结肠癌多药耐药性相关基因筛选与鉴定

目的筛选结肠癌多药耐药相关基因，探讨结肠癌多药耐药的机制。方法通过逐步递增氟尿嘧啶（5-FU）浓度孵育结肠癌Lovo细胞，建立耐药细胞株Lovo／5-FU。M1Tr法检测Lovo／5-FU细胞对5-FU和顺铂（CDDP）的敏感性。采用二维电泳-质谱联用技（2DE—MS）术比较Lovo和Lovo／5-FU两株细胞间差异表达蛋白，并采用Western blot法鉴定筛选蛋白。结果与Lovo相比，Lovo／5-FU细胞对5-Fu和CDDP的IC。值分别升高31倍和3倍（均P〈0.01）。通过2DE—MS筛选得到。在耐药株中CAP—G和RhoGD12表达上调，6-PGL、DCI、Prdx-6和Maspin表达下调。Western blot检测显示．Lovo／5-FU细胞中RhoGD12和CAP-G较Lovo增高6．14倍和2．98倍（均P〈0．01）．而Maspin则明显下降至Lovo中的5．2％（P〈0．01）。结论结肠癌耐药性的产生是多基因、多通路改变的结果。CAP—G、RhoGD12、Masoin是潜在的结肠癌多药耐药相关基因。

Screening and identification of genes associated with multi-drug resistance in colonic cancer

Objective To identify novel multi-drug resistance-related genes, and to explore the mechanisms of multi-drug resistance. Methods Multi-dmg resistant cell line Lovo/5-FU was established by incubation with increasing dose of 5-FU. The sensitivity to 5-FU and cis-diaminodichloroplatinum （CDDP） was measured by MTT assay. Two dimensional electrophoresis plus mass spectrum（2-DE/MS） was used to identify the differentially expressed protein between Lovo and Lovo/5-FU. The identified protein was then verified by Western blot analysis. Results The ICSO concentrations of Lovo/5-FU to 5-Fu and CDDP were increased by 31 and 3 times, compared with Lovo （both P〈0.01）. 2DE-MS showed that CAP-G and RhoGDI2 were up-regulated, whereas 6-PGL, DCI, Prdx-6 and Maspin were down-regulated in Lovo/5-FU. Western blot analysis confirmed that the expression levels of RhoGDI2 and CAP-G in Lovo/5-FU were increased by 6.14 and 2.98 fold respectively （both P〈0.01）, whereas Maspin was decreased to 5.2% of Lovo （P〈0.01）. Conlusions Muhi-gene and multi-pathway are involved in the development of muhi-drug resistance of colorectal cancer cells. CAP-G, RhoGD12 and Maspin are potential multi-drug resistant genes.