氨氯地平对C57小鼠黑色素瘤B16抗肿瘤转移作用的研究

发布时间：2018-05-02 14:57

本文选题：氨氯地平 + 小鼠黑色素瘤B16　；参考：《重庆医科大学》2011年硕士论文

【摘要】：目的:探讨钙通路阻滞剂(calcium channel blocker, CCB)氨氯地平对C57小鼠黑色素瘤B16的抗肿瘤转移作用以及其相关机制的研究,为氨氯地平治疗高转移株的治疗提供了相应的理论基础。方法:将B16细胞接种于C57小鼠右腹股沟皮下(2×106/只)将64只小鼠随机分成对照组,以及三个剂量治疗组。对照组从肿瘤种植第2天起灌胃给予生理盐水0.2ml/只;治疗组灌胃给予氨氯地平其剂量分别为1 mg/kg,3 mg/kg和10 mg/kg,每日1次,每次0.2ml,共21天,给药期间观察小鼠成瘤,进食,及身体情况。于肿瘤种植第22天断颈处死小鼠,观察对照组和治疗组的肺转移情况,计数肺转移结节数以及各组的抑瘤率;体外进行抑制血小板聚集实验研究;建立血小板免疫粘附小鼠黑色素细胞瘤B16细胞实验模型,比较药物作用前后粘附能力的差异;免疫组织化学检测治疗组和对照组肿瘤组织IL-8蛋白的表达水平;RT-PCR检测各个实验组肿瘤组织的IL-8mRNA的表达情况; Western Blot检测各组肿瘤组织的ERK1/2 ,磷酸化ERK1/2(P-ERK1/2)表达水平。结果:体内实验结果显示,与对照组相比氨氯地平可明显抑制高转移株黑色素瘤B16的增殖和自发性肺转移作用,并成剂量依赖性; 体外实验结果显示,与对照组相比氨氯地平可显著抑制小鼠黑色素瘤B16诱导的小鼠血小板聚集以及血小板与小鼠黑色素瘤B16细胞的粘附作用; 免疫组织化学检验结果显示,与对照组相比IL-8蛋白表达明显减弱(P0.05),随着药物剂量的增加IL-8蛋白表达逐渐减弱;PCR结果显示,各个治疗组的IL-8mRNA表达也明显低于对照组(P0.05),且随着药物剂量增加IL-8mRNA的表达逐渐减弱; Western Blot检测结果表明治疗组与对照组相比p-ERK1/2表达减弱,并且随着药物剂量增加p-ERK1/2表达成逐渐减弱趋势,而ERK1/2的表达各个组之间没有显著性差异。结论:氨氯地平可剂量依赖性的抑制小鼠黑色素瘤B16的体内增殖和自发性肺转移;其作用机制可能通过两个方面抑制转移:一对血行转移的影响,也就是抑制肿瘤细胞诱导的血小板聚集作用,以及抑制肿瘤细胞与血小板的粘附作用;二抗转移作用可能与影响IL-8的表达有关,通过抑制MARK中ERK1/2的磷酸化,使MARK激活受抑制,AP-1与IL-8启动子结合减少,而下调IL-8的转录,使IL-8介导的一系列肿瘤转移过程受到一定程度的抑制。
[Abstract]:Objective: to investigate the anti-metastasis effect of calcium channel blocker, CCB) amlodipine on melanoma B16 in C57 mice and its related mechanism, and to provide a theoretical basis for the treatment of high metastatic strain with amlodipine. Methods: B16 cells were inoculated into 2 脳 10 6 / mouse right groin of C57 mice. 64 mice were randomly divided into control group and three dose treatment groups. The control group was given normal saline 0.2ml/ from the second day of tumor implantation, and the treatment group was given amlodipine at the dose of 1 mg / kg 3 mg/kg and 10 mg / kg, once a day, 0.2 ml per day for 21 days. And physical condition. The mice were killed on the 22nd day of tumor implantation, the lung metastasis of control group and treatment group was observed, the number of metastatic nodules of lung and the inhibition rate of tumor in each group were counted, and the inhibition of platelet aggregation was studied in vitro. To establish the experimental model of B16 cells of mouse melanocytoma immunized with platelets and compare the difference of adhesion ability before and after drug treatment. The expression of IL-8 protein in tumor tissues of treatment group and control group was detected by immunohistochemistry. The expression of IL-8mRNA in tumor tissues of each experimental group was detected by RT-PCR. The expression of ERK1/2 and phosphorylated ERK1 / 2 (P-ERK1 / 2) in each group were detected by; Western Blot. Results: compared with the control group, amlodipine significantly inhibited the proliferation and spontaneous lung metastasis of melanoma B16 in a dose-dependent manner. The results of in vitro experiments showed that amlodipine significantly inhibited the platelet aggregation induced by mouse melanoma B16 and the adhesion of platelet to mouse melanoma B16 cells compared with the control group. The results of immunohistochemistry showed that the expression of IL-8 protein was significantly decreased compared with the control group, and the expression of IL-8 protein gradually decreased with the increase of drug dosage. The expression of IL-8mRNA in each treatment group was significantly lower than that in the control group (P 0.05), and the expression of IL-8mRNA gradually decreased with the increase of drug dosage. The results of Western Blot showed that the expression of p-ERK1/2 decreased in the treatment group compared with the control group, and the expression of p-ERK1/2 decreased gradually with the increase of drug dosage, but there was no significant difference in the expression of ERK1/2 among the different groups. Conclusion: amlodipine can inhibit the proliferation and spontaneous lung metastasis of murine melanoma B16 in a dose-dependent manner. In other words, the inhibition of platelet aggregation induced by tumor cells and the adhesion of tumor cells to platelets may be related to the inhibition of IL-8 expression by inhibiting the phosphorylation of ERK1/2 in MARK. The binding of AP-1 to IL-8 promoter was decreased and the transcription of IL-8 was down-regulated, which inhibited a series of tumor metastasis mediated by IL-8 to some extent.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.5

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