【作者】 王莉；

【导师】 姚智；

【作者基本信息】 天津医科大学 ， 免疫学， 2009， 博士

【摘要】 目的：观察三肽化合物酪丝亮肽(tyroserleutide, YSL)对人肝癌BEL-7402细胞的细胞周期的影响,并初步探讨其影响细胞周期调控因子阻滞细胞周期运转的可能作用机制。方法：1.应用流式细胞术观察YSL对体外人肝癌BEL-7402细胞的G0/G1期和S期细胞周期时相的影响。2.本研究应用流式细胞术和单细胞凝胶电泳法观察YSL对人肝癌BEL-7402细胞DNA损伤的作用。3.应用比色法定量检测人肝癌BEL-7402细胞的细胞周期G0/G1期CDK4/cyclinD激酶的活性；应用Western blot和RT-PCR方法从蛋白水平和mRNA水平,观察YSL对体外培养人肝癌BEL-7402细胞的CDK4和cyclin D1蛋白表达的影响。4.应用比色法定量检测人肝癌BEL-7402细胞的细胞周期G0/G1期CDK2/cyclinE激酶的活性；应用Western blot和RT-PCR方法从蛋白水平和mRNA水平,观察YSL对体外培养人肝癌BEL-7402细胞的CDK2和cyclin E蛋白表达的影响。5.应用比色法定量检测人肝癌BEL-7402细胞的细胞周期S期CDK2/cyclin A激酶的活性；应用Western blot和RT-PCR方法从蛋白水平和mRNA水平,观察YSL对体外培养人肝癌BEL-7402细胞的CDK2和cyclin A蛋白表达的影响。结果：1.血清饥饿法使细胞同步化于G0/G1期,YSL(6.4mg/ml、3.2mg/ml)作用14hrs可使体外培养的BEL-7402细胞G0/G1期细胞百分比增高,S期细胞百分比降低。双胸苷阻断法使细胞同步化于G1/S期,YSL明显增加S期细胞百分比,减少G2/M期细胞百分比。2. YSL (6.4mg/ml、3.2mg/ml)作用1、2、3、4hrs均能明显增加人肝癌BEL-7402细胞的磷酸化组蛋白H2A.X阳性细胞的百分比,诱导DNA损伤。单细胞凝胶电泳检测显示YSL(6.4mg/ml、3.2mg/ml)作用2、4hrs能够引起人肝癌BEL-7402细胞的DNA损伤。3. YSL(6.4mg/ml、3.2mg/ml)能够明显抑制BEL-7402细胞G0/G1期CDK4/cyclinD激酶的活性,并随给药剂量的增加抑制作用明显增强。YSL能够显著下调体外培养人肝癌BEL-7402细胞的CDK4、cyclin D1蛋白表达,但对其mRNA水平没有影响。4. YSL(6.4mg/ml、3.2mg/ml)能够明显抑制BEL-7402细胞G0/G1期CDK2/cyclinE激酶的活性,并随给药剂量的增加抑制作用明显增强。YSL能够显著下调体外培养人肝癌BEL-7402细胞的p-CDK2、CDK2、cyclin E蛋白表达,但对其mRNA水平没有影响。5. YSL (6.4mg/ml、3.2mg/ml)能够明显抑制BEL-7402细胞S期CDK2/cyclin A激酶的活性,并随给药剂量的增加抑制作用明显增强。YSL能够显著下调体外培养人肝癌BEL-7402细胞的p-CDK2、CDK2、cyclin A蛋白表达,但对其mRNA水平没有影响。结论：YSL能够将人肝癌BEL-7402细胞阻滞在G0/G1期和S期,该作用可能是由于YSL引起肿瘤细胞DNA损伤,CDK/cyclin激酶复合物感受到了DNA损伤这一信号,使得相应细胞周期的CDK/cyclin激酶复合物活性降低,最终引起细胞周期阻滞,细胞走向凋亡。更多还原

【Abstract】 Objective:To explore the inhibition effects of YSL on the cell cycle of human hepatocellular carcinoma BEL-7402 cells, and to approach its preliminary mechanisms by affecting cell cycle control factors in tumor cells.Methods:1. The effect of YSL on cell cycle of G0/G1 phase and S phase of human hepatocellular carcinoma BEL-7402 cells was assayed by FCM method.2. H2A.X phosphorylation flow cytometry assay and comet assay were used to estimate the double-stranded DNA breaks of BEL-7402 cells induced by YSL.3. The activity of CDK4/cyclin D kinase of G0/G1 phase was determined by colorimetric methods; Western blot was applied for CDK4 and cyclin D protein expression of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro; and Real-time PCR was applied for CDK4 and cyclin D mRNA level of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro.4. The activity of CDK2/cyclin E kinase of G0/G1 phase was determined by colorimetric methods; Western blot was applied for CDK2 and cyclin E protein expression of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro; and Real-time PCR was applied for CDK2 and cyclin E mRNA level of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro.5. The activity of CDK2/cyclin A kinase of S phase was determined by colorimetric methods; Western blot was applied for CDK2 and cyclin A protein expression of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro; and Real-time PCR was applied for CDK2 and cyclin A mRNA level of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro.Results: 1. Cells were synchronized on G0/G1 phase by serum starvation method, YSL (6.4mg/ml、3.2mg/ml) could increase the percentage of Go/G1 phase cells, decreasing the percentage of S phase cells. Cells were synchronized on G1/S phase by double thymidine blocking method, YSL could increase the percentage of S phase cells, decreasing the percentage of G2/M phase cells.2. YSL(6.4mg/ml、3.2mg/ml) could increase the percentage of positive cells of phosphorylation histone H2A.X. By comet assay YSL also could induce DNA damage in human hepatocellular carcinoma BEL-7402 cells.3. YSL (6.4mg/ml、3.2mg/ml) could remarkably inhibit the activity of CDK4/cyclin D kinase. YSL could significantly inhibited CDK4 and cyclin D protein expression, but could not affect mRNA level of CDK4 and cyclin D.4. YSL (6.4mg/ml、3.2mg/ml) could remarkably inhibit the activity of CDK2/cyclin E kinase. YSL could significantly inhibited CDK2 and cyclin E protein expression, but could not affect mRNA level of CDK2 and cyclin E.5. YSL (6.4mg/ml、3.2mg/ml) could remarkably inhibit the activity of CDK2/cyclin A kinase. YSL could significantly inhibited CDK2 and cyclin A protein expression, but could not affect mRNA level of CDK2 and cyclin A.Conclusion:YSL could arrest the cell cycle of human hepatocellular carcinoma BEL-7402 cells on G0/G1 phase and S phase. The mechanisms maybe by DNA damage caused by YSL, CDK/cyclin kinase complexes firstly experienced the signal of DNA damage, then the activity of CDK/cyclin kinase complexes were decreased, which induce the cell cycle arrested. And at last, YSL induces tumor cells apoptosis.更多还原