脐血CIK细胞对耐药白血病细胞体外杀伤效应及其机制的研究

In vitro tumor killing effect of CIK cells in cord blood on drug-resistant leukemia cells and its mechanism

目的:探索干预或克服白血病细胞耐药的新策略。方法:采用抗CD3McAb联合多种细胞因子诱导的脐血杀伤细胞(CB-CIK)体外作用于K562耐药细胞株(K562/A02),用MTT比色法检测其杀伤效应;用免疫组化染色法检测杀伤前后K562/A02细胞表面多药耐药基因mdr1表达产物P170水平;采用DNA凝胶电泳进行CB-CIK细胞诱导白血病细胞凋亡的检测。结果:CB-CIK细胞杀伤K562/A02细胞活性(73.647±5.72)与杀伤K562细胞活性(75.124±4.36)相比差异无统计学意义,P>0.05;其杀瘤活性显著高于CB-LAK细胞、CB-CD3AK细胞,P<0.05,与成人CB-CIK细胞相比差异无统计学意义,P>0.05。②经CIK细胞杀伤后,K562/A02细胞表面mdr1表达产物P170含量明显减少。③K562/A02细胞在CIK细胞作用20h后,其DNA结构断裂,在DNA凝胶电泳上呈现凋亡特有的梯形图谱(DNA Ladder)。结论:CB-CIK细胞对K562细胞及其耐药株K562/A02细胞均有较强的杀伤作用,其杀伤机制可能与CIK细胞能下调白血病细胞表面多药耐药基因mdr1表达产物P170水平,以及诱导白血病细胞凋亡有关。提示脐血CIK细胞在抗白血病多药耐药性方面具有独特的优势,若与化疗联合使用,有可能成为克服白血病细胞多药耐药性的一种可供选择的新策略,因脐血的来源较广,采制相对简便,该研究方法可能具有广泛的应用前景。

Objective：To explore the new strategy of intervening and overcoming multi-drug resistance （MDR） of leukemia cells. Method： The killer cells in cord blood （CB-CIK） were induced by CD3 monoclonal antibody （CD3 McAb） combined with multi-cytokines, and subsequently used to kill drug-resistant leukemia cells（K562/A02） in vitro. Cytotoxic effect was detected by MTT colourimetry. Expression of P-glycoprotein （P170） on surface of K562/A02 cells was tested by immuno-histochemical dyeing method. Apoptosis of K562/A02 cells was measured by DNA gel electrophoresis. Result：（1） Cytotoxic effect of CB-CIK cells on K562/A02 cells was not statistically different from that on K562 cells. Tumor-killing effect of CB-CIK cells on K562/A02 cells was significantly higher than that on CB-LAK cells and CB-CD3 AK cells（ P 〈0.05）. But no significant difference in tumor-killing effect was found between CB-CIK group and PB-CIK group（ P 〉0.05）. （2） After treated with CB-CIK cells, expression of P170 on surface of K562/A02 cells reduced obviously. （3）After 20 hours co-culturing with CB-CIK cells and K562/A02 cells, apoptotic K562/A02 cells displayed clear DNA ladder band on DNA gel electrophoresis. Conclusion：Tumor-killing effect of CB-CIK ceils on K562/A02 cells was as efficient as that on K562. The tumor-killing mechanism of CB-CIK cells may be decrease of expression of P170 and induce of apoptosis in leukemia cells. Therefore combination of CB-CIK ceils with chemotherapy might be one of new strategy for overcoming MDR in leukemia cells.