调宁蛋白基因siRNA重组腺病毒载体构建及其对体外子宫平滑肌细胞的作用

Construction of adenoviral vector encoding Calponin-1 siRNA and its effect on human myometrium cells in vitro

目的:构建siRNA重组腺病毒载体,为研究Calponin-1基因在分娩发动中的作用机制提供实验基础。方法:采用消化酶法对足月妊娠人子宫平滑肌细胞原代培养,并用免疫细胞化学方法进行鉴定。构建腺病毒siRNA-Calponin-1质粒转染原代培养的子宫平滑肌细胞,采用RT-PCR和Western印迹检测Calponin-1的表达。结果:构建的Calponin-1siRNA腺病毒载体可以显著抑制子宫平滑肌细胞中Calpo-nin-1mRNA和蛋白的表达。实验组、空白对照组、空载体组的Calponin-1mRNA表达的灰度值分别为316.3±39.2,1048.5±126.4,1027.2±127.5,Calponin-1蛋白表达的灰度值分别为323.3±43.2,1021.5±143.4,1019.2±144.5,实验组与空白对照组、空载体组比较,差异有统计学意义(P<0.05),空白对照组与空载体组比较,差异无统计学意义(P>0.05)。结论:构建的pAdEasy-pShuttle-U6-Calponin-1siRNA质粒能抑制体外子宫平滑肌细胞Calponin-1的表达,为研究Calponin-1在分娩发动中的作用提供了实验基础。

Objective To investigate the effect of Calponin-1 suppression on human myometrium cells through adenovirus mediated siRNA.Methods Human uterine smooth muscle tissues were digested with enzymes,cultured and confirmed with immunocytochemistry.Aadenovirus siRNA-Calponin-1 plasmid was transfected into primary cultured uterine smooth muscle cells in vitro.The expressions of Calponin-1 mRNA and protein were analyzed by RT-PCR and Western blot,respectively.Results The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid was successfully constructed,and Calponin-1 siRNA mediated by recombinant adenovirus resulted in markedly reduced expression of Calponin-1 mRNA and protein in human myometrium cells.The gray values of Calponin-1 mRNA in the uterine smooth muscle cells in the experimental,blank control,and empty vector groups were 316.3±39.2,1048.5±126.4 and 1027.2±127.5,respectively.The gray values of Calponin-1 protein were 323.3±43.2,1021.5±143.4,and 1019.2±144.5,respectively.The difference between the experimental group and the blank control group as well as the empty vector group was significant（P〈 0.05）.There was no significant difference between the empty vector group and the blank control group（P〉0.05）.Conclusion The pAdEasy-pShuttle-U6-Calponin-1 siRNA plasmid can inhibit the expression of Calponin-1 in human myometrium cells in vitro,which may be a useful approach to determine the role of Calponin-1 in delivery.