Objective To investigate the expression of T cell receptor (TCR) Vβ8.3 gene on CD4+ T lymphocytes in the rats with experimental autoimmune uveoretinitis (EAU). Methods Eighteen Lewis rats were divided into EAU, complete Freund′s adjuvant, and the control group. Inter photoreceptor retinoid-binding protein (IRBP) R16 peptide was synthesized using Fmoc procedure for induction of EAU. Magnetic absorption cell sorting (MACS) me thod was used to isolate the CD4+T lymphocytes from the spleen of the rats. Flow cytometry was used to monitor the efficiency of isolation. The expression of TCR Vβ8.3 gene segment on CD4+T lymphocytes was determined by fluorescent quantitative polymerase chain reaction. Results EAU was successfully induced in the Lewis rats immunized with IRBP R16 peptide. The proportion of CD4+T lymphocytes isolated by means of MACS was statistically higher than that before isolation (P＜0.001). The expression of TCR Vβ8.3 gene segment on CD4+ T lymphocytes in EAU rats was significantly higher than that in the control (P＜0.05). Conclusions There is a predominant usage of antigen-specific TCR Vβ 8.3 gene in EAU rats induced by IR BP R16 peptide, which may serve as a target for immunotherapy of EAU. (Chin J Ocul Fundus Dis，2004，20：165-167)
OBJECTIVE To study the biocompatibility of skin reproductive membrane. METHODS According to ISO’s standards, the extractions of the skin reproductive membrane were prepared, and the acute systematic toxicity test, primary skin irritant test, cytotoxicity test, gene expression of type I collagen and fibronectin were detected to evaluate the biocompatibility of skin reproductive membrane. RESULTS All of those tests showed negative results. CONCLUSION The skin reproductive membrane has excellent biocompatibility in the level of the systematic, cellular and molecular biology.
Purpose To investigate the expression of intercellular adhesion molecules ICAM-1 and Mac-1，in epiretinal membanes (ERM) of eyes wi th proliferative vitreoretinopathy (PVR)． Methods Twenty epiretinal membranes were obtained from eyes undergone vitrectomy for retinal detachment complicated with PVR and observed by immunohistochemical examination． Results Expressions of ICMA-1 and Mac-1 were observed in 18 and 15 membranes respectively．Expression of both adhesion molecules in 12 membranes． Conclusion The findings indicate that adhesion molecules might be involved in the development of PVR． (Chin J Ocul Fundus Dis，2000，16：71-138)
OBJECTIVE To probe the possibility of direct transfer of exogenous gene into peripheral nerve and its following expression in vivo. METHODS The PCMV beta plasmid containing cytomegalovirus (CMV) promoter and Escherichia Coli (E. Coli), beta-Galactosidease (beta-Gal) structural gene (lacZ gene) was constructed and injected into the rabbit sciatic nerve. The control group was injected PBS solution. The injected nerves were sampled and tested by beta-Gal enzyme activity assay of the 5-bromo-4-chloro-3-indolyl-beta-D-galactoside and beta-Gal histochemical stain. RESULTS In the control group, no beta-Gal enzyme activity was detected in the different stages after operation, and beta-Gal histochemical stains showed positive. In the experimental group, enzyme activity could be detected from 2 days to 30 days after operation, and the histochemical stains showed negative. CONCLUSION The exogenous gene can be transferred into peripheral nerve and expressed with bioactivity, thus the gene therapy to accelerate the recovery of nerve is practical.
ObjectiveTo screen compounds or drugs can affect the hypoxia induced-gene expression of retinal vascular endothelial cell based on gene expression microarrays and connectivity map (CMAP) technology. MethodsTotally 326 up-regulated and down-regulated genes of hypoxic human embryonic retinal microvascular endothelial cells minduced by cobalt chloride in the previous study were converted into query signature format documents. Gene profile of the disease characteristics was then compared with that of control in CMAP website database, positive and negative compounds related to retinopathy of prematurity (ROP) were finally screened out. Results44 and 18 compounds or drugs have positive and negative relationship with ROP respectively by searching CMAP database with differentially expressed genes. Ciclopirox, cobalt chloride, gossypol and withaferin A have positive relationship with ROP. Cyclic adenosine monophosphate, harmalol, naringin and probenecid have a negative effect on ROP. ConclusionsCiclopirox, cobalt chloride, gossypol and withaferin A have a positive effect on ROP. However, cyclic adenosine monophosphate, harmalol, naringin and probenecid have a negative effect.
ObjectiveTo analyze the clinicopathologic characteristics and prognosis of human epidermal growth factor receptor 2 (HER2)-negative breast cancer patients with different expression status of estrogen receptor (ER). MethodsThe patients with HER2-negative breast cancer met the inclusion and exclusion criteria and were treated in the Affiliated Hospital of Southwest Medical University from January 1, 2017 to December 31, 2019 were retrospectively collected, and then were assigned into 3 groups according to the ER expression status: ER-negative (ER expression positive rate <1%) group, ER-low expression (ER expression positive rate 1%–10%) group, and ER expression positive rate >10% group. The differences of clinicopathologic characteristics, therapy, and prognosis among the 3 groups were compared. And the risk factors affecting recurrence and metastasis of patients with ER-low expression were analyzed by Cox proportional hazards regression model. ResultsA total of 610 patients with HER2-negative breast cancer were included in this study, including 130 patients in the ER-negative group, 48 patients in the ER-low expression group, and 432 patients in the ER expression positive rate >10% group. The Bonferroni method was used to correct the test level after pairwise comparison, it was found that the histological grade was later (P<0.001, P=0.023) and the Ki-67 expression was higher (P<0.001, P=0.023) in the ER-negative group and ER-low expression group as compared with the ER expression positive rate >10% group; The proportion of the patients receiving chemotherapy in the ER-negative group was higher than that of the ER expression positive rate >10% group (χ2=10.310, P=0.001), while which had no statistical difference between the ER-low expression group and the ER-negative group or the ER expression positive rate >10% group (Fisher exact probability method, P=1.000; χ2= 3.585, P=0.058); The proportion of patients receiving endocrine therapy in the ER-low expression group was higher than that in the ER-negative group (χ2=36.333, P<0.001) and lower than the ER expression positive rate >10% group (χ2=246.996, P<0.001). The difference in disease-free survival (DFS) curves among 3 groups was statistically significant (χ2=46.805, P<0.001); There were no statistical differences in the overall survival (OS) curve and DFS curve between the ER-negative group and the ER-low expression group (Two stage test, P=0.786; χ2=1.141, P=0.286), and which in the ER expression positive rate >10% group were significantly better than thoses in the ER-negative group (χ2=10.137, P=0.001; χ2=39.344, P<0.001) and the ER-low expression group (χ2=4.075, P=0.044; χ2=31.911, P<0.001). The results of multivariate Cox proportional hazards regression analysis showed that N1 and N2 [N0 as reference: RR (95%CI)=7.740 (1.939, 30.897), P=0.004; RR (95%CI)=9.513 (1.990, 45.478), P=0.005) and T3 [T1 as reference: RR (95%CI)=27.357 (2.188, 342.041), P=0.010] increased the probabilities of recurrence and metastasis HER2-negative breast cancer patients with ER-low expression. ConclusionsAccording to results of this study, patients with HER2-negative breast cancer showed certain differences in histological grade and Ki-67 expression among patients with three different ER expression status, but no statistical difference is found between ER-low expression and ER-negative breast cancer, and the prognoses of both are worse than that of ER expression positive rate >10% breast cancer patients. Lymph node metastasis and larger tumor are risk factors affecting recurrence and metastasis in ER-low expression breast cancer patients.
【Abstract】Objective To investigate the role of interleukin-10（IL-10） and interleukin-18 （IL-18） in the pathogenesis of acute lung injury in experimental severe acute pancreatitis.Methods Forty-eight SD rats were divided into control group and SAP group by the random data table. The model of experimental severe acute pancreatitis was established by injection of 3.5% sodium taurocholate into the bili-pancreatic duct. Lung wet weight index, ascities and level of serum amylase, IL-10 and IL-18 were quantitatively measured in different time. Intrapulmonary expressions of IL-10 mRNA and IL-18 mRNA were detected by semiquantitative RTPCR. The histopathology of pancreas and lung were observed under the light microscope.Results Lung wet weight index, ascities, level of serum amylase, IL-10 and IL-18, intrapulmonary expressions of IL-10 mRNA and IL-18 mRNA were significantly increased in SAP group (P＜0.01). The level of serum IL-18 and intrapulmonary expression of IL-18mRNA are positively correlated with lung wet weight index （r=0.68,P＜0.01; r=0.72,P＜0.01） and lung injury score （r=0.74,P＜0.01; r=0.79,P＜0.01) respectively, whereas the level of serum IL-10 and intrapulmonary expression of IL-10 mRNA are negatively correlated with lung wet weight index（r=-0.62,P＜0.01; r=-0.69,P＜0.01） and lung injury score（r=-0.66,P＜0.01; r=-0.60,P＜0.01）. Conclusion IL-18 may play a key role in the pathogenesis of acute lung injury in experimental severe acute pancreatitis, and IL-10 exerts the protection role in this process.
Objective To investigate the effects of expression of TNFα mRNA on glucose uptake in both the liver and skeletal muscle after endotoxemia. Methods In the mice with intraperitoneal injection of lipopolysaccharide (LPS), the changes of TNFα level of plasma and uptake of 2-deoxyglucose (2-DG) in the isolated soleus muscle and hepatic tissues were determined, then the reinstatement of glucose uptake by injecting TNF-McAb for 3 days was also observed. In addition, changes of TNFα mRNA expression of liver were evaluated. Results The expression of TNFα mRNA in the liver showed markedly increased in the first 3 hours post endotoxemia and remaind high for 3 days, and the plasma TNFα level paralleled with TNFα mRNA expression of liver also was elevated. The basal uptake of 2-DG both in muscle and liver were markedly increased, but the stimulated 2-DG uptake with insulin was greatly reduced as compared with the control. In addition, these abnormalities of 2-DG uptake can be partially corrected by neutralization of the circulatory TNFα by administration of TNF-McAb. Conclusion The disorders of glucose uptake of the liver and the muscle due to the overexpression of TNFα mRNA and elevated circulatory TNFα level may be the mechanism of insulin resistance after endotoxemia.
ObjectiveTo detect the induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in immunostimulated retinal pigment epithelial (RPE) cells to seek for the supplying of the arginine, a substrate for NOS; as well as the effects of produced NO on the tight junction of RPE-J cells. MethodsRat′s RPE-J cells were treated with interferon-γ(INF-γ), tumor necrosis factor-α(TNF-α) and lipopolysaccharide (LPS), and Northern and Western blotting were applied to analyze the expression of the citrulline-NO cycle enzymes and related enzymes and the effect of dexamethasone and cyclic adenosine monophosphate (camp) on the expression of iNOS. Immunocytochemistry reaction and Western blotting were used to evaluate the effect of produced NO on the tight junctions of RPE-J cells.ResultsiNOS and argininosuccinate synthetase (AS) were highly induced at both mRNA and protection levels in immunostimulated RPE cells while arginiosuccinate lyase (AL) was not induced. NO was produced by cells after stimulation with TNFα, IFNγ and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. And the produced NO impaired the tight junction of RPE-J cells, decreased the production of tight junction related protein ZO-1.ConclusionIn activated RPE-J cells, citrullinearginine recycling is important for NO production, and the produced NO weakened the function of tight junction of RPE-J cells.(Chin J Ocul Fundus Dis, 2005,21:32-36)
An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum. In the analytical process applied with coupled-enzyme, the quality control of sarcosine oxidase (SOX) as a key enzyme is the first problem to be solved. In order to establish an efficient and laboratory-scale production of SOX, the recombinant sarcosine oxidase (r-SOX) gene was a high-level expression in E.coli induced with lactose on a large-scale fermentation in 300 L fermenter. The results suggested that the biomass concentration reached OD600 of 22 and the expression of recombinant sarcosine oxidase in E. coli accounted for about 25% of total soluble protein in culture after fermentation. The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography. The sarcosine oxidase with 97% purity, 25 U/mg specific activity and 92.4% activity recovery was obtained. The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography. This sarcosine oxidase was found to be a conjugated protein, yellow enzyme, which combined with FAD as prosthetic group by covalent linkage. The contaminant of catalase was not detected in the sample pool of this enzyme. In addition, a further test to the thermal stability of sarcosine oxidase was done. According to the above results, the development and utilization of this enzyme has been set up on a reliable foundation.