血根碱对紫杉醇耐药卵巢癌细胞的作用及机理研究
发布时间：2018-08-20 13:52
【摘要】：[目的]探讨血根碱对紫杉醇耐药卵巢癌A2780/Taxol细胞生长及化疗敏感性的影响和机制,为血根碱用于紫杉醇耐药卵巢癌的治疗提供实验基础和理论依据。[方法]培养紫杉醇耐药卵巢癌细胞株A2780/Taxol细胞,MTT比色法检测不同浓度的血根碱、紫杉醇对A2780/Taxol细胞的抑制率,确定血根碱及紫杉醇的最适工作浓度、时间。A2780/Taxol细胞分为对照组、血根碱组、紫杉醇组、血根碱联合紫杉醇组,对照组加生理盐水,血根碱组加2. 0μ mol/L血根碱,紫杉醇组加2μg/mL紫杉醇,血根碱联合紫杉醇组同时加2. 0μ mol/L血根碱及2μg/mL紫杉醇。培养48h,应用MTT法、细胞克隆形成实验、流式细胞仪和Western blot法检测血根碱对A2780/Taxol细胞增殖、克隆形成、凋亡、细胞周期的影响及Bax、Caspase-3 及β -tubulin Ⅲ和 TGF-β /Smad 信号通路的影响。[结果]1.血根碱可显著抑制A2780/Taxol细胞增殖,并呈剂量、时间依赖性。根据血根碱剂量、时间关系,确定血根碱工作浓度为2. 0μ mol/L,作用时间为48h,用于后续实验。紫杉醇的工作浓度为2μg/mL,A2780/Taxol细胞紫杉醇的耐药系数(IR)为25.07。2.细胞增殖实验显示,与对照组相比,紫杉醇组、血根碱组及血根碱联合紫杉醇组均能显著抑制A2780/Taxol细胞增殖,血根碱联合紫杉醇组抑制率最高,差异具有统计学意义(P0. 05)。3.克隆形成结果显示,与对照组相比较,紫杉醇组、血根碱组及血根碱联合紫杉醇组克隆形成率均明显降低,血根碱联合紫杉醇组降低最为明显,差异有统计学意义(P0. 05)。4.流式细胞检测细胞凋亡结果显示,与对照组相比较,紫杉醇组、血根碱组及血根碱联合紫杉醇组细胞凋亡率明显增加,紫杉醇联合血根碱组细胞凋亡率增加最为明显,差异性显著(P0.05)。5.流式细胞检测细胞周期结果显示,与对照组相比,血根碱组细胞周期百分比无差异性(P0.05);紫杉醇组G0/G1期、S期细胞百分比显著减少,G2/M期细胞百分比显著增加,差异均有统计学意义(P0. 05);血根碱联合紫杉醇组G0/G1期细胞百分比含量降低、G2/M期细胞百分比含量增加,均具有差异性(P0. 05);血根碱联合紫杉醇组与紫杉醇组比较发现,G2/M细胞百分比明显减少,而G0/G1期细胞百分比增加,差异具有统计学意义(P0. 05)。6. Western blot结果显示,与对照组相比,紫杉醇组、血根碱组、血根碱联合紫杉醇组Caspase-3、Bax与Smad7蛋白表达均显著增高,血根碱联合紫杉醇组表达量最高,差异具有统计学意义(P0.05);与对照组相比,紫杉醇组、血根碱组、血根碱联合紫杉醇组TGF-β 1、Smad3、β-tubulinⅢ表达均明显降低,血根碱联合紫杉醇组表达量最低,差异具有统计学意义(P0. 05)[结论]1.血根碱可抑制紫杉醇耐药卵巢癌A2780/Taxol细胞生长,增加紫杉醇药物敏感性,逆转紫杉醇耐药;2.血根碱通过抑制TGF-β /Smad信号通路,上调紫凋亡相关蛋白Bax及Caspase-3的表达,促进A2780/Taxol细胞凋亡,发挥抗肿瘤作用;3.血根碱通过抑制TGF-β /Smad信号通路,降低β-tubulinⅢ表达,促进紫杉醇化疗敏感性,逆转卵巢癌紫杉醇耐药。
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
本文编号：2193847
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31
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