摘要
目的探讨汉防己甲素（Tet）对离体兔角膜基质细胞的抑制作用及其作用机制。方法 选用原代及传代兔角膜基质细胞，实验组加入含不同质量浓度Tet的培养液，对照组加入含等量PBS的培养液，同时用不同质量浓度氟美童作比较。采用四甲基偶氮唑蓝（MTT）比色法检测对细胞增生的抑制；流式细胞仪（FCM）测定细胞周期和凋亡率的变化；透射电子显微镜观察其超微结构变化；免疫细胞化学检测其凋亡指标的改变。结果 Tet质量浓度为1～10μg／ml时，作用24、48、72h均明显抑制角膜基质细胞的增生（P〈0．01），并具有剂量-反应关系，其各时间点IC50均低于氟美童；FCM结果显示，实验组G0／G1期细胞增加，并可见细胞凋亡峰和凋亡率的升高；免疫细胞化学显示，实验组bax、bcl-2表达均增加，但bax增加更为明显。结论 Tet对角膜基质细胞的增生具有明显抑制作用，并呈现剂量-反应关系，其抑制作用强于氟美童。Tet可能通过诱导细胞凋亡和使细胞周期停滞在G0／G1期而发挥其抗增生作用。
Objective The keratocyte plays an important role in haze formation after refractive operation including photorefractive keratectomy（PRK） ,LASEK and Epi-LASIK. In this study ,we discussed the effect of tetrandrine on inhibition of rabbit keratocyte and explored the mechanism. Methods The primary cultured and subcultured keratocytes from New Zealand white rabbit were exposed to different concentrations of tetrandrine （1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μg/ml）and fluorometholone （16.67, 25.00, 33.33, 41.67, 50.00, 58.33, 75.00, 83.33 μg/ml）in experimental groups and the culture media containing 20 ILl PBS in contrast group. The inhibitory characteristics of tetrandrine to the proliferation of cells were measured with methyl thiazolyl thiazolium （MTT） assay. The changes of cells cycles and the apoptosis rate were observed with flow cytometer（ FCM ）. The ultrastructure of cell was observed with transmission electron microscope. The changes of the apoptosis indexes were measured by immunocytochemistry. Results After the treatment of 1 - 10 μg/ml tetrandrine,keratocyte showed the marked inhibition in 24,48 and 72 hours（ P 〈 0. 01 ） in a dose-depend manner and its IC50 at each time point was lower than fluorometholone treatment group. FCM result showed that the cells of G0/G1 phase were obviously increased（ 85.41% vs 91.89% and 96. 61% ,P 〈 0. 01 ） and the apoptosis rate also was increased （0 vs 2. 93% and 4. 46% ,P 〈0. 01 ）. The brown positive expression of bax and bc1-2 in rabbit corneal stromal cells（RCSCs） was displayed in cytoplasm. There were only few positive cells of bax and bcl-2 in contrast groups and many positive cells in experimental groups. The staining of the positive cells in bax experimental group was stronger than that in bcl-2 experimental group. Conclusion Tetrandfine has obvious inhibitory effect on the proliferation of rabbit keratocytes by inducing apoptosis and ceasing cells cycles. It＇ s inhibitory effect is more dominant in comparison with fluorometholon
出处
《眼科研究》
CSCD
北大核心
2007年第1期29-32,共4页
Chinese Ophthalmic Research
