【摘要】：目的:目前我國胃癌的發(fā)病率以及死亡率均居高不下。中藥瓜蔞的塊根含天花粉蛋白(trichosanthin,TCS),具有潛在的抗腫瘤作用。本文通過研究天花粉蛋白對人胃癌細胞誘導凋亡的作用,以期發(fā)現(xiàn)通過誘導細胞凋亡來抑制胃癌的新型中成藥。同時,我們將在細胞內(nèi)研究天花粉蛋白賴氨酸(K)173/精氨酸(R)174和賴氨酸(K)177的突變體對核糖體蛋白合成功能的抑制作用,進一步探討天花粉蛋白抗腫瘤的分子機制。研究方法:取處于對數(shù)生長期的胃癌SGC7901細胞,用不同濃度的天花粉蛋白處理后,分別從細胞形態(tài)學及細胞生長增殖兩方面來檢測天花粉蛋白對胃癌SGC7901細胞的影響。應用流式細胞儀測定天花粉蛋白對胃癌SGC7901細胞周期及凋亡的作用。應用Western blotting 觀察胃癌 SGC7901 細胞 caspase-9、caspase-3;PARP;pERK;ERK 的變化。同時,通過定點突變、質(zhì)粒轉(zhuǎn)化、擴增、提取、測序、轉(zhuǎn)染等方法構(gòu)建天花粉蛋白K173A/R174A和K177A的突變體,最后通過測定熒光素酶活性,對比野生型天花粉蛋白及突變K173A/R174A和K177A后的天花粉蛋白對核糖體蛋白合成的抑制作用。研究結(jié)果:(1)天花粉蛋白處理后的胃癌SGC7901細胞出現(xiàn)了皺縮、變形,對胃癌SGC7901細胞的生長有明顯的抑制作用。(2)天花粉蛋白可以將胃癌SGC7901細胞阻滯于S期。(3)天花粉蛋白誘導胃癌SGC7901細胞發(fā)生凋亡,并且細胞凋亡呈現(xiàn)出濃度依賴性。(4)Western blotting 法檢測結(jié)果顯示:caspase-9,caspase-3,PARP 表達降低,呈濃度依賴性。并且隨著天花粉蛋白濃度的升高,ERK表達降低,天花粉蛋白處理(7.0和14 μM)激活ERK,磷酸化ERK升高。(5)在細胞內(nèi),野生種天花粉蛋白能抑制熒光素酶活性,突變種K173A/R174A對熒光素酶活性抑制作用強于野生種天花粉蛋白,突變種K177A對熒光素酶活性抑制作用最強。研究結(jié)論:天花粉蛋白可以抑制胃癌SGC7901細胞的生長,誘導細胞凋亡。天花粉蛋白能激活ERK信號通路,說明天花粉蛋白誘導細胞凋亡與ERK信號通路有關(guān)。野生種天花粉蛋白及突變后的天花粉蛋白均具有抑制細胞核糖體蛋白合成的作用,且K177A突變體抑制核糖體蛋白合成的能力強于K173A/R174A突變體,K173A/R174A突變體抑制核糖體蛋白合成的能力強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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2
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本文編號：2218462