【作者】 王炎；

【导师】 李一；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 硕士

【摘要】 细胞周期是从细胞分裂结束到下一次分裂结束的过程。包括G1、S、G2和M等四期。其基本功能是确保在S期合成的DNA忠实复制和在M期同源染色体平均分配到两子细胞中。这些过程通常受到信号传导途径和反馈环路的精确调节,其正常与否和个体细胞的生长、分化、衰老及癌变密切相关。肿瘤细胞与正常细胞通过细胞周期的时间基本相同,但肿瘤细胞的增殖失控,一旦进入有丝分裂期,便不停止的循环,无止境的生长。而正常细胞周期中的某些限制点可抑制细胞反常的增殖分裂,抑或是引起细胞凋亡。近年来研究发现,许多抗肿瘤药通过引起肿瘤细胞凋亡发挥治疗作用。而肿瘤细胞凋亡与抑癌基因表达状态密切相关,其中以P53和Rb起主要作用。Rb基因的产物Rb蛋白在控制细胞进入或脱离细胞周期的过程中起重要作用,并以高磷酸化和低磷酸化形式存在,去磷酸化Rb蛋白为其活性形式。本实验以人乳腺癌细胞为研究对象,探讨吡柔比星(THP)作用后乳腺癌细胞凋亡与去磷酸化Rb蛋白之间的关系。结果表明去磷酸化Rb蛋白表达量与THP抑制乳腺癌MCF-7细胞增殖作用呈正相关,去磷酸化Rb蛋白可以使乳腺癌细胞MCF-7阻滞于G1期,阻断DNA合成,导致细胞凋亡。本研究深入探讨了抗肿瘤药物调控磷酸化Rb蛋白量和功能及其诱导肿瘤细胞凋亡的因果关系,或许能为抗肿瘤药物筛选提供一个新的作用靶点。更多还原

【Abstract】 People think highly of regulating the cell cycle in the tumor generation. It’s known to all, the cell cycle is the process from cell cleavage ending to next cleavage ending. Cell cycle essential function are to ensure DNA that composite in S phases faithful copy, to ensure that homologous chromosome can be divided equally to two daughter cells. If cell cycle regulated aberration, cells will have a tendence of uninterrupted cycling, to have no limit cleavage and cell differentiation aberration, and ultimately lead to tumor generation.To follow people understand uninterruptedly profound to cell cycle regulation , people discover that in fact tumor is aclass diseases of cell cycle, and tumor cells are a cluster cell cycle abnormal cells, especially these cells have loss of control in cell cycle G1/S check point. pRb protein have important roles in the regulation of cell cycle G1/S check point. A great quantity investigation demonstrated that pRb protein participated the negative regulation of cell cycle and educed the function to restrain the development of tumor. Many cell cycle positive and negative regulation factor, such as cyclins, CDKs ,CDK1s, they could form“Rb pathway”with pRb and regulate cell normal cleavage and differentiation. If the pathway lost balance, it could be one reason of cell malignant proliferation.Recently some investigations discovered that many antineoplastic agents educed therapeutical effect through causing the tumor cells apoptosis. Therefore tumor cells apoptosis had closely relation with anti-oncogene expression, but P53 and Rb possessed major effect. Rb protein, the production of Rb gene, educed major effect in controlling cell access or leaving cell cycle. In our experiment we made use of breast cancer cells as study object, and approached the relationbetween breast cancer cells apoptosis and dephosphorylated Rb protein after THP treatment. Consequence demonstrated that the expression of dephosphorylated Rb protein and the inhibition breast cancer cell MCF-7 proliferation effect of THP had positive correlation, and dephosphorylated Rb protein made MCF-7 cell cycle stoppage in G1 phases and blocked DNA synthesis, therefore led to cell apoptosis.Experiment objectiveTo investigate the relation between breast cancer cells apoptosis and dephosphorylated Rb protein after THP treatment. Study concents1. THP has effect to the proliferation of MCF-7.In order to study the change of proliferation of MCF-7 after THP treatment, we detected different concentration THP to effect the proliferation of MCF-7 through MTT assay. MTT assay revealed that 1,2.5,5,7.5,10ug/ml THP had different effect to the proliferation of MCF-7. THP significantly inhibited the proliferation of MCF-7 and with the THP concentration’s increasing, the survival rate of MCF-7 obviously decreased.2. THP has effect to the expression of Rb protein of MCF-7. In order to study the expression of dephosphorylated Rb protein of MCF-7 before treatment and after treatment, we detected the expression of dephosphorylated Rb protein of MCF-7 before treatment and after treatment through immunohistochemistry SP assay, positive cell was that cell nucleus were stained buffy; and negative cell was cell nucleus were stained blue. The results of immunocytochemical staining were achieved: Before treatment of THP, phosphorylated Rb protein was positive and dephosphorylated Rb protein was negative. After 24 hours treatment of different concentration THP(1,2.5,5,7.5,10ug/ml), Rb protein located intranuclear, especially Rb protein expressed comparative conspicuously in the treatment of 5ug/ml THP, whlie phosphorylated Rb protein was negative.3. THP has effect to the apoptosis of MCF-7.In order to study THP to the effect of tumor cells apoptosis, we detected the cells apoptosis through FACS with PI staining. Detecting the rate of apoptosis of MCF-7 by THP induced withFCM assays.FCM assays indicated: the nature rate of apoptosis of MCF-7 was 0.0417, MCF-7 cells apoptosis increased by the treatment of THP, especially in the treatment of 5ug/ml THP, apoptosis was very significant and the rate of apoptosis was 0.2321.4. The relationship of the effect of THP inhibiting breast cancer cell proliferation and the expression of dephosphorylated Rb protein.The correlative analysis by SPSS 10 for windows showed the correlation coefficient of THP inducing the apoptosis rate of MCF-7/S cells and the expression levels of dephosphorylated Rb protein was 0.0972, It indicates there were positive correlation (P<0.05) between the expression levels of dephosphorylated Rb protein and the apoptosis of THP inducement MCF-7 cells.5. THP has effect to the cell cycle of MCF-7.After 24 hours treatment of THP with 0.25, 1, 2.5, 5 ug/ml, we analyzed the cell cycle made clear : sensitive breast cancer MCF-7 were accumulated in G1 phases, and sensitive breastcancer MCF-7 were decrease in S phases, that indicated THP made MCF-7 block in G1 phase by dephosphorylated Rb protein.Conclusion1. We detected different concentration THP to effect the proliferation of MCF-7 through MTT assay. With the THP inhibition function increasing, the expression of dephosphorylated Rb protein was gradually rised. It indicates there were positive correlation between the expression levels of dephosphorylated Rb protein and the function of THP inhibiting MCF-7 cells proliferation.2. We detected the expression of dephosphorylated Rb protein of MCF-7 before treatment and after treatment through immunohistochemistry SP assay, It indicates there were positive correlation between the expression levels of dephosphorylated Rb protein and the apoptosis of THP inducement MCF-7/S cells.3. We detected different concentration THP to effect of the cells apoptosis and cell cycle through FACS with PI staining. Itindicated THP made MCF-7 block in G1 phase by dephosphorylated Rb protein.更多还原