was constructed, thegrowth of homograft tumor was observed, the distribution of immune cells in tumor tissueswas detected by FCM, furthermore the expression of chemokine was detected by real-timefluorescent quantitative PCR.Results: The levels of AREG expression were relatively low in mouse colon carcinoma CT26cells, melanoma B16 cells and hepatoma LPC-Akt cells. AREG overexpression did notmarkedly affect the proliferation, colony-forming abilities and cell cycle progression of thesethree types of tumor cells in vitro (all P > 0.05). However, in the homograft mouse model ofCT26 cells, AREG overexpression significantly promoted the growth of tumor cells in vivo (P <0.01), decreased the percentage of CD8+ T cells (P < 0.05), and reduced the mRNA level ofCC chemokine 5 ligand (CCL5) (P < 0.05) which was related to CD8+ T cell recruitment.Conclusion: AREG promotes the growth of mouse colon carcinoma CT26 cells in vivo . AREGmay affect the tumor microenvironment by regulating the production of chemokine which isrelated to CD8+ T cell recruitment.

Inflammatory-promoting roles of thymic stromal lymphopoietin and interleukin-4 in allergic conjunctivitis mice / 中华实验眼科杂志

Background Researches showed that thymic stromal lymphopoietin (TSLP) is an interleukin-17-like inflammatory factor and plays important roles in the pathogenesis and development of allergic diseases.However,the study whether TSLP plays roles in allergic conjunctivitis is rare.Objective This study was to investigate the expression change of TSLP and IL-4 in ocular surface tissue and cervical lymph node in the mice with allergic conjunctivitis induced by artemisia annua,a common plant in China,and to explore the role of TSLP and IL-4 in the pathogenesis and development of allergic conjunctivitis.Methods Twenty female BALB/c mice aged 6-8 weeks were randomized into normal control group and model group.Antigen solution was prepared using 400 μl extracting solution of artemisia annua pollen with 400 μl antigen solvent.The mouse models of allergic conjunctivitis were established by injection of 50 μl antigen solution in footpad followed by ocular topical administration of extracting solution once a day from day 10 to 12 after injection,and no any intervention was given in the normal control group.The mice were sacrificed and eyeballs were obtained 13 days after modeling,and corneal epithelium,conjunctiva and cervical lymph nodes were harvested for the detection of TSLP mRNA and IL-4 mRNA by real-time PCR.Immunochemistry was employed to assay the expression of TSLP and IL-4 proteins in corneal,conjunctival and subconjunctival tissues.Results Ocular inflammatory signs appeared 0.5 hours after ocular topical administration of extracting solution,including eyelid edema,conjunctival congestion,tears,scratching eyelids,etc.The symptoms lasted for 6-24 hours,with the model successful rate 80％.Real-time PCR indicated that the expression of IL-4 was absent in corneal epithelium in both model group and normal control group.The relative expression levels of TSLP mRNA in the corneal epithelium,conjunctiva and cervical lymph nodes in the model group were more (1.63±0.20)times,(2.71±0.48) times and (1.48 ±0.05) times than the normal control group,showing significant differences between the two groups (t =4.44,14.16,5.01,all at P＜0.05).Compared with the normal control group,the relative expression levels of IL-4 mRNA in the model group increased (2.94±0.39) times and (1.74±0.09) times,with significant differences between the two groups (t =9.92,14.54,both at P＜0.05).Immunochemistry assay showed that the expression of TSLP protein in the corneal and conjunctival epithelial cells were enhanced in the model group compared with the normal control group.In addition,an intensive expression of IL-4 protein was seen in subconjunctival tissue in the model group,while the expression of IL-4 was absent in the normal control group.Conclusions TSLP is mainly expressed in the cornea,conjunctiva and cervical lymph nodes of the mice with allergic conjunctivitis,suggesting that TSLP promotes the inflammatory process of cornea and conjunctiva;IL-4 is mainly expressed in conjunctiva,showing IL-4 participates in conjunctival inflammatory process.TSLP and IL-4 play synergistic roles in promoting the inflammatory process of ocular surface in the mice with allergic conjunctivitis,which may be new therapeutic targets.

Experimental study on inhibiting graft rejection following high-risk cornea transplatation by resolvinE1 in mice / 中华实验眼科杂志

Background The conventional drugs for preventing and treating graft rejection have the risks of inducing adverse responses.Researches showed that resolvinE1 (RvE1) can regulate Th1 cell-mediated immunoreaction.However,whether RvE1 has an inhibit effect on high-risk corneal graft is unclear now.Objective This study was to investigate the effects of RvE1 on immune rejection in high-risk corneal grafting mouse models.Methods SPF BALB/c mice were used as recipients,C57BL/6 mice were as donors.Ninety BALB/c mice were divided into corneal allograft group,corneal allograft+RvE1 group and corneal autograft group according to random number table.High-risk corneal graft models were established by corneal suturing for 14 days and followed by penetrating keratoplasty in recipients.Allograft keratoplasty was performed on the right eyes in the mice of corneal allograft group and corneal allograft+RvE1 group,and self-corneal graft rotated 180°was transplanted on the right eyes in the mice of autograft group.Normal saline solution of 10 μl was subconjunctivally injected after surgery once per day for 7 days in the corneal allograft group and corneal autograft group,and 10 μl RyE1 (1 μg) was used in the same way in the corneal allograft+RvE1 group.The recipient eyes were examined for potential rejection signals with slit lamp microscope and calculated the mean survival time and rejection index (RI).The histopathology was examined 21 days after modeling by hemotoxylin and eosin staining.The expressions of CD4 and interferon-γ (IFN-γ)in the corneas were detected by immunohistochemistry.Th1 cell (CD3+CD8a-IFN-γ+) percentage in draining lymph nodes were measured by flow cytometry.The mRNA expression levels of interleukin-2 (IL-2),tumor mecrosis factor-α (TNF-α),IFN-γ and T-bet were detected by real-time fluorescence quantitative PCR.Results The mean survival time of grafts was (28.5± 1.7) days in the corneal allograft group,and that in the corneal allograft+RvE1 group was (14.0±1.6) days,showing a significant difference between them (t =4.14,P＜0.001),while the survival rate was 100％ at 50 days after modeling in the corneal autograft group.Corneal edema and inflammatory cell infiltration were slight in the corneal allograft+RvE1 group and corneal autograft group compared with corneal allograft group.CD4 was positively expressed in corneal tissue,and IFN-γ was expressed in corneal epithelium.The CD4+ and IFN-γ+ cell number was decreased in the corneal allograft+RvE1 group and corneal autograft group compared with corneal allograft group under the fluorescence microscope.The percentages of Th1 cells in lymph cells of corneal allograft +RvE1 group and corneal autograft group were (1.07 ±0.25) ％,(0.85 ±0.12) ％,respectively,which were significnatly lower than (1.56±0.20) ％ in the corneal allograft group (both at P＜0.05).The expressions of IL-2,TNF-α,IFN-γ and T-bet mRNA in the corneal tissue in the corneal allograft group were higher than those in the corneal allograft+RvE1 group and corneal autograft group (all at P＜0.05).Conclusions RyE1 inhibits graft rejection in high-risk allograft mouse models probably by down-regulating the Th1 cell percentage in lymph cells and the expression of inflammationrelated cytokines in corneal grafts.

Bone marrow mesenchymal stem cell transplantation via different approaches in treatment of liver cirrhosis in mice / 临床肝胆病杂志

ObjectiveTo investigate the clinical effects of bone marrow mesenchymal stem cell (BMSC) transplantation via different approaches in the treatment of liver cirrhosis in mice. MethodsA total of 46 BALB/c mice were randomly divided into normal control group with 5 mice and liver cirrhosis model group with 41 mice. Subcutaneously injected carbon tetrachloride olive oil was used to establish the mouse model of liver cirrhosis. A total of 36 mice with liver cirrhosis were randomly divided into control group, caudal vein BMSC transplantation group, and spleen BMSC transplantation group, with 12 mice in each group. Whole bone marrow adherent culture was performed to obtain the third-generation BMSCs, and flow cytometry was used for cell surface identification. BMSCs were injected into the mice through the caudal vein or spleen. Blood samples were collected at 4 weeks after transplantation to measure liver function. HE and Masson staining and α-smooth muscle actin (α-SMA) immunohistochemistry were performed for liver sections. Liver injury and fibrosis in mice were examined. A one-way analysis of variance was used for comparison between groups. ResultsAt 8 weeks after the establishment of the model, the mice in the model group had sparse and dark yellow hair, reduced food consumption and activity, and a reduction in body weight. After transplantation, compared with the model control group, the caudal vein BMSC transplantation group and spleen BMSC transplantation group showed a significant increase in albumin and significant reductions in alanine aminotransferase and aspartate aminotransferase (all P＜0.01). There were no significant differences between the two transplantation approaches (P＞0.05). After transplantation, there were significant changes in diseased tissue, alleviated liver cirrhosis, reduced collagen fiber and necrotic area, and a good structure. Immunohistochemistry showed both transplantation groups showed significant reductions in the number of cells with positive α-SMA. ConclusionBMSC transplantation via the tail vein or spleen can improve liver function and diseased tissue in mice with liver cirrhosis and these two approaches have comparable clinical effects.

Efeito do inibidor de proteinase de origem vegetal EcTI, sobre a inflamação pulmonar alérgica crônica em camundongos Balb/c / Effect of proteinase inhibitor of plant origin EcTI in an experimental model of chronic allergic pulmonary inflammation in Balb/c mice

A prevalência de asma tem crescido e a maioria dos pacientes com asma grave não obtém o controle total dos sintomas com as terapias disponíveis, fazendo-se necessária a busca por novas alternativas terapêuticas. Inibidores de proteinases têm sido estudados como tratamento de processos inflamatórios, dentre eles o Enterolobium contortisiliquum Tripsin Inhibitor (EcTI) OBJETIVO: Avaliar se o inibidor de proteinase EcTI modula a hiperresponsividade brônquica à metacolina, inflamação, remodelamento e estresse oxidativo nas vias aéreas e septos alveolares em um modelo experimental de inflamação pulmonar alérgica crônica. MÉTODOS: Vinte e quatro camundongos Balb/c machos, entre seis e sete semanas de vida, pesando em media 25 g foram divididos em quatro grupos: C (controle), OVA (sensibilizados com ovalbumina, 50 ug intraperironeal (i.p) nos dias 0 e 14 e desafiados nos dias 22, 24, 26, 28); C+EC (controle tratados com EcTI (2 mg/kg/i.p) nos dias 22 a 28); OVA+EC (sensibilizados e desafiados com ovalbumina e também tratados com EcTI (2 mg/kg -i.p) nos dias 22 a 28). No dia 29, foram realizadas realizadas: (i) hiperresponsividade à metacolina e obtidas as respostas máximas de resistência e elastância do sistema respiratório; (ii) análise histopatológica do pulmão para quantificação de eosinófilos, fibras colágenas e elásticas nas vias aéreas (VA) e nos septos alveolares (SA); e (iii) imunohistoquímica para quantificação de células positivas para IFN-y, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-beta, iNOS, NF-kB e fração de volume de isoprostano nas VA e nos SA. Uma semana após o dia 29 foi realizada a técnica de anafilaxia cutanea passiva(PCA) para quantificar IgE e IgG1. A significância foi considerada quando p < 0,05. RESULTADOS: Houve aumento de todos os parâmetros avaliados no grupo OVA em relação ao grupo controle (p < 0,05). Houve atenuação da resposta máxima de Rrs e Ers no grupo OVA+EC comparado as grupo OVA (p < 0,05). O tratamento...

The number of cases of asthma has grown in recent decades. People who have severe asthma are likely to have more attacks and are at greater risk of a fatal attack, which propose to keep up global attention and keep approaching for advances in asthma care. Proteinase inhibitors of vegetable origin have been studied as a modulator of inflammatory responses and diseases. Among these inhibitors is Enterolobium contortisiliquum Trypsin Inhibitor (EcTI). AIMS: To evaluate the effects of EcTI in pulmonary mechanical, eosinophilic recruitment, inflammatory cytokines, remodeling of extracellular matrix and oxidative stressin an experimental model of chronic allergic pulmonary inflammation. METHODS: Twenty-four young adult male pathogen-free mice BALB/c (6-7 weeks old, 25-30g) were divided into 4 groups: C (control), OVA (sensitized with ovalbumin, 50 ug intraperitoneal (i.p), on days 0 and 14 and challenged with ova 1%, on days 22, 24, 26, 28); C+EC (control treated with EcTI- 2 mg/kg/i.p. from days 22 to 28); OVA+EC (sensitized and challenged with ovalbumin and treated with EcTI (2 mg/kg/i.p) from days 22 to 28). At day 29, we performed: (i) Bronchial hyperresponsiveness to methacholine and obtained the maximum response of resistance (Rrs) and elastance (Ers) of the respiratory system; (ii) lung histopathological analysis by morphometry to quantify eosinophils, collagen and elastic fibers volume fraction in airways; and (iii) immunohistochemistry to quantify IFN-y, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-, iNOS, NF-kB positive cells and isoprostane volume fraction in airways. One week after the day 29 we performed PCA technique to quantify IgE and IgG1 antibodies. Significance was considered at p < 0.05. RESULTS: The EcTI treatment in the ovalbumin-sensitized animals attenuated the maximal response of resistance and elastance of respiratory system after methacholine, the number of eosinophils, IL-4, IL-5, IL-13, IFN-y, NF-kB and iNOS-positive cells,...

Isolation and culture of corneal neovascular endothelial cells and expression of chemokine receptors / 中华实验眼科杂志

Background The pathogenesis and mechanism research of corneal neovascularization is of important significance for the prevention and management of corneal neovascularization.Some relative researches are being performed on non-corneal neovascularization-derived vascular endothelial cells, so the results are affected to a certain extent.Objective This study was to isolate and culture vascular endothelial cells from experimental corneal neovascularization tissue and detect the expression of chemokine receptors in vitro.Methods Corneal neovascularization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas, and then the immunofluorescence technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling.Corneal pieces were made in 2 weeks after alkali burn and then were digested by collagenase type D.Vascular endothelial cells were isolated from neovascularized tissue by affinity purification using magnetic beads coated with anti-CD31.The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry.Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells.The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee.Results Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescence exhibited the green network-like fluorescence for CD31 antibody in corneas.The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage.The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm,in contrast,the expression of CD31 was absent in corneal stromal cells.Chemokine receptors were positively expressed in the cells with the strongest expression levels in CCR1 ,CCR2,CCR3 and CCR4 mRNA and the weakest expression levels in CCR9,CXCR4 and CXCR5 mRNA,while CXCR3, CCR6, CCR10 and CX3CR1 mRNA were expressed with the moderate intensity.Conclusions Vascular endothelial cells can be obtained from experimental neovascularized corneas by affinity purification and express chemokine receptors,which facilitate the study of their biological properties.

Ganshuang granules protect mouse liver from chronic injury induced by CCl4 via autophagy / 临床肝胆病杂志

ObjectiveTo investigate the role and action mechanism of Ganshuang granules in the protection against CCl4-induced chronic liver injury in mice via autophagy, and to provide a basis for its clinical application. MethodsWe established a chronic liver injury model in mice by intraperitoneal injection of CCl4, and a cell model of liver damage in cell line HL-7702 induced by CCl4 in vitro. The animals were divided into three groups, Ganshuang granule intervention group, normal control group, and CCl4 group without receiving Ganshuang granules. In addition, we exposed the cells to 3-methyladenine (3-MA), an autophagy inhibitor, and observed the effects of Ganshuang granules on cell apoptosis. Liver tissue injury was evaluated by HE staining; serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined by biochemical assays. Autophagy was assessed by immunofluorescence staining and Western blotting. Liver apoptosis was analyzed by flow cytometry and Annexin V/PI double staining. Comparisons between groups were performed using analysis of variance. ResultsHE staining showed that liver tissue injury was significantly milder in the Ganshuang granule intervention group than in the CCl4 group. Serum ALT and AST levels were significantly differences between Ganshuang granule intervention group, normal control group, and CCl4 group (F=1576、1335，P＜005). Both in vivo and in vitro tests showed that autophagy increased significantly in cells treated with Ganshuang granules than in cells exposed to CCl4 alone, while apoptosis was significantly reduced in the former. The administration of 3-MA weakened the protective effect of Ganshuang granules and increased apoptosis. Flow cytometry showed that apoptosises were significiantly differences between five groups (F=25637,P＜001). ConclusionGanshuang granules have protective effects against chronic liver injury by inhibiting apoptosis, possibly through enhanced autophagy.

Technique improvement in establishing mice orthotopic tracheal transplantation surgery model / 天津医药

Objective To explore a simple and effective way of establishing a mice orthotopic tracheal transplantation model in studying bronchiolitis obliterans to reduce operation time. Methods Tracheas from C57BL/6 mice (n=30) were im?planted into Balb/c mice (n=30). Grafts and hosts were matched according to body weight.Orotracheal intubation and endo-stentwas used for trachea anastomose and animal model was establish. Operating time was also noted. Grafts were harvest?ed on Days 15, 30 and 60 after transplantation, using HE staining to observe pathological changes of allograft. Trachea block?ing rate was also calculated. Results Until survival of trachea transplantation, the operative time of donor′s and the recipi?ent′tracheas resection were (7.0±1.0) min and (7.0±1.0) min respectively.Orotracheal intubation the endo-stentestablish?ment cost(1 ± 0.5)min. Trachea anastomose took(20.0 ± 4.0)min and the operation time last(43.0 ± 10.0)min. Conclusion The improvement of orthotopic tracheas transplantation usingOrotracheal intubation the endo-stenfor trachea anastomose is esay-operated and did required complicate skills and instruments with high successful rate. So it is an ideal and stable model in studying bronchiolitis obliterans.

Construction of murine model of TAO by immunification with recombinant extracellular domain of human thyrotropin receptor in cationic liposomes / 中华实验眼科杂志

Background Thyroid-associated ophthalmopathy (TAO) is a kind of clinically common and incurable ocular disease,and its incidence is at top place.The etiology and pathologic mechanism of TAO are still unknown because of shortness of replicative animal models and difficulty to acquire the ocular tissues in the early stage of the disease.To better understand the pathogenesis of TAO and investigate effective treatable measures, an appropriate animal model should be developed.Objective This study was to immunize female BALB/c mice with the recombinant plasmid of human thyroid-stimulating hormone receptor (TSHR) extracellular domain in cationic liposomes for the establishement of TAO models.Methods Thirty-two 6-to 8-week-old female BALB/c mice were randomly assigned to four groups according to computer random allocation.pcDNA3.1 +/hTSHR289 of 100 μg in an adjuvant cationic liposomes was injected via anterior tibialis muscle and peritoneal cavity separately in the recombinant plasmid injection group in 0, 3,6 weeks, and pcDNA3.1 or cationic liposomes was injected in the liposomes injection group or the blank plasmid group in the same way, respectively, and normal saline solution was injected in the blank control group.Body weight of the mice was measued before and 1 month,2,3 and 4 months after initial injection.The manifestations were observed after modeling.The mice were sacrificed 17 weeks after initial injection,and the histopathology examination was carried out on the thyroid gland and orbital tissue.The heart blood was collected from the mice,and serum contents of total thyroxin 4 (TT4) and thyroid-stimulating hormone (TSH)were assayed by ELISA.Results Protrusion, eyelid swell and keratitis occurred in 12 eyes of 6 mice in the recombinant plasmid injection group after immunization.A significant difference in the body weight of the mice was found among the blank control group, blank plasmid group, liposomes injection group and recombinant plasmid injection group (Fgroup =3.425, P =0.028), and the body weight was considerably reduced in the recombinant plasmid injection group in comparison with the blank control group, blank plasmid group,liposomes injection group (Ftime =0.838 ,P=0.023).The serum levels of TT4 were (7.75±1.00), (7.96±0.76), (6.76±1.10) and (4.43±2.88) μg/dlin the blank control group, liposomes injection group, blank plasmid group, and recombinant plasmid injection group, and those of TSH were (6.36±2.58),(4.83±3.96),(6.63±1.71) and (1.60 ±1.76) ng/ml, showing significant differences among the groups (F =7.150, P＜0.001;F =5.521, P＜0.01) , and the serum levels of TT4 and TSH were remarkably lower in the recombinant plasmid injection group than those of the blank control group,liposomes injection group and blank plasmid group (all at P ＜ 0.05).Histopathology revealed the lymphocyte infiltration of thyroid gland in 6 mice and proliferation of orbital adipose tissue, infiltration of lymphocytes and mastocytes,deposition of hyaluronic acid as well as swell, breakage and inflammatory cell infiltration of extraocular muscle in 15 eyes of the recombinant plasmid injection group.Conclusions A murine model of TAO can be successfully induced by immunization with recombination plasmid pcDNA3.1 +/hTSHR289 and cationic liposomes.The histopathology characteristics and ocular findings of the animal models are similar to human TAO.

Effect of Hypoxia-Inducible Factor(HIF)-2 Alpha Silencing on Osteosarcoma MG-63 Cells / 天津医药

Objective To investigate the effect of HIF-2a silencing by transfection of siRNA into MG-63 cells un-der hypoxia. Methods HIF-2αexpression level in MG-63 cells under hypoxia was determined by Western Blot. Small in-terfering RNA (siRNA) was used to construct MG-63/siHIF-2α(siHIF-2α)cells and control MG-63/scramble (NC) cells. The expression levels of HIF-2α, Vascular endothelial growth factor (VEGF), p-Erk/ErK and Mcl-1 in MG-63, NC and si-HIF-2αcells was determined by Western Blot. NC and siHIF-2αcells were cultured under hypoxia. Cell viability was as-sessed by MTT assay. Migration was identified by scratch migration assay. Tumor formation was identified by clone formation assay. Nude mouse subcutaneous xenograft model was used to investigate tumor development in vivo. Results Hypoxia im-proved HIF-2αexpression in MG-63 cells in a time-dependent manner (F=2 037.412,P<0.001). HIF-2αexpression un-der hypoxia in siHIF-2αcells was lower than that in NC cells (P<0.01). Cell viability of siHIF-2αcells under hypoxia for 12 h and 24 h were lower than that in NC cells (P<0.05 or P<0.01). The relative width of scratch in siHIF-2αgroup under hypoxia for 12 h and 24 h were larger than that in NC group (P<0.01 or P<0.01). When cell counts reach 1 000-5 000, the clone formation rates of siHIF-2αcells were lower than that in NC cells (P<0.05 or P<0.01). The expression of VEGF, p-Erk/Erk and Mcl-1 protein under hypoxia in siHIF-2αcells was lower than that in NC cells(P<0.01). Tumor sizes, weights and density of siHIF-2α group in nude mice were suppressed compared with those in NC group (P<0.01). Conclusion Blocking HIF-2αsignal pathway warrants its investigation as a potential strategy in osteosarcoma treatment.

Pathogenicity of Ureaplasma urealyticum serotype 1 and 8 in the genital tract of female BALB/c mice / 中华皮肤科杂志

Objective To compare the pathogenicity between Ureaplasma urealyticum serotype 1 (Up1)and 8 (Uu8) in the genital tract of BALB/c mice.Methods A total of 48 BALB/c mice were randomly and equally divided into four groups:blank control group receiving no treatment,estradiol group pretreated with intramuscular injection of estradiol followed by intravaginal inoculation with sterial liquid culture media,Up1 and Uu8 groups pretreated with intramuscular injection of estradiol followed by intravaginal inoculation with suspensions of Up1 and Uu8 respectively.Three mice were randomly selected from each group to be sacrificed after the collection of vaginal lavage fluid on day 3,7,14 and 21 after the inoculation.Vaginal and uterine tissue specimens were obtained from these sacrificed mice and underwent hematoxylin and eosin (HE) staining.Vaginal lavage fluid samples were subjected to culture of Uu and measurement of tumor necrosis factor-α (TNF-α).Results No evidences were observed for Uu growth in either the blank control group or estradiol group at any of the time points after the inoculation,with the average level of TNF-α in vaginal lavage fluid being (4.17 ± 0.85) pg/ml at these time points in both groups.Uu grew in all the vaginal lavage fluid samples from the Up1 and Uu8 groups at the four time points,with the color change unit (CCU) value decreasing with time.The level of TNF-α in vaginal lavage fluid peaked on day 14 after the inoculation in the Up 1 ((14.93 ± 1.11) pg/ml) and Uu8 ((27.04 ± 24.26) pg/ml) groups.Both Up1 and Uu8 infection caused acute and chronic inflammatory responses in the mice,which were mainly located in the uterus,and Up1 might cause intrauterine adhesion.Conclusions At the same inoculation concentration,no significant difference is found in the pathogenicity between Up1 and Uu8,both of which appear to mainly cause cervicitis.Upl might be partially responsible for intrauterine adhesion in mice.

Study on inhibitory effect of baicalein on mycoplasma pneumonia and protection mechanism of pulmonary epithelial cells of mice / 中国医师杂志

Objective To investigate the inhibitory effect of baicalein on mycoplasma pneumonia and its the protective mecha -nism in body , and to provide scientific experimental basis for prevention and treatment of mycoplasma pneumoniae infection .Methods The mycoplasma pneumoniae and baicalein treated BALB /c mice lung tissues were stained with hematoxylin eosin (HE), and his-topathological grading .Minimum inhibitory concentration ( MIC) of baicalein on mycoplasma pneumonia was determined by real-time quantitative polymerase chain reaction ( PCR) .The expression of P 1 adhesion molecules mRNA and protein in lung tissue of BALB /c mice was determined with reverse transcription-PCR and Western blot .The expression of epidermal growth factor ( EGF) mRNA and protein in lung tissue was detected by quantitative RT-PCR and immunofluorescence .Results Baicalein significantly reduced the my-coplasma treated mice's lung tissue pathological score .The minimal inhibitory concentration of baicalein was 32 μg/ml.Baicalein sig-nificantly downregulated P 1 gene transcription and protein translation , and upregulated EGF gene transcription and protein expression . Conclusions Baicalein shows significant resistance to mycoplasma pneumoniae , and can protect the body against mycoplasma damage by inhibiting the expression of P 1 protein and promoting the expression of EGF protein .

Isolation, culture and identification of adipose-derived stem cells from mouse epididymis / 中国组织工程研究

BACKGROUND:As a new kind of adult stem cells, adipose-derived stem cells get more and more attention, because of rich source, drawing materials easily and powerful proliferation. OBJECTIVE:To isolate and culture adipose-derived stem cells from the epididymal adipose tissue in mice, and to identify their biological characteristics. METHODS:Adipose tissue was obtained from epididymis in mice by aseptical y cutting. The tissue was digested using col agenase. Adipose-derived stem cells were separated and purified by using one digestion, multiple col ection method and differential adhesion method. The morphology of adipose-derived stem cells was observed using inverted microscopy and transmission electron microscopy. Growth curve of adipose-derived stem cells was drawn. Immunophenotype of adipose-derived stem cells was identified by flow cytometry. Adipose-derived stem cells were induced to differentiate into adipocytes and osteocytes using cellinductors. Compatibility of adipose-derived stem cells and col agen scaffold material was observed using scanning electron microscope. RESULTS AND CONCLUSION:Adipose-derived stem cells exhibited long spindle-like or fibroblast-like appearance, grew intensively and arranged in scrol and fascicular shape. In vitro, adipose-derived stem cells could be passaged to passage 9 under the inverted microscope. Under the transmission electron microscope, adipose-derived stem cells showed abundant microvil i on the cellsurface. The nuclei were big in size. Some organel es were seen in cytoplasma, such as mitochondria and rough endoplasmic reticulum. Adipose-derived stem cells expressed CD44 and CD29, did not express CD34. After inducing by inductor, many smal lipid droplets were seen in the cytoplasm of adipose-derived stem cells. The smal lipid droplets were dyed red with oil red O. After induction of osteogenic inductor, the boundary line among adipose-derived stem cells was not clear and the structure of cells was fuzzy in the growth-intensive areas. There were many strong refractive granular material deposits at that field after dyeing with alizarin red. Scanning electron microscope revealed that adipose-derived stem cells were spread on the col agen scaffold. Results suggested that adipose-derived stem cells isolated by this method could amplify in vitro and stably subcultured. Under a certain inducing condition, adipose-derived stem cells could differentiate into osteoblasts and adipocytes, which showed a good compatibility with col agen scaffold.

Pathological features of chronic liver injury induced by Helicobacter hepaticus in BALB/cCr mice / 中华临床感染病杂志

Objective To observe pathological features of liver injury induced by Helicobacter hepaticus ( H.hepaticus) and the difference between male and female BALB/cCr mice.Methods Fifty SPF-class BALB/cCr mice (25 males and 25 females) were administrated by gavage with 0.2 mL bacterial suspension (1 ×108 CUF/ml) of H.hepaticus standard strain ATCC 51450 for 3 times with 48 h intervals. The control group (25 males and 25 females) received same volume of phosphate buffered saline (PBS). Mice were sacrificed in batches ( n=5) after fasting for 12 h at month 1, 3, 6, 9 and 12.Enzyme linked immunosorbent assay ( ELISA) was used to determine the serum level of H.hepaticus IgG antibodies.Liver tissue samples were taken for histopathology examiantion, micro-aerobic bacteria isolation, culture and identification.t test was used to analyze the differences in serum levels of H.hepaticus IgG antibody and liver histopathologic scores between different time points and groups. Results The seroprevalance of H. hepaticus-IgG antibody in male BALB/cCr mice infected with H.hepaticus were all positive, peaked at 6 month, and then gradually declined.H.hepaticus-IgG antibody levels at 3, 6, 9 and 12 month were higher than that at 1 month (t=2.828, 4.300, 3.536 and 4.500, P0.05).Conclusion Compared with female mice, H.hepaticus colonization and histopathologic changes in liver are more significant in male BALB/cCr mice infected with H.hepaticus, and the histological scores are increased as infection time extended.

Effect of RNA Interference Targeting HIF-1αGene on the Proliferation of Human Tongue Cancer Cells / 天津医药

Objective To explore the effect of RNA interference targeting hypoxia-inducible factor-1α(HIF-1α) gene on the proliferation of human tongue cancer cells (Tca8113 and Tscca). Methods HIF-1α-specific short hairpin RNA (shRNA) expression vector and empty shRNA vector were respectively transferred into two kinds of cultured human tongue cancer cells. The expression of HIF-1αgene was detected by RT-PCR and Western blot method. The clone forma-tion assay was used to observe the cell proliferation. The cell counting was used to monitor the number of living cells. Flow cytometry was applied to detect cell cycle distribution. Additionally, the cells were injected into nude mice, and tumor form-ing condition was observed. Results After the shRNA HIF-1αplasmid was transfected into tongue cancer cells, the expres-sions of both HIF-1αmRNA and protein were significantly decreased in tongue cancer cells under hypoxia condition. The number of colony formation was significantly lower in HIF-1αsmall interfering RNA (siRNA) group than that in empty and negative control group. The cell counting revealed that the number of living cells was remarkably lower in HIF-1αsiRNA group than that in negative control group. Also, the cell cycle in HIF-1α siRNA group was arrested at G0/G1 phase after transfection and 48 hours hypoxia culture. Besides, the tumor volume was remarkably smaller in HIF-1αsiRNA group than that in negative control group after the tongue cancer cells transplanted into nude mice for several days. The statistical analy-sis indicated that the difference was significant (P＜0.05). Conclusion RNA interference targeting HIF-1α gene could suppress the proliferation of human tongue cancer cells effectively. HIF-1αgene could be researched as a new therapeutic target of tongue cancer in the future.