Calponin-1表达沉默对子宫平滑肌细胞生物学行为的影响

Effects of calponin-1 gene silencing on the biological behavior of uterine smooth muscle cells

目的研究抑制calponin-1表达对人子宫平滑肌细胞增殖、侵袭、凋亡和细胞骨架的影响,探讨妊娠时人子宫平滑肌细胞中calponin-1蛋白表达上调在分娩发动中的作用机制。方法构建腺病毒siRNA-calponin-1质粒,转染原代培养的人子宫平滑肌细胞,分别用四甲基偶氮唑蓝(MTT)、Transwell侵袭实验和流式细胞术(AnnexinV/PI双染法)检测转染siRNA-calponin-1前后对子宫平滑肌细胞增殖、侵袭、凋亡的影响;同时用Rhodamine-Phalloidin标记子宫平滑肌细胞骨架蛋白(F-actin),荧光显微镜观察其变化并进行荧光定量检测。结果实验组与空载体组、空白对照组比较,子宫平滑肌细胞的运动能力明显下降(P<0.05),细胞增殖能力及凋亡率无明显差异(P>0.05);空载体组与空白对照组比较,差异无统计学意义(P>0.05)。实验组子宫平滑肌细胞中F-actin的含量减少,排列松散、不规则、微丝较细;空载体组、空白对照组子宫平滑肌细胞中F-actin微丝明显增多增粗、呈束状平行排列。结论体外实验表明抑制calponin-1的表达可以抑制子宫平滑肌细胞的运动而不影响其增殖和凋亡,并可引起子宫平滑肌细胞F-actin的形态变化与重排。此结果提示:妊娠时子宫平滑肌细胞迁移能力的改变及F-actin的重排,可能是导致分娩发动的重要机制之一。

Objective To study the effects of calponin-1 expression inhibition on the proliferation,invasiveness,apoptosis and cytoskeleton of uterine smooth muscle cells,and explore the molecular mechanism of calponin-1 in the uterine smooth muscle cells for labor onset.Methods siRNA-calponin-1 adenovirus plasmid was constructed and transfected into primarily cultured uterine smooth muscle cells.The proliferation,invasiveness and apoptosis of the cells were determined by MTT assay,matrigel invasion assays and flow cytometry,respectively.Rhodamine-Phalloidin was used for labeling filamentous actin （F-actin）,and the morphology and the distribution of F-actin was observed under fluorescence microscopy and analyzed quantitatively.Results The motor ability of uterine smooth muscle cells decreased significantly after transfection with siRNA-calponin-1 adenovirus plasmid（P0.05）.The transfected cells showed thinner,loosened and irregular F-actin microfibers,and the cells in the empty vector and blank control groups showed thicker and longer F-actin microfibers.Conclusion Inhibition of calponin-1 expression can inhibit uterine smooth muscle cell migration and cause the morphological change and rearrangement of F-actin without affecting its proliferation and apoptosis in vitro,suggesting that the morphological change and rearrangement of F-actin of uterine smooth muscle cell may be one of the important mechanisms in the labor onset.