缝隙连接蛋白43在骨关节炎软骨及细胞中表达及shRNA慢病毒载体的构建

Expression of connexin 43 in cartilage and chondrocyte of osteoarthritis and construction of shRNA lentiviral vector targeting connexin 43

背景:缝隙连接蛋白43在骨关节炎的发生发展中具有重要作用,但具体机制尚不明确。目的:通过体内和体外实验检测缝隙连接蛋白43在骨关节炎软骨和细胞中的表达,验证骨关节炎软骨及软骨细胞(SW1353)中缝隙连接蛋白在连接蛋白家族的主导地位;构建缝隙连接蛋白基因的shRNA慢病毒载体,建立SW1353细胞的稳定转染细胞株。方法:C57BL/6小鼠6只,通过前交叉韧带横切术建立小鼠骨关节炎动物模型,通过免疫组织化学染色检测骨关节炎与正常关节中缝隙连接蛋白表达水平的差异。采用RT-PCR技术检测SW1353细胞中缝隙连接蛋白43 mRNA的表达,同时检测SW1353细胞中缝隙连接蛋白37、40、45和46基因的表达水平作为对照。将缝隙连接蛋白43 mRNA连接到含有增强型绿色荧光蛋白基因的慢病毒载体中,重组获得慢病毒质粒,将其与辅助包装质粒共转染293T细胞。所获慢病毒载体感染SW1353细胞后,检测缝隙连接蛋白43的表达。实验方案经口腔疾病研究国家重点实验室伦理委员会批准(批准号为SKLODLL2013A172)。结果与结论:①缝隙连接蛋白43在小鼠骨关节炎软骨组织中的表达较健康关节明显增加;②缝隙连接蛋白43 mRNA在SW1353细胞中的表达明显高于缝隙连接蛋白37、40、45和46的基因表达,SW1353细胞中的缝隙连接蛋白43在连接蛋白家族中具有主导地位;③缝隙连接蛋白43 shRNA慢病毒载体可抑制SW1353细胞中缝隙连接蛋白43 mRNA的表达;④实验筛选出稳定转染的SW1353细胞株,为验证缝隙连接蛋白43在骨关节炎中的作用机制奠定了基础。

BACKGROUND:Connexin 43(Cx43)plays an important role in occurrence and development of osteoarthritis.However,the specific mechanisms involved remain unclear.OBJECTIVE:To verify the possibility of the dominant position of Cx43 in connexin family in osteoarthritis by detecting the expression of Cx43 in articular cartilage and chondrocyte cell line,and to construct shRNA lentivirus vector of Cx43 gene and establish a stable transfer cell line of chondrocyte(SW1353).METHODS:Animal models of osteoarthritis were established in six C57BL/6 mice by anterior cruciate ligament transection.The differences of Cx43 expression between osteoarthritic and normal knees were investigated by immunohistochemistry.Expression of Cx43 mRNA in chondrocyte(SW1353)was detected by RT-PCR,and the expression levels of Cx37,Cx40,Cx45 and Cx46 in SW1353 cells were detected as control.Cx43 were connected to the lentiviral vector carrying the EGFP gene,to reconstruct the lentiviral vector plasmid.The viral particles were generated by co-transfection of 293T cells with Cx43-shRNA.After transfection of Cx43-shRNA lentiviral vector into chondrocytes(SW1353),the expression level of Cx43 was detected by western blot assay and RT-PCR.The study protocol was approved by the Ethics Committee of State Key Laboratory of Oral Diseases,approved No.SKLODLL2013A172.RESULTS AND CONCLUSION:The expression level of Cx43 was significantly increased in the articular cartilage of osteoarthritic knees.The expression level of Cx43 mRNA was significantly higher than that of Cx37,Cx40,Cx45 and Cx46 in chondrocytes(SW1353).In SW1353 cells,Cx43 occupied the dominant position in connexin family.Cx43 shRNA lentiviral vector could inhibit the expression of Cx43 mRNA in SW1353 cells.The stably transfected SW1353 cell line was screened,laying a foundation for verifying the role of Cx43 in osteoarthritis.