结肠癌耐药细胞株LoVo/5-FU的建立及其差异表达基因的筛选

Establishment and selection of genes expressing differently in a human colon carcinoma LoVo/5-FU cell line

目的:建立结肠癌细胞耐药模型LoVo/5-FU并初步筛选可能的耐药相关基因.方法:采用5-FU浓度递增法建立人结肠癌细胞耐药模型LoVo/5-FU,观察其生长规律并绘制细胞生长曲线:用MTT法鉴定耐药细胞株耐药性并计算耐药指数(RI);用基因芯片技术检测耐药细胞株LoVo/5-FU与其亲本细胞株LoVo中的差异表达基因,从中筛选出可能的耐药相关基因;用半定量RT-PCR方法对筛选出的部分耐药相关基因在耐药细胞及其亲本细胞中的表达情况进行验证.结果:LoVo/5-FU细胞与LoVo细胞相比,生长缓慢,细胞体积增大.LoVo/5-FU对5-FU、ADM、MMC耐药,而对CDDP无耐药性(RI:7.69,2.78,1.43和0.96).通过基因芯片筛选出差异表达基因425个,可能的耐药相关基因包括CYP1B1,NAT2,RNF20,SNAI2,MAP3K2,其中CYP1B1在LoVo/5-FU与LoVo中的表达有明显差异(Cy5/Cv3=5.877).结论:LoVo/5-FU细胞株耐药性稳定,耐药机制可能与CYP1B1,NAT2等耐药相关基因有关.

AIM： To establish a Lovo/5-FU of human drugresistance colon carcinoma cell line, and to screen possible drug resistance-associated genes. METHODS： LoVo/5-FU of a human drug-resistance colon carcinoma cell line was induced by continuously exposing human colon carcinoma cells to gradually increasing concentrations of 5-FU. The growth law was observed and the growth curve protracted. The drug resistance of LoVo/5-FU was measured by MTT assay and the drug resistant index （RI） was calculated. Genes expressing differently between LoVo/5-FU and its parent cell line Lovo were screened using a gene chip, and several selected associated drug resistance genes were confirmed by reverse transcription-polyrnerase chain reaction （RT-PCR）. RESULTS： Compared with parental cells, the resistance cell line had a slower growth rate and larger morphology. The LoVo/5-FU cell line showed drug resistance to 5-FU, adriarnycin （ADM）, mitomycin （MMC）, but not to cisplatin （CDDP） （RI： 7.69, 2.78, 1.43, and 0.96）. There were 425 differently expressed genes associated with drug resistance by the gene chip screening method. The possible drug resistance-related genes included CYPIB1, NAT2, RNF20, SNAI2, and MAP3K2. The expression of CYPIB1 had a significant difference between the LoVo/5- FU cell line and the parent cell line （Cy5/Cy3 = 5.877）. RT-PCR results were consistent with the gene chip results. CONCLUSION： The altered biological properties of LoVo/5-FU may be related to its drug resistance phenotype. Several genes, such as CY- PIB1, are possibly involved in the mechanism of drug resistance.