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[摘要]
目的：研究羟基喜树碱(HCPT)对人眼Tenon's囊成纤维细胞(human Tenon's capsule fibroblasts， HTFs)的细胞周期、细胞毒性的影响，探讨其抗纤维化作用机制。
方法：取本院眼库新鲜眼球(<6h)的Tenon's囊组织，用组织块培养法进行成纤维细胞体外培养； 流式细胞仪测定HCPT和丝裂霉素C(MMC)对HTFs细胞周期的影响； 台盼蓝染色法鉴定HCPT和MMC对HTFs的抑制作用是否由药物的细胞毒性引起； RT-PCR检测HCPT、MMC作用24h后HTFs的Smad7 mRNA基因表达。
结果：HTFs 体外生长良好，可用于实验研究。不同浓度HCPT(0，0.25，1，4mg/L)作用后的HTFs细胞周期与对照组相比，G2期细胞百分数的组间差异均有统计学意义(P<0.05)； 不同浓度MMC(0，0.025，0.1，0.4mg/L)作用后的HTFs细胞周期与对照组相比，G1期细胞百分数的组间差异均有统计学意义(P<0.05)。不同浓度HCPT和MMC作用后的HTFs活细胞率和空白对照组比较，差异均无统计学意义(P>0.05)。4mg/L HCPT，0.4mg/L MMC作用HTFs 24h后，Smad7 mRNA表达水平与空白对照组比较差异有统计学意义(P<0.05)。
结论：HCPT将HTFs阻滞于G2期，MMC将HTFs阻滞于G1期； HCPT，MMC抑制HTFs的作用和药物的细胞毒性无关； HCPT抑制HTFs的增殖可能是通过上调Smad7 mRNA的表达阻断TGF-β信号通路实现的。
[Key word]
[Abstract]
AIM: To investigate the effect of different concentrations of 10-hydroxycamptothecin(HCPT)on the cell cycle and cytotoxicity of human Tenon's capsule fibroblasts(HTFs), and to explore its mechanism in anti-fibrosis.
METHODS:The Tenon's capsule tissue of fresh eyes(<6h)of our hospital eye bank was taken, in vitro culture of fibroblasts was done by tissue block culture; A flow cytometry(FCM)was used to evaluate the effect of different concentrations of HCPT(0, 0.25, 1, 4mg/L)and mitomycin C(MMC: 0, 0.025, 0.1, 0.4mg/L)on HTFs cell cycle; Trypan blue staining was adopted to determine whether the inhibitory effect of HCPT and MMC on HTFs was caused by their cytotoxicity; RT- PCR was employed to detect the level of Smad7mRNA gene expression of HTFs after HCPT and MMC were used for 24h.
RESULTS:HTFs were cultured successfully in vitro, and can be used in our study; compared with the control groups, the cell cycle of HTFs which was affected by different concentrations of HCPT was significantly different in G2 phase(P<0.05). As for MMC, there were significant differences in G1 phase between groups with different concentrations of MMC and the control group(P<0.05). There was no significant difference in the rate of HTFs living cells between HCPT group(or MMC group)to which HCPT and MMC were added for 24h and the control group(P>0.05). After HFTs was affected by 0.4mg/L MMC or 4mg/L HCPT for 24h, RT PCR found that the level of Smad7 mRNA expression was significantly increased(P<0.05).
CONCLUSION: HCPT mainly blocks HTFs in G2 phase, MMC mainly impacts the G1 phase. The inhibitory effect of HCPT and MMC on HTFs proliferation is not relevant to cytotoxicity induced by the two drugs. The mechanism that HCPT inhibits the proliferation of HTFs may be due to the increasing Smad7 mRNA expression to block TGF-β signaling pathway.
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