五味子脂A增强紫杉醇和卡铂对Skov3的抑瘤作用及机制研究

发布时间：2018-04-15 01:23

本文选题：五味子脂A + 紫杉醇　；参考：《吉林大学》2014年硕士论文

【摘要】：卵巢癌是病死率最高的妇科恶性肿瘤，以紫杉醇（paclitaxel, PTX）和卡铂（carboplatin, CBP）联合化疗的标准方案是治疗卵巢癌的重要手段，但具有选择性差、毒副作用强、易产生耐药等缺点。中药治疗以其毒性低、效率高的优势成为卵巢癌的辅助治疗手段之一。近年来研究表明，五味子有效成分具有逆转耐药、抑制增殖、促进凋亡、抗炎、抗氧化、提高免疫力等作用。本实验选取五味子有效成份——五味子脂A(GomisinA, GA)作为研究对象，观察GA分别与紫杉醇和卡铂联合应用对卵巢癌Skov3细胞凋亡的影响，并初步探讨其作用机制，为更加合理、有效的临床化疗联合用药方案提供实验依据。本研究应用体外培养的人浆液性囊腺性卵巢癌细胞Skov3，分别设空白对照组、五味子脂A组、紫杉醇组、卡铂组、五味子脂A联合紫杉醇组和五味子脂A联合卡铂组，MTT法检测各给药组细胞的增殖抑制率。选取抑制率在30%左右、IC50值下游的浓度作为给药浓度，作用48小时后，相差显微观察细胞形态；流式细胞仪检测细胞凋亡率；TUNEL染色观察细胞凋亡指数；JC-1染色检测细胞线粒体膜电位；克隆形成实验检测细胞形成克隆能力；Transwell侵袭实验检测细胞侵袭能力；Real time PCR检测Bax、Caspase3、Bcl-2、Stat3、MMP2、MMP9基因的转录；Western-blot检测细胞Bax、Bcl-2、Caspase3、MMP2、MMP9、STAT3、AKT1、pAKT1、Cytc蛋白的表达。实验结果显示，五味子脂A单独应用可轻度抑制Skov3，紫杉醇、卡铂可明显抑制Skov3细胞增殖，且其作用呈剂量依赖效应。五味子脂A联合紫杉醇、五味子脂A联合卡铂对Skov3细胞均具有协同抑制作用。通过流式细胞仪检测细胞凋亡率、TUNEL染色检测细胞凋亡指数、JC-1检测细胞线粒体膜电位变化，发现与单独用药相比较，五味子脂A可分别增强紫杉醇、卡铂诱导Skov3细胞凋亡的作用，，其中五味子脂A与紫杉醇联合应用效果更为显著。Real time PCR结果显示，五味子脂A与紫杉醇、卡铂联合应用后，Bax、Caspase3转录水平上调，Bcl-2、Stat3、MMP2、MMP9转录水平下调。Western blot检测结果显示：五味子脂A与紫杉醇、卡铂联合应用后，Bax、Caspase3蛋白表达上调，Bcl-2、MMP2、MMP9、STAT3、AKT1、pAKT1及Cytc蛋白表达下调，联合给药组上调更明显；单独给药组均不同程度下调蛋白表达，联合给药组下调更明显。本研究结果提示，五味子脂A可抑制卵巢癌Skov3细胞增殖、促进其凋亡；五味子脂A与紫杉醇、卡铂联合应用对卵巢癌Skov3细胞可发挥协同抑制作用，增强紫杉醇和卡铂的促凋亡作用，联合应用可能发挥化疗的减毒增效作用。其可能的分子机制是：通过下调Bcl-2、AKT1基因表达，上调Bax、Caspase3、Cytc基因表达来促进凋亡；通过下调MMP2、MMP9的表达，抑制Skov3细胞的侵袭和转移；抑制STAT3蛋白的表达，从而减少p-STAT3的生成，阻断JAK-STAT信号转导通路，抑制癌细胞增殖。本研究为五味子脂A与化疗药联合应用治疗卵巢癌提供理论依据和实验基础。
[Abstract]:Ovarian cancer is a gynecologic malignant tumor with the highest mortality. The standard regimen of paclitaxel (PTX) and carboplatin (CBPPC) chemotherapy is an important method in the treatment of ovarian cancer, but it has the disadvantages of poor selectivity, strong toxicity and resistance.Because of its low toxicity and high efficiency, traditional Chinese medicine has become one of the adjuvant treatments for ovarian cancer.Recent studies have shown that Schisandra chinensis has the effects of reversing drug resistance, inhibiting proliferation, promoting apoptosis, anti-inflammation, anti-oxidation, enhancing immunity and so on.In this experiment, the active component of Schisandra chinensis, Schisandra chinensis Gomisin A (GA), was selected as the research object, to observe the effect of GA combined with paclitaxel and carboplatin on apoptosis of ovarian cancer Skov3 cells, and to explore its mechanism.Effective clinical chemotherapy combined with drugs to provide experimental basis.In this study, Skov3 cells were cultured in vitro and were divided into blank control group, Schisandrin A group, paclitaxel group and carboplatin group, respectively.The proliferation inhibition rates of Schisandrin A combined with paclitaxel group and Schisandrin A combined with carboplatin group were detected by MTT assay.The concentration downstream of IC50 of 30% or so was selected as the administration concentration. After 48 hours of treatment, the cell morphology was observed microscopically.Apoptosis rate was detected by flow cytometry and Tunel staining was used to detect the mitochondrial membrane potential by JC-1 staining.Detection of Clone formation ability by Transwell invasion Assay Real time PCR was used to detect the expression of BaxanCaspase3Bcl-2Stat3MMP2MMP9 gene and the expression of Bax-Bcl-2Caspase3 / MMP9STAT3AKT1 pAKT1 / Cytc protein in the cell line Baxan Bcl-2Caspase3 / MMP9STAT3AKT1pAKT1 / Cytc protein.The results showed that Schisandrin A alone could slightly inhibit Skov3, paclitaxel and carboplatin on Skov3 cell proliferation in a dose-dependent manner.Schisandrin A combined with paclitaxel and Schisandrin A combined with carboplatin had synergistic inhibitory effects on Skov3 cells.Apoptosis rate was detected by flow cytometry and Tunel staining was used to detect cell apoptosis index (JC-1). It was found that Schisandrin A could enhance paclitaxel and carboplatin induced apoptosis of Skov3 cells.The effect of Schisandrin A combined with paclitaxel was more significant. Real time PCR showed that Schisandrin A and paclitaxel,The results of Western blot analysis showed that Schisandrin A and paclitaxel, carboplatin combined with Caspase3 protein up-regulated Bcl-2MMP2MMP9STAT3STAT1 and Cytc protein expression, especially in combination group.The expression of protein was down-regulated in different degree in the single administration group, especially in the combined administration group.The results suggest that Schisandrin A can inhibit the proliferation and promote apoptosis of ovarian cancer Skov3 cells, and the combination of Schisandra A and paclitaxel, carboplatin can play a synergistic effect on ovarian cancer Skov3 cells.The combination of paclitaxel and carboplatin may play the role of antitoxic and synergistic effect of chemotherapeutics by enhancing the apoptotic effect of paclitaxel and carboplatin.Its possible molecular mechanism is to promote apoptosis by down-regulating the expression of Bcl-2AK T1 gene and up-regulating the expression of Bax-Caspase3Ctc gene; inhibiting the invasion and metastasis of Skov3 cells by down-regulating the expression of MMP2mMP9; and inhibiting the expression of STAT3 protein, thereby reducing the production of p-STAT3.The JAK-STAT signal transduction pathway was blocked and the proliferation of cancer cells was inhibited.This study provides a theoretical and experimental basis for the treatment of ovarian cancer with Schisandrin A combined with chemotherapeutic agents.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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