發(fā)布時(shí)間：2018-04-15 01:23

  本文選題：五味子脂A + 紫杉醇　； 參考：《吉林大學(xué)》2014年碩士論文

【摘要】：卵巢癌是病死率最高的婦科惡性腫瘤，以紫杉醇（paclitaxel, PTX）和卡鉑（carboplatin, CBP）聯(lián)合化療的標(biāo)準(zhǔn)方案是治療卵巢癌的重要手段，但具有選擇性差、毒副作用強(qiáng)、易產(chǎn)生耐藥等缺點(diǎn)。中藥治療以其毒性低、效率高的優(yōu)勢成為卵巢癌的輔助治療手段之一。近年來研究表明，五味子有效成分具有逆轉(zhuǎn)耐藥、抑制增殖、促進(jìn)凋亡、抗炎、抗氧化、提高免疫力等作用。本實(shí)驗(yàn)選取五味子有效成份——五味子脂A(GomisinA, GA)作為研究對(duì)象，觀察GA分別與紫杉醇和卡鉑聯(lián)合應(yīng)用對(duì)卵巢癌Skov3細(xì)胞凋亡的影響，并初步探討其作用機(jī)制，為更加合理、有效的臨床化療聯(lián)合用藥方案提供實(shí)驗(yàn)依據(jù)。 本研究應(yīng)用體外培養(yǎng)的人漿液性囊腺性卵巢癌細(xì)胞Skov3，分別設(shè)空白對(duì)照組、五味子脂A組、紫杉醇組、卡鉑組、五味子脂A聯(lián)合紫杉醇組和五味子脂A聯(lián)合卡鉑組，MTT法檢測各給藥組細(xì)胞的增殖抑制率。選取抑制率在30%左右、IC50值下游的濃度作為給藥濃度，作用48小時(shí)后，相差顯微觀察細(xì)胞形態(tài)；流式細(xì)胞儀檢測細(xì)胞凋亡率；TUNEL染色觀察細(xì)胞凋亡指數(shù)；JC-1染色檢測細(xì)胞線粒體膜電位；克隆形成實(shí)驗(yàn)檢測細(xì)胞形成克隆能力；Transwell侵襲實(shí)驗(yàn)檢測細(xì)胞侵襲能力；Real time PCR檢測Bax、Caspase3、Bcl-2、Stat3、MMP2、MMP9基因的轉(zhuǎn)錄；Western-blot檢測細(xì)胞Bax、Bcl-2、Caspase3、MMP2、MMP9、STAT3、AKT1、pAKT1、Cytc蛋白的表達(dá)。 實(shí)驗(yàn)結(jié)果顯示，五味子脂A單獨(dú)應(yīng)用可輕度抑制Skov3，紫杉醇、卡鉑可明顯抑制Skov3細(xì)胞增殖，且其作用呈劑量依賴效應(yīng)。五味子脂A聯(lián)合紫杉醇、五味子脂A聯(lián)合卡鉑對(duì)Skov3細(xì)胞均具有協(xié)同抑制作用。通過流式細(xì)胞儀檢測細(xì)胞凋亡率、TUNEL染色檢測細(xì)胞凋亡指數(shù)、JC-1檢測細(xì)胞線粒體膜電位變化，發(fā)現(xiàn)與單獨(dú)用藥相比較，五味子脂A可分別增強(qiáng)紫杉醇、卡鉑誘導(dǎo)Skov3細(xì)胞凋亡的作用，，其中五味子脂A與紫杉醇聯(lián)合應(yīng)用效果更為顯著。Real time PCR結(jié)果顯示，五味子脂A與紫杉醇、卡鉑聯(lián)合應(yīng)用后，Bax、Caspase3轉(zhuǎn)錄水平上調(diào)，Bcl-2、Stat3、MMP2、MMP9轉(zhuǎn)錄水平下調(diào)。Western blot檢測結(jié)果顯示：五味子脂A與紫杉醇、卡鉑聯(lián)合應(yīng)用后，Bax、Caspase3蛋白表達(dá)上調(diào)，Bcl-2、MMP2、MMP9、STAT3、AKT1、pAKT1及Cytc蛋白表達(dá)下調(diào)，聯(lián)合給藥組上調(diào)更明顯；單獨(dú)給藥組均不同程度下調(diào)蛋白表達(dá)，聯(lián)合給藥組下調(diào)更明顯。 本研究結(jié)果提示，五味子脂A可抑制卵巢癌Skov3細(xì)胞增殖、促進(jìn)其凋亡；五味子脂A與紫杉醇、卡鉑聯(lián)合應(yīng)用對(duì)卵巢癌Skov3細(xì)胞可發(fā)揮協(xié)同抑制作用，增強(qiáng)紫杉醇和卡鉑的促凋亡作用，聯(lián)合應(yīng)用可能發(fā)揮化療的減毒增效作用。其可能的分子機(jī)制是：通過下調(diào)Bcl-2、AKT1基因表達(dá)，上調(diào)Bax、Caspase3、Cytc基因表達(dá)來促進(jìn)凋亡；通過下調(diào)MMP2、MMP9的表達(dá)，抑制Skov3細(xì)胞的侵襲和轉(zhuǎn)移；抑制STAT3蛋白的表達(dá)，從而減少p-STAT3的生成，阻斷JAK-STAT信號(hào)轉(zhuǎn)導(dǎo)通路，抑制癌細(xì)胞增殖。本研究為五味子脂A與化療藥聯(lián)合應(yīng)用治療卵巢癌提供理論依據(jù)和實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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