探讨GGTI-2133对前列腺癌细胞的增殖、迁移、凋亡、化疗敏感性的影响及其作用机制

发布时间：2018-08-31 09:46

【摘要】：目的：通过实验来观察GGTaseI酶抑制剂GGTI-2133对体外培养的人前列腺癌细胞增殖、凋亡、迁移的影响，并检测其在肿瘤细胞对化疗药物敏感性上的影响，以及探讨可能的机制；方法：第一部分：（1）体外培养人前列腺癌DU145细胞，建立细胞实验模型；（2）分别用不同浓度的GGTI-2133干预培养DU145细胞相同的时间或者相同浓度GGTI-2133干预培养不同的时间，采用倒置光学显微镜观察及应用MTT法检测GGTI-2133对DU145细胞增殖的影响，并FACS法检测不同浓度GGTI-2133干预培养DU145细胞24h后的细胞周期的情况，并计算细胞增殖指数PI值；（3）不同浓度GGTI-2133干预培养DU145细胞24h后，应用TUNEL法检测DU145细胞凋亡情况；（4）采用transwell chamber检测对照组及实验组DU145细胞的迁移情况；（5）应用MTT法检测单用及联用GGTI-2133（5μmol/L）时阿霉素对DU145细胞半抑制浓度IC50值。第二部分：不同浓度的GGTI-2133干预培养DU145细胞24h后：（1）应用western blot检测RALA-GTP、RALB-GTP、RALBP1、REPS2/POB1、TBK1、NF-κB蛋白的表达；（2）应用RT-PCR检测Bcl-2、Bax基因的表达。结果：第一部分：（1）DU145细胞增殖受抑制，倒置光学显微镜观察及MTT检测结果显示，随着GGTI-2133浓度（在5～20μmol/L范围内）的升高、作用时间的延长，细胞增殖受抑制情况愈明显，，且GGTI-2133能诱导DU145细胞周期G0/G1期阻滞，实验组细胞增殖指数与对照组比较差异明显（P0.05）；（2）GGTI-2133能促进DU145细胞凋亡，实验组细胞凋亡率增高与对照比较差异具有统计学意义（P0.05）；（3）实验组细胞迁移受抑制，其迁移到transwell板下室面的细胞数（37±16.29）明显少于对照组（327±22.13），差异显著（P0.01）；（4）阿霉素单用时C50值为10.1190±0.1220，联合GGTI-2133（5μmol/L）时IC50值为5.7600±0.0580，两者比较差异显著（P0.05）第二部分：（1）随着GGTI-2133浓度（在5～20μmol/L范围内）的升高，RALA-GTP、RALB-GTP、RALBP1、TBK1、NF-κB蛋白的表达量减少，而REPS2/POB1蛋白的表达量则增加，均与对照组比较差异显著（P0.05）；（2）GGTI-2133可影响凋亡基因Bcl-2、Bax的表达，可使Bcl-2基因表达下调、Bax基因表达上调，且呈浓度依赖性。结论： 1、一定的浓度和时间范围内，GGTI-2133能够抑制体外培养的人前列腺癌细胞增殖、迁移，促进细胞凋亡，并可提高阿霉素对人前列腺癌细胞化疗敏感性； 2、GGTI-2133对人前列腺癌细胞增殖、凋亡、迁移及化疗药物敏感性的影响的可能机制是：GGTI-2133阻断了RalGEF-Ral信号通路的激活过程，主要是抑制Ral-GDP转化为Ral-GTP，从而影响了下游相关蛋白（如RalBP1、REPS2、TBK1、NF-κB）的表达及下游信号通路（如NF-κB信号通路）的激活；另外在前列腺癌细胞的凋亡上，GGTI-2133也可通过下调Bcl-2基因表达及上调Bax基因表达来促进细胞的凋亡。
[Abstract]:Aim: to observe the effects of GGTaseI enzyme inhibitor GGTI-2133 on the proliferation, apoptosis and migration of human prostate cancer cells in vitro, and to detect the effect of GGTI-2133 on the sensitivity of tumor cells to chemotherapeutic agents. Methods: (1) Human prostate cancer DU145 cells were cultured in vitro and the cell model was established. (2) DU145 cells were incubated with different concentrations of GGTI-2133 for the same time or the same concentration of GGTI-2133 for different time. The effects of GGTI-2133 on the proliferation of DU145 cells were observed by inverted optical microscope and MTT assay. The cell cycle and cell proliferation index (PI) of cultured DU145 cells were measured by FACS method after 24 hours of DU145 cell culture with different concentrations of GGTI-2133. (3) TUNEL assay was used to detect the apoptosis of DU145 cells after 24 hours of GGTI-2133 treatment with different concentrations of GGTI-2133. (4) transwell chamber was used to detect the migration of DU145 cells in the control group and experimental group, and (5) MTT assay was used to detect the IC50 value of the semi-inhibitory concentration of adriamycin on DU145 cells when GGTI-2133 was used alone or in combination with GGTI-2133 (5 渭 mol/L). The second part: after 24 hours of DU145 cell culture with different concentrations of GGTI-2133: (1) western blot was used to detect the expression of RALA-GTP,RALB-GTP,RALBP1,REPS2/POB1,TBK1,NF- 魏 B protein, and (2) RT-PCR was used to detect the expression of Bcl-2,Bax gene. Results: (1) the proliferation of DU145 cells was inhibited. The results of inverted optical microscope and MTT showed that with the increase of GGTI-2133 concentration (in the range of 5 ~ 20 渭 mol/L), the action time was prolonged. The inhibition of cell proliferation was more obvious, and GGTI-2133 could induce G0/G1 phase arrest of DU145 cell cycle. Compared with the control group, the cell proliferation index of the experimental group was significantly different from that of the control group (P0.05); (2) GGTI-2133 could promote the apoptosis of DU145 cells. The increase of apoptosis rate in the experimental group was significantly higher than that in the control group (P0.05); (3). The number of cells migrating to the subplate of transwell (37 卤16.29) was significantly lower than that of the control group (327 卤22.13) (P0.01). (4) the C50 value of adriamycin alone was 10.1190 卤0.1220, and the IC50 value in combination with GGTI-2133 (5 渭 mol/L) was 5.7600 卤0.0580. There was a significant difference between the two groups (P0.05) in the second part: (1) the expression of RALA-GTPRALB-GTPRALB-GTPRALB-GTPRALB-GTPRALB-GTPRALB1TBK1- 魏 B protein decreased, while the expression of REPS2/POB1 protein increased with the increase of GGTI-2133 concentration (within the range of 5 ~ 20 渭 mol/L). Compared with the control group, GGTI-2133 (P0.05); (2) could affect the expression of apoptotic gene Bcl-2,Bax and down-regulate the expression of Bcl-2 gene in a dose-dependent manner. Conclusion: 1. GGTI-2133 can inhibit the proliferation, migration and apoptosis of human prostate cancer cells in vitro, and increase the chemosensitivity of adriamycin to human prostate cancer cells. The possible mechanism of the effects of GGTI-2133 on the proliferation, apoptosis, migration and chemotherapeutic susceptibility of human prostate cancer cells is that the activation of the RalGEF-Ral signaling pathway is blocked by: GGTI-2133. It mainly inhibited the transformation of Ral-GDP into Ral-GTP, which affected the expression of downstream related proteins (such as RalBP1,REPS2,TBK1,NF- 魏 B) and activation of downstream signaling pathways (such as NF- 魏 B signaling pathway). In addition, GGTI-2133 can promote the apoptosis of prostate cancer cells by down-regulating the expression of Bcl-2 gene and up-regulating the expression of Bax gene.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.25

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