整合常规技术探索肿瘤差异表达基因

The exploration of differential gene expression of cancer with integrated techniques

目的 建立一种简单实用的、探索肿瘤差异表达基因的方法。方法 从骨髓瘤细胞株U2 6 6中分离出CD4 5 +和CD4 5 -细胞 ,结合应用减法杂交、抑制性PCR、T/A克隆、测序等技术 ,寻找CD4 5 +和CD4 5 -细胞间的差异表达基因。结果 减法杂交可缩减约 2 0倍的高丰度表达基因 ,意即低丰度表达基因富聚约 2 0倍。用正向或负向减法探针 ,分别和来源于CD4 5 +细胞克隆的质粒DNA杂交 ,筛选 5 0 0个表达克隆 ,发现了几个差异表达基因 ,并经RT PCR证实。结论 采用常规分子生物学方法建立起来的综合技术 ,可以用来研究肿瘤性疾病的差异表达基因 ,且简单实用 。

Objective To develop a method for identification of differential gene expression between different cell populations. Methods Several convenient techniques of molecular biology including subtractive hybridization, suppression PCR, T/A cloning and sequencing, were used to identify genes expressed differentially in CD45 + and CD45 - cells isolated from U266 cells line of multiple myeloma.Results The level of abundant gene scales down 20 times through subtractive hybridization. Plasmid DNA from CD45 + cell clones was hybridized with forward or backward cDNA probes synthesized from CD45 + and CD45 - cells, respectively. A few of differential expressed genes reconfirmed by RT PCR were found among 500 expressed clones of CD45 + cells. Conclusion A strategy for gene identification developed from convenient molecular biological methods can be used at any scale of laboratory.