玻璃体液诱导视网膜色素上皮细胞间质转分化作用

Epithelial-mesenchymal transdifferentiation of retinal pigment epithelial cells induced by vitreous humor in vitro

目的观察人玻璃体液体外培养诱导人视网膜色素上皮（RPE）细胞间质转分化作用。方法将3～5代传代人RPE细胞分为普通培养组和玻璃体液培养组，分别应用普通培养液和25％玻璃体液进行培养。采用相差显微镜观察两组细胞形态变化，细胞爬片检测肌动蛋白细胞骨架的变化。分别用细胞划痕法、Transwell小室侵袭法及Ⅰ型胶原凝胶收缩实验检测细胞迁移、侵袭及收缩力的变化。应用逆转录聚合酶链反应（RT—PCR）检测α-平滑肌肌动蛋白（α—SMA）和SnaillmRNA的表达。结果相差显微镜观察发现，普通培养组细胞保持扁平形态，细胞接触广泛；玻璃体液培养组细胞一端呈扇形，另一端呈锥形拖尾形或梭形，细胞生长呈彼此分离的方式。细胞爬片结果显示，普通培养组肌动蛋白骨架主要分布于RPE细胞周边部，形如细胞周边的条带状；玻璃体液培养组肌动蛋白纤维在细胞的一端重排，形成扇形扁平状伪足突起，而在细胞另一端形成锥形拖尾改变。玻璃体液培养组与普通培养组比较，RPE迁移及侵袭能力显著增强（t=14．190、22．630）、细胞收缩能力显著下降（t=6．221），差异均有统计学意义（P〈0．05）。RT—PCR检测结果显示，玻璃体液培养组与普通培养组比较，SnaillmRNA表达显著上升（t=3．218，P=0．032），α-SMAmRNA表达显著下降（t=3．990，P=0．016），差异均有统计学意义。结论玻璃体液培养可诱导RPE细胞间质转分化。这一作用通过增强RPE细胞迁移及侵袭能力，抑制RPE细胞收缩力；提高RPE细胞SnaillmRNA表达，下调α-SMAmRNA表达实现。

Objective To observe the effect of epithelial-mesenehymal transdifferentiation （EMT） of human retinal pigment epithelial （RPE） cells induced by vitreous humor in vitro. Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10% serum-containing Dulbecco minimum essential medium （DMEM）/F12 medium （group A）, or the same medium supplemented with 25% human vitreous （group B）. The morphological changes were observed with a phase contrast microscrope. Cell migration, invasion and contractility were tested using a scratch wound assay, Transwell invasion assay and collagen gel contraction analysis. The expression levels of α-smooth muscle actin （SMA） and SnaiI1 were detected by reverse transcriptase polymerase chain reaction （RT-PCR）. Results The RPE ceils in group A were flat and gathered together. The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge, or spindle-shaped, and appeared to separate. In group A, filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape. In group B, filamentous actin reorganized and formed fan-like fiat pseudopodia at one edge of the ceils. Compared to group A, the migration and invasion of the ceils increased significantly （t= 14. 190, 22. 630; P〈0.05）, but contractility decreased remarkably （t=6. 221, P〈0.05） in group B. Compared to group A, the expression level of Snail1 mRNA increased significantly （t= 3. 218, P = 0. 032）, but the expression level of α-SMA mRNA decreased （t=3. 990, P=0. 016）. Conclusions Vitreous humor can induce the EMT of RPE cells. Increasing cell migration, cell invasion, and expression of Snail1 mRNA as well as up-regulated cells contractility and expression of α-SMA mRNA may be the mechanism.