本文選題：天花粉蛋白　切入點：喉癌細(xì)胞株　出處：《復(fù)旦大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究天花粉蛋白(trichosanthin, TCS)單獨或聯(lián)合順鉑用于對喉癌(HEp-2細(xì)胞株和AMC-HN-8細(xì)胞株)增殖抑制,以及研究TCS激活的JNK/MAPK信號通路與其發(fā)揮藥理作用之間的關(guān)系。方法：設(shè)立不同的實驗組,包括不同濃度TCS組(1、5、9、13μg/ml),低濃度順鉑組(3μg/ml)、高濃度順鉑組(10μg/ml)、及TCS和順鉑聯(lián)合作用組(5pg/ml TCS和3μg/ml頤鉑),陰性對照組；連續(xù)5天使用CCK-8法(cell counting kit-8, CCK-8)和乳酸脫氫酶法檢測不同實驗組對喉癌細(xì)胞的抑制作用和細(xì)胞毒性；流式細(xì)胞儀和Hochest33258染色觀察細(xì)胞凋亡和細(xì)胞周期；Western blot檢測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的變化。結(jié)果：TCS通過誘導(dǎo)細(xì)胞凋亡和細(xì)胞周期停滯的方式來抑制喉癌細(xì)胞增殖,TCS通過時間和濃度依賴的方式激活JNK/MAPK信號通路,從蛋白水平上阻斷JNK/MAPK信號通路,TCS對喉癌細(xì)胞的增殖抑制作用和誘導(dǎo)細(xì)胞凋亡的作用減弱,與TCS和低劑量順鉑單獨作用相比,TCS和順鉑聯(lián)合作用可明顯抑制喉癌細(xì)胞的生長(P0.01),凋亡細(xì)胞數(shù)量增加；聯(lián)合作用組的抑制效果與高濃度順鉑組差異無統(tǒng)計學(xué)意義,細(xì)胞毒性同TCS和順鉑單獨作用相比差異無統(tǒng)計學(xué)意義(P0.05),而毒性低于高濃度順鉑組(P0.01)；Western blot顯示順鉑增強(qiáng)NF-κB轉(zhuǎn)錄因子活性,抑制JNK信號通路,TCS組主要激活JNK/MAPK信號通路而抑制NF-κB, TCS和順鉑聯(lián)合作用后磷酸化JNK保持高水平抑制NF-κB活性。結(jié)論：TCS通過激活JNK/MAPK信號通路,發(fā)揮其抑制喉癌細(xì)胞增殖作用,同時抑制NF-κB轉(zhuǎn)錄因子活性,增強(qiáng)順鉑誘導(dǎo)喉癌細(xì)胞凋亡比例,從而降低其對細(xì)胞的毒性并增強(qiáng)其抗腫瘤療效,為中藥天花粉蛋白聯(lián)合順鉑應(yīng)用于喉癌臨床治療奠定了實驗基礎(chǔ)。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R739.65

【共引文獻(xiàn)】

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