| 殷洁烽,徐统球,张喆,孙元水.TAT-E4orf4融合蛋白对胃癌细胞迁移和凋亡的影响[J].浙江中西医结合杂志,2021,31(2): |
| TAT-E4orf4融合蛋白对胃癌细胞迁移和凋亡的影响 |
| The effect of TAT-Eorf4 fusion protein on the migration and apoptosis of Gastric cancer cells |
| 投稿时间：2020-05-16 修订日期：2021-01-20 |
| DOI： |
| 中文关键词: 胃癌 凋亡 细胞穿膜肽 腺病毒早期区4第4编码蛋白 迁移 |
| 英文关键词:Gastric cancer Apoptosis Cell penetrating peptide Adenovirus early region 4 open reading frame 4 protein Migration |
| 基金项目:浙江省医药卫生科技计划项目(2018KY329) |
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| 中文摘要: |
| 目的 构建融合细胞穿膜肽的腺病毒早期区4第4编码蛋白的pET28-TAT-E4orf4原核表达载体,分析TAT-E4orf4融合蛋白对胃癌AGS细胞凋亡的影响及其作用机制。方法 PCR法构建pET28-his-TAT-E4orf4表达载体,经PCR酶切鉴定后,诱导表达。并分别用pET28、pET28-his-E4orf4和pET28-his-TAT-E4orf4处理胃癌AGs细胞,分别采用MTT法和Transwell小室检测TAT-E4orf4对胃癌细胞增殖和迁移的影响,以流式细胞仪检测刺激后胃癌细胞的凋亡,以Western blot检测Caspases和Src信号轴上关键蛋白的表达变化。结果 PCR和Western blot实验结果证实成功构建pET28-TAT-E4orf4表达载体,MTT法显示pET28-his-E4orf4和pET28-his-TAT-E4orf4刺激后,胃癌AGS细胞增殖活性下降,且后者下降更为明显。与MTT实验结果趋势相似,Transwell实验的结果,pET28-his-TAT-E4orf4刺激后胃癌细胞迁移数目显著减少,与前三组比较,差异具有统计学意义。Western blot实验结果表明,Caspase-3和Caspase-9的表达水平无明显变化,而Src信号通路上的蛋白均出现规律性变化。结论TAT-E4orf4融合蛋白能抑制胃癌细胞增殖和迁移,促进胃癌细胞凋亡,主要依赖于Src信号通路。 |
| 英文摘要: |
| Objective: This study was to construct an pET28-TAT-E4orf4 expression plasmid, which contains cell penetrating peptide and adenovirus early region 4 open reading frame protein. And further investigated the effect and molecular mechanism of TAT-E4orf4 fusion protein on the apoptosis of gastric cancer cells. Method An expression plasmid containing pET28-his-TAT-E4orf4 was constructed by PCR. After being identified by PCR, the expression was induced. AGS cells were treated with pET28, pET28-his-E4orf4 and pET28-his-TAT-E4orf4, respectively. The effects of TAT-E4orf4 on the proliferation and migration of gastric cancer cells were detected by MTT and Transwells assays, respectively. The apoptosis of gastric cancer cells was detected by flow cytometry. The expression of crucial proteins on the signaling pathways of Caspases and Src were detected by Western blot. Results PCR and Western blot confirmed the successful construction of pET28-TAT-E4orf4 expression vector. MTT assays displayed that the proliferation activity of AGS cells decreased obviously after simulating with pET28-his-E4orf4 and pET28-his-TAT-E4orf4. Moreover, the latter decreased more significantly than the former. Similarly transwell assays demonstrated that pET28-his-TAT-E4orf4 inhibited the migration of gastric cancer cell migration. Furthermore, there is significantly difference comparing with the other groups. Western blot showed that there is no significantly difference on the expression of Caspase-3 and Caspase-9. While the crucial proteins on the pathway of Src were both changed regularly. Conclusion TAT-E4orf4 fusion protein can inhibit the proliferation and migration of gastric cancer cells and promote the apoptosis of gastric cancer cells, and it was mainly depended on Src signaling pathways. |
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