中期因子过表达对肝癌细胞多药耐药的影响

The effect of MK gene overexpression in hepatic carcinoma cells on multidrug resistance

目的探讨中期因子（midkine，MK）过表达对肝癌细胞多药耐药（multidrug resistance，MDR）的影响。方法将肝癌SMMC7721细胞转染pIRES2-EGFP-MK重组质粒后，分别以实时荧光定量PCR、蛋白质印迹法及流式细胞术检测MK基因mRNA和蛋白水平，通过流式细胞术检测细胞内DNR蓄积。MTT法检测细胞对不同化疗药物的敏感性了解MK对肝癌细胞多药耐药的影响。结果pIRES2-EGFP—MK重组质粒转染肝癌SMMC7721细胞后，MK基因mRNA和蛋白产物MK的表达水平明显增高。提示MK重组质粒达到了对MK转录的有效增强。中期因子转染组细胞胞内DNR蓄积量显著降低（4．06±0．88，P〈0．05），对ADM、5-FU的Ic。显著增加（15±3，27±4，P〈0．05）。结论经pIRES2-EGFP—MK重组质粒转染肝癌细胞后．细胞内中期因子表达的增高可增强肝癌细胞对不同化疗药物的耐药性。

Objective To study the effect of enhanced MK gene expression in hepatic carcinoma cells. Methods The recombinant plasmid pIRES2-EGFP-MK was transfected into SMMC 7721 cells. The mRNA and protein expression levels of MK gene in these cells were determined by real-time PCR, Western blotting and /low eytometry. The intracellular DNR accumulation of these cells was measured by flow cytometry. To investigate the effect of MK gene mediated muhidrug resistance, MTT assay was employed to determine the cellular sensitivity of different chemotherapeutic drugs in MK-overexpressed SMMC 7721 cells. Results The mRNA and protein expression levels of MK gene significantly increased after the recombinant plasmid pIRES2-EGFP-MK transfected into SMMC 7721 cells, suggesting that the recombinant plasmid pIRES2-EGFP-MK can enhance the transcription of MK effectively. The DNR accumulation of MK transfected cells decreased significantly （4. 06±0. 88, P 〈 0. 05 ）, and IC50 of MK transfected cells to ADM/5-FU increased significantly （ 15 ±3, 27 ± 4, P 〈 0. 05 ）. Conclusions After the recombinant plasmid pIRES2-EGFP-MK transfected into hepatic carcinoma cells, expression of midkine increased, enhancing the resistance of hepatic carcinoma cells to chemotherapeutic drugs.