天花粉蛋白应用于喉癌化疗及机制的研究

发布时间：2018-03-09 17:12

本文选题：天花粉蛋白　切入点：喉癌细胞株　出处：《复旦大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的：研究天花粉蛋白(trichosanthin, TCS)单独或联合顺铂用于对喉癌(HEp-2细胞株和AMC-HN-8细胞株)增殖抑制,以及研究TCS激活的JNK/MAPK信号通路与其发挥药理作用之间的关系。方法：设立不同的实验组,包括不同浓度TCS组(1、5、9、13μg/ml),低浓度顺铂组(3μg/ml)、高浓度顺铂组(10μg/ml)、及TCS和顺铂联合作用组(5pg/ml TCS和3μg/ml颐铂),阴性对照组；连续5天使用CCK-8法(cell counting kit-8, CCK-8)和乳酸脱氢酶法检测不同实验组对喉癌细胞的抑制作用和细胞毒性；流式细胞仪和Hochest33258染色观察细胞凋亡和细胞周期；Western blot检测JNK/MAPK,磷酸化JNK/MAPK,核因子κB(nuclear factor κB, NF-κB)、p38磷酸化p38、I-κB及磷酸化I-κB的变化。结果：TCS通过诱导细胞凋亡和细胞周期停滞的方式来抑制喉癌细胞增殖,TCS通过时间和浓度依赖的方式激活JNK/MAPK信号通路,从蛋白水平上阻断JNK/MAPK信号通路,TCS对喉癌细胞的增殖抑制作用和诱导细胞凋亡的作用减弱,与TCS和低剂量顺铂单独作用相比,TCS和顺铂联合作用可明显抑制喉癌细胞的生长(P0.01),凋亡细胞数量增加；联合作用组的抑制效果与高浓度顺铂组差异无统计学意义,细胞毒性同TCS和顺铂单独作用相比差异无统计学意义(P0.05),而毒性低于高浓度顺铂组(P0.01)；Western blot显示顺铂增强NF-κB转录因子活性,抑制JNK信号通路,TCS组主要激活JNK/MAPK信号通路而抑制NF-κB, TCS和顺铂联合作用后磷酸化JNK保持高水平抑制NF-κB活性。结论：TCS通过激活JNK/MAPK信号通路,发挥其抑制喉癌细胞增殖作用,同时抑制NF-κB转录因子活性,增强顺铂诱导喉癌细胞凋亡比例,从而降低其对细胞的毒性并增强其抗肿瘤疗效,为中药天花粉蛋白联合顺铂应用于喉癌临床治疗奠定了实验基础。
[Abstract]:Aim: to study the inhibitory effects of trichosanthin (TCSs) on the proliferation of laryngeal carcinoma cell line Hep 2 and AMC-HN-8 cell line, either alone or in combination with cisplatin. And to study the relationship between the JNK/MAPK signaling pathway activated by TCS and its pharmacological function. Methods: different experimental groups were established. It included 13 渭 g / ml of TCS, 3 渭 g / ml of low concentration of cisplatin, 10 渭 g / ml of high concentration of cisplatin, and 5 PG / ml of TCS and 3 渭 g / ml of Aplatin in combination of TCS and cisplatin, negative control group. CCK-8 counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) were used to detect the inhibitory effect and cytotoxicity of different experimental groups on laryngeal cancer cells for 5 days. Flow cytometry and Hochest33258 staining were used to detect the changes of apoptosis, phosphorylated Hochest33258, nuclear factor- 魏 B nuclear factor 魏 B, NF- 魏 B p38 phosphorylated p38 I- 魏 B and phosphorylated I- 魏 B.Results the expression of P38-I- 魏 B and phosphorylated I- 魏 B were detected by inducing apoptosis and cell cycle arrest. To inhibit the proliferation of laryngeal cancer cells and activate the JNK/MAPK signaling pathway in a time-and concentration-dependent manner. The inhibitory effect of JNK/MAPK on the proliferation and apoptosis of laryngeal carcinoma cells was decreased by blocking the JNK/MAPK signaling pathway at the protein level. Compared with TCS and low dose cisplatin alone, TCS and cisplatin could significantly inhibit the growth of laryngeal cancer cells and increase the number of apoptotic cells, but there was no significant difference between the combined treatment group and the high concentration of cisplatin group. There was no significant difference in cytotoxicity between TCS and cisplatin alone, but the cytotoxicity was lower than that in high concentration cisplatin group. Western blot showed that cisplatin enhanced the activity of NF- 魏 B transcription factor. Inhibition of JNK signaling pathway mainly activates JNK/MAPK signaling pathway and inhibits NF- 魏 B, and phosphorylated JNK maintains a high level of inhibition of NF- 魏 B activity after combined action of TCS and cisplatin. Conclusion: TCS can inhibit the proliferation of laryngeal cancer cells by activating JNK/MAPK signaling pathway. At the same time, it inhibited the activity of NF- 魏 B transcription factor, enhanced the percentage of apoptosis induced by cisplatin, reduced its cytotoxicity and enhanced its anti-tumor effect, which laid an experimental foundation for the clinical treatment of laryngeal carcinoma with trichosanthin combined with cisplatin.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.65

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