蛋白酶体抑制剂对卵巢癌细胞的作用研究

发布时间：2018-02-27 12:32

本文关键词： 蛋白酶体抑制剂卵巢癌乳胞素 SKOV3细胞免疫组化　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：实验目的:因为卵巢癌早期无明显症状,加之其缺乏早期的诊断方法和依据、病情进展快等特点,大部分卵巢癌预后很差,高居死亡率首位。而在所有原发性卵巢癌中,70%-90%的病理类型属于上皮性卵巢癌,是临床上最为常见的一种病理类型。本文通过探讨蛋白酶体抑制剂乳胞素对卵巢癌SKOV3细胞体外存活率及增殖率的影响,并检测细胞内相关物质信号水平的变化,初步探讨其可能的作用机制,为卵巢癌的治疗研究提供新的理论思路与研究方向,希望能够改变临床上卵巢癌死亡率居高不下的状况,造福于患者。实验方法:MTT染色、流式细胞仪检测从细胞层面检测蛋白酶体抑制乳胞素作用于体外生长的卵巢癌SKOV3细胞后,对其在细胞增殖及凋亡方面产生的影响Western Blot,RT-PCR则分别检测细胞内蛋白质及基因水平的变化,从微观的层面检测乳胞素引起细胞损伤后,细胞内相关物质信号表达水平发生的变化。实验结果:(1)显微镜观察细胞形态变化:对照组SKOV3细胞生长状态良好,细胞形态饱满,呈多边形样式,以贴壁的方式生长,各细胞之间边界清晰、连接紧密。而经不同浓度的乳胞素作用后的各组细胞,生长状态较差,细胞萎缩,形状多变为圆形或椭圆形,细胞与细胞之间的界限也不十分清楚,细胞体内因充满大量颗粒状物质而使其透光变差,培养基中漂浮的无活性细胞明显增多。随着乳胞素浓度的增高,上述差别表现的更加明显。(2)MTT染色检测结果显示:对比于对照组,经不同浓度的乳胞素作用后,体外生长的SKOV3细胞的生长受到明显抑制,增殖率降低[0.5umol/L组(65.5±0.10%)、1.0umol/L组(54.4±0.11%),2.5umol/L组(42.5±0.09%),5.0umol/L组(29.2±0.12%)及10.0umol/L组(18.9±0.13%)](差异有统计学意义:P0.05)。(3)流式细胞仪检测结果表明,对比于对照组,乳胞素可以促使SKOV3细胞凋亡的发生,并且在单位时间内,其凋亡率随着药物作用浓度的增高而增加,与药物浓度呈正相关,浓度依赖性明显(差异有统计学意义:P0.05)。(4)Western Blot检测结果表明:对比于对照组,随着乳胞素浓度的增高,在相同作用时间条件下,卵巢癌细胞内Bax蛋白的表达水平逐渐增强,而Bcl-2蛋白的表达水平逐渐减弱(差异有统计学意义:P0.05)。(5)RT-PCR电泳结果表明:对比于对照组,随着乳胞素浓度的增加,在相同作用时间条件下,卵巢癌细胞内Bax m RNA的表达水平明显增加,而Bcl-2 m RNA的表达水平逐渐减少(差异有统计学意义:P0.05)。实验结论:蛋白酶体抑制剂乳胞素对体外生长的卵巢癌SKOV3细胞,具有抑制其增殖和诱导其凋亡的作用,推断其主要内部机制可能是通过抑制泛素-蛋白酶体通路的活性,从而导致细胞内Bax、bcl-2等与细胞凋亡密切相关的基因及蛋白的表达水平的失衡,进而影响线粒体途径参与的细胞凋亡,诱导卵巢癌SKOV3细胞凋亡的发生。
[Abstract]:Objective: because ovarian cancer has no obvious symptoms at the early stage and lacks of early diagnostic methods and evidence, the prognosis of most ovarian cancer is poor. The highest mortality rate. And in all primary ovarian cancer, 70 to 90 percent of the pathological types belong to epithelial ovarian cancer, In this paper, we studied the effects of proteasome inhibitor lactin on the survival rate and proliferation rate of ovarian cancer SKOV3 cells in vitro, and detected the changes of intracellular signal level of related substances. In order to provide a new theoretical approach and research direction for the treatment of ovarian cancer, we hope to change the high mortality rate of ovarian cancer in clinic and benefit the patients. Flow cytometry was used to detect the inhibitory effect of proteasome on ovarian cancer SKOV3 cells in vitro. The effects of Western blottir RT-PCR on cell proliferation and apoptosis were detected by reverse transcription-polymerase chain reaction (RT-PCR). The changes of protein and gene levels in cells were detected by RT-PCR, and the changes of cell damage induced by lactin were detected at the microcosmic level. Changes in signal expression level of related substances in cells. Results: microscopic observation of cell morphology: SKOV3 cells in the control group were in good growth state, full shape, polygonal pattern, and grew in the form of adherent to the wall. The boundary between the cells is clear and the connection is tight. However, the cells treated with different concentrations of lactin have a poor growth state, the cells atrophy, the shape is varied into a round or oval shape, and the boundary between the cells is not very clear. When the cells were filled with a large number of granular substances, the light transmittance of the cells became worse, and the number of inactive cells floating in the culture medium increased obviously with the increase of the concentration of lactin. The results of MTT staining showed that the growth of SKOV3 cells in vitro was significantly inhibited after the treatment of different concentrations of lactin, as compared with the control group. The proliferation rate was decreased [0.5umolr / L group 65.5 卤0.10umol.L group 54.4 卤0.11mg / L + 2.5umoll / L group 42.5 卤0.09umoll / L group 29.2 卤0.12cm / L, and 10.0umolol / L group 18.9 卤0.135mm / L respectively] (the difference was statistically significant (P 0.05 / L).) flow cytometry analysis showed that compared with the control group, lactin could induce apoptosis of SKOV3 cells, and in a unit time, lactin could induce apoptosis of SKOV3 cells. The apoptosis rate increased with the increase of drug concentration, and was positively correlated with drug concentration. The results of Western Blot analysis showed that compared with the control group, the apoptosis rate increased with the increase of the concentration of lactin. At the same time, the expression of Bax protein in ovarian cancer cells was gradually increased, while the expression level of Bcl-2 protein was gradually decreased (the difference was statistically significant). The results of RT-PCR electrophoresis showed that: compared with the control group, the expression of Bax protein in ovarian cancer cells was significantly lower than that in control group. At the same time, the expression level of Bax m RNA in ovarian cancer cells increased significantly with the increase of the concentration of lactin. However, the expression of Bcl-2 m RNA was gradually decreased (P 0.05). Conclusion: the proteasome inhibitor lactin can inhibit the proliferation and induce apoptosis of ovarian cancer SKOV3 cells in vitro. It is inferred that its main internal mechanism may be to inhibit the activity of the ubiquitin proteasome pathway, resulting in the imbalance of the expression level of genes and proteins closely related to cell apoptosis, such as Baxanbcl-2. The apoptosis of ovarian cancer SKOV3 cells was induced by mitochondrial pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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