中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (50): 9357-9363.doi: 10.3969/j.issn.2095-4344.2012.50.009

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

人端粒酶反转录酶小发夹RNA质粒表达载体的构建及鉴定

胡凡果1，史玉荣2，牛瑞芳2，刘 彤1，只向成3   

  • 出版日期:2012-12-09 发布日期:2012-12-09

Construction and identification of human telomerase reverse transcriptase-targeted small hairpin RNA-expressing plasmid

Hu Fan-guo1, Shi Yu-rong2, Niu Rui-fang2, Liu Tong1, Zhi Xiang-cheng3   

  • Online:2012-12-09 Published:2012-12-09

PDF

504

被引次数

6

摘要：

背景：端粒酶反转录酶是端粒酶的活性亚基，已成为肿瘤研究的热点。RNA干扰技术作为一种基因沉默方法，具有高效、特异等优点，现已广泛应用于肿瘤、病毒等研究领域。
目的：构建针对人端粒酶反转录酶的小发夹RNA质粒表达载体，并观察其对乳腺癌T47D细胞人端粒酶反转录酶基因的表达和端粒酶活性的影响。
方法：以Genbank中人端粒酶反转录酶基因的mRNA序列为基础，设计人端粒酶反转录酶基因的小干扰RNA序列，将其连接到具有G418抗性的质粒pBAsi-hU6-Neo(Bam HⅠ/ Hind Ⅲ)中，应用基因测序加以验证，扩增提取质粒，以脂质体转染表达小发夹RNA的质粒到乳腺癌T47D细胞，抗生素G418筛选出转染成功的各组细胞。
结果与结论：实验所构建的人端粒酶反转录酶的小发夹RNA质粒表达载体，经测序验证无误。将pBAsi-hU6-Neo重组质粒转染入T47D细胞，经G418筛选获得了转染成功的细胞。经RT-PCR和Western blot检测，转染后的人端粒酶反转录酶基因在mRNA和蛋白水平的表达均明显降低(P < 0.01)，经TRAP-ELISA法检测实验组细胞端粒酶活性出现显著下降(P < 0.01)。结果证实，实验成功构建人端粒酶反转录酶的小发夹RNA质粒表达载体，实验所设计的小干扰RNA能有效抑制肿瘤细胞人端粒酶反转录酶基因的表达，进而降低细胞的端粒酶活性。

Abstract:

BACKGROUND: The activation of telomerase is closely related to the occurrence and development of malignant tumor. While telomerase reverse transcriptase is the critical subunit of telomerase and now it is a hot point in the field of cancer research. RNA interference which is a kind of effective and specific gene blocking method has been widely used in cancer and virus research area.
OBJECTIVE: To construct the human telomerase reverse transcriptase-targeted small hairpin RNA-expressing plasmid (hTERT-targeted shRNA-expressing plasmid) system, and to observe its effect on hTERT expression and telomerase activity in breast cancer cells T47D.
METHODS: Sequence of hTERT-targeted shRNA was designed based on the mRNA sequence of hTERT which was obtained from the Genbank. They were recombined with the plasmid pBAsi-hU6-Neo (BamHI/HindⅢ) which is immune to antibiotic G418, and then those plasmids were identified by gene sequencing to make sure they were correctly connected. Then those plasmids were transfected into breast cancer cells T47D with liposome and those cells expressing shRNA were selected by G418.
RESULTS AND CONCLUSION: The hTERT-targeted shRNA-expressing plasmids were successfully constructed, which was proved by gene sequencing. Then the pBAsi-hU6-Neo recombined plasmids were transfected into T47D cells, and the successfully transfected cells were selected with G418. Reverse transcription-PCR and western blot showed that the expression of the trasfected hTERT was significantly decreased both on mRNA and protein levels (P < 0.01), and tartrate resistant acid phosphatase-enzyme-linked immunosorbent assay showed that the telomerase activity of the TERT in the experimental group was decreased significantly (P < 0.01). It showed that the hTERT-targeted shRNA-expressing plasmids were successfully constructed, and the designed small interferencing RNA can effectively block the expression of hTERT and then inhibit the telomerase activity.