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目的构建舌鳞癌Tca8113细胞系中高、低醛脱氢酶活性(high/low aldehyde dehydrogenase activity,ALDHhigh/ALDHlow)细胞差异表达基因cDNA文库。方法用流式细胞仪检测ALDEFLUOR染色的Tca8113细胞中干细胞标志物ALDH的表达,并收集ALDHhigh和ALDHlow细胞;用Trizol分别提取两亚群细胞的总RNA;用抑制性消减杂交(SSH)对2组RNA进行差异基因筛选和扩增,扩增产物与pMD18-T载体连接并转化大肠杆菌DH5α。文库扩增后,每库随机挑取24克隆进行酶切、测序及同源性分析。结果分选得到ALDHhigh和ALDHlow两亚群细胞,其中ALDHhigh细胞的比例为2.5%;分光光度计测得ALDHhigh和ALDHlow的RNA D(260)/D(280)的比值分别为1.93和1.92;构建了ALDHhigh和ALDHlow细胞差异基因cDNA双向文库,每个库约500个克隆菌落,每库随机挑选24个菌落进行PCR分析,克隆片段均匀分布在200～700 bp,无假阳性克隆。测序结果经同源性比对分析和PubMed检索,其中与肿瘤有关的基因有SLC25A13、KLHL2、NPC1、WAPL、BARD1、Notch2、EEF2K。结论成功构建ALDHhigh和ALDHlow细胞差异基因cDNA文库。
Objective To construct a cDNA library of differentially expressed genes in high and low aldehyde dehydrogenase activity (ALDHhigh / ALDHlow) cells of tongue squamous cell carcinoma Tca8113 cell line. Methods ALDEFLUOR staining of Tca8113 cells by flow cytometry ALDH expression of stem cell markers and ALDHhigh and ALDHlow cells were collected; Trizol were extracted from the two subpopulations of total RNA; suppression subtractive hybridization (SSH) of 2 Group RNA was selected for differential gene amplification and amplification. The amplified product was ligated into pMD18-T vector and transformed into E. coli DH5α. After the library was amplified, 24 clones were randomly picked per library for restriction analysis, sequencing and homology analysis. Results Two subpopulations of ALDHhigh and ALDHlow were selected and the proportion of ALDHhigh cells was 2.5%. The ratios of RNA D (260) / D (280) of ALDHhigh and ALDHlow measured by spectrophotometer were 1.93 and 1.92 respectively. ALDHhigh and ALDHlow cells differentially expressed cDNA bi-directional library, each library about 500 colonies, 24 colonies randomly selected for PCR analysis, cloned fragments evenly distributed in the 200 ~ 700 bp, no false positive clones. Sequencing results were compared by homology analysis and PubMed search. Among them, the tumor related genes were SLC25A13, KLHL2, NPC1, WAPL, BARD1, Notch2 and EEF2K. Conclusion The cDNA library of differential genes in ALDHhigh and ALDHlow cells was successfully constructed.