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)
【摘要】 目的 观察不同种培养基中重组人色素上皮衍生因子(rPEDF)融合蛋白的表达。 方法 将前期研究已构建的pET28aPEDF原核表达重组体转化E.coli BL21大肠杆菌表达宿主菌,酶切鉴定阳性菌落后,分别在M9和LB培养基中用异丙基βD硫代半乳糖(IPTG,IsopropylbetaDthiogalactoside)诱导表达,SDSPAGE电泳检测表达的PEDF蛋白, 美国ImagePro Plus 分析系统进行蛋白定量分析。结果 LB和M9培养基中均获得相对分子质量约54×103的rPEDF融合蛋白。但LB培养基获得的是rPEDF融合蛋白的包涵体,目的蛋白占总蛋白含量为21046%,M9培养基获得的是可溶性的rPEDF的融合蛋白,目的蛋白占总蛋白含量的1231%。结论 不同种培养基中均有rPEDF 融合蛋白的表达。【Abstract】 Objective To observe the express of recombinant pigment epithelial derivative facto (rPEDF) in the different medium. Methods The pET28aPEDF was transformed into E.coli BL21. After the colonies were positive identification which were induced by IsopropylbetaDthiogalactoside in medium M9 and LB. The PEDF protein were detected by SDSPAGE and analyzed by American ImagePro Plus system. Results LB and M9 medium obtained the relative molecular mass about 54×103 rPEDF fusion protein. But LB medium obtained the inclusion bodys of rPEDF fusion protein，the purpose protein account for 21.046%；LB medium obtained the soluble rPEDF fusion protein，the purpose protein account for 12.31%. Conclusion The rPEDF protein was expressed in the different medium.
ObjectiveTo detect the expression of motilin in gastric cancer tissues and to explore the relationship between motilin protein expression and clinicopathologic characteristics of gastric cancer. MethodsThe immunohistochemical staining was used to detect the expression of motilin protein in gastric cancer, paracancerous tissues, and normal gastric mucosa tissues. The relationship between motilin protein expression and clinicopathologic characteristics of gastric cancer was analyzed. ResultsThe expression of motilin protein in gastric cancer tissues (1 206.43±631.67) was significantly higher than that in normal gastric mucosa tissues and paracancerous tissues, respectively (Plt;0.01). The difference of motilin protein expression between normal gastric mucosa tissues and paracancerous tissues was not significant (Pgt;0.05). The expression of motilin protein in gastric cancer was correlated with the site of tumor, differentiation degree, and lymph node metastasis (Plt;0.05). ConclusionMotilin may participate in the carcinogenesis of gastric cancer, and correlated with the invasion and metastasis of gastric cancer.
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 investigate the changes in the expression level of PDGF in the bone callus of rats with femoral fracture and brain injury to explore the effect of brain injury on the fracture heal ing and the related mechanism. Methods Sixty-four 12-week-old SD rats weighing (356 ± 25) g were randomly divided into 8 groups with 8 rats in each. The rats in groups A1, B1, C1 and D1 had a femoral fracture and a brain injury for 1, 2, 3 and 4 weeks, respectively; the rats in groups A2, B2, C2 and D2 had a mere fracture without a brain injury for 1, 2, 3 and 4 weeks, respectively. After the CR films were taken, the bone callus was obtained 1, 2, 3 and 4 weeks after operation, respectively. Then, the bone callus and its histology were examined by HE staining, the expressions and changes in the level of PDGF were examined by the immunohistochemical staining, and the level of PDGF mRNA was measured by in situ hybridization. Results The CR films showed that the callus formation in the A1-D1 groups was earl ier and greater than that in the A2-D2 groups at the same time point. The HE staining indicated that more fibroblasts and early-stage chondrocytes were found in group A1; some fibroblasts in the fracture interspace and few early-stage chondrocytes were found in group A2; some newly-formed trabecular bones were found at the end of the fracture in group B1; but no trabecular bone formation was found in group B2; woven bone formation and a few chondrocytes between trabecular bones in the fracture interspace were found in group C1; only a few trabecular bones in the fracture interspace were found in group C2；woven bones turned to lamellar bones in group D1；and more immature trabecular bones in the fracture interspace were found in group D2. The positive expression of PDGF and PDGF mRNA was b in the cytoplasms of fibroblasts, mesenchymal cells, vascular endothel ial cells, early-stage chondrocytes, osteoblasts and osteoclasts. The percentage of the positive cells for PDGF and PDGF mRNA in the callus was significantly higher in groups A1-D1 than in groups A2-D2 at the same time point (P lt; 0.05). Conclusion Brain injury can promote the fracture heal ing process, which is probably related to an increase in the expression level of PDGF after the brain injury.
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 systematically review the correlation of pSTAT3 overexpression and prognosis in lung cancer patients.MethodsWe searched from PubMed, EMbase, Web of Science, CNKI, VIP and WanFang Data databases to collect relevant studies about the correlation of pSTAT3 overexpression and prognosis in lung cancer patients from inception to November 2016. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies. Then, meta-analysis was performed by using RevMan 5.2 software.Results A total of thirteen studies were enrolled. The results of the meta-analysis showed that the overall survival (HR=1.23, 95%CI 1.04 to 1.46, P=0.02) of pSTAT3 overexpression group was shorter than that of low expression group. In terms of clinical prognostic characteristics, pSTAT3 overexpression rate in stage Ⅲ to Ⅳ group was significantly higher than stage Ⅰ to Ⅱ (OR=1.92, 95%CI 1.13 to 3.27, P=0.02). pSTAT3 overexpression rate of lung cancer patients with lymphatic node metastasis was also significantly higher than lung cancer patients without lymphatic node metastasis (OR=1.81, 95%CI 1.20 to 2.72, P=0.004). However, there was no statistical difference of pSTAT3 overexpression between well-moderately differentiation and poorly differentiation group (OR=0.82, 95%CI 0.44 to 1.53, P=0.54).ConclusionpSTAT3 overexpression is associated with poorer overall survival of lung cancer patients, as well as with more and advanced TNM grade and lymph node metastasis. It may be an indicator poor biomarker in lung cancer patients. Due to limited quality and quantity of the included studies, more high quality studies are needed to verify above conclusion.
Objective To research the transfer of adenovirus human bone morphogenetic protein 4 (Ad-hBMP-4) to human degenerative lumbar intervertebral disc cells in vitro and analyze its effect on the proteoglycan, collagen type II, and Sox9 of intervertebral disc cells. Methods Identified Ad-hBMP-4 was amplified and detected. Degenerative lumbar intervertebral disc cells were aspirated from the degenerative lumbar intervertebral disc of patients with Modic III level disc protrusion (aged, 27-50 years). The expressing position of collagen type II was identified in the intervertebral disc cells through the laser confocal microscope. The intervertebral disc cells at passage 1 were transfected with Ad-hBMP-4 as experimental group. After 3 and 6 days of transfection, RT-PCR was used to detect the mRNA expressions of proteoglycan, collagen type II, and Sox9, and Western blot to detect the expressions of proteoglycan and collagen type II proteins. Non-transfected cells at passage 1 served as control group. Results The virus titer of Ad-hBMP-4 was 5 × 106 PFU/mL. No morphological changes in the cells after transfection by Ad-hBMP-4. Collagen type II mainly expressed in the cell cytoplasm. The mRNA expressions of the proteoglycan, collagen type II, and Sox9 in experimental group at 3 and 6 days after transfection were significantly higher than those in control group by RT-PCR (P lt; 0.05), and the expressions of proteoglycan and collagen type II proteins were significantly higher than those in contorl group by Western blot (P lt; 0.05). There were significant differences between 3 days and 6 days in experimental group (P lt; 0.05). Conclusion Ad-hBMP-4 could transfect human degenerative lumbar intervertebral cells with high efficiency and promote collagen type II, proteoglycan, and Sox9 expressions. hBMP-4 may play an important role in the repair process during early disc degeneration.
ObjectiveTo analyze the regulative rule of mRNA of vascular endothelial growth factor (VEGF) in mice with oxygen-induced retinopathy, and to elucidate the possible mechanism of occurrence of neovascularization in retinopathy of prematurity (ROP).MethodsSixty 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group. In oxygen-induced retinopathy group, 36 mice were exposed to 75% oxygen for 5 days and then to room air for 5 days; in control group, 24 mice were raised in room air. Vascular perfusion of fluorescein and retinal stretched preparation were used to observe the morphologic changes of retinal vessels. Reversal transcriptionpolymerase chain reaction (RT-PCR) was used to observe changes of VEGF mRNA in each group. ResultsIn oxygen-induced retinopathy group, the morphologic characteristics of retinal vessels were the unperfused area at the center of superficial and deepseated vessels, and the neovascularization appeared at mid-peripheral retina after 2 days in relative hypoxia condition. The results of RT-PCR showed space-time corresponding relation between expression of VEGF and neovascularization, which meant that the transcription of VEGF mRNA decreased in hyperxia conditionand increased in relative hypoxia condition. ConclusionHypoxia is the main reason of occurrence of retinal neovascularization; increased expression of VEGF caused by relative hypoxia after hyperxia might be effective in reducing the occurrence of neovascularization in ROP.(Chin J Ocul Fundus Dis, 2005,21:292-295)