汉防己甲素对兔眼角膜基质细胞增殖和凋亡作用的比较研究

An investigation about antiproliferative effect of tetrandrine on keratocyte

目的探讨汉防己甲素(Tet)对离体兔眼角膜基质细胞抑制增殖作用,及其对增殖率和凋亡率的作用程度比较。方法选用原代及传代兔角膜基质细胞,实验组加入含不同浓度Tet的培养液,对照组加入含等量PBS的培养液。通过免疫细胞化学技术了解细胞的PCNA表达来检测细胞增殖情况,计算增殖率并测定变化;流式细胞仪(FCM)测定细胞周期和凋亡率的变化。结果Tet质量浓度为2μg/ml,3μg/ml,4μg/ml时,作用48h,随着Tet浓度的增大,PCNA阳性细胞数明显减少,角膜基质细胞的增殖率呈递减趋势,存在剂量一效应关系(P<0.05);FCM结果显示,随着Tet的浓度增大,实验组G0/G1期细胞增加,细胞周期阻滞在G1期,见细胞凋亡峰,凋亡率也随浓度增大而升高〔1〕(P<0.05);Tet对增殖率影响的曲线斜率绝对值明显大于对凋亡率作用的斜率绝对值。结论Tet对角膜基质细胞的PCNA的表达具有明显抑制作用,随Tet浓度增大,角膜基质细胞的增殖率下降,Tet通过将细胞阻滞在G1期而发挥其抗增殖作用,且存在剂量效应关系;Tet诱导细胞凋亡,且也存在剂量效应关系,随浓度增大,凋亡率增加;在2μg/ml,3μg/ml,4μg/ml的浓度内,Tet对角膜基质细胞同时具有抑制增殖和诱导凋亡作用,但以抑制增殖作用为主。

Objective：The keratocyte＇ s abnonnal proliferation plays an important role in haze formation after refractive operation , including photorefractive keratectomy（PRK）, LASEK and Epi - LASIK. In this study,we plan to explore the antipmliferative effect about tet and clear out the extent of the effect, which one is more important, antiproliferation or apoptosis. Methods： The primary cultured and subcuhured keratocytes from NewZealand white rabbits were exposed to different concentrations of tetrandrine（2μg/ml, 3μg/ml,4μg/ml） in experimental groups and the culture medium containing 20μml PBS in contrast group. We detect the expression of PCNA by immunoeytochemistry, observe the cell cycle and ratio of apoptosis by FCM. Results： The immunocytochemistry showed that with the treatment of tetrandrine,48 hours later, the expression of PCNA depressed with the concentrations of tetrandrine＇ s increasing, and the index of proliferation also dcprssed. FCM result showed that the cell cycle was blocked at G0/G1 phase , the cells at G1 phase muhed with the concentrations of tetrandrine＇ s increasing and the index of apoptosis muhed too. The slope rate of the curve about index of proliferation larger than slope rate of the curve about index of apoptosis . Conclusion：Tetrandrine obversly depressed the expression of PCNA, and blocked cells at GO/G1 phase to antiproliferat. And at the concentrations of tetrandrine （2μg/ml, 3μg/ml,4μg/ml）, the main action of Tet was not apoptosis but antiproliferative.