KBv_(200)细胞株耐药逆转前后HLA、B7抗原的表达

Expressions of HLA and B7 in KBv_(200) cell line before and after reversion of multidrug resistance

目的 探讨肿瘤多药抗性细胞的免疫逃避机制。方法 利用特异性切割mdr1的核酶为工具，以表达Mdr1的耐药细胞株KBv200为靶细胞，采用脂质体转染技术，将含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo导入KBv200及其亲本KB细胞内，运用Northern-blotting、免疫组化方法观察核酶对mdr1 mRNA及P-gp的影响，运用流式细胞仪技术检测各种不同细胞亚株HLA-Ⅰ、HLA-Ⅱ、B7-1、B7-2的表达。 结果 含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo可以在KB、KBv200细胞中稳定表达，核酶可以特异性地切割mdr1，导致KBv200/5mR3的mdr1 mRNA含量下降，P 糖蛋白（P-gp）表达减低，各种不同细胞亚株均表达较强的HLA-Ⅰ类抗原，而HLA-Ⅱ、B7-1、B7-2的表达较低。各亚株HLA-Ⅰ表达无明显差异，但HLA-Ⅱ、B7-1、B7-2的表达变化较大，KB的HLA-Ⅱ、B7-1、B7-2的表达较KBv200强，经化疗药物作用后KB的HLA-Ⅱ、B7-2进一步表达增强，核酶逆转后，KBv200/5mR3 HLA-Ⅱ、B7-1、B7-2的表达趋于接近KB水平。 结论 mdr1-核酶在细胞内具有一定的逆转肿瘤多药抗性的生物学效应；多药耐药细胞和敏感株细胞有着不同的免疫逃避特点，与敏感株相比，KBv200较易逃避机体的免疫反应。

Objective To investigate the mechanism by which multidrug-resistant (MDR) tumor cells evade the host immune surveillance, the expressions of HLA and B7 in KBv200 cell line before and after the reversion of MDR were studied. Methods By using lipofectin-mediated transfection method, eukaryotic expression plasmids incorporated with an anti-MDR ribozyme were transfected into KBv200 cell line. RNA dot blotting assay was used to detect the expression of ribozyme in the transfected cell line. Northern blotting assay and immunohistochemistry assay were employed to examine the expression of mdr1 mRNA and P-glycoprotein in cell lines. The expressions of HLA-Ⅰ, HLA-Ⅱ, B7-1, B7-2 in the above cell lines were measured by flow cytometry. Results After being screened by 400 (g/ml antibiotic G418, the transformed colonies, KB/Vec, KBv200/Vec, KB/5mR3 and KBv200/5mR3 were obtained. The ribozyme was stably expressed in the cell line and could decrease the level of mdr1 mRNA expression by 83% and inhibit the formation of P-glycoprotein. Most tumor cells expressed MHC class Ⅰ molecules, and a small portion of the cells also expressed MHC-Ⅱ, B7-1 and B7-2 molecules. Similar HLA Ⅰ expression levels in every cell colonies were observed. The expressions of HLA-Ⅱ, B7-1, B7-2 were higher in KB cells than those in KBv200 cells, and were lowered after anti-mdr1-ribozyme transfection. The expressions of HLA-Ⅱ, B7-2 in KB cells were further enhanced after vincristine treatment. Conclusions Anti-mdr1-ribozyme is able to inactivate mdr1 mRNA and reverse the multidrug resistance phenotype in MDR cell lines. Compared with its parental sensitive cell line, KBv200 cells are easier to escape the host immune surveillance.