摘要
目的建立人肝癌HepG2/VCR耐药细胞株,检测ZNF300基因在HepG2/VCR中的表达并初步分析其在肝癌多药耐药(MDR)中发挥的功能。方法采用体外低浓度梯度递增的诱导方法建立长春新碱(VCR)获得性HepG2/VCR耐药细胞株。MTT法检测确定HepG2/VCR耐药细胞株对VCR的耐药性,用Western blot方法检测人锌指蛋白ZNF300基因编码的ZNF300及多药耐药基因编码的P糖蛋白(P-gp)在HepG2和HepG2/VCR细胞中的表达差异;在HepG2/VCR细胞中转染ZNF300基因正向或反向cDNA质粒后,MTT法检测VCR对耐药细胞IC50值的变化,Westernblot方法检测细胞内P-gp表达的影响。结果 MTT检测确认HepG2/VCR耐药细胞构建成功,Western blot检测发现耐药细胞中ZNF300及P-gp的表达相对于HepG2细胞明显增高。在HepG2/VCR细胞中转染正向ZNF300 cDNA质粒后,MTT和Western blot检测发现ZNF300过表达可使VCR对耐药细胞的IC50值增高,并使细胞内P-gp表达上调;在转染反向cDNA质粒Knockdown ZNF300基因表达后得到相反的结果。结论 ZNF300基因在HepG2/VCR耐药细胞中表达明显增高,并能通过上调耐药蛋白P-gp的表达促进肝癌细胞耐药性,可以作为逆转肝癌多药耐药的分子作用靶点。
Aim To establish human hepatic cancer drug resistant cell line HepG2/VCR, to detect the ex- pression of human ZNF300 gene in HepG2/VCR cells, and to primarily analyze the possible roles of ZNF300 gene in hepatic cancer multi-drug resistance （MDR）. Methods HepG2/VCR cell line was established by using treatment with vincristine （VCR） of low concen- tration gradually increased. MTT analysis was per- formed to confirm the VCR resistance feature of HepG2/VCR. The expression variances of ZNF300 and multi-drug resistant protein P-glycoprotein （P-gp） between HepG2 and HepG2/VCR were detected by Western blot. Then a transient transfection to HepG2/ VCR cells was performed using plasmids containing ZNF300 sense or antisense cDNA. After plasmid trans- fection, MTT analysis was used to detect the effect of ZNF300 gene overexpression or knockdown on VCR- ICs0 value alternation of HepG2/VCR, and Western blot analysis was used to detect the expression alterna- tion of P-gp. Results MTT analysis showed the suc- cessful construction of HepG2/VCR. Western blot a- nalysis showed that the expression of ZNF300 and P-gp was significantly elevated in HepG2/VCR cells, com- pared with HepG2 cells. After plasmid transient trans- fection, MTT and Western blot analysis showed that ZNF300 overexpression increased the VCR-ICs0 value of HepG2/VCR cells, and promoted the expression of P-gp, whereas ZNF300 gene knockdown had an oppo- site effect. Conclusions ZNF300 gene expression is evaluated in hepatic cancer drug resistant cell line HepG2/VCR, and it can promote the drug resistance feature of HepG2/VCR through up-regulating the ex- pression of P-gp. It is suggested that ZNF300 may be a molecular target of reversing the hepatic cancer MDR.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2013年第7期951-955,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81001455)
安徽医科大学博士科研基金资助项目(No XJ200912)
关键词
ZNF300
肝癌
多药耐药
P-糖蛋白
化疗
锌指蛋白
ZNF300
hepatic cancer
multi-drug re- sistance
P-gp
chemotherapy
zinc finger protein
