摘要
人端粒酶逆转录酶基因(human telomerase reverse transcriptase,hTERT)的表达与端粒酶活性密切相关,而多项研究表明,端粒酶在肿瘤发生、发展、细胞永生化方面起重要作用.为探讨单细胞技术检测卵巢癌SKOV3细胞转导hTERT前后其mRNA表达的差异,前期已通过慢病毒转染,将携带hTERT基因的表达质粒载体LV4-pGLV-EF1a-EGFP-hTERT转入卵巢癌SKOV3细胞系,构建了稳定表达hTERT蛋白的细胞株(SKOV3 h),同时设空载体感染组(SKOV3 b)和未处理组(SKOV3)为对照组,采用流式细胞术,分选不同数量的细胞进行实时PCR.结果显示,初步摸索的细胞数在50个以内,且随着细胞数目的增多,Ct值逐渐增大(P<0.05);SKOV3 h组hTERT基因mRNA表达最高,SKOV3 b组次之,SKOV3组表达最低(P<0.05).提示SKOV3细胞转导hTERT基因后可促进该基因的mRNA表达.同时用该法检测了单个卵裂球内hTERT基因的表达,为利用单细胞单基因遗传学诊断研究及肿瘤学研究提供基础.
The expression of human telomerase reverse transcriptase （hTERT） defines the cellular activity of telomerase. A number of studies have shown that telomerase closely related to tumorigenesis and cell immortalization. To detect the hTERT mRNA expression at single cell level, SKOV3 cells infected with lentiviral vector LV4-pGLV-EFla-E＇GFP-hTERT （SKOV3h） was used and compared with empty vector infected （SKOV3b） and non-infected （SKOV3） cells. Less than 50 ceils were sorted by flow cytometry for each group, and the hTERT mRNA expression were detected by real-time PCR. The results showed that the Ct value was increased as the cells increased （P 〈 0.05）. The hTERT mRNA level of SKOV3h was higher than those of SKOV3b and SKOV3. The method was able to detect the hTERT expression of a single blastomere, thus can be useful in applications of gene diagnosis for oncology research.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2013年第4期383-388,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
广西自然科学基金资助项目(No.0992032-12)~~
