RESUMO
Objective To observe the effects of Bushen Zhuyun Prescription on regulating mitochondrial function and endometrial angiogenesis;To explore its mechanism of improving endometrial receptivity.Methods The mouse model of controlled ovarian hyperstimulation(COH)was established,and the mice were randomly divided into normal group,model group,and Bushen group,with 20 mice in each group.Bushen group received Bushen Zhuyun Prescription for gavage for 11 d,and the normal group and model group received normal saline for gavage.The number of embryo implantation was counted,the endometrial morphology was observed by HE staining,α-smooth muscle actin(α-SMA)expression was observed by immunofluorescence staining.Human endometrial microvascular endothelial cells(HEMECs)were cultured in vitro,they were divided into control group,VEGFA group,Bushen group and VEGFA + Bushen group,and were intervened with VEGFA and/or Bushen Zhuyun Prescription medicated serum.The activities of mitochondrial respiratory chain complexes Ⅰ-Ⅳ,the content of ATP,the expression of PCNA and Caspase-3 were detected.Results Animal experiment showed that,compared with the normal group,the number of embryo implantation in model group significantly decreased(P<0.05),α-SMA protein expression in endometrial tissue significantly decreased(P<0.05);compared with the model group,the number of embryo implantation in Bushen group significantly increased(P<0.05),α-SMA protein expression in endometrial tissue significantly increased(P<0.05).Cell experiment showed that,Bushen Zhuyun Prescription medicated serum could increase the activity of mitochondrial respiratory chain complexes Ⅰ-Ⅳ and ATP content in HEMECs,promote PCNA protein expression,and inhibit Caspase-3 protein expression(P<0.05,P<0.01).Conclusion Bushen Zhuyun Prescription can promote endometrial angiogenesis through improving mitochondrial function.
RESUMO
Parkinson's disease (PD) is a kind of chronic progressive neurodegenerative disease that has a high prevalence rate in recent years, especially in the elderly. PD belongs to the category of "tremor disease" and "tremor" in traditional Chinese medicine, and Tianma Goutengyin is a classic prescription contained in the Synopsis of The New Significance of Patterns and Treatment in Miscellaneous Diseases(《中医内科杂病证治新义》). This article explored the theory of Tianma Goutengyin in the treatment of PD, and based on network pharmacological research, the article summarized relevant research on Tianma Goutengyin and its single herb in the treatment of PD. Moreover, it discussed the clinical applications and mechanisms of Tianma Goutengyin and its single herb in the treatment of PD. It is found that the mechanisms of Tianma Goutengyin in treating PD may be related to resisting oxidative stress, inhibiting inflammatory response, regulating neurotransmitters, and protecting dopamine (DA) neurons. Besides, the main components of the single herb in Tianma Goutengyin for treating PD are gastrodin, rhynchophylline, geniposide, gardenial alcohol, eucommitol glycoside, motherwort alkaloid, baicalin, pachyman, and achyranthes bidentata sterol. They can improve the related symptoms of PD patients by inhibiting inflammatory response, resisting oxidative stress, affecting calcium ion concentration, restoring mitochondrial function, and and protecting DA neurons. This article summarized the research progress of Tianma Goutengyin and its single herb in treating PD, so as to provide a reference for the prescription and medication of Tianma Goutengyin in the treatment of PD and subsequent research on the mechanisms of Tianma Goutengyin and its single herb in the treatment of PD and give play to the advantages of traditional Chinese medicine in the treatment of PD.
RESUMO
Heart failure with preserved ejection fraction (HFpEF) displays normal or near-normal left ventricular ejection fraction, diastolic dysfunction, cardiac hypertrophy, and poor exercise capacity. Berberine, an isoquinoline alkaloid, possesses cardiovascular benefits. Adult male mice were assigned to chow or high-fat diet with L-NAME ("two-hit" model) for 15 weeks. Diastolic function was assessed using echocardiography and noninvasive Doppler technique. Myocardial morphology, mitochondrial ultrastructure, and cardiomyocyte mechanical properties were evaluated. Proteomics analysis, autophagic flux, and intracellular Ca2+ were also assessed in chow and HFpEF mice. The results show exercise intolerance and cardiac diastolic dysfunction in "two-hit"-induced HFpEF model, in which unfavorable geometric changes such as increased cell size, interstitial fibrosis, and mitochondrial swelling occurred in the myocardium. Diastolic dysfunction was indicated by the elevated E value, mitral E/A ratio, and E/e' ratio, decreased e' value and maximal velocity of re-lengthening (-dL/dt), and prolonged re-lengthening in HFpEF mice. The effects of these processes were alleviated by berberine. Moreover, berberine ameliorated autophagic flux, alleviated Drp1 mitochondrial localization, mitochondrial Ca2+ overload and fragmentation, and promoted intracellular Ca2+ reuptake into sarcoplasmic reticulum by regulating phospholamban and SERCA2a. Finally, berberine alleviated diastolic dysfunction in "two-hit" diet-induced HFpEF model possibly because of the promotion of autophagic flux, inhibition of mitochondrial fragmentation, and cytosolic Ca2+ overload.
Assuntos
Masculino , Camundongos , Animais , Insuficiência Cardíaca/tratamento farmacológico , Volume Sistólico/fisiologia , Função Ventricular Esquerda/fisiologia , Berberina/uso terapêutico , Modelos Animais de Doenças , Dinâmica Mitocondrial , Miocárdio , HomeostaseRESUMO
Clinical application of doxorubicin (DOX) is heavily hindered by DOX cardiotoxicity. Several theories were postulated for DOX cardiotoxicity including DNA damage and DNA damage response (DDR), although the mechanism(s) involved remains to be elucidated. This study evaluated the potential role of TBC domain family member 15 (TBC1D15) in DOX cardiotoxicity. Tamoxifen-induced cardiac-specific Tbc1d15 knockout (Tbc1d15CKO) or Tbc1d15 knockin (Tbc1d15CKI) male mice were challenged with a single dose of DOX prior to cardiac assessment 1 week or 4 weeks following DOX challenge. Adenoviruses encoding TBC1D15 or containing shRNA targeting Tbc1d15 were used for Tbc1d15 overexpression or knockdown in isolated primary mouse cardiomyocytes. Our results revealed that DOX evoked upregulation of TBC1D15 with compromised myocardial function and overt mortality, the effects of which were ameliorated and accentuated by Tbc1d15 deletion and Tbc1d15 overexpression, respectively. DOX overtly evoked apoptotic cell death, the effect of which was alleviated and exacerbated by Tbc1d15 knockout and overexpression, respectively. Meanwhile, DOX provoked mitochondrial membrane potential collapse, oxidative stress and DNA damage, the effects of which were mitigated and exacerbated by Tbc1d15 knockdown and overexpression, respectively. Further scrutiny revealed that TBC1D15 fostered cytosolic accumulation of the cardinal DDR element DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Liquid chromatography-tandem mass spectrometry and co-immunoprecipitation denoted an interaction between TBC1D15 and DNA-PKcs at the segment 594-624 of TBC1D15. Moreover, overexpression of TBC1D15 mutant (∆594-624, deletion of segment 594-624) failed to elicit accentuation of DOX-induced cytosolic retention of DNA-PKcs, DNA damage and cardiomyocyte apoptosis by TBC1D15 wild type. However, Tbc1d15 deletion ameliorated DOX-induced cardiomyocyte contractile anomalies, apoptosis, mitochondrial anomalies, DNA damage and cytosolic DNA-PKcs accumulation, which were canceled off by DNA-PKcs inhibition or ATM activation. Taken together, our findings denoted a pivotal role for TBC1D15 in DOX-induced DNA damage, mitochondrial injury, and apoptosis possibly through binding with DNA-PKcs and thus gate-keeping its cytosolic retention, a route to accentuation of cardiac contractile dysfunction in DOX-induced cardiotoxicity.
RESUMO
Objective:To explore the team construction and treatment strategy of the Diabetic Foot-Multidisciplinary Team.Methods:The clinical data of 19 patients with severe ischemic diabetic foot treated by our Diabetic Foot-Multidisciplinary Team Center from Apr 2021 to Mar 2022 were collected, and the overall amputation rate, above-ankle major amputation rate, minor amputation rate and mortality, Diabetic Foot-Multidisciplinary Team consultation discipline participation rate and treatment participation degree were retrospectively analyzed.Results:Nineteen patients (15 males and 4 females) were enrolled, aged 26 to 94 (68.6±14.2). All were with severe ischemic diabetic foot ulcer:Rutherford grade 5 or up and dysfunction in 2 or more organs. Complications included arteriosclerosis obliterans of the lower extremities in 18 cases, heart diseases in 18, hypertension in 15, and renal insufficiencies in 10. The overall amputation rate was 36.8%, major amputation rate in 21.1%, minor amputation rate in 15.8%, and mortality rate was 15.8%. A total of 16 disciplines participated in Diabetic Foot-Multidisciplinary Team; the main participating disciplines were vascular surgery (19 times), endocrinology (12 times), and cardiology (11 times). The main treatment disciplines were vascular surgery (14 times), plastic surgery (3 times), and cardiology (2 times).Conclusion:For the diagnosis and treatment of diabetic foot, it is necessary to set up a multidisciplinary team as early as possible to control the causes of diabetic foot ulcer, prevent the recurrence of diabetic foot ulcer, reduce the mortality and amputation rate, and improve the quality of life of patients.
RESUMO
Objective@#To detect the effect and regulatory mechanism of human ether à go-go related gene 1 (Herg 1) knockdown on the proliferation and invasion of osteosarcoma (OS).@*Methods@#We constructed a recombinant adenovirus vector (Ad5-Herg1-shRNA) expressing short hair RNA (shRNA) against Herg1 and tested the knockdown efficiency. Then, the effects of Herg 1 knockdown on the proliferation, growth and invasion of osteosarcoma were measured by using cell counting kit-8 (CCK-8), wound healing assay, Transwell assay and xenograft model of nude mice, respectively. Tandem affinity purification, mass spectrometry and dual luciferase reporter assay were used to find out the molecules interacted with Herg1. Western blot was used to detect the expressions of large tumor suppressor gene (LATS1), p-LATS1, Yes-associated protein (YAP) and p-YAP in cells after infection of Ad5-Herg1-shRNA.@*Results@#Compared to Ad5-control-shRNA, Ad5-Herg1-shRNA dramatically inhibited the expression of Herg1 in OS cells. The result of CCK8 array demonstrated that 143B cell vitalities of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (65.47±3.90)% and (79.90±1.52)%, significantly lower than (100.00±6.14)% of Ad5-control-shRNA group. Meanwhile, U2OS cell vitality of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (69.69±1.36)% and (76.72±2.75)%, significantly lower than (100.00±3.01)% of Ad5-control-shRNA group (all P<0.001). The results of wound healing array showed that 143B cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (33.03±2.88)% and (36.47±4.16)%, significantly lower than (97.78±2.28)% of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (68.07±0.90)% and (73.97±1.25)%, significantly lower than (96.50±1.12)% of Ad5-control-shRNA group (all P<0.001). The results of Transwell showed that 143B cell invasion numbers of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 36.50±12.15 and 44.83±7.62, significantly lower than 195.33±19.68 of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 21.83±7.99 and 22.85±7.08, significantly lower than 83.33±12.36 of Ad5-control-shRNA group (all P<0.001). The results of xenograft model of OS showed that the tumor volume and weight of Ad5-Herg1-shRNA group were significantly smaller than of Ad5-control-shRNA group after 14 days and 5 weeks of inoculation, respectively (P<0.001). Moreover, knockdown of Herg1 inhibited the metastasis of OS cells. In mechanism, Herg1 protein interacted with NF2 protein. Knockdown of Herg1 significantly suppressed the expression levels of LATS1 and YAP protein, and promoted the phosphorylation of LATS1 and YAP in OS cells (all P<0.001).@*Conclusion@#Our findings suggest that Herg1 participates in the proliferation and motility of OS cells and may serve as a potential therapeutic target for osteosarcoma patients.
RESUMO
Objective To study the infection rate of fusobacterium nucleatum cancer re appeared in patients with colorectal cancer before and after radiotherapy,and the changes after cancer recarrence.Methods A total of 20 persons receiving physical examination were recruited in the control group and collected the stool specimens,and 40 colorectal cancer patients were selected in the study group.All of the subjects in the study group were collected stool specimens before operation 3 days and after operation 5 day,after radiation therapy 7 days and 30 days.The patients were followed-up 1 year.The bacterial fluid was collected by filtration,and real-time fluorescence quantitative PCR was used to detect the expression of fusobacterium nucleatum gene in feces.Results The positive rate of fecal fusobacterium fusiformis was 30% in the study group and 5% in the control group.The gene relative expression of 12 colorectal cancer patients before operation 3 days and after operation 5 days,after radiation therapy 7 days and 30 days were 5.20±0.34,8.50±0.45,1.20±0.22,0.20±0.15.The fusobacterium nucleatum gene expression of 12 patients with positive fusobacterium after operation 5 days was significantly increased compared with that before operation 3 days(t=10.419,P=0.001),which after radiation therapy 7 days and 30 days was significant lower than that before operation 3 days(t=12.728,P=0.001;t=25.889,P=0.001).Six patients recurred among 1 year,the fusobacterium nucleatum gene expression was 7.2±0.56,which was significant higher than that after radiation therapy 7 days.Conclusion The infection of fusobacterium nucleatum might be a risk factor for colorectal cancer,and the gene relative expression might be an early warning indicator of recurrence.
RESUMO
Tumor derived microparticles are released by activated or apoptotic tumor cells.They are ex-tracellular vesicles which are 0.1~1.0μm in diameters.Tumor derived microparticles carry abundant bioactive molecules,such as nucleic acids and proteins,which resemble that of the parental cell.In this review,we summa-rize the role of tumor derived microparticles in the occurrence,development,diagnosis and treatment of tumor.
RESUMO
OBJECTIVE To investigate the arsenic trioxide(As2O3)-induced oxidative damage to mitochondrial DNA (mtDNA) in mouse oocytes and possible mechanisms. METHODS ① For in vitro assay,the mouse oocytes were denuded from ovaries of normal mice and incubated in medium for 20 h in different treatment groups:control,As2O3 1 and 2 μmol · L- 1,N-acetylcysteine (NAC) 5 mmol · L-1, 2,2,6,6-tetramethyl-1-piperidinyloxy(Tempo)1 mmol · L-1, As2O3(1 and 2 μmol · L-1)+NAC 5 mmol · L-1,As2O3 (1 and 2 μmol · L-1)+Tempo 1 mmol · L-1. ② For in vivo assay,mice were subjected to ip injection with physiological saline (normal control),As2O3 1 and 2 mg · kg-1,or As2O3 (1 and 2 mg · kg-1)+NAC 200 mg · kg-1, respectively. After 60 d,all the mice were sacrificed and their ovaries were quickly excised. Intracellular reactive oxygen species(ROS) levels were determined by 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The oxidative damage to mtDNA was induced using enzyme-linked immunosorbent assay(ELISA)for 8-hydroxy-2′-deoxyguanosine(8-OHdG). The expression of DNA polymerase γ(Polγ)and mitochondrial transcription factor A(mtTFA)was detected by Western blotting and the vitality of lysosomes was monitored by β-galactosidase(β-Gal)Assay Kit. RESULTS ①In vitro experiments,As2O3 elevated 8-OHdG levels of mtDNA in mouse oocytes accom? panied by increased levels of ROS (P<0.05),but co-treatment with NAC or Tempo significantly reduced ROS and 8- OHdG levels (P<0.05). Meanwhile, the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05),but were markedly elevated by the addition of NAC or Tempo (P<0.05). ②In vivo assay,As2O3 elevated ROS as well as 8-OHdG levels of mtDNA in mouse oocytes,while the expression levels of Pol γ and mtTFA were down-regulated by As2O3(P<0.05). Co-treatment with NAC significantly reduced ROS and 8-OHdG levels,but markedly elevated Pol γ and mtTFA levels(P<0.05). Besides,a notable increase in β-Gal activity was shown in As2O3-treated mouse oocytes in vitro (P<0.05),while antioxidants efficiently reduced the activity (P<0.05). However,no significant changes were observed in the in vivo study. CONCLUSION The oxidative damage to mtDNA induced by As2O3 in mouse oocytes may be mediated by ROS and associated with down-regulation of protein levels of Pol γ and mtTFA as well as increment of lysosomal activity.
RESUMO
Objective To summarize the nursing methods and effect of artificial femoral head replacement in treatment of osteoporotic interttochanteric fracture in elderly patients. Methods 55 patients with esteoporotic intertrochanteric fracture were selected in our hospital from February 2007 to February 2011.All patients were cared by targeted preoperative and postoperative nursing,helping them conducting functional training in time,giving discharge counseling and follow-up.The hip functional recovery of patients was evaluated by using the Harris evaluation standard,and the excellent rate of surgery was also evaluated. Results After the targeted care,55 patients went through the perioperative period safely,the average length of stay was ( 14.0 ± 3.1 )days,a total of 5 cases (9.09%)patiants suffered varying degrees of complications which were improved after symptomatic treatment.Patients were followed up for 3 to 36 months,only 2 patients died of cerebral vascular accident,all patients gained good recovery of hip function.Harris score was (84.45 ± 9.38),the excellent rate of surgery reached 78.18%. Conclusions Atificial femoral head replacement proves significant effect in treatment of osteoporotic intertrochanteric fractures in elderly patients,but with the accompanying surgical compliuations,special care and timely functional exercise methods should be paid attention to.
RESUMO
Epidemiology,as a major subject in the field of public health science,plays a pivotal role in the construction and development of disease prevention and control system.It is also vital for the public health system to improve the emergency response ability and to cultivate high-quality talents.After analyzing current situation of graduate education of epidemiology,we found some problems.In our research,deepgoing dissection was carried out and possible solution was provided.
RESUMO
Objective To investigate the reason,diagnosis and treatment of tension-free gastric disease after subtotal gastrectomy (delayed gastric emptying). Methods To analyze the diagnosis and treatment of 32 cases gastric remnant after resection of gastric emptying in our surgery department from 1990 to 2007. Results Residual postoperative delayed gastric emptying occurred in 4~8d;27 patients were cured with conservative treatment of 8 ~ 24d,13d on average;5 eases of hopelessness were transmitted to conservative treatment surgery and their residues found delayed gastric emptying were related to adhesion,and four eases of omental adhesions and mass oppression anastomotic jejunum were related to output the relevant paragraph. Conclusion Subtotal gastrectomy complicated by delayed gastric emptying residue are functional most of the time,which can be cured by conservative treatment,but if it not yet effective after more than 3 weeks of conservative treatment,the existence of mechanical obstruction should be taken into account,and stomach barium meal(GI) and gastroscopy help diagnosis clearly. Transit operations should be carried out as early as possible.
RESUMO
Objective To study the relationship between the oncosis of pancreatic acinar cells and activa-tion of macrophage in rat model of acute pancreatitis (AP). Methods The pancreatic acinar cells were isolated by two-step enzyme digestion, and then they were divided into control group, AP group and test group. Pancreatic acinar cells were cultured with caerulein in AP group, with caerulein and endothelin in test group, and with culture medium in control group. The oncesis rate of the pancreatic acinar cells was detected after acridine orange and ethidium bromide fluorescent staining. The supernatant was collected to detect the release of amylase and lactate dehydrogenase (LDH). The macrophages were cultured with 1 ml of supematant for 6 hours, and then the protein level of tumor necrosis factor-α (TNF-α) was measured by ELISA. Results Few oncotic pancreatic acinar cells were observed in the control group, and the levels of amylase and LDH secreted by pancreatic acinar cells and TNF-α secreted by macrophage were (1175±165)kU/L, (846±118)U/L and (36±5)μg/L, respectively. Oncotic pancreatic acinar cells were observed in AP group, and the levels of amylase, LDH and TNF-α were (7130±680) kU/L, (4262±626) U/L and (155±18) μg/L, respectively, which were significantly higher than those in control group (t = 5.184, 4.277, 3.665, P < 0.05). The levels of amylase, LDH and TNF-α were even higher in test group, and they were (9240±1177) kU/L, (6937±893)U/L and (268±35)μg/L, respectively, which were significantly higher than those in AP group (t = 2.251, 2.825, 2.843, P < 0.05). Conclusions The release of amylase was changed as the oncosis of pancreatic acinar cells occurred. The secretion of TNF-α was along with the degree of oncosis of pancreatic acinar cells. The results of the study indicate that a relationship exists among the inflammatory response of macrophage, the release of contents of pancreatic acinar cells and the oncosis of the pancreatic acinar cells.
RESUMO
BACKGROUND AND OBJECTIVE: A variety of cytokines are involved in the incidence and development of bronchial asthma. This study was designed to reveal the relationship among genetic polymorphism of cytokine genes, expression levels of cytokines and incidence of bronchial asthma. METHODS: We analyzed 14 single nucleotide polymorphism loci in the 10 major cytokine genes plus plasma protein levels of 7 proteins in the bronchial asthma patients (n = 108) and the healthy population (n = 88) of the Han people in northern China. The polymorphism-based genotypes were identified by the sequence-specific primer-polymerase chain reaction. The plasma protein levels were determined by enzyme-linked immunoassay. Bioinformatics analysis was applied to further data processing. RESULTS: Data presented here showed that the 6 polymorphism loci were significantly correlated with the incidence of asthma (p < 0.05). Two of them, IL-2 (-330) and IL-2 (+166), constituted a linkage disequilibrium block. The GG haplotype of this block gave a relatively higher correlation with asthma (p = 0.0767). The plasma protein levels of IgE, IL-6 and IL-1 beta correlated with a number of polymorphism loci tested (p < 0.05), of which IgE gave the most significant correlation. The plasma IL-10 and IL-12 levels of the patients in the asthma group were significantly lower than those of the healthy adults in the control group (p < 0.05), while IgE gave the opposite result (p < 0.0001). We constructed a prototype of the metabolic and regulatory network composed of bronchial asthma-related proteins. In this network, IL-6 and TNF-alpha, were found with a high degree (D = 343 and 235, respectively) and IL-1beta with a moderate degree of connection (D = 155). CONCLUSIONS: We have found that various degrees of correlation with cytokine genes and protein expression of single nucleotide polymorphism in bronchial asthma. IL-6 and IL-1beta appear to play important biological roles in the pathogenesis of asthma. During the analysis of correlation between genetic polymorphism and a complex disease, the effects of environmental factors should be taken into account. The information at the protein level should be fully developed and the bioinformatics techniques can be used for the comprehensive analysis, to have a deep understanding of molecular mechanisms of incidence and development of diseases.
Assuntos
Asma/epidemiologia , Asma/genética , Proteínas Sanguíneas/análise , Citocinas/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , China/epidemiologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Predisposição Genética para Doença , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Objective To express human chemokine-like factor 1 (CKLF1) in Drosophila S2 cells and study its function. Methods The pMT/V5-His-CKLF1 expression plasmid was constructed and transfected into Drosophila S2 cells. The positive clones were selected through PCR and RT-PCR. The culture medium was analyzed by Western blot with anti-CKLF1 polyclonal antibody. Chemotaxis and MTT assays on human peripheral blood and C2C12 cells, respectively, were then carried out with the medium. Results CKLF1 was transcribed efficiently in S2 cells. The expressed CKLF1 protein could be detected in the culture supernatant by Western blot, which showed weak chemotactic activity on both human peripheral blood neutrophils and lymphocytes as well as enhancing effect on the proliferation of C2C12 cells. Conclusion CKLF1 was expressed successfully in Drosophila S2 cells and secreted into the culture medium. The recombinant CKLF1 expressed in Drosophila cells can chemoattract leucocytes and promote the proliferation of C2C12 cells.
RESUMO
Objective: To develop a reporter gene system based on transient transfections with a NF-?B responsive reporter gene to detect the bioactivity of IL-1? and IL-1 receptor antagonist.Methods: NF-?B reporter and Dual-Luciferase assays were applied to measure the bioactivity of IL-1? and IL-1 receptor antagonist in mouse EL4 cells(some subclones of EL4 cells expressed high level of IL-1 receptor on cell surface).pNF-?B-luc and pRL-TK, used as an internal control,were co-transfected into EL4 cells and then the IL-1? was added.Results: The results indicated that IL-1? was able to induce the expression of this luciferase,which could be blocked by IL-1 receptor antagonist. The optimal dose of IL-1? was 5(?g/L) in Dual-Luciferase assay,whose bioactivity can be effectively inhibited by IL-1ra at 50 ?g/L.Conclusion: We have established a new method to detect the bioactivity of IL-1? and IL-1 receptor antagonist,which can give repeatable results.
RESUMO
Objective:To set up an effective and simple purification method to obtain highly purified prokaryotic protein of PDCD5 and study its stability. Methods:Recombinant PDCD5 protein expressed in E. coli was accumulated as an inclusion body. After washing, the inclusion body was denatured, renatured, digested with thrombine and then purified by two steps of chromatography. The purity of the products was analyzed by capillary electrophoresis and the stability was identified by SDS-PAGE. Results:Capillary electrophoresis showed that the purity of protein was 100%, and molecular weight was 15 800 with pI 5.9. Further bioactivity assay indicated that the purified PDCD5 could enhance the apoptosis of HL 60 cells withdrawing cytokine, which was in a dose dependent manner. Stability analysis showed that the PDCD5 protein was sensitive to temperature and easy to degrade at 4 ℃ and 25 ℃. However, it was relatively stable at -20 ℃ or lyophilized. Conclusion:Highly purified and stable recombinant PDCD5 protein was obtained, which lays a foundation for the functional study and application investigation of PDCD5 .
RESUMO
<p><b>BACKGROUND</b>To investigate expression of IL-6 in non?replicating vaccinia virus and its immune effects on recombinant virus.</p><p><b>METHODS</b>The recombinant non replicating vaccinia virus RVJ123 delta CK11 beta 75IL6 was constructed with non?replicating vaccinia virus vector pNEOCK11beta75IL6 and replicating vaccinia virus RVJ123. In animal model, immunization with the recombinant virus was carried out and its immune response was studied.</p><p><b>RESULTS</b>The recombinant virus could express IL-nd HBsAg simultaneously. Southern blot analysis demonstrated that the genes between vaccinia virus Hind? C and K fragments were deleted and IL-6 gene was integrated stably. Given intranasal inocula of the virus to immunize BALB/c mouse and New Zealand Rabbit, the elevated anti-HBsAg IgA and IgG antibody secreting cells in mouse lung lymphoid to vectors expressing IL-6 was at about two?fold higher level than those elicited by control virus at day 14 after immunization. Authors also could detect elevated anti-HBsAg IgA and IgG antibody conversion in mouse serum and lung fluid, rabbits serum, lung fluid, saliva, vagina and nasal washing samples.</p><p><b>CONCLUSIONS</b>IL-6 expressed by non-replicating recombinant vaccinia virus could enhance the induced?immune effects, it could serve as the effective adjuvant for recombinant vector vaccine.</p>
Assuntos
Animais , Embrião de Galinha , Feminino , Humanos , Camundongos , Coelhos , Adjuvantes Imunológicos , Células Cultivadas , Vetores Genéticos , Imunoglobulina A , Imunoglobulina G , Interleucina-6 , Genética , Alergia e Imunologia , Pulmão , Alergia e Imunologia , Camundongos Endogâmicos BALB C , Recombinação Genética , Vaccinia virus , Alergia e Imunologia , Metabolismo , Fisiologia , Replicação ViralRESUMO
<p><b>OBJECTIVE</b>To explore the effects of a novel human chemokine-like factor 1 (CKLF1) on stem cell/progenitor cells.</p><p><b>METHODS</b>Human bone marrow mononuclear cells were separated by Ficoll (1.077 g/ml), and cultured in suspension and semisolid colony culture. The effects of CKLF1 on CFU-GM and CFU-Mix were observed.</p><p><b>RESULTS</b>The number of CFU-GM was significantly increased in 10 groups by addition of CKLF1 plasmid supernatant. The mean value was 234.81 +/- 98.71/1 x 10(5) cells, 2.42 fold of control group (P < 0.05). The mean value of CFU-Mix in groups of negative control, CKLF1, PHA, GM-CSF and G-CSF were 82.00 +/- 20.25, 105.76 +/- 36.70, 146.63 +/- 27.09, 143.33 +/- 60.23 per 1 x 10(5) cells, respectively, no statistical differences were seen between control and CKLF1 groups. The CD(34)(+) cells were detected by FACS. The average percentage in the groups of normal control, CKLF1, PHA and GM-CSF were (0.75 +/- 0.62)%, (1.32 +/- 0.87)%, (0.15 +/- 0.02)%, and (0.29 +/- 0.23)%, respectively. Compared with control, no significant differences were seen between each group (P > 0.05).</p><p><b>CONCLUSION</b>Novel chemokine-like factor 1 can increase the proliferation of CFU-GM, which indicated that CKLF1 could increase the proliferation of human bone marrow hematopoietic progenitor cells and colony formation.</p>
Assuntos
Adulto , Humanos , Células Cultivadas , Quimiocinas , Farmacologia , Células-Tronco Hematopoéticas , Biologia Celular , Proteínas com Domínio MARVELRESUMO
Objective: in order to provide rapid and reliable method. Methods: Encoded Annexin Ⅴ cDNA was amplifyed from U937 cDNA libary by PCR and then subcloned into E coli expression vector. MS2-Annexin Ⅴ fusion protein could be overexpressed in E coli. The MS2 bacteria protein could be removed by thrombin digestion.The mature Annexin Ⅴ was obtained by ion exchange chromatography and the FITC labled Annexin Ⅴ could be used in the detection of apoptosis. Results:Up to 37% of the total bacterial proteins was rhAnnexin Ⅴ as showed by SDS-PAGE. The purification of Annexin Ⅴ is over 99%. The FITC labled Annexin Ⅴ could efficiently detect apoptosis. Conclusion: We successfully established the technique procedure of obtaining a large quantity of Annexin Ⅴ and provided the basic routine for popularizing the detection of apoptosis' with high effciency.