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目的:构建磷酸化Akt1(Thr308)位点突变真核表达载体,并瞬时转染胃癌耐药细胞,对其生物学功能进行初步检测。方法:以带有pc DNA3.0-Flag标签的Akt1质粒为模板,采用重组PCR技术扩增得到Akt1(T308A)(苏氨酸突变成丙氨酸)、Akt1(T308E)(苏氨酸突变成谷氨酸)位点突变编码区序列,将其插入pc DNA3.0-Flag载体,双酶切和测序验证后瞬时转染人胚肾293T细胞,Western印迹检测其表达情况;将突变质粒与空载体分别转染人胃癌耐药细胞BGC-823/c DDP,通过Western印迹和实时定量PCR检测Notch1蛋白和m RNA水平的变化,通过CCK-8法检测对耐药细胞生长曲线的影响。结果:双酶切和测序结果表明Akt1(T308A)、Akt1(T308E)的真核表达质粒构建成功,转染293T细胞后获得表达;转染人胃癌耐药细胞BGC-823/c DDP后,利用实时定量PCR和Western印迹证明去磷酸化Akt1(T308A)可抑制Notch1的转录和蛋白水平,模拟持续磷酸化Akt1(T308E)升高Notch1的转录和蛋白表达;细胞生长曲线结果显示,转染Akt1(T308A)较空载体耐药细胞生长慢,而Akt1(T308E)促进耐药细胞生长。结论:PI3K/Akt与Notch1信号通路的激活在胃癌耐药的发生、发展过程中发挥重要作用。
OBJECTIVE: To construct eukaryotic expression vector of phosphorylated Akt1 (Thr308) site and transiently transfected into drug-resistant gastric cancer cells for preliminary detection of its biological function. Methods: The Akt1 (T308A) (threonine to alanine), Akt1 (T308E) (threonine mutation) were amplified by PCR using Akt1 plasmid with pcDNA3.0-Flag tagged as template. Into glutamic acid) site mutation coding sequence, insert it into pcDNA3.0-Flag vector, double digestion and sequencing verified transiently transfected human embryonic kidney 293T cells, Western blot detection of its expression; the mutant plasmid The expression of Notch1 protein and m RNA in gastric cancer cell line BGC-823 / c was detected by Western blotting and real-time quantitative PCR respectively. The cell growth curve was detected by CCK-8 assay. Results: The double digestion and sequencing results showed that the eukaryotic expression plasmids of Akt1 (T308A) and Akt1 (T308E) were successfully constructed and transfected into 293T cells. After transfection with human gastric cancer cell line BGC-823 / c DDP, Real-time PCR and Western blotting demonstrated that dephosphorylation of Akt1 (T308A) inhibited Notch1 transcription and protein levels and mimicked the sustained phosphorylation of Akt1 (T308E) to upregulate Notch1 transcription and protein expression. The cell growth curve showed that Akt1 T308A) grew slower than empty vector-resistant cells, whereas Akt1 (T308E) promoted drug-resistant cell growth. Conclusion: The activation of PI3K / Akt and Notch1 signaling pathways play an important role in the development of gastric cancer drug resistance.