玻璃体对视网膜色素上皮细胞收缩作用的研究

Study on contractility of retinal pigment epithelial cells induced by vitreous in vitro

目的探讨人玻璃体体外培养对人视网膜色素上皮细胞收缩性的影响。方法低传代(3-5代)人视网膜色素上皮细胞分两组体外培养,一组用普通培养液(含体积分数10%胎牛血清的DMEM/F12)培养,一组用体积分数25%玻璃体培养,应用I型胶原凝胶收缩实验检测收缩力的变化,应用免疫荧光技术检测细胞骨架蛋白F-actin、α-SMA和vinculin的定位染色,应用免疫印迹技术检测α-SMA和vinculin蛋白的表达。结果与普通培养液培养相比,低传代的人视网膜色素上皮细胞玻璃体中培养48 h,胶原凝胶面积为(22 127±2072)像素,明显大于在普通培养液中的凝胶面积(16 084±1154)像素,细胞收缩能力显著下降,两组比较差异有统计学意义(t=4.412,P=0.012)。在普通培养液中,Factin、vinculin主要分布于细胞周边,vinculin形成的粘连斑粗大;而在玻璃体中培养后,Factin重组,在细胞的一端形成扁平伪足结构,vinculin主要分布于细胞扁平伪足端和细胞中央,形成的粘连斑明显变小。玻璃体培养后α-SMA蛋白表达显著下降,与普通培养液相比差异有统计学意义(t=2.845,P=0.047);vinculin蛋白表达无显著变化(t=0.198,P=0.852)。结论玻璃体培养可导致视网膜色素上皮细胞收缩性下降,这一作用可能与α-SMA蛋白表达下调及vinculin重新分布有关。

Objective To investigate the effects of vitreous cultured in vitro on contractility of human retinal pigment epithelial （RPE） cells. Methods The low-passage cultured RPE cells divided into two groups of treatment by volumn fraction 10% serum-containing Dulbecco minimum essential medium （DMEM）/F12 medium （normal medium） or the same medium supplemented with volumn fraction 25% human vitreous （25% vitreous medium）. Cell contractility was tested with collagen gel contraction analysis. Staining location of F-actin,α-smooth muscle actin （α-SMA） and vinculin were detected by immunofluorescence. The expression levels of α-SMA and vinculin were detected by Western blotting. Results Compared to cells cultured in normal medium, low-passage number RPE cells cultured in 25% vitreous medium for 48 hours, the collagen gel area of （22 127 ± 2072） pixels in 25% vitreous medium was significantly greater than the gel area of （ 16 084± 1154） pixels in normal medium,contractility of the cells decreased remarkably（ t = 4.412 ,P = 0.012）. In normal medium cultured RPE cells, filamentous actin （F-actin） and vinculin-forming focal adhesions mainly at margin of cell, the focal adhesions were bigger. But in 25% vitreous treated cells, F-actin reorganized and formed pseudopodia at one side of cells, vinculin-fonning focal adhesions redis-tributed mainly from margin to center and the side of actin polymerization, and became small. The expression level of α-SMA was down-regulated significantly （ t = 2. 845 ,P = 0. 047）, the expression level of vinculin was not significantly different between the two media （ t = 0. 198, P = 0. 852 5 ）. Conclusion Vitreous decrease the contractility of RPE cells, this effect may be related to the down-regulated expression of α-SMA and the redistribution of vinculin.