紫杉醇抑制原代培养人视网膜色素上皮细胞增殖的实验研究

Taxol inhibits proliferation of primarily cultured human retinal pigment epithelial cells

  • 摘要: 目的 研究紫杉醇(Taxol)对体外原代培养人视网膜色素上皮(human Retinal Pigment Epithelium,hRPE)细胞增殖的抑制作用及机制。方法 体外分离、原代培养hRPE细胞,采用免疫细胞化学方法对其进行鉴定。不同浓度紫杉醇(0、0.005、0.05、0.5、5mg/L)处理hRPE细胞一定时间,采用形态学观察、细胞生长曲线及MTT法检测药物对hRPE细胞的生长抑制效应,流式细胞术检测药物对hRPE细胞的细胞周期阻滞作用及凋亡诱导作用。结果 原代培养的hRPE细胞胞浆富含色素,随传代次数增加,黑色素颗粒逐渐减少直至消失。用抗人细胞角蛋白抗体进行免疫细胞化学鉴定hRPE细胞呈特异的阳性反应。细胞生长曲线及MTT法显示:紫杉醇作用于hRPE细胞24h和72h,其抑制细胞生长增殖的IC50值分别为5.24和3.24mg/L。流式细胞分析术检测结果显示:0.5mg/L紫衫醇作用细胞48h即可显著延迟hRPE细胞G2/M期进展并诱导凋亡(P<0.05)。透射电镜观察显示:0.5mg/L紫杉醇作用后细胞表面微绒毛减少,电子密度增加,细胞器减少,异染色质聚集成团、边聚等。结论 紫杉醇通过阻滞G2/M期进展和诱导凋亡显著抑制hRPE细胞生长增殖。

     

    Abstract: Objective To study the inhibitory effect of Taxol on proliferation of primarily cultured in vitro human retinal pigment epithelial(hRPE) cells and its mechanism. Methods After in vitro isolation and primary culture, hRPE cells were identified with immunocytochemistry method. Inhibitory effects of Taxol at different concentrations(0, 0.005, 0.05, 0.5, and 5mg/L) on growth of hRPE cells were observed by morphology, growth curve and MTT assay, respectively, while its cell cyclearresting and apoptosis-inducing effects were detected by flow cytometry. Results Rich pigment was found in the hRPE cells after primary culture, which increased with the number of passages. However, black pigment granules decreased gradually and even completely disappeared. Specific positive reactions were observed in hRPE cells by immunocytochemistry using anti-human keratin. Cell growth curve and MTT assay demonstrated that the IC50 value of Taxol for the growth inhibition of hRPE cells was 5.24mg/L and 3.24mg/L, after 24h and 72h, respectively. Flow cytometric analysis displayed that 0.5mg/L Taxol could significantly delay the G2/M arrest and induce apoptosis of hRPE cells 48h after it was used(P<0.05). Transmission electron microscopy revealed that 0.5mg/L Taxol decreased the microvilli on cell surface and cell organs, and increased the electron density in hRPE cells with marginal accumulation of heterochromatin masses. Conclusion Taxol can exert its direct effects on the proliferation of hRPE cells in vitro by inducing G2/M arrest and apoptosis.

     

/

返回文章
返回