人大肠癌HCT-8/5-FU耐药细胞株的建立及P-gp测定  

Preparation of HCT-8/5-fluorouracil multidrug resistant cell line and detection of P-glycoprotein

作　　者：布立民[1] 孙淑红[2] 华建平[1] 韩英[3] 赖靖 鲍文漪[1] 

机构地区：[1]天津市第一中心医院消化科,天津市300000 [2]承德医学院外科教研室,河北省承德市063700 [3]北京军区总医院消化科,北京市100700 [4]解放军464医院内二科,天津市300810

出　　处：《世界华人消化杂志》2006年第21期2082-2086,共5页World Chinese Journal of Digestology

摘　　要：目的：建立人大肠癌多药耐药细胞株HCT-8/5- FU及并对其耐药机制进行探讨．方法：先采用较大剂量间歇诱导法进行筛选,再采用浓度梯度递增法作用,历时7mo,至HCT8细胞可长期在5-FU浓度为2．0mg/L的细胞培养液中稳定生长．电镜、HE染色观察2种细胞形态结构差异．体外细胞毒性实验观察他对5-FU ADM,DDP的耐药性．绘制亲本细胞和耐药细胞的体外生长曲线．罗丹明染色法检测其两种细胞P-gp功能表达．结果：HCT-8细胞株经7mo诱导,可在5-FU 2．0 g/L的培养液中稳定增殖,具有多药耐药性,命名为HCT-8/5-FU,该细胞株对5-FU的耐药指数为16．6,并对ADM,DDP有交叉耐药性．该细胞株体外群体倍增时间与亲本细胞差别不显著．HE染色观察耐药细胞胞体较亲本细胞大,细胞核不规则,可见双核、多形核,细胞形态不规则,呈多角形、细长形改变,可见巨核细胞．透射电镜下耐药细胞胞质内线粒体、内质网、溶酶体增多．流式细胞仪罗丹明染色法观察荧光强度曲线左移,提示耐药细胞有过度P-gp表达．结论：成功建立HCT-8/5-FU多药耐药细胞株．先采用较大剂量间歇诱导进行筛选,再采用浓度梯度递增法作用是诱导大肠癌耐药细胞株的较好方式．HCT-8/5-FU细胞株的耐药机制与P-糖蛋白表达有关．AIM： To develop HCT-8/5-fluorouracil multidrug resistant cell line and explore the mechanism its drug resistance. METHODS： High concentration of 5-FU was used in the beginning to induce drug resistance of HCT-8 cell line, and thereafter, the concentration of 5-FU was increased in gradient. About 7 mo later, the cells could stably grow in 2.0 mg/L 5-FU, which was named HCT-8/5-FU multidrug resistant cell line. The resistant index of HCT- 8/5-FU cells to 5-FU, adriamycin （ADM）, and cisplatin （DDP） was examined with MTT assay, and growth curve was drawn. The morphological changes were observed by both light and electron microscopy. The function of P-glycoprotein （P-gp） was detected by flow cytometry using rhodamine staining. RESULTS： The resistant index of HCT-8/5-FU cells to 5-FU is 16.6, and a cross-resistance to ADM and DDP was also noticed. In comparison with that of the parental cells, the growth rate of HCT-8/5-FU cells did not changed significantly, but the morphology of the cells was remarkably changed. Irregular nucleus, double nucleus and polymorphic nucleus appeared in HCT-8/5-FU cells, and the cells shaped as polygon-like and leptosomatic. The numbers of mitochondria, endoplasmic reticulum and lysosome were increased, and over-expression of P-gp was observed in HCT-8/5-FU cells. CONCLUSION： HCT-8/5-FU multidrug resistant cell line is successfully developed. The drug-resistance mechanism of HCT-8/5-FU cell line may be related to the over-expression of P-gp.

关 键 词：肿瘤细胞 细胞培养 大肠癌 耐药细胞株 多药耐药 P-糖蛋白 

分 类 号：R735.34[医药卫生—肿瘤]