uPA-siRNA重组慢病毒载体感染兔软骨细胞对其增殖情况初步研究

发布时间：2018-06-08 07:54

本文选题：RNA干扰 + 慢病毒载体　；参考：《石河子大学》2015年硕士论文

【摘要】：目的:构建靶向特异尿激酶型纤溶酶原激活物(uPA)-siRNA慢病毒表达载体后并筛选出高效靶向序列,转染兔软骨细胞,观察其对软骨细胞增殖、u PA、MMP-3基因及蛋白表达水平的影响。方法:取原代生长良好的兔膝关节软骨细胞传代接种培养,根据Gen Bank中兔u PA基因序列,参照si RNA靶点设计原则,设计、合成构建靶向兔u PA-si RNA序列,利用RT-PCR筛选高效靶向序列,按照不同的感染复数(MOI)值加入慢病毒颗粒液,共同培养后确定最佳感染复数(MOI)值,并测定分析病毒感染效率,高效靶向序列经慢病毒包装后,通过Lipofectamine 2000转染兔软骨细胞,并按实验进行随机分组,分为实验组(转染u PA-si RNA慢病毒载体组)、空载体组(转染空慢病毒载体组)、空白对照组(未予任何处理组)、药物对照组(加载MMP特异性抑制剂TIMP)。细胞离体培养96h后,应用实时定量逆转录-聚合酶链反应(RT-PCR)及蛋白免疫印迹法(Western blot)分别检测软骨细胞u PA-si RNA对细胞内u PA、MMP-3 m RNA和蛋白的表达水平,并通过CCK-8法检测u PA-si RNA对软骨细胞增殖的影响。结果:慢病毒载体成功转染原代软骨细胞,通过si RNA载体质粒测序与预期si RNA序列一致,DNA测序序列正确无突变,从中筛选出高效靶向序列(即沉默效果最佳)。转染后软骨细胞培养可见,早期软骨细胞呈多角形,细胞贴壁生长;传2代时,细胞呈现梭形,并聚集生长;传5代时,细胞呈现典型纤维细胞样;符合软骨细胞生长特点,可进行后续实验。随着感染复数(MOI)的增加,慢病毒的感染效率也随之增加,当感染复数(MOI)为100时感染率超过85%,符合后续实验要求。通过CCK-8检测si RNA对软骨细胞增殖能力的影响发现,给予转染u PA-si RNA慢病毒载体后的软骨细胞增殖并未受到抑制。实验组u PA m RNA、MMP-3 m RNA及u PA、MMP-3蛋白的表达水平显著低于空载体租、空白对照组和药物对照组(P0.05),而空载体组、空白对照组及药物对照组u PA m RNA、MMP-3 m RNA及u PA、MMP-3蛋白的表达水平差异无统计学意义(P0.05)。结论:成功构建高效靶向u PA-si RNA慢病毒载体,其可稳定转染兔软骨细胞并能高效抑制u PA基因及蛋白表达,也可对MMP-3基因及蛋白表达呈抑制作用,对软骨细胞增殖起到促进作用。
[Abstract]:Aim: to construct a plasminogen activator targeting urokinase type plasminogen activator (UPA) -siRNA lentivirus expression vector and screen out a highly efficient target sequence for transfection into rabbit chondrocytes and observe its effect on the expression of MMP-3 gene and protein in chondrocytes. Methods: rabbit knee articular chondrocytes were cultured and cultured. According to the gene sequence of rabbit u PA gene in GenBank, according to the principle of si RNA target design, the target rabbit u PA-si RNA sequence was designed and synthesized. The highly efficient target sequences were screened by RT-PCR, and the lentivirus particles were added to different infected plural moi values. After co-culture, the optimal infection complex moi values were determined, and the efficiency of virus infection was analyzed, and the highly efficient target sequences were packaged by lentivirus. Rabbit chondrocytes were transfected with Lipofectamine 2000. They were divided into experimental group (transfection of UPA-si RNA lentivirus vector group), empty vector group (transfection group of empty lentivirus vector group, blank control group (no treatment group), drug control group (loaded with TIMP, a specific inhibitor of MMP). After cultured in vitro for 96 h, the expression of u PA-si mRNA in chondrocytes was detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotanalysis, respectively, and the expression of uPA-MMP-3 mRNA and protein in chondrocytes were detected. The effect of u PA-si RNA on the proliferation of chondrocytes was detected by CCK-8 method. Results: the lentivirus vector was successfully transfected into the primary chondrocytes. The siRNA plasmid sequencing was consistent with the expected si RNA sequence. After transfection, the chondrocytes were found to be polygonal and adherent to the wall; at passage 2, the cells were fusiform and aggregated; at passage 5, the cells were typical fibrocyte-like, which was consistent with the characteristics of chondrocyte growth. Further experiments can be carried out. The infection efficiency of lentivirus increased with the increase of the number of infected moi. The infection rate of lentivirus was more than 85 when the infection number of moi was 100, which met the requirements of subsequent experiments. The effect of si RNA on the proliferation of chondrocytes was detected by CCK-8. It was found that the proliferation of chondrocytes transfected with lentivirus vector of uPA-si RNA was not inhibited. The expression levels of MMP-3 mRNA and UPA-MMP-3 protein in the experimental group were significantly lower than those in the empty vector group, while in the blank control group and the drug control group, the expression levels of MMP-3 mRNA and uPA-MMP-3 protein were significantly lower than those in the empty vector group. There was no significant difference in the expression level of UPA m RNA-MMP-3 mRNA and uPA-MMP-3 protein between blank control group and drug control group (P 0.05). Conclusion: the highly efficient targeting uPA-si RNA lentivirus vector can be successfully constructed, which can stably transfect rabbit chondrocytes and inhibit the expression of UPA gene and protein, and also inhibit the expression of MMP-3 gene and protein. Promote the proliferation of chondrocytes.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684.3

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