槐定碱联合顺铂对宫颈腺癌Hela细胞增殖抑制作用的研究

发布时间：2018-04-09 19:15

本文选题：宫颈腺癌　切入点：Hela细胞　出处：《兰州大学》2014年硕士论文

【摘要】：目的：探讨槐定碱联合顺铂对宫颈腺癌Hela细胞增殖的影响,以期对宫颈腺癌的临床治疗提供新的思路和实验室理论依据。方法：体外培养宫颈腺癌细胞系Hela细胞：采用细胞增殖与毒性试验法即MTT法检测不同浓度的槐定碱及其联合顺铂对Hela细胞的增殖抑制率的影响,并计算半数抑制率IC50,且用金正均Q值法判断两药的联合效应,倒置相差显微镜下观察Hela细胞的形态学改变：Hoechst33342染色法观察不同浓度的槐定碱联合顺铂作用24h后Hela细胞的形态学改变及凋亡情况；应用流式细胞术测定不同浓度的槐定碱联合顺铂作用24h后对Hela细胞周期分布及凋亡率的影响；应用Western Blot法检测不同浓度的槐定碱联合顺铂作用24h后Hela细胞凋亡相关蛋白Bcl-2、Bax、P53、Caspase-3的表达情况。结果：(1)MTT结果表明：槐定碱能够明显抑制宫颈腺癌Hela细胞的体外增殖(P0.05),其作用呈明显的剂量-时间依赖性,计算24h、48h和72h的半数抑制率IC50分别为5.5mg/ml、4.2mg/ml及2.1mg/ml,应用金正均Q值法判断两药的联合效应为：槐定碱联合顺铂作用于Hela细胞24h后,槐定碱浓度1.0mg/ml,两药为拮抗作用(Q0.85)；槐定碱浓度≥1.0mg/ml两药为协同作用(Q0.85)；槐定碱联合顺铂作用于Hela细胞48h及72h后,不同浓度的槐定碱联合顺铂组均表现出协同作用(Q0.85),各组两两比较差异有统计学意义(P0.05)；槐定碱及其联合顺铂处理24h后,Hela细胞的形态发生显著变化,且随着槐定碱浓度的增加形态学变化愈加明显。 (2)Hoechst33342染色证实槐定碱联合顺铂可以诱导宫颈腺癌Hela细胞凋亡,并可见典型的凋亡小体； (3)流式细胞术检测细胞周期结果表明：槐定碱联合顺铂可影响宫颈腺癌Hela细胞周期分布,随着槐定碱浓度的增加,G0/G1期细胞的百分比逐渐降低而S期细胞的百分比逐渐增高,呈现S期阻滞,差异具有统计学意义(P0.05)；与顺铂组相比较,联合用药组①的G0/G1期细胞的百分比高于顺铂组,S期细胞的百分比低于顺铂组,差异有统计学意义(P0.05)；联合用药组②③④的G0/G1期细胞的百分比低于顺铂组,S期细胞的百分比高于顺铂组,与顺铂组及各组间比较均具有统计学差异(P0.05)。 (4)流式细胞术检测细胞凋亡率结果表明：槐定碱联合顺铂能够诱导宫颈腺癌Hela细胞发生凋亡,与阴性对照组相比,顺铂组及联合用药组的凋亡率升高,差异有统计学意义(P0.05)；与顺铂组相比较,联合用药组①组的凋亡率低于顺铂组,差异有统计学意义(P0.05)；联合用药组②③④的凋亡率高于顺铂组,协同顺铂促凋亡效应,与顺铂组及各组间比较均具有统计学差异(P0.05)。 (5) Western Blot结果证实：槐定碱联合顺铂作用于宫颈腺癌Hela细胞24h后,与阴性对照组相比较,顺铂组及槐定碱联合顺铂组均可使Bcl-2蛋白表达下调,P53蛋白、Bax蛋白及Caspase-3蛋白表达上调,差异有统计学意义(P0.05)；与顺铂组相比较,联合用药组①组的Bcl-2蛋白表达率高于顺铂组,Bax蛋白、P53蛋白及Caspase-3蛋白表达率低于顺铂组,差异有统计学意义(P0.05),联合用药组②③④随着槐定碱浓度的增加,Bcl-2蛋白表达逐渐降低,Bax、P53及Caspase-3蛋白表达逐渐增加,与顺铂组及各组间相比较均具有统计学意义(P0.05)。结论：(1)槐定碱可以时间-剂量依赖性的抑制宫颈腺癌Hela细胞的增殖。槐定碱与顺铂联合应用具有协同抗肿瘤作用。 (2)槐定碱联合顺铂抑制宫颈腺癌Hela细胞的增殖作用的机制可能与诱导Hela细胞凋亡,S期阻滞,下调Bcl-2蛋白的表达,上调Bax、P53、Caspase-3蛋白的表达及下调Bcl-2/Bax的比值有关,因此槐定碱联合顺铂引起Hela细胞的凋亡可能是通过P53依赖的凋亡途径完成的。
[Abstract]:Objective: To investigate the effects of sophoridine combined with cisplatin on the proliferation of cervical carcinoma cell line Hela, to provide ideas and laboratory new theoretical basis for the clinical treatment of cervical adenocarcinoma.
Methods: Hela cells of cervical adenocarcinoma cell lines cultured in vitro: the effects of proliferation and cytotoxicity test method of detecting Huai MTT of different concentration by proliferation inhibition rate of alkali and cisplatin on Hela cells, and calculate the half inhibition rate of IC50, and Jin Zhengjun Q value method to determine the combined effect of the two drugs, inverted to observe the morphological changes of Hela cells under the microscope: Observation of different concentrations of Hoechst33342 staining, sophoridine morphological changes and apoptosis of Hela cells after 24h alkali combined cisplatin; different concentrations were determined by flow cytometry of sophoridine combined with cisplatin 24h effect on cell cycle distribution and apoptosis rate of Hela detection; different Huai the application of Western Blot concentration Hela apoptosis related protein Bcl-2, 24h Bax after alkali combined with cisplatin, P53, Caspase-3 expression.
Results: the results showed that: (1) MTT sophoridine could significantly inhibit the proliferation of cervical carcinoma cell line Hela in vitro (P0.05), the effect was dose and time-dependent, calculation of 24h, half 48h and 72h inhibition rate of IC50 were 5.5mg/ml, 4.2mg/ml and 2.1mg/ml, the application of Nintaus Q value judgment method the combined effects of two drugs: sophoridine combined with cisplatin in Hela cells after 24h, sophoridine concentration 1.0mg/ml, the two drugs for antagonism (Q0.85); sophoridine concentration higher than two 1.0mg/ml drug synergy (Q0.85); sophoridine combined with cisplatin on Hela cell 48h and 72h, different the concentration of sophoridine combined with cisplatin group showed synergistic effect (Q0.85), the 22 groups was statistically significant difference (P0.05); sophoridine and cisplatin after 24h treatment, the morphology change of Hela cells, and with the increase of the concentration of sophoridine more morphological changes Obviously.
(2) Hoechst33342 staining confirmed that sophoridine combined with cisplatin can induce apoptosis of cervical carcinoma cell line Hela, and the typical apoptotic bodies;
(3) flow cytometry results showed that sophoridine combined with cisplatin can affect the cervical gland cancer Hela cell cycle distribution, with the increase of sophoridine concentration, the percentage of cells in G0/G1 phase decreased and the percentage of S phase cells increased gradually, showed S arrest, the difference was statistically significant (P0.05); compared with cisplatin group, combination group the percentage of G0/G1 cells was higher than that of cisplatin group, the percentage of cells in S phase was lower than that of cisplatin group, the difference was statistically significant (P0.05); the combination group the G0/G1 phase cells percentage lower than that of cisplatin group, the percentage of cells in S phase was higher than that of cisplatin group and cisplatin group and between groups were statistically different (P0.05).
(4) flow cytometry results showed that the apoptosis rate of sophoridine combined with cisplatin can induce cervical adenocarcinoma Hela cell apoptosis, compared with the negative control group, the apoptosis of cisplatin group and the combination group increased, the difference was statistically significant (P0.05); compared with cisplatin group, combination group, apoptosis the group was lower than that of cisplatin group, the difference was statistically significant (P0.05); apoptosis in combination group was higher than that of the 4 cisplatin group and cisplatin induced apoptosis, and cisplatin group and between groups were statistically different (P0.05).
(5) Western Blot results confirmed that sophoridine combined with cisplatin on cervical carcinoma cell line Hela after 24h, compared with the negative control group, cisplatin group and sophoridine combined with cisplatin group can make the down-regulation of Bcl-2 protein expression of P53 protein, up-regulated expression of Bax protein and Caspase-3 protein, the difference was statistically significant (P0.05); compared with cisplatin group, combined treatment group 1 group the expression rate of Bcl-2 protein was higher than that of cisplatin group, Bax protein, P53 protein expression and Caspase-3 protein was lower than that of cisplatin group, the difference was statistically significant (P0.05), combination group and with the increase of the concentration of sophoridine, Bcl-2 protein expression decreased gradually the expression of Bax, P53 and Caspase-3 protein gradually increased, compared with DDP group and between groups were statistically significant (P0.05).
Conclusion: (1) sophoridine can time dose dependent inhibition of cervical carcinoma cell line Hela proliferation. Sophoridine has anti-tumor effect co application of alkali with cisplatin.
(2) the proliferation mechanism of sophoridine alkali combined with cisplatin inhibiting cervical adenocarcinoma Hela cells may be associated with the induction of Hela cell apoptosis, S phase arrest, down-regulation of Bcl-2 protein expression, up regulation of Bax, P53, Caspase-3 protein expression and reducing the ratio of Bcl-2/Bax, so the sophoridine combined with cisplatin induced apoptosis in Hela cells may is the completion of apoptosis via a P53 dependent pathway.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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