ROCK蛋白表达和活性改变对视网膜母细胞瘤细胞运动表型的影响研究

发布时间：2018-05-19 02:09

本文选题：视网膜母细胞瘤 + ROCK蛋白　；参考：《上海交通大学》2013年博士论文

【摘要】：研究目的和内容:视网膜母细胞瘤（Retinoblastoma，RB）是小儿最常见的原发性眼内恶性肿瘤，严重危害患儿的视力和生命，而RB的颅内或全身转移是RB患儿死亡的主要原因。有研究表明Rho/ROCK蛋白活性的改变与许多肿瘤细胞的增殖与侵袭密切相关，作为Rho的主要下游效应蛋白，ROCK-1和ROCK-2对活体或离体肿瘤细胞的运动和侵袭能力有重要调节作用。但是，关于ROCK蛋白在RB细胞增殖与侵袭中的作用目前尚不清楚。方法:采用RT-PCR和western blot方法检测RB细胞株（Y79）中ROCK-1和ROCK-2的mRNA和蛋白的表达情况；应用Lipofectamine2000将ROCK小分子干扰RNA (Small interfering RNA，siRNA)转染到Y79细胞中，，观察RNA干扰（RNA interference，RNAi）后细胞中的ROCK-1和ROCK-2的mRNA和蛋白的表达抑制情况；运用ROCK抑制剂（Y-27632）和siRNA抑制ROCK蛋白的活性后，通过CCK-8细胞活性试验、matrigel细胞粘附试验和Transwell细胞侵袭试验，观察ROCK蛋白对RB细胞增殖、转移和侵袭能力的影响。结果:实验结果显示：在Y79细胞中有ROCK-1和ROCK-2mRNA和蛋白的表达，而且ROCK-1的表达要高于ROCK-2；siRNA介导的RNAi可以有效地抑制Y79细胞中ROCK-1和ROCK-2mRNA的表达，进而抑制ROCK-1和ROCK-2蛋白的表达；ROCK抑制剂（Y-27632）对Y79细胞的增殖能力没有影响，但可以显著地增加Y79细胞的粘附能力并降低其侵袭能力；ROCK-1siRNA也可以显著地增加Y79细胞的粘附能力并降低其侵袭能力，但ROCK-2siRNA对Y79细胞的粘附能力和侵袭能力没有影响。结论: ROCK-1蛋白在RB细胞转移与侵袭中起重要作用，很有可能成为治疗RB的新靶点。
[Abstract]:Objective and content: Retinoblastoma RBB is the most common primary intraocular malignant tumor in children, which seriously endangers the visual acuity and life of children. The intracranial or systemic metastasis of RB is the main cause of death in children with RB. Some studies have shown that the change of Rho/ROCK protein activity is closely related to the proliferation and invasion of many tumor cells. As the main downstream effector of Rho, Rock-1 and ROCK-2 play an important role in regulating the motion and invasiveness of tumor cells in vivo or in vitro. However, the role of ROCK protein in RB cell proliferation and invasion is unclear. Methods: RT-PCR and western blot were used to detect the expression of mRNA and protein of ROCK-1 and ROCK-2 in RB cell line Y79, and Lipofectamine2000 was used to transfect ROCK small interfering RNAs into Y79 cells. To observe the inhibition of mRNA and protein expression of ROCK-1 and ROCK-2 in the cells after RNA interference RNAi, and to observe the inhibition of ROCK protein activity by ROCK inhibitor, ROCK inhibitor, Y-27632) and siRNA. The cell adhesion test and Transwell cell invasion test were carried out by CCK-8 cell activity test. To observe the effect of ROCK protein on the proliferation, metastasis and invasion of RB cells. Results: the results showed that ROCK-1, ROCK-2mRNA and protein were expressed in Y79 cells, and the expression of ROCK-1 was higher than that of RNAi mediated by ROCK-2siRNA, which could effectively inhibit the expression of ROCK-1 and ROCK-2mRNA in Y79 cells. Furthermore, inhibition of the expression of ROCK-1 and ROCK-2 protein and the inhibition of the proliferation of Y79 cells by the inhibitor of rock Y-27632 did not affect the proliferation of Y79 cells. But the adhesion ability and invasion ability of Y79 cells were significantly increased and decreased by ROCK-2siRNA, but the adhesion and invasion ability of Y79 cells were not affected by ROCK-2siRNA. Conclusion: ROCK-1 protein plays an important role in the metastasis and invasion of RB cells and may be a new target for the treatment of RB.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.7

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