蛋白激酶抑制剂AT13148对胃癌细胞生物学活性的影响及其作用机制研究

发布时间：2018-07-04 09:17

本文选题：蛋白激酶抑制剂 + AT13148　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分蛋白激酶抑制剂AT13148对胃癌细胞生物学活性的影响目的：通过分组实验研究蛋白激酶抑制剂AT13148对不同类型胃癌细胞增殖和凋亡的影响。对比分析不同浓度的蛋白激酶抑制剂AT13148与胃癌细胞凋亡的相关性。探索AT13148对胃癌细胞增殖的抑制作用是否通过促进细胞凋亡来实现。方法：分别用不同浓度的蛋白激酶抑制剂AT13148 (0.1,1,5,10μmol/L)处理不同类型的人胃癌细胞(包括HGC27、AGS、SNU-601、MKN-28、N87)和正常胃上皮细胞GEC-1,通过MTT, Brdu染色和台盼蓝染色分析蛋白激酶抑制剂AT13148处理后的胃癌细胞系各型细胞株的增殖能力,并通过TUNEL. Annexin V和caspase-3活性检测试验来验证AT13148对胃癌细胞的抑制作用是否通过促进细胞凋亡实现的。结果：不同浓度的蛋白激酶抑制剂AT13148 (0.1,1,5,10μmol/L)处理不同类型的胃癌细胞后,MTT, Brdu染色和台盼蓝染色实验证明胃癌细胞系HGC27、AGS、SNU-601、MKN-28、N87和胃上皮细胞GEC-1的增殖能力与空白对照组相比均有不同程度地降低,且与作用时间呈正相关,在48h之后的影响更为明显,同时实验结果显示AT13148抑制胃癌细胞增殖和促进细胞死亡的作用呈浓度依赖性。TUNEL, Annexin V和caspase-3活性检测试验显示：AT13148处理胃癌HGC27细胞后,细胞凋亡数和caspase-3活性明显增加；使用caspase和caspase-3抑制剂阻断caspase-3途径后,再用TUNEL, MTT和台盼蓝染色实验检测,结果显示细胞凋亡和增殖指数都有所恢复。结论：蛋白激酶抑制剂AT13148能够抑制胃癌细胞增殖并且促进肿瘤细胞凋亡,其抑制细胞增殖和促进凋亡的作用呈浓度依赖性。AT13148抑制胃癌细胞增殖的作用可以通过促进肿瘤细胞凋亡来实现。第二部分AT13148通过PI3K/Akt信号通路调控胃癌细胞增殖及凋亡的作用机制目的：探索蛋白激酶抑制剂AT13148调控胃癌细胞增殖及凋亡作用的可能机制,通过实验来验证其是否通过PI3K/Akt信号通路来完成对胃癌细胞的调控作用。方法：分别用不同浓度的蛋白激酶抑制剂AT13148 (0.1,1,5,10μmol/L)处理胃癌细胞HGC27和AGS细胞1h并通过western blot方法测定PI3K/Akt信号通路中几个重要的下游因子(比如Akt、GSK3β、p70S6K等相关激酶)的磷酸化水平和总的蛋白水平,并进行相关性分析。结果：胃癌细胞HGC27和AGS细胞经过不同浓度的AT13148 (0.1,1,5, 10μmol/L)处理后1h,测得Akt、GSK3β、p70S6K和RSK等激酶的磷酸化水平与空白对照组相比均有不同程度地降低,且有明显的浓度依赖性,以10μmol/L的作用最为明显,但是各组总的蛋白水平无明显改变。结论：蛋白激酶抑制剂AT13148抑制胃癌细胞HGC27和AGS细胞的细胞增殖和促进凋亡的作用是通过抑制PI3K/Akt信号通路中与生长相关的蛋白激酶(比如Akt、GSK3β、p70S6K等)的活化来实现的。第三部分蛋白激酶抑制剂AT13148对胃癌细胞裸鼠移植瘤的调控作用目的：建立裸鼠胃癌移植瘤模型,观察无干预状态下裸鼠移植瘤的生长,对比研究蛋白激酶抑制剂AT13148对胃癌细胞裸鼠移植瘤的调控作用。方法：用人胃癌细胞HGC27建立裸鼠移植瘤模型,选取30只造模成功的BALB/c-nu雌性裸鼠,随机分为3组,每组10只。对照组10只每天经口腔灌胃适量生理盐水；A组10只每天按照15 mg/kg经口腔灌胃AT13148；B组10只每天按照45 mg/kg经口腔灌胃AT13148。开始治疗后每三天测定各组裸鼠移植瘤的成瘤体积以及荷瘤裸鼠的体重,三周后处死各组裸鼠,留取肿瘤组织标本,用免疫印迹法(western blot)检测移植瘤组织中Akt、GSK3β、p70S6K等蛋白激酶的表达及磷酸化水平；通过免疫组织化学实验检测p-GSK3β在移植瘤瘤体中的表达,并将三组数据进行统计学分析比较。结果：成功建立人胃癌细胞HGC27裸鼠移植瘤模型。A、B两组荷瘤裸鼠的移植瘤体积在各个时间点均明显小于对照组移植瘤体积。三组荷瘤裸鼠体重无明显差异。瘤体的Western blotting结果显示A、B两个实验组移植瘤组织中磷酸化水平均降低,且与AT13148的浓度呈负相关。免疫组化实验结果显示p-GSK3β在AT13148处理后的裸鼠移植瘤组织中的表达较对照组明显降低,表现为棕黄色颗粒减少。结论：蛋白激酶抑制剂AT13148可以抑制裸鼠体内Akt、GSK3β、p70S6K等激酶的磷酸化,从而阻断PI3K/Akt信号传导通路过度活化,进而发挥抑制肿瘤细胞恶性增殖的作用,但对裸鼠本身没有药物毒性。
[Abstract]:The effect of protein kinase inhibitor AT13148 on biological activity of gastric cancer cells in part 1: the effect of protein kinase inhibitor AT13148 on the proliferation and apoptosis of different types of gastric cancer cells was studied by grouping experiments. The correlation between AT13148 and apoptosis of gastric cancer cells was compared and analyzed, and AT13148 was explored. Whether the inhibitory effect of gastric cancer cell proliferation is achieved by promoting apoptosis. Methods: different types of human gastric cancer cells (including HGC27, AGS, SNU-601, MKN-28, N87) and normal gastric epithelial cells are treated with different concentrations of protein kinase inhibitor AT13148 (0.1,1,5,10 micron mol/L), and GEC-1 in normal gastric epithelial cells by MTT, Brdu staining and trypan blue. The proliferation ability of various cell lines of gastric cancer cell lines treated with protein kinase inhibitor AT13148 and the test of TUNEL. Annexin V and caspase-3 activity test to verify whether the inhibitory effect of AT13148 on gastric cancer cells is realized by promoting cell apoptosis. Results: AT13148 (0.1,1,5,10 u mol) of protein kinase inhibitor at different concentrations (0.1,1,5,10 u mol). /L) after the treatment of different types of gastric cancer cells, MTT, Brdu staining and trypan blue staining showed that the proliferation ability of gastric cancer cell lines HGC27, AGS, SNU-601, MKN-28, N87 and gastric epithelial cells decreased in varying degrees compared with the blank control group, and had a positive correlation with the action time, and the effect was more obvious after 48h, at the same time the experimental knot was found. The results showed that AT13148 inhibited the proliferation of gastric cancer cells and promoted cell death in a concentration dependent.TUNEL. The test of Annexin V and caspase-3 activity showed that the number of apoptosis and the activity of Caspase-3 increased significantly after AT13148 treatment of gastric cancer HGC27 cells, and the caspase-3 pathway was blocked by caspase and caspase-3 inhibitors. The results of TT and trypan blue staining showed that the apoptosis and proliferation index were all recovered. Conclusion: protein kinase inhibitor AT13148 can inhibit the proliferation of gastric cancer cells and promote the apoptosis of tumor cells. The inhibitory effect of.AT13148 on the proliferation of gastric cancer cells in a concentration dependent manner can inhibit the proliferation of gastric cancer cells. The second part AT13148 regulates the proliferation and apoptosis of gastric cancer cells through PI3K/Akt signaling pathway: explore the possible mechanism of protein kinase inhibitor AT13148 to regulate the proliferation and apoptosis of gastric cancer cells, and verify whether it completes the stomach through the PI3K/Akt signaling pathway. The regulation of cancer cells. Methods: the HGC27 and AGS cells 1h of gastric cancer cells were treated with different concentration of protein kinase inhibitor AT13148 (0.1,1,5,10 mol/L), and the phosphorylation level and total protein of several important downstream factors (Akt, GSK3 beta, p70S6K and other related kinases) in the PI3K/Akt signaling pathway were measured by Western blot method. Results: the HGC27 and AGS cells of gastric cancer cells were treated with different concentrations of AT13148 (0.1,1,5, 10 mu mol/L) after 1h. The phosphorylation levels of Akt, GSK3 beta, p70S6K and RSK were decreased in varying degrees compared with those of the blank control group, with a significant concentration dependence, and the most important effect was 10 mu mol/L. Obviously, the total protein level of each group was not significantly changed. Conclusion: the inhibitory effect of protein kinase inhibitor AT13148 on the proliferation and apoptosis of gastric cancer cells HGC27 and AGS cells is achieved by inhibiting the activation of the growth related protein kinase (such as Akt, GSK3 beta, p70S6K, etc.) in the PI3K/Akt signaling pathway. The third part of the egg Regulatory effect of white kinase inhibitor AT13148 on xenografts in nude mice of gastric cancer cells: to establish a nude mouse model of gastric cancer transplantation, to observe the growth of xenografts in nude mice without intervention, and to compare the regulatory effect of protein kinase inhibitor AT13148 on xenografts in nude mice of gastric cancer cells: a nude mouse transplantation with human gastric cancer cell HGC27 30 BALB/c-nu female nude mice were randomly divided into 3 groups, 10 in each group, 10 in the control group, and 10 in the oral administration of normal saline every day in the oral cavity, and 10 in the A group by 15 mg/kg by the oral gavage of the stomach every day; 10 in the B group were treated with the oral gavage of the stomach by the mouth of 45 mg/kg every day for the treatment of nude mice every three days. The tumor volume and the weight of the tumor bearing nude mice were killed three weeks after the tumor tissue specimens were left. The expression of Akt, GSK3 beta, p70S6K and other protein kinase in the transplanted tumor tissues were detected by immunoblotting (Western blot). The expression of p-GSK3 beta in the transplanted tumor was detected by immunohistochemistry and the expression of p-GSK3 beta in the tumor transplanted tumor was detected. Three groups of data were compared statistically. Results: human gastric cancer cell HGC27 nude mice transplanted tumor model.A was successfully established. The volume of transplanted tumor in B two group of nude mice was significantly smaller than that of the control group. There was no significant difference between the three groups of tumor bearing nude mice. The Western blotting results of the tumor showed A, B and the two experimental groups. The level of phosphorylation in the tumor tissue decreased and was negatively correlated with the concentration of AT13148. The immunohistochemical results showed that the expression of p-GSK3 beta in the transplanted tumor tissues of nude mice after AT13148 treatment was significantly lower than that of the control group, showing a decrease in brown granules. Conclusion: protein kinase inhibitor AT13148 can inhibit Akt, GSK3 beta, p70 in nude mice. The phosphorylation of S6K and other kinases, thus blocking the excessive activation of the PI3K/Akt signal transduction pathway, thus inhibits the malignant proliferation of tumor cells, but has no drug toxicity to nude mice.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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