【摘要】：[目的]探討血根堿對紫杉醇耐藥卵巢癌A2780/Taxol細(xì)胞生長及化療敏感性的影響和機(jī)制,為血根堿用于紫杉醇耐藥卵巢癌的治療提供實驗基礎(chǔ)和理論依據(jù)。[方法]培養(yǎng)紫杉醇耐藥卵巢癌細(xì)胞株A2780/Taxol細(xì)胞,MTT比色法檢測不同濃度的血根堿、紫杉醇對A2780/Taxol細(xì)胞的抑制率,確定血根堿及紫杉醇的最適工作濃度、時間。A2780/Taxol細(xì)胞分為對照組、血根堿組、紫杉醇組、血根堿聯(lián)合紫杉醇組,對照組加生理鹽水,血根堿組加2. 0μ mol/L血根堿,紫杉醇組加2μg/mL紫杉醇,血根堿聯(lián)合紫杉醇組同時加2. 0μ mol/L血根堿及2μg/mL紫杉醇。培養(yǎng)48h,應(yīng)用MTT法、細(xì)胞克隆形成實驗、流式細(xì)胞儀和Western blot法檢測血根堿對A2780/Taxol細(xì)胞增殖、克隆形成、凋亡、細(xì)胞周期的影響及Bax、Caspase-3 及β -tubulin Ⅲ和 TGF-β /Smad 信號通路的影響。[結(jié)果]1.血根堿可顯著抑制A2780/Taxol細(xì)胞增殖,并呈劑量、時間依賴性。根據(jù)血根堿劑量、時間關(guān)系,確定血根堿工作濃度為2. 0μ mol/L,作用時間為48h,用于后續(xù)實驗。紫杉醇的工作濃度為2μg/mL,A2780/Taxol細(xì)胞紫杉醇的耐藥系數(shù)(IR)為25.07。2.細(xì)胞增殖實驗顯示,與對照組相比,紫杉醇組、血根堿組及血根堿聯(lián)合紫杉醇組均能顯著抑制A2780/Taxol細(xì)胞增殖,血根堿聯(lián)合紫杉醇組抑制率最高,差異具有統(tǒng)計學(xué)意義(P0. 05)。3.克隆形成結(jié)果顯示,與對照組相比較,紫杉醇組、血根堿組及血根堿聯(lián)合紫杉醇組克隆形成率均明顯降低,血根堿聯(lián)合紫杉醇組降低最為明顯,差異有統(tǒng)計學(xué)意義(P0. 05)。4.流式細(xì)胞檢測細(xì)胞凋亡結(jié)果顯示,與對照組相比較,紫杉醇組、血根堿組及血根堿聯(lián)合紫杉醇組細(xì)胞凋亡率明顯增加,紫杉醇聯(lián)合血根堿組細(xì)胞凋亡率增加最為明顯,差異性顯著(P0.05)。5.流式細(xì)胞檢測細(xì)胞周期結(jié)果顯示,與對照組相比,血根堿組細(xì)胞周期百分比無差異性(P0.05);紫杉醇組G0/G1期、S期細(xì)胞百分比顯著減少,G2/M期細(xì)胞百分比顯著增加,差異均有統(tǒng)計學(xué)意義(P0. 05);血根堿聯(lián)合紫杉醇組G0/G1期細(xì)胞百分比含量降低、G2/M期細(xì)胞百分比含量增加,均具有差異性(P0. 05);血根堿聯(lián)合紫杉醇組與紫杉醇組比較發(fā)現(xiàn),G2/M細(xì)胞百分比明顯減少,而G0/G1期細(xì)胞百分比增加,差異具有統(tǒng)計學(xué)意義(P0. 05)。6. Western blot結(jié)果顯示,與對照組相比,紫杉醇組、血根堿組、血根堿聯(lián)合紫杉醇組Caspase-3、Bax與Smad7蛋白表達(dá)均顯著增高,血根堿聯(lián)合紫杉醇組表達(dá)量最高,差異具有統(tǒng)計學(xué)意義(P0.05);與對照組相比,紫杉醇組、血根堿組、血根堿聯(lián)合紫杉醇組TGF-β 1、Smad3、β-tubulinⅢ表達(dá)均明顯降低,血根堿聯(lián)合紫杉醇組表達(dá)量最低,差異具有統(tǒng)計學(xué)意義(P0. 05)[結(jié)論]1.血根堿可抑制紫杉醇耐藥卵巢癌A2780/Taxol細(xì)胞生長,增加紫杉醇藥物敏感性,逆轉(zhuǎn)紫杉醇耐藥;2.血根堿通過抑制TGF-β /Smad信號通路,上調(diào)紫凋亡相關(guān)蛋白Bax及Caspase-3的表達(dá),促進(jìn)A2780/Taxol細(xì)胞凋亡,發(fā)揮抗腫瘤作用;3.血根堿通過抑制TGF-β /Smad信號通路,降低β-tubulinⅢ表達(dá),促進(jìn)紫杉醇化療敏感性,逆轉(zhuǎn)卵巢癌紫杉醇耐藥。
[Abstract]:[Objective] To investigate the effect and mechanism of haematogenine on the growth and chemosensitivity of paclitaxel-resistant ovarian cancer cell line A2780/Taxol, and to provide experimental and theoretical basis for the use of haematogenine in the treatment of paclitaxel-resistant ovarian cancer. The inhibiting rate of alkaloids and paclitaxel on A2780/Taxol cells was determined, and the optimum concentration of haematogen and paclitaxel was determined. A2780/Taxol cells were divided into control group, haematogen alkaloids group, paclitaxel group, haematogen alkaloids combined with paclitaxel group, control group with saline, haematogen alkaloids group with 2.0 micromol/L haematogen alkaloids, paclitaxel group with 2 microgram/mL paclitaxel and haematogen alkaloids combined. Paclitaxel group was treated with 2.0 micromol/L haemorrhizine and 2 micromol/mL paclitaxel at the same time. The effects of haemorrhizine on proliferation, cloning, apoptosis, cell cycle and Bax, Caspase-3, beta-tubulin III and TGF-beta/Smad signaling pathway of A2780/Taxol cells were detected by MTT assay, cell cloning assay, flow cytometry and Western blot. [Results] 1. Haematogenine significantly inhibited the proliferation of A2780/Taxol cells in a dose-and time-dependent manner. According to the dose-and time-dependent relationship of haematogenine, the working concentration of haematogenine was 2.0 micromol/L for 48 hours. The working concentration of paclitaxel was 2 ug/mL, and the resistance coefficient (IR) of A2780/Taxol cells was 25.07.2. Cell proliferation test showed that compared with the control group, paclitaxel group, haematogenine group and haematogenine combined with paclitaxel group could significantly inhibit the proliferation of A2780 / Taxol cells. The inhibition rate of haematogenine combined with paclitaxel group was the highest, and the difference was statistically significant (P 0.05). 3. Cloning results showed that compared with the control group, paclitaxel group, haematogenine group and haematogenine group were significantly inhibited. The results of flow cytometry showed that compared with the control group, the cell apoptosis rate of paclitaxel group, haematogenine group and haematogenine combined paclitaxel group increased significantly, and the combination of paclitaxel and paclitaxel group increased significantly. Flow cytometry showed that there was no significant difference in the percentage of cell cycle between the two groups (P The percentage of G0/G1 phase cells decreased and the percentage of G2/M phase cells increased in the combination group (P 0.05). Compared with the taxol group, the percentage of G2/M phase cells decreased significantly, but the percentage of G0/G1 phase cells increased significantly (P 0.05). Tern blot results showed that compared with the control group, the expression of Caspase-3, Bax and Smad7 protein in paclitaxel group, haemorrhizine group, haemorrhizine combined with paclitaxel group were significantly increased, and the expression of TGF-beta in haemorrhizine combined with paclitaxel group was the highest (P 0.05); compared with the control group, the expression of TGF-beta in paclitaxel group, haemorrhizine group, haemorrhizine combined with paclitaxel group was statistically significant 1. The expression of Smad3 and beta-tubulin III was significantly decreased, and the expression of serum rhizonine combined with paclitaxel was the lowest, with significant difference (P 0.05)[Conclusion]1. Serum rhizonine could inhibit the growth of paclitaxel-resistant ovarian cancer A2780/Taxol cells, increase the drug sensitivity of paclitaxel and reverse the drug resistance of paclitaxel; 2. Serum rhizonine could inhibit the TGF-beta/Smad signaling pathway. Upregulate the expression of Paclitaxel-related proteins Bax and Caspase-3, promote apoptosis of A2780/Taxol cells, play an anti-tumor role; 3. Haemorrhizine can inhibit the TGF-beta/Smad signaling pathway, reduce the expression of beta-tubulin III, promote the chemosensitivity of paclitaxel, and reverse the resistance of ovarian cancer to paclitaxel.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 張瑞;張靖;王歌;陸允敏;朱金水;;血根堿通過下調(diào)DUSP4/ERK通路抑制胃癌細(xì)胞生長及侵襲[J];上海交通大學(xué)學(xué)報(醫(yī)學(xué)版);2016年01期

2 王琳;余瑞雙;楊文亮;欒淑娟;秦本凱;龐曉斌;杜冠華;;紫杉醇載藥膠束對人肺癌A549細(xì)胞增殖與凋亡的影響[J];藥學(xué)學(xué)報;2015年10期

3 杜培;張曉薇;陳禮全;陳國勤;;原發(fā)性上皮性卵巢癌患者中β-tubulin Ⅲ 表達(dá)與紫杉醇化療耐藥的相關(guān)性研究[J];實用醫(yī)學(xué)雜志;2014年07期

4 劉兵;白洋;張立;李向輝;王鴻艷;張涵亮;余冬陽;;紫杉醇聯(lián)合順鉑對人卵巢癌耐藥細(xì)胞株SKOV3/DDP耐藥性的逆轉(zhuǎn)作用[J];中國婦幼保健;2013年18期

5 楊舒;劉巖;楊千帆;向俊鋒;唐亞林;徐廣智;;博落回抗腫瘤作用及誘導(dǎo)人體端粒DNA形成G-四鏈體分子機(jī)制研究[J];中草藥;2011年04期

6 董西林;周晶;陳真真;王雅娟;李家芝;;LRP及β-Tubulin在非小細(xì)胞肺癌組織中表達(dá)及其與腫瘤耐藥的關(guān)系[J];實用癌癥雜志;2010年05期

7 邵澤勇;辛建;;腫瘤細(xì)胞多藥耐藥化學(xué)逆轉(zhuǎn)劑研究進(jìn)展[J];中外醫(yī)療;2008年22期

相關(guān)博士學(xué)位論文 前3條

1 郭通航;葛根素影響人卵巢癌細(xì)胞生長及逆轉(zhuǎn)耐藥機(jī)制的實驗研究[D];山東大學(xué);2016年

2 郝潤;幾種原小檗堿類生物堿與G-四鏈體結(jié)構(gòu)相互作用的研究[D];北京工業(yè)大學(xué);2014年

3 徐加英;血根堿抗宮頸癌作用及其機(jī)制的研究[D];蘇州大學(xué);2013年

相關(guān)碩士學(xué)位論文 前4條

1 梁宏;基于生物信息學(xué)挖掘卵巢癌順鉑耐藥機(jī)制及潛在治療藥物[D];昆明醫(yī)科大學(xué);2016年

2 姚雯;血根堿抗氧化活性及其對MKN-45和TE-1癌細(xì)胞凋亡的研究[D];西北農(nóng)林科技大學(xué);2014年

3 禚銀玲;下調(diào)βⅢ-tubulin逆轉(zhuǎn)非小細(xì)胞肺癌細(xì)胞株A549/Taxol紫杉醇耐藥的研究[D];濟(jì)南大學(xué);2012年

4 張鴻;血根堿對細(xì)胞的增殖抑制和凋亡誘導(dǎo)作用研究[D];內(nèi)蒙古工業(yè)大學(xué);2010年

，

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