CD3AK、LAK对KB、KBv_(200)细胞株耐药逆转前后的杀伤作用

CD3AK and LAK's susceptibility and multi-drug resistance in KB and KBv_(200) cell line

目的 探讨免疫活性细胞对肿瘤细胞的杀伤活性与mdr1的关系。方法 利用特异性切割mdr1的ribozyme为工具 ,以表达mdr1的耐药细胞株KBv2 0 0 为靶细胞 ,采用脂质体转染技术 ,将含ribozyme的质粒pHβApr 1neo/ 5mR3及空载体pHβApr 1neo导入KBv2 0 0 及其亲本KB细胞内 ,运用North ernblotting、免疫组化方法观察ribozyme对mdr1mRNA及P 170的影响 ,采用MTT法检测了CD3AK、LAK对KB、KBv2 0 0 细胞株耐药逆转前后杀伤活性的变化。结果 含ribozyme的质粒pHβApr 1neo/ 5mR3及空载体pHβApr 1neo可以在KB、KBv2 0 0 细胞中稳定表达 ,ribozyme可以特异性地切割mdr1,导致KBv2 0 0 / 5mR3的mdr1mRNA含量下降 ,P 170表达减低 ,LAK和CD3AK对KBv2 0 0 的杀伤活性较对KB的强 ,MDR逆转后杀伤活性降至敏感株水平 ,LAK和CD3AK对KB/ 5mR3、KB/vec、KBv2 0 0 /vec的杀伤活性也较KB及KBv2 0 0 / 5mR3强。结论 mdr1 ribozyme在细胞内具有一定的逆转肿瘤多药抗性的生物学效应 。

Objective To study the characteristics of CD3AK and LAK cell-mediated killing of MDR and its parental sensitive tumor cells, the cytotoxicity of CD3AK and LAK to KB and KBv 200 cell line variants was studied. Methods By lipofectin mediated transfection, eukaryotic expression plasmids which have been incorporated in anti-MDR ribozyme were transfected into KBv 200 cell line. The expression of ribozyme in the transfected cell lines was detected by RNA dot blotting. Northern blotting assay and immunocytochemistry assay was employed to examine the expression of mdr1 mRNA and P-glycoprotein in cell lines. The cytotoxicity of LAK and CD3AK cells to KB, KBv 200 and other transformed cell lines were examined by MTT method. Results The transformed colonies, KB/vec, KBv 200/vec, KB/5mR3 and KBv 200/5mR3 were obtained through antibiotic selection. The ribozyme was stably expressed in the cell line and expressed ribozymes decreased the level of mdr1 mRNA expression by 83% and inhibit the formation of P-glycoprotein. The cytotoxicity of LAK and CD3AK cells to KBv 200 cells was much higher than those to KB cells. Though the cytotoxicity of LAK and CD3AK cells to KBv 200/5mR3 became lower, their cytotoxicity to KB/vec, KBv 200/vec and KB/5mR3 was still high. Conclusions Anti-mdr1-ribozyme is a useful tool which may inactivate mdr1 mRNA and reverse the multi-drug resistance phenotype in MDR cell lines. The cytotoxicity of LAK and CD3AK to MDR cells is associated with the expression of P-gp.