Construction of differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity from tongue squamous carcinoma Tca8113 cell line

孙守娟; 季平; 邓诚; 李颖; 邹波; 漆小娟

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Construction of differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity from tongue squamous carcinoma Tca8113 cell line

更新时间：2026-01-09

- Construction of differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity from tongue squamous carcinoma Tca8113 cell line
- Journal of Army Medical University Vol. 34, Issue 3, Pages: 226-230(2012)
- 作者机构：
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  CLC： R394-33;R394.2;R739.86
- Published：2012
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孙守娟,季平,邓诚,李颖,邹波,漆小娟.基于舌鳞癌Tca8113细胞醛脱氢酶活性不同构建差异表达基因cDNA文库[J].陆军军医大学学报(原第三军医大学学报),2012,34(03):226-230.

　Sun Shoujuan,Ji Ping,Deng Cheng,Li Ying,Zou Bo,Qi Xiaojuan.Construction of differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity from tongue squamous carcinoma Tca8113 cell line[J].J Amry Med Univ (J Third Mil Med Univ),2012,34(03):226-230.
孙守娟,季平,邓诚,李颖,邹波,漆小娟.基于舌鳞癌Tca8113细胞醛脱氢酶活性不同构建差异表达基因cDNA文库[J].陆军军医大学学报(原第三军医大学学报),2012,34(03):226-230. DOI：

　Sun Shoujuan,Ji Ping,Deng Cheng,Li Ying,Zou Bo,Qi Xiaojuan.Construction of differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity from tongue squamous carcinoma Tca8113 cell line[J].J Amry Med Univ (J Third Mil Med Univ),2012,34(03):226-230. DOI：

摘要

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，ALDH

high

/ALDH

low

)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ALDH

high

和ALDH

low

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ALDH

high

和ALDH

low

两亚群细胞，其中ALDH

high

细胞的比例为2.5%；分光光度计测得ALDH

high

和ALDH

low

的RNA D（260）/D（280）的比值分别为1.93和1.92；构建了ALDH

high

和ALDH

low

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ALDH

high

和ALDH

low

细胞差异基因cDNA文库。

Abstract

Objective To construct the differentially expressed cDNA libraries of aldehyde dehydrogenase with high and low activity (ALDH

high

/ALDH

low

) from tongue squamous carcinoma Tca8113 cell line. Methods

Expression of stem cell marker ALDH was detected

and ALDH

high

and ALDH

low

cells were collected by Aldefluor assay combined with flow cytometry. Differentially expressed genes of total RNA that was extracted from the two cell subpopulations by Trizol were screened and amplified by suppressing subtractive hybridization (SSH)

and the PCR products were connected with pMD18-T vector and then transfected into E.coli DH5α for amplification. Enzyme digestion

gene sequencing and homology analysis were performed in 24 positive clones that were randomly picked from each library. Results Two subproportions of ALDH

high

and ALDH

low

were screened out

and ALDH

high

cells in Tca8113 cells accounted for 2.5%. RNA D(260)/D(280) of ALDH

high

and ALDH

low

were 1.93 and 1.92

respectively. Two-directional subtractive cDNA libraries of ALDH

high

and ALDH

low

were constructed

and each library comprised 500 clones. PCR analysis of 24 clones randomly picked from each library showed that insert-fragments distributed in 200-700 bp

and no false positive clones were detected. Gene sequencing result that was analyzed and indexed by PubMed showed that cancer related genes included SLC25A13

KLHL2

NPC1

WAPL

BARD1

Notch2 and EEF2K. Conclusion Two-directional subtractive cDNA libraries of ALDH

high

and ALDH

low

cells were successfully constructed.

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