【作者】 王宇峰；

【导师】 常雅萍；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 硕士

【摘要】 本研究检测Stat3mRNA在肿瘤细胞中的表达,并探讨JAK激酶抑制剂AG490作为JAK/STAT通路的阻断剂阻断Stat3信号转导通路对肿瘤细胞增殖、凋亡及凋亡相关因子mRNA表达的影响,研究Stat3信号通路与凋亡相关因子之间可能存在的联系。采用半定量RT-PCR法对人6种肿瘤细胞进行Stat3mRNA表达检测;应用AG490作用于肿瘤细胞及小鼠脾细胞, MTT法检测细胞增殖;流式细胞术等动态观察AG490阻断Stat3信号转导通路对人宫颈癌HeLa细胞的细胞周期、凋亡状况的影响;RT-PCR法检测Stat3、bcl-2、bax因子的mRNA表达情况以及AG490阻断Stat3信号分子对HMGA1表达的影响。结果表明,在所检测的人肿瘤细胞中,MCF-7、PC-3m、Smmc7721、BGC823及Hela细胞株中均有Stat3mRNA的表达; AG490作用人宫颈癌HeLa细胞,表明HeLa细胞增殖抑制水平随作用时间延长而增强;Stat3mRNA表达随AG490作用时间延长而减弱;凋亡相关因子bcl-2、bax等的mRNA表达根据AG490作用时间的不同有相应的改变;而对HMGA1的mRNA表达则无影响。结果提示Stat3在肿瘤细胞增殖、凋亡过程中起关键性作用, AG490阻断Stat3信号途径可抑制肿瘤细胞stat3的持续活化,诱导肿瘤细胞凋亡。上述结果为JAK激酶抑制剂AG490治疗肿瘤提供了实验依据。更多还原

【Abstract】 Tumor, especially maligenant tumor, seriously threaten human health. The scientists from different fields have been searching therapic measure for maligenant tumor diseases for decades, but little effect has been received. In the past ten years, with rapid development and intensive reseach in theory and technology of cytobiology and molecular biology, researchers discovered that Stat3(signal transducer and activator of transcription-3) involved in some physiological processes in cell growth, differentiation , proliferation, and survival. It was reported that Stat3 was overactiviated in different kinds of maligenant tumor cells, and regarded as one of oncogenes. Cell apoptosis is distinct from cell necrosis in morph and biochemical character in that cell apoptosis is active cell death, which is controlled by expressions of some genes. Death of tumor cells induced by apoptosis can not cause fervent inflammatory reaction, but act as protective effect on normal tissues and cells by killing tumor cells meanwhile. So, researchers pay close attention to presumption that apoptosis of tumor cells mey been induced by inhibiting expression of oncogenes.[Objective]To investigate feasibility and molecular mechanism of Stat3 signal transduction pathway ,we explored effect of Stat3 signal transduction pathway with AG490 inhibitor on proliferation and apoptosis of tumor cells during treating tumor. [Methods]RT-PCR was performed to detect expressions of Stat3m- RNA in tumor cells of human; MMT was used to explore proliferative state of Hela cells, Smmc7721 cells and spleen cells of mice; generation cycle and apoptosis were determined by using flow cytometer and we also investigated expressions of Stat3,bax and bcl-2 Mrna after Stat3 signal transduction pathway was inhibited by AG490- JAK inhibitor in Hela cell.[Results]1 Stat3 was markly expressed in tumor cell lines of human. RT-PCR assay showed that Stat3 mRNA was expressed in 5of 6 tumor cells, including MCF-7, PC-3m, Smmc7721, BGC823 and Hela cells .2 Expressions of Stat3 mRNA were gradually attenuated with time went on after Stat3 signal transduction pathway was inhibited by AG490 at different timepoints . However, expressions of HMGA1 mRNA were intact following Stat3 signal transduction pathway being inhibited.3 Depressant effect of AG490 on proliferation of Hela cells and smmc7721 cells. Compared with control group, experimental group cell proliferation was obviously decreased4 Flow cytometer assay showed AG490 action Hela cells 72h that the ratio of cells in G1 cycle increased by from 64.06% to 70.12% in G1 cycle, decreased by from 28.93% to 14.39% in S cycle, and the ratio cell apoptosis increased by 0.49% to 3.79%. AG490 action smmc7721 cells 72h that the ratio of cells in G1 cycle increased by from 54.19% to 63.51% in G1 cycle, decreased by from 23.09% to 0.03% in S cycle, and AG490 action smmc7721 cells 48h the ratio cell apoptosis increased by 5.96% to 20.51%.5 Change of expressions for tumor related apoptotic factors mediated by Stat3 signal transduction pathway was determined following AG490 treating in Hela cells and Smmc7721 cells. Expressions of bax mRNA were enhanced and bcl-2 mRNA were attenuated, even eliminated after Stat3 signal transduction pathway was blocked in Hela cells; Expressions of bcl-2 mRNA were decreased after Stat3 signal transduction pathway was blocked on Smmc7721 cells.[Conclusions]1 Stat3 mRNA was generally expressed in tumor cells of human.2 Expressions of Stat3 mRNA were downregulated with time went on after tumor cells were treated with AG490.3 Cell proliferation was markly inhibited in tumor cells HeLa and smmc7721, and cell apoptosis was promoted with AG490 treatment at dose of 65μmol/L .4 Expressions of bcl-2 were downregulated, while bax mRNA were upregulated after Stat3 signal transduction pathway was blocked with AG490, JAK inhibitor.To sum up, Stat3 was generally expressed in tumor cells of human, and AG490, one of JAK inhibitors, effectively blocked Stat3 signal transduction pathway, which promoted cell apoptosis through inhibiting cell proliferation . The present study shows that expressions of tumor related apoptotic factors, including bcl-2,bax, are controlled by Stat3 signal transduction pathway. Apoptosis of tumor cells maybe occur following Stat3 signal transduction pathway being blocked, which will lay experimental foundation on treating tumor diseases.更多还原