雷公藤红素在前列腺癌细胞中增敏多西紫杉醇的研究

发布时间：2018-06-08 02:23

本文选题：雷公藤红素 + 多西紫杉醇　；参考：《哈尔滨工业大学》2016年硕士论文

【摘要】：前列腺癌(Prostate Cancer,PCa)是在男性中发病率和死亡率都非常高的癌症之一。虽然随着检测手段的更新以及治疗方式的多样化,但是大部分病人一旦发展出转移瘤,进而转变为CRPC(Castration-resistant PCa),最终产生药物抗性时,其生存期将大大降低。CRPC被认为与AR(Androgen Receptor)通路的再激活关系密切,AR通路的再激活是导致肿瘤存活的关键。AR剪切变异体AR-V7不需要雄激素激活转运入核,能够激活AR通路调控下游基因。因此,AR-V7已成为治疗CRPC前列腺癌的肿瘤治疗的研究热点。多西紫杉醇作为治疗CRPC的一线化疗药物,能够通过抑制微管解聚进而抑制AR核转入并激活细胞凋亡。然而CRPC最终会发展出药物抗性,抗性来源可能是凋亡通路的改变以及AR-V7的表达。雷公藤红素作为一种天然的蛋白酶体抑制剂,能够通过诱导细胞凋亡、抑制AR的表达进而抑制前列腺癌细胞系的生长。因此,本研究利用AR-V7阳性细胞系22Rv1作为模型,并将AR-V7阴性细胞系LNCa P作为对照,分析并解释多西紫杉醇与雷公藤红素对前列腺癌细胞的生长抑制作用差异以及其机理。本研究通过MTT实验确证了22Rv1为多西紫杉醇抗性细胞系、LNCa P则为敏感细胞系,同时与雷公藤红素联合用药后细胞存活率相似,联合用药计算显示雷公藤红素能够恢复22Rv1对多西紫杉醇的敏感性,而LNCa P经过药物联用处理后则表现出拮抗效果。凋亡实验显示,0.5 nmol/L的多西紫杉醇与0.8μmol/L的雷公藤红素单独用药对AR-V7表达差异的两株前列腺癌细胞诱导的凋亡结果无明显差异,药物联用下22Rv1的凋亡率上升至50.62%,LNCa P则仅为32.6%。这说明了雷公藤红素在AR-V7阳性细胞系中能恢复多西紫杉醇诱导凋亡的能力。同时Western Blot结果显示,雷公藤红素能够恢复多西紫杉醇在多西紫杉醇抗性细胞系中的PARP切割,并且在多西紫杉醇处理下22Rv1细胞系较LNCa P细胞PARP切割表达量少,这说明了雷公藤红素能够通过恢复多西紫杉醇抗性细胞系内已改变的凋亡通路产生增敏作用。q RT-PCR结果显示,雷公藤红素能够从m RNA水平上抑制AR、AR-V7的表达,而多西紫杉醇不能够影响其表达。这说明,雷公藤红素能够通过抑制AR、AR-V7表达抑制细胞内的AR通路。综上,本研究初步分析雷公藤红素与多西紫杉醇对前列腺癌细胞生长抑制差异,阐明雷公藤红素抑制AR-V7阳性细胞生长、药物增敏的部分分子机制。
[Abstract]:Prostate Cancer is one of the cancers with high morbidity and mortality in men. Although most patients develop metastases and become resistant to CRPC-Castration-resistant PCA, when they develop a metastatic tumor and eventually develop drug resistance, as testing methods are updated and treatment options are diversified, Its survival time will be greatly reduced. CRPC is considered to be closely related to the reactivation of the ARAndrogen receptor) pathway. The key factor leading to tumor survival is that AR-V7 does not require androgen activation and transport into the nucleus, and can activate the downstream genes of AR pathway. Therefore, AR- V 7 has become the focus of cancer therapy for CRPC prostate cancer. Docetaxel, as a first-line chemotherapeutic agent for CRPC, can inhibit AR nuclear transfer and activate apoptosis by inhibiting microtubule depolymerization. However, CRPC may eventually develop drug resistance, which may be due to changes in apoptosis pathway and the expression of AR-V7. As a natural proteasome inhibitor, Tripterygium wilfordii can inhibit the expression of AR and inhibit the growth of prostate cancer cell line by inducing apoptosis. Therefore, using AR-V7 positive cell line 22Rv1 as model and AR-V7 negative cell line LNCa P as control, this study analyzed and explained the difference between docetaxel and tripterygium on the growth inhibition of prostate cancer cells and its mechanism. In this study, we confirmed that 22Rv1, a docetaxel resistant cell line, was a sensitive cell line by MTT assay, and the cell survival rate was similar to that of tripterine. The combined drug calculation showed that tripterine could restore the sensitivity of 22Rv1 to docetaxel, while LNCAP showed antagonistic effect after combined treatment. Apoptosis test showed that there was no significant difference in apoptosis induced by docetaxel (0.5 nmol / L) and tripterygium (0.8 渭 mol / L) alone in two prostate cancer cell lines with different expression of AR-V7, but the apoptosis rate of 22Rv1 increased to 50.62LNCa P only 32.6%. This suggests that tripterine can restore the ability of docetaxel to induce apoptosis in AR-V 7 positive cell line. At the same time, Western blot showed that Tripterygium wilfordii could restore the PARP cleavage of docetaxel resistant cell line, and the expression of PARP in 22Rv1 cell line treated with docetaxel was lower than that in LNCa P cell line. The results indicated that tripterine could increase the sensitivity by restoring the altered apoptotic pathway in docetaxel-resistant cell lines. The results of RT-PCR showed that tripterin could inhibit the expression of AR-V7 at the mRNA level. Docetaxel did not affect its expression. This suggests that tripterine can inhibit AR pathway by inhibiting AR-V 7 expression. In conclusion, this study preliminarily analyzed the difference between tripterine and docetaxel in inhibiting the growth of prostate cancer cells and elucidating the molecular mechanism of tripterine inhibiting the growth of AR-V7 positive cells and enhancing drug sensitivity.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.25

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