天花粉蛋白抑制胃癌细胞生长及其分子机制研究

发布时间：2018-09-02 05:55

【摘要】：目的:目前我国胃癌的发病率以及死亡率均居高不下。中药瓜蒌的块根含天花粉蛋白(trichosanthin,TCS),具有潜在的抗肿瘤作用。本文通过研究天花粉蛋白对人胃癌细胞诱导凋亡的作用,以期发现通过诱导细胞凋亡来抑制胃癌的新型中成药。同时,我们将在细胞内研究天花粉蛋白赖氨酸(K)173/精氨酸(R)174和赖氨酸(K)177的突变体对核糖体蛋白合成功能的抑制作用,进一步探讨天花粉蛋白抗肿瘤的分子机制。研究方法:取处于对数生长期的胃癌SGC7901细胞,用不同浓度的天花粉蛋白处理后,分别从细胞形态学及细胞生长增殖两方面来检测天花粉蛋白对胃癌SGC7901细胞的影响。应用流式细胞仪测定天花粉蛋白对胃癌SGC7901细胞周期及凋亡的作用。应用Western blotting 观察胃癌 SGC7901 细胞 caspase-9、caspase-3;PARP;pERK;ERK 的变化。同时,通过定点突变、质粒转化、扩增、提取、测序、转染等方法构建天花粉蛋白K173A/R174A和K177A的突变体,最后通过测定荧光素酶活性,对比野生型天花粉蛋白及突变K173A/R174A和K177A后的天花粉蛋白对核糖体蛋白合成的抑制作用。研究结果:(1)天花粉蛋白处理后的胃癌SGC7901细胞出现了皱缩、变形,对胃癌SGC7901细胞的生长有明显的抑制作用。(2)天花粉蛋白可以将胃癌SGC7901细胞阻滞于S期。(3)天花粉蛋白诱导胃癌SGC7901细胞发生凋亡,并且细胞凋亡呈现出浓度依赖性。(4)Western blotting 法检测结果显示:caspase-9,caspase-3,PARP 表达降低,呈浓度依赖性。并且随着天花粉蛋白浓度的升高,ERK表达降低,天花粉蛋白处理(7.0和14 μM)激活ERK,磷酸化ERK升高。(5)在细胞内,野生种天花粉蛋白能抑制荧光素酶活性,突变种K173A/R174A对荧光素酶活性抑制作用强于野生种天花粉蛋白,突变种K177A对荧光素酶活性抑制作用最强。研究结论:天花粉蛋白可以抑制胃癌SGC7901细胞的生长,诱导细胞凋亡。天花粉蛋白能激活ERK信号通路,说明天花粉蛋白诱导细胞凋亡与ERK信号通路有关。野生种天花粉蛋白及突变后的天花粉蛋白均具有抑制细胞核糖体蛋白合成的作用,且K177A突变体抑制核糖体蛋白合成的能力强于K173A/R174A突变体,K173A/R174A突变体抑制核糖体蛋白合成的能力强于野生型天花粉蛋白。
[Abstract]:Objective: the morbidity and mortality of gastric cancer in China are very high. Trichosanthin (trichosanthin,TCS) in the root of Trichosanthes trichosanthes has potential anti-tumor effect. In this paper, we studied the effect of trichosanthin on apoptosis of human gastric cancer cells in order to find out a new Chinese patent medicine which can inhibit gastric cancer by inducing apoptosis. At the same time, we will study the inhibitory effect of Trichosanthin lysine (K) 173 / arginine (R) 174 and lysine (K) 177 mutants on ribosomal protein synthesis in cells, and further explore the molecular mechanism of trichosanthin antitumor. Methods: the effects of trichosanthin on gastric cancer SGC7901 cells were detected from cell morphology and cell growth and proliferation after different concentrations of trichosanthin were treated with different concentrations of trichosanthin in logarithmic growth phase of gastric cancer SGC7901 cells. The effects of trichosanthin on SGC7901 cell cycle and apoptosis in gastric cancer were determined by flow cytometry. The changes of caspase-9,caspase-3;PARP;pERK;ERK in SGC7901 cells of gastric cancer were observed by Western blotting. At the same time, Trichosanthin K173A/R174A and K177A mutants were constructed by site-directed mutation, plasmid transformation, amplification, extraction, sequencing and transfection. Finally, luciferase activity was determined. The inhibition of ribosomal protein synthesis by wild type trichosanthin and mutant K173A/R174A and K177A was compared. The results were as follows: (1) the SGC7901 cells of gastric cancer treated with trichosanthin showed shrinkage and deformation. (2) trichosanthin could block gastric cancer SGC7901 cells from S phase. (3) Trichosanthin induced apoptosis of gastric cancer SGC7901 cells. (4) the results of) Western blotting assay showed that the expression of 10 caspase-9 and caspase-3 was decreased in a concentration-dependent manner. With the increase of Trichosanthin concentration, Trichosanthin treatment (7.0 渭 M and 14 渭 M) activated ERK, phosphorylation of ERK. (5) Trichosanthin could inhibit luciferase activity in the cells. The inhibitory effect of mutant K173A/R174A on luciferase activity was stronger than that of wild species Trichosanthin, and the mutant K177A had the strongest inhibitory effect on luciferase activity. Conclusion: trichosanthin can inhibit the growth of gastric cancer SGC7901 cells and induce apoptosis. Trichosanthin can activate the ERK signaling pathway, suggesting that trichosanthin induces cell apoptosis related to ERK signaling pathway. Trichosanthin in wild species and trichosanthin after mutation can inhibit the synthesis of ribosomal proteins. Moreover, the ability of K177A mutant to inhibit ribosomal protein synthesis was stronger than that of K173A/R174A mutant K173A / R174A, which was stronger than wild-type Trichosanthin.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
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本文编号：2218462