摘要
背景:多基因联合转染具有增强功能基因协同效应、消除短板差异等作用,因而目前被受到广泛重视。目的:观察以逆转录病毒(RV)为载体,介导白介素-1受体拮抗蛋白(IL-1Ra)基因单独转染及其与转化生长因子-β1(TGF-β1)基因共同转染兔膝软骨细胞后的表达,及对其增殖代谢的影响。方法:构建逆转录病毒载体PLNCX2-IL-1Ra-GFP 与 PLNCX2-TGF-β1-RFP,将其转染至包装细胞 PT67,待形成阳性克隆后扩增培养,收集病毒上清,并计算病毒上清滴度。将体外培养的软骨细胞分为空白转染组、PLNCX2空载体转染组、IL-1Ra单基因转染组及IL-1Ra +TGF-β1双基因转染组。酶联免疫吸附分析法(ELISA)进行瞬时基因表达(细胞转染后48 h)及稳定基因表达(筛选后4周)的检测;免疫组织化学染色法检测各组IL-1Ra、TGF-β1及Ⅱ型胶原的表达;流式细胞仪检测各组细胞生长周期比例。结果:PLNCX2-IL-1Ra-GFP 与 PLNCX2-TGF-β1-RFP 转染包装细胞后分别有绿色荧光与红色荧光表达;空白组与PLNCX2空载体转染组的上述指标无统计学差异(P>0.05);ELISA检测示基因转染组有明显基因表达;与空白组及PLNCX2空载体转染组相比,双基因转染组IL-1Ra、TGF-β1、Ⅱ型胶原含量明显提高,IL-1Ra基因转染组IL-1Ra含量提高明显,但TGF-β1、Ⅱ型胶原含量提高不明显;双基因转染组软骨细胞位于S期的比例明显增高,与其他组比较有统计学差异(P<0.05)。结论:逆转录病毒载体构建成功,基因转染软骨细胞后可获得稳定表达,双基因转染的软骨细胞生物学活性优于单基因转染,为基因治疗骨关节炎(OA)的研究提供实验基础。
The multi-gene transfection has been studied extensively because of its cooperative action within the multiple genes Objective： To investigate the effects of transfection of the IL-IRa with or without TGF-β1 on rabbit chondrocytes mediated by retrovirus vector. Methods： Recombination gene PLNCX2-IL-1 Ra-GFP and PLNCX2-TGF-β1-RFP was constructed and transfected into park-ing cells PT67, and the positively cloned cells were amplified and cultivated, virus supernatant was collected and the virus ti-tre was calculated. Monolayer cultures of rabbit articular chondrocytes were infected with recombinant plasmid PLNCX2 carrying genes encoding IL-IRa and TGF-β1. The synthesis of IL-1Ra, TGF-β1 and type Ⅱ collagen was measured by en-zyme-linked immunosorbent assay（ELISA）, immunohistochemistry and flow cytometry, respectively. Results： The parking cells infected with PLNCX2-IL-1Ra-GFPand PLNCX2-TGF-β1-RFP expressed green fluorescence and red fluorescence, respectively. There was no significant difference between the blank group and the blank vector group （P〉 0.05）. The positive expression was detected in the cell culture supematant. The content of IL-1Ra, TGF-β1 and type Ⅱ colla-gen in double gene transfeetion group was highly elevated, while the expression of TGF-β1 and type II collagen in single gene transfection group was lower than in the double gene transfection group. The chondrocyte ratio at stage S in double gene transfection group was obviously higher than in other groups （P〈 0.05）. Conclusions： The retrovirus vectors are successfully constructed. IL-1Ra and TGF-β1 genes can be expressed effectively af-ter being transfected into chondrocytes. The biologic activity of chondrocytes transfected with double gene is superior to that with single gene. The results are helpful for the gene therapy for osteoarthritis.
出处
《中国骨与关节外科》
2013年第1期50-55,共6页
Chinese Journal of Bone and Joint Surgery
基金
国家自然基金项目(30371440
30500515)
山西省自然科学基金资助项目(20041117
2006021045
2010011050-2)
