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N 6-methyladenosine (m 6A) is the most abundant epigenetic modification in eukaryotic messenger RNA (mRNA), which could be catalyzed by m 6A methyltransferase (Writers), recognized by methylation recognition enzymes (Readers), and removed by demethylase (Erasers). RNA splicing, translation, and stability could be modulated by m 6A methylation modification. The m 6A methylation modification is involved in the biological regulation of a variety of important functional genes in cellular activities. Importantly, abnormal m 6A modification affects the occurrence, development, metastasis and recurrence of tumors. Ionizing radiation can affect the level of m 6A and m 6A methylation-related enzymes. Recently, m 6A methylation is reported to regulate the efficacy of tumor radiotherapy by affecting DNA damage and radiosensitivity of tumor cells. In addition, ionizing radiation can also affect the level of m 6A modification in normal cells to regulate the progress of radiation-induced injuries. This review summarizes the research progress on the roles of m 6A methylation in tumor radiosensitivity and radiation-induced injuries, with the aim of providing novel strategies for the development of clinical tumor radiosensitizers and radioprotective agents.
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Objective:To investigate the effects and mechanisms of copper transporter 1 (CTR1) in radiation induced intestinal injury in vitro. Methods:Human small intestinal epithelial cells (HIEC) were irradiated with 2, 4, 6, 8 Gy of X-rays and rat intestinal epithelial cells (IEC-6) were irradiated with 5, 10, 15, 20 Gy of X-rays. At 2, 4, 8, 24, and 48 h after irradiation, the expression of CTR1 was detected by Western blot assay. In some experiments, HIEC and IEC-6 cells were transfected with CTR1 shRNA and then exposed to X-rays. Copper levels were detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The radiosensitivity of cells was verified by colonogenic assay, the cellular reactive oxygen species (ROS) level and DNA damage were detected to further explore the related mechanism. In addition, Western blot was applied to detect the expressions of antioxidants and cuproptosis associated proteins in enterocytes after silencing CTR1 or irradiation.Results:The expression of CTR1 was increased by X-ray irradiation in a dose-dependent manner ( t=3.53, 3.45, 6.37, 11.11, 11.13, P<0.05). CTR1 expression was successfully diminished by CTR1 shRNA adenovirus vectors. According to the survival curves, the enhancement ratios of the radiosensitivity of HIEC and IEC-6 cells with CTR1 knocking-down were 1.146 and 1.201, respectively. Radiation-induced copper accumulation was alleviated after CTR1 silencing in IEC-6 cells ( t=3.10, P<0.05). At 0.5 h after irradiation, the ROS production in the CTR1 knockdown group was significantly lower than that in the control group ( t=5.23, 2.96, P<0.05). At 1 h after irradiation, the protein expression of γ-H2AX in the CTR1 knockdown group was obviously lower than that in the control group ( t=7.50, 4.29, P<0.05). The expressions of Nrf2 and HO-1 were increased after irradiation, which could be further increased after CTR1 silencing. In addition, cuproptosis associated protein DLAT, LIAS and FDX1 were reduced post-irradiation, which were recovered after CTR1 silencing. Conclusions:The radioresistance of HIEC and IEC-6 cells was enhanced after CTR1 silencing, possibly through the intracellular ROS and cuproptosis pathway.
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Objective To investigate the prevalence and genotypes of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality. Methods A total of 145 fresh fecal samples were collected from pet dogs and cats in a pet hospital in Shanghai Municipality during the period from November 2021 to June 2022, including 99 dog fecal samples and 46 cat fecal samples. The small subunit ribosomal ribonucleic acid (SSU rRNA) gene of Cryptosporidium and the triose phosphate isomerase (TPI) gene of G. lamblia were amplified using nested PCR assay, and the positive amplification products were sequenced from both directions. The sequence assembly was performed using the software Clustal X 2.1, and sequence alignment was conducted using BLAST. A phylogenetic tree was created with the Neighbor-Joining method using MEGA 11.0 to identify parasite species or genotype. Results The overall prevalence of Cryptosporidium and G. lamblia was 20.00% (29/145) in 145 pet dog and cat fecal samples, with the prevalence of 0.69% (1/145) and 19.31% (28/145) in Cryptosporidium and G. lamblia, respectively. G. lamblia was only detected in dog fecal samples, with prevalence of 18.18% (18/99), while the detection rates of Cryptosporidium and G. lamblia were 2.17% (1/46) and 21.74% (10/46) in cat fecal samples. Nucleotide sequence analysis showed that one Cryptosporidium positive sample was characterized as C. felis, and 28 G. lamblia positive samples were all characterized as Giardia assemblage A, which showed 100% sequence homology with human isolates of Giardia. Phylogenetic analysis revealed that the sequences obtained in this study belonged to the same branch with the reported Giardia assemblage A. Conclusions Cryptosporidium and G. lamblia infection was prevalent in pet dogs and cats from the study pet hospital in Shanghai Municipality, and there is a zoonotic risk for the species and genotype. Intensified surveillance of Cryptosporidium and Giardia infection is recommended in pets and their owners, and improved management of pet keeping is required.
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Liver sinusoidal endothelial cells (LSECs) locate on the surface of hepatic sinusoids. As the first line of defense between the liver and blood, LSECs are the most abundant non-parenchymal cells in the liver. Under physiological conditions, LSECs may induce liver immune tolerance through participating in substance transport and metabolic waste removal, thereby maintaining liver homeostasis, and under pathological conditions, LSECs may promote liver immune response via antigen presentation. LSECs have been found to play a crucial regulatory role in maintaining the balance between liver regeneration and liver fibrosis. This article reviews the progress of researches on LSECs functions, LSECs changes in liver injury, signal pathways associated with regulation of LSECs functions, and the interaction between LSECs and other types of cells in the liver, aiming to elucidate the function of LSECs and their roles in liver diseases.
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Objective:To investigate the effects of rapamycin on the autophagy activation of M2 macrophages and the radiosensitivity in colorectal cancer xenograft.Methods:THP-1 cells were induced into Type-Ⅱ macrophages with PMA and/or IL-4. Rapamycin and Bafilomycin A1 were uesd to activate and suppress autophagy of M2 macrophage, respectively. Colorectal cancer LoVo cells were inoculated on BALB/c-nu/nu nude mice. After the xenograft tumor size approached to 10 mm in diameter, the nude mice were divided into the following groups randomly: M2 macrophage autophagy inactive group and active group, autophagy downregulation of the activated group, and nontreatment control group. The tumors in mice were irradiated with 8 Gy X-rays in two fractions, and the radiosensitivity of colorectal cancer xenograft in each group was analyzed.Results:The expression levels of M2 macrophage markers Arg-1 and CCL-22 were significantly higher than those in M0 macrophage. The tumor weight, volume [(1.93±0.05)g, (2.14±0.06)cm 3] and micro-vessel density (36.37±1.04) in M2 autophagy inactive group were higher than those in control group [(1.35±0.05)g, (1.77±0.02)cm 3, 25.69±1.34] ( t=20.07, 14.56, 10.92, P < 0.05). After activation of M2 autophagy, the tumor weight, volume and micro-vessel density were significantly decreased to (0.89±0.03)g, (1.24±0.01)cm 3, and 13.60±1.52 ( t=44.37, 40.32, 21.43, P < 0.05). After down-regulation of M2 autophagy with bafilomycin A1, the tumor weight, volume and micro-vessel density were increased to (1.02±0.07)g, (1.37±0.02)cm 3, and 21.06±1.41 ( t=4.67, 13.79, 6.23, P < 0.05). Autophagy inaction suppressed the expression of Livin and Survivin in tumor ( t=2.64, 7.90, P < 0.05), and the activation of M2 autophagy further down-regulated the expression of Livin, Survivin ( t=5.43, 9.39, P < 0.05). The expression levels of Livin and Survivin were increased after the treatment with bafilomycin A1 ( t=2.80, 3.17, P<0.05). Conclusions:M2 macrophagy promoted the growth of colorectal cancer xenograft by inducing the formation of micro-vessels in the tumor, which is one of the mechanisms of tumor-associated macrophages participating in the radiotherapy resistance of colorectal cancer. Activation of M2 autophagy by rapamycin inhibited the ability of M2 macrophagy in promoting tumor growth, and induced apoptosis of colorectal cancer cells after radiotherapy by down-regulating the expression of anti-apoptotic genes Livin and Survivin, thus increased the radiosensitivity of colorectal cancer.
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Objective:To investigate whether irradiated U251 glioma cells can induce bystander effects in unexposed neural stem cells (NSCs) thus affecting its proliferation, stemness and differentiation.Methods:The cells were divided into NSCs group, NSCs+ U251 group (co-cultured with U251) and NSCs+ IR U251 group (co-cultured with 10 Gy irradiated U251). Glioma cells and NSCs were co-cultured in a transwell insert set. Cell counting and neurosphere diameter measuring were carried out to evaluate the proliferation and neurosphere formation ability of NSCs. Immunofluorescence assay was performed to detect the expression of Nestin protein to evaluate the stemness maintenance of NSCs, and to measure the expression levels of Tuj1 and GFAP proteins, the number of neuronal dendrites, synaptic length, the number of glial protrusions, as well as the length of glial protrusions.Results:The number of NSCs cultured with irradiated U251 cells was obviously smaller than that of NSCs cultured with sham-irradiated U251 cells ( t=2.52, P<0.05). The neurosphere formation ability of NSCs and the percentage of Nestin positive NSCs after co-culture with irradiated U251 cells significantly reduced in comparison with those after co-culture with sham-irradiated U251 cells ( t=-3.50, P<0.05). The percentages and the extent of NSCs differentiating into neuronal cells and glial cells( t=6.09, P<0.05)decreased obviously after co-culture with irradiated U251 cells in comparison with those after co-culture with sham-irradiated U251 cells. Conclusions:Irradiated glioma cells can significantly inhibit the proliferation, stemness and differentiation of unexposed NSCs due to bystander effect.
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Objective:To make comparisons of treatment outcomes between video-assisted thoracoscopic surgery (VATS) lobectomy and stereotactic body radiotherapy (SBRT) for early stage non-small cell lung cancer (NSCLC).Methods:Overall survival (OS), cancer specific survival (CSS), locoregional control (LRC), and disease-free survival (DFS) were retrospectively compared between early stage NSCLC patients who underwent VATS lobectomy and SBRT at our institution from January 2012 to December 2016. Propensity score matching (PSM) was carried out to reduce selection bias between two groups based on age, gender, Karnofsky performance score (KPS), Charlson comorbidity index (CCI), pulmonary function, and tumor diameter.Results:A total of 567 patients treated with VATS lobectomy ( n=458) or SBRT ( n=109) were included. 104 patients were matched for further analysis (52 in VATS lobectomy group and 52 in SBRT group). The median follow-up time was 44 months. the 3- and 5-year OS were 94.2% and 91.6% for VATS lobectomy and 88.6% and 79.9% for SBRT ( P=0.097), respectively. No statistically significant differences were noted in 5-year CSS (91.6% vs. 83.7%, P=0.270). The cumulative incidence of LRC was comparable between two group (94.0% and 85.9% vs. 93.5% and 93.5% at 3, 5 years, P=0.621). Differences in the DFS were not statistically significant (80.5% and 79.0% at 5 years, P=0.624). In the VATS lobectomy group, 10% patients ( n=5) experienced ≥ grade 3 CTCAE toxicity. One patient died of septicemia due to severe lung infection within 30 d after VATS lobectomy. In the SBRT group, one patient suffered from grade 3 radiation pneumonitis. There were no grade 4 or 5 toxicities in SBRT group. Conclusions:This propensity matched analysis suggests that SBRT can be an alternative option to VATS lobectomy for stage I-II NSCLC. Randomized trials are needed to evaluate the outcomes.
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Objective:To analyze the clinical efficacy of prednisone, hydroxychloroquine (HCQ) combined with plasmapheresis (PE) or not for the treatment of systemic lupus erythematosus (SLE) during pregnancy.Methods:Fourteen patients with SLE during pregnancy were analyzed. Totally 7 patients in the non-PE group were given prednisone and HCQ only while 7 patients in PE group were given prednisone and HCQ combined with PE. The fetus outcomes and clinical data, such as erythrocyte sedimentation tate (ESR), urine protein level, blood cell count and systemic lupus erythematosus disease activity index (SLEDAI) score before and after treatment at 3, 6, 12 months were used to evaluate the efficacy between the two groups. The comparison between groups was performed by repeated measures analysis of varianc (ANOVA).Results:Totally 11 patients delivered successfully in both groups while three of the 7 patients in the non-PE group had stillbirth. The 11 fetuses developed well and were born with an Apgar score of 8 or more at birth in both groups. There was a significant difference in ESR and platelet counts between the two groups ( F=7.838, P<0.05 ; F=32.269 , P<0.05). The ESR of the PE group was lower than that in the non-PE group at 3, 6 and 12 months after delivery, while the platelet count was higher than that in the non-PE group. Although there was no significant difference in the SLEDAI scores between the two groups ( F=2.816, P=0.119), the average of SLEDAI scores in the PE group was lower than that in the non-PE group at 3, 6 and 12 months after delivery. In addition, the urine protein of 7 patients in the PE group turned negative at 6, 12 months after delivery. In the non-PE group, urinary protein-positive patients were present in 3, 6, 12 months after delivery. Conclusion:PE in combination with oral prednisone and HCQ is a more effective than oral prednisone and HCQ alone for patients with active SLE during pregnancy, which reduces pregnancy loss and promote the patient's outcome.
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Objective@#To evaluate the parabiotic tissue protection concept in the repairment of acute radiation-induced skin injury.@*Methods@#Seven patients(3 males and 4 females) with acute radiation injury treated in the Department of Plastic Surgery of the Second Affiliated Hospital of Soochow University from February 2014 to January 2018. The ages of patients ranged from 45 to 76 years. The wound areas include perineum and buttock (n=3), chest(n=2), and thigh(n=2). In the early stage, subregional " sandwich" surgical dressing was used to protect the probiotic tissue. Two months later, the necrotic tissue was clearly demarcated, the debridement was underwent, and the parabiotic tissue was preserved as far as possible. Vacuum sealing drainage(VSD)was applied to cover and soak wound with normal saline to moisturize the wound and promote the benign transformation of ecological tissue. Ten days later, the granulation grown well, and the skin flaps and myocutaneous flaps with good blood supply were designed to repair the wounds. The VSD device was continued to be used, to drain effusion under flap and promote the growth of cystic cavity granulation, with the purpose to promote blood supply of the skin flap, perform the final biological cleaning effect on the parabiotic tissue of the wound surface, promote the benign transformation of parabiotic tissue, and reduce the further necrosis.@*Results@#Seven patients with Ⅳ degree acute radiation-induced injury wounds were treated 6-10 weeks for surgery preparation, and 2-4 weeks for VSD-application after debridement. Except for part of flap was necrotized on 10th day after the first operation in one patient, all the other patients achieved satisfied outcome in a surgery. There was no further radiation-induced ulcer occurred during the 0.5-3 years of follow-up.@*Conclusions@#The concept of parabiotic tissue protection during preoperative, intraoperative and postoperative recovery phase can promote parabiotic tissue transformed to a good result after acute radiation injury, and reduce the size and depth of soft tissue necrosis, which can provide a good foundation for the secondary repair with flap and reduce complications.
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Cyclospora cayetanensis is a foodborne and waterborne pathogen that causes endemic and epidemic human diarrhea worldwide. A few epidemiological studies regarding C. cayetanensis infections in China have been conducted. During 2013, a total of 291 stool specimens were collected from patients with diarrhea at a hospital in urban Shanghai. C. cayetanensis was not detected in any of the stool specimens by traditional microscopy, whereas five stool specimens (1.72%, 5/291) were positive by PCR. These positive cases confirmed by molecular technology were all in the adult group (mean age 27.8 years; 2.94%, 5/170) with watery diarrhea. Marked infection occurred in the rainy season of May and July. Sequence and phylogenetic analyses of the partial 18S rRNA genes of C. cayetanensis isolated showed intra-species diversity of this parasite. This study showed, for the first time, that C. cayetanensis is a pathogen in outpatients with diarrhea in Shanghai, albeit at a low level. However, the transmission dynamics of this parasite in these patients remain uncertain.
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Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , China , Epidemiology , Cyclospora , Genetics , Cyclosporiasis , Epidemiology , Diarrhea , Parasitology , Feces , Parasitology , Outpatients , Phylogeny , Polymerase Chain Reaction , RNA, Ribosomal, 18S , Retrospective StudiesABSTRACT
Myofibrillogenesis regulator-1 (MR-1) is a novel protein involved in cellular proliferation, migration, inflammatory reaction and signal transduction. However, little information is available on the relationship between MR-1 expression and the progression of atherosclerosis. Here we report atheroprotective effects of silencing MR-1 in a model of Ang II-accelerated atherosclerosis, characterized by suppression focal adhesion kinase (FAK) and nuclear factor kappaB (NF-κB) signaling pathway, and atherosclerotic lesion macrophage content. In this model, administration of the siRNA-MR-1 substantially attenuated Ang II-accelerated atherosclerosis with stabilization of atherosclerotic plaques and inhibited FAK, Akt, mammalian target of rapamycin (mTOR) and NF-kB activation, which was associated with suppression of inflammatory factor and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in Ang II-treated vascular smooth muscle cells (VSMCs) and macrophages: siRNA-MR-1 inhibited the expression levels of proinflammatory factor. These studies uncover crucial proinflammatory mechanisms of Ang II and highlight actions of silencing MR-1 to inhibit Ang II signaling, which is atheroprotective.
Subject(s)
Animals , Mice , Angiotensin II , Angiotensins , Arteries , Atherosclerosis , Cell Proliferation , Focal Adhesion Protein-Tyrosine Kinases , Gene Expression , In Vitro Techniques , Macrophages , Muscle Development , Muscle, Smooth, Vascular , NF-kappa B , Plaque, Atherosclerotic , RNA, Small Interfering , Signal Transduction , SirolimusABSTRACT
Objective To observe the destroyed architecture of splenic lymphoid follicles in mice infected with Schistosoma japonicum by immunohistochemistry. Methods The mice infected with S. japonicum(20 cercariae/mouse)for 8 weeks were sacrificed,and the splenic samples were paraffin embedded and sliced. The sections were first stained by hematoxylin and eosin to observe the massive structure of splenic lymphoid follicles,and then B cells,follicular dendritic cells(FDC)and germinal center cells were labeled with anti-B220,anti-CD21 or anti-Ki67 antibodies respectively by immunohistochemistry to observe the distribution of the specific cells of lymphoid follicles. Results The results of HE staining showed that the structure of lym-phoid follicles in spleens of infected mice was blurred,the number and area of follicles were significantly reduced compared to those of the normal mice. The immunohistochemical staining showed that the splenic T/B lymphocyte segregation ,FDC network and germinal centers of the infected mice all disappeared. Conclusion The structure of splenic lymphoid follicles in the mice infected with S. japonicum is obviously damaged.
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Cryptosporidium spp.are protozoan parasites that infect the epithelial cells of the gstrointestinal tract of hosts.In humans,cryptosporidiosis is usually a self-limiting infection in immunocompetent individuals,but severe diarrhea and dissemination to extra-intestinal sites can occur in high-risk individuals,such as the very young,the elderly,immunedeficiency individuals,particularly in HIV-positive patients.So far,molecular epidemiological data have confirmed the presence of 30 species and over 40 genotypes with genus Cryptosporidium,with 21 species and genotypes being found in humans.The majority of human cryptosporidiosis cases are responsible for C.hominis and C.parvum.Human cases caused by C.meleagridis,C.ubiquitum,C.felis and C.canis have been increasing.Besides that,with data accumulation of molecular epidemiology of human cryptosporidiosis,some more Cryptosporidium species and genotypes were newly identified in humans.This paper mainly reviews epidemiology status of these new emerging Cryptosporidium species and genotypes in humans.
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@#Giardia lamblia is an important intestinal protozoan which can cause diarrhea in humans. The detection of Giardia infection is performed through the detection methods of pathogen，immunoassay and molecular biology. Currently，the immunodiagnostic methods have good application and development prospect because of high sensitivity and specificity，simple and convenient，and time saving. In this article，we review the main progress and application of immunodiagnostic methods for Giardia infection.
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@#To preliminarily study the pro⁃angiogenic activity of Echinococcus granulosus hydatid cysts against hu⁃ man umbilical vein endothelial cells in vitro and the transcriptional level of potential pro⁃angiogenic factors. Methods The hydatid cysts and protoscolex derived from experimentally infected mice were collected and cultured in vitro，then the human umbilical vein endothelial cells were stimulated by the supernatant and cyst fluid respectively，and the angiogenesis was observed and analyzed through a microscope and the angiogenesis mode of the software NIH Image J. Meanwhile，the mouse homologous proteins of matrix metalloproteinase⁃9（MMP⁃9）and high mobility group box B1（HMGB1）were identified in E. granulosus genome through sequence alignment，and their transcriptional levels in the cyst wall and protoscolex were analyzed. Results The culture supernatant of hydatid cysts significantly promoted human umbilical vein endothelial cells into tubes（F = 73.03，P < 0.001），the transcriptions of MMP⁃9 and HMGB1 were detected in the cyst wall and protoscolex，and the transcriptional level of MMP⁃9 was higher in protoscolex（t = -11.65，P < 0.001），while the level of HMGB1 was higher in hydatid cysts（t = 6.43，P = 0.003）. Conclusion Some parasite⁃derived pro⁃angiogenic molecules may exist in the supernatant of E. granulosus hydatid cysts，while further researches are required into their exact mechanisms.
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Objective To clone and express the thioredoxin(Trx)from RH strain tachyzoites of Toxoplasma gondii,estab?lish the prokaryotic expression vector and purify the recombinant protein,then produce the polyclonal anti?Trx antibody in rab?bits. Methods Trx fragment was amplified by PCR and cloned into the pET?28a(+)vector,and the recombinant protein was in?duced with IPTG and purified by Ni?NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting. Results The trx gene was amplified from T. gondii cDNA by PCR. The recombinant plasmid trx/pET?28a(+)was use?fully constructed,and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also ob?tained. The specific band of TRX was detected by Western blotting. Conclusion Western blotting can detect the specificity of polyclonal anti?Trx antibody,which will facilitate the biological functions of Trx.
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Objective To explore the role of p16 gene methylation in fibroblasts in the occurrence and development of keloid.Methods Skin tissue specimens were resected from the lesions of patients with keloid and normal skin of healthy human controls.Fibroblasts were isolated from these tissue specimens and subjected a primary culture.An immunohistochemical analysis was performed to measure the expression of p16 protein in tissue specimens,real-time fluorescence-based quantitative PCR to determine the mRNA expression level (expressed as 2-△△Ct) of p 16 and DNA methyltransferases (DNMTs) in fibmblasts,and bisulfite sequencing PCR (BSP) to estimate the methylation status of p16 gene in the tissue specimens and primary fibroblasts.Results The keloid fibroblasts (KFbs) showed significandy lower mRNA expression of p16 gene (0.64 ± 0.18 vs.1.92 ± 0.23,t =10.54,P＜ 0.05),but significantly higher mRNA expressions of 3 DNMTs (DNMT1:2.58 ± 0.23 vs.1.13 ± 0.21,t =11.22,P ＜ 0.05; DNMT3A:4.87 ± 0.46 vs.2.38 ± 0.32,t =10.81,P＜ 0.05; DNMT3B:1.57 ± 0.12 vs.0.57 ± 0.16,t =12.45,P＜ 0.05) compared with the normal fibmblasts (NFbs).The DNA methylation rate in the p16 gene promoter region was significantly increased in keloid tissue (1.81％ ± 0.46％) and KFbs (3.15％ ± 0.94％) compared with normal skin tissue (0.90％ ± 0.35％,F =14.23,P＜ 0.01) and NFbs (0.17％ ± 0.29％,F=37.62,P＜ 0.01).Conclusions The methylation and low expression of p16 gene in KFbs may be associated with the uncontrolled growth of keloid,and DNMTs may play a role in the pathogenesis of keloid.
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Objective To separate and purify intrahepatic macrophages from Microtus fortis Mf and identify its phagocy?tosis. Methods The intrahepatic macrophages from Mf were separated and purified by perfusion collagenase digestion and density gradient centrifugation. The function of the cells was identified by FACS analysis and ink phagocytosis activity. Results The macrophage cells from the liver of Mf were obtained. These cells were bright and circular and grew adhering to the wall. The proportion of the living cells was 95%. The binding rate of these cells from Mf with anti?mouse CD14 antibody Clone Sa2?8 was about 50%of the rate of macrophage from C57BL/6 mice with this monoclonal antibody. The result of ink?phagocytosis ex?periment of macrophage cells from the liver of Mf was positive. Conclusion The method above mentioned is useful to separate and purify macrophage from the liver of Mf. The study builds the foundation for further research on macrophages of Mf against Schistosoma japonicum.
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Objective To evaluate the therapeutic effect of NeuroD protein on radiation-induced intestinal injuries.Methods The expression and purification of NeuroD-enhanced green fluorescent protein (EGFP) fusion protein was performed in prokaryotic expression system.The efficiency of the fusion protein transduction into cells was monitored under fluorescence microscope.C57BL/6J mice were randomly divided into four groups with 10 mice in each group:normal control group,PBS group,EGFP group,and NeuroD-EGFP group.Besides the normal control group,the other three groups of mice received 9 Gy γ-ray total body irradiation.Intestinal tissues were collected,frozen sections were prepared to monitor the distribution of NeuroD in mice intestinal tract under fluorescence microscope,and pathological sections were prepared for H&E staining to evaluate the therapeutic effect of NeuroD protein.Results The NeuroD-EGFP fusion protein was purified by Ni-NTA column and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Visible green fluorescence gathered within the cells after NeuroD-EGFP fusion protein was added in the culture medium,suggesting that NeuroD-EGFP could penetrate the cell membrane into the cells.Five hours after intraperitoneal injection of NeuroD-EGFP,visible green fluorescence gathered within the intestinal epithelial cells in villi.At 3.5 d after irradiation,NeuroD-EGFP treated mice showed significantly higher villus (F =49.49,P ＜ 0.01) and crypt depth (F =16.72,P ＜ 0.01) and more crypts per circumference (F =10.32,P ＜ 0.01) compared with PBS and EGFP groups.Conclusion NeuroD protein can accelerate the post-irradiation recovery of injured villi and crypt of intestinal tract and could be used to treat radiation-induced intestinal injuries.
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Objective To study the proliferation,collagen production and related gene expression in keloids and normal skin fibroblast.Methods Isolated primary cells of keloid fibroblasts (KFb,n=12) and normal human dermal fibroblasts (NFb,n=12) were identified,the cell viability and proliferating potential and the cell cycle were detected,and the difference on the collagen synthesis between KFb and NFb were compared.The expression of cell cycle-associated genes such as p21,p16,and p27 was dectected by real-time fluorescent quantitative PCR.Results The phase contrast optical microscopy imaging showed that both KFb isolated from keloid tissues and NFb from normal skin tissues possessed classic and similar fibroblast morphology.But there was a significant difference between cell proliferation,Hyp [(2.30±0.10) μg/ml vs.(1.66±0.13) μg/ml,P＜0.05] and collagen levels [(17.19±0.75) μg/ml vs.(12.37±0.94) μg/ml,P＜0.05].Compared with NFb,KFb exhibited more percentage of G2/M phase cells [(5.90±0.62)％ vs.(16.94 ％±1.93)％,P＜0.05]and less percentage of G0/G1 phase cells [(90.24 ±2.27)％ vs.(75.65±1.92)％,P＜0.05].Cell cycle related genes p16,p21 and p27 were low expressed.Collagen type Ⅰ was highly expressed at mRNA levels in KFb than that in NFb [0.84±0.11,1.32±0.2,1.69±0.12,4.33±0.27 in KFb vs.1.43±0.13,2.56±0.26,2.89±0.37,1.40±0.12 in NFb,P＜0.05].Conclusions There are cell dysfunction and abnormal cellular dynamics in keloid fibroblasts.The formation of keloid likely involves aberrant interactions of some genes that affected its development at different extents.