Bax基因诱导鼻咽癌细胞中相关基因的mRNA差异显示分析

中国生物工程杂志

研究简报

Bax基因诱导鼻咽癌细胞中相关基因的mRNA差异显示分析

董加喜李运南马涧泉

广州市微生物研究所广州市微生物研究所

In CNE2 cell after bax gene transfection analysed by mRNA defference to demonstrate

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摘要： 目的：应用差异显示技术，筛选鼻咽癌相关基因的异常表达。方法：选用连接有bax基因的真核表达质粒pSFFV-bax-neo, 采用脂质体转染法, 将其转染到CNE2细胞中, 以转染有空载的pSFFV-neo 质粒的CNE2细胞为对照, 采用Trizol试剂快速抽取法, 提取mRNA经逆转录成cDNA,用锚式引物和随机引物进行PCR扩增, 加入同位素, 用6%的测序变性聚丙烯酰胺凝胶上电泳PCR产物进行分离。切下聚丙烯酰胺凝胶上明显的6条差异带, 回收差异cDNA, 进行第二次PCR扩增, 经低溶点琼脂凝胶回收, 获取大量PCR扩增的差异cDNA片段，同位素标记作为探针, 抽取RNA, 进行点样, 杂交, 洗膜放射自显影等步骤, 进行细胞RNA的Northern印迹斑点杂交。结果：表明4条cDNA片段均为CNE2细胞表达片段。结论：发现bax可诱导CNE2细胞中有某些相关基因表达，并抑制了CNE2细胞某些相关基因表达。

Abstract: bjective ：To look for the correlated gene of CNE2 cell after bax gene transfection. Method: In CNE2 cell after bax gene transfection analysed by mRNA defference to demonstrate, and DNA sequencing was performed, and small amount of PCR products were used as probes for Northern blot after labeled with radioisotope. Result: By mRNA differential display technique, ten differential expression bands were found in CNE2 cell, four bands of which were induced by pSFFV-bax-neo and the others inhibited. The four cDNA framents were to reamplify by PCR selected from the inhibited bands and expressions in CNE2 cell were found by Northern blot. Conclusion：Bax gene can induce the expression of some genes in CNE2 cell, whereas it can also inhibit the expression of some genes. By selecting two lengthened primers and adding T7RNA polymerase promoter to the nondT anchoring primers, it can overcome the disadvantags of excessive PCR products and pseudopositive bands in mRNA differential display technique, Thus PCR products can be sequenced directly.

收稿日期: 2006-02-09 出版日期: 2006-02-25

通讯作者: 董加喜

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引用本文:

董加喜,李运南,马涧泉. Bax基因诱导鼻咽癌细胞中相关基因的mRNA差异显示分析[J]. 中国生物工程杂志, .

. In CNE2 cell after bax gene transfection analysed by mRNA defference to demonstrate. China Biotechnology, .

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2006/V26/I02/69

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