论文部分内容阅读
目的构建人端粒酶蛋白催化亚单位(Human telomerase reverse transcriptase,hTERT)逆转录病毒真核表达质粒,并检测其在人乳腺癌相关的成纤维细胞(Cancer-associated fibroblast,CAF)中的表达及其对CAF增殖的影响。方法扩增pIRES-EGFP-hTERT质粒,酶切回收hTERT片段,正向克隆至逆转录病毒载体pBABE-puro上,构建重组质粒pBABE-puro-hTERT,转染PT67细胞,进行逆转录病毒的包装。将PT67细胞上清感染原代培养的人乳腺癌CAF,荧光定量PCR和Westernblot分别检测hTERT基因在人乳腺癌CAF中的转录和表达,流式细胞术检测细胞的增殖情况。结果重组质粒pBABE-puro-hTERT经酶切和测序证实构建正确;与未处理的人乳腺癌CAF和感染pBABE-puro的细胞相比,感染重组质粒的人乳腺癌CAF中hTERT基因mRNA的转录水平及蛋白的表达水平均明显增强,其增殖指数也明显增加(P<0.05)。结论成功构建了hTERT基因逆转录病毒真核表达质粒,其能增强人乳腺癌CAF的增殖能力,为乳腺癌微环境CAF的研究提供了良好的细胞模型。
Objective To construct eukaryotic expression plasmid of human telomerase reverse transcriptase (hTERT) retrovirus and detect its expression in human breast cancer-associated fibroblast (CAF) Its effect on proliferation of CAF. Methods The plasmid pIRES-EGFP-hTERT was amplified. The hTERT fragment was recovered by restriction enzyme digestion and cloned into retroviral vector pBABE-puro. The recombinant plasmid pBABE-puro-hTERT was constructed and transfected into PT67 cells for retroviral packaging. The supernatant of PT67 cells were infected with primary cultured human breast cancer CAF. Fluorescent quantitative PCR and Western blot were used to detect the transcription and expression of hTERT gene in human breast cancer CAF. Flow cytometry was used to detect cell proliferation. Results The recombinant plasmid pBABE-puro-hTERT was confirmed by restriction enzyme digestion and sequencing. Compared with untreated human breast cancer CAF and pBABE-puro-infected cells, the transcriptional level of hTERT mRNA in CAF of the recombinant plasmid And protein expression levels were significantly increased, and its proliferation index also increased significantly (P <0.05). Conclusion The eukaryotic expression plasmid of hTERT gene retrovirus was successfully constructed, which can enhance the proliferation of human breast cancer CAF and provide a good cell model for the study of breast cancer microenvironment CAF.