中性鞘糖脂在KBv_(200)细胞的表达及其与多药耐药性的关系

Investigation on the relationship between MDR and expression of Nglycosphingolipids in vincristine resistant KBv_(200) cell line

目的 研究肿瘤细胞mdr1与其细胞表面中性鞘糖脂 (N GSLs)表达的关系。方法 利用特异性切割mdr1的核酶 (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 ,将含核酶的质粒pHβApr 1neo/ 5mR3及空载体pHβApr 1neo导入KBv2 0 0 及其亲本KB细胞内 ,运用Northern blotting、免疫组化方法观察核酶对mdr1mRNA及P gp的影响 ,用改良的Hakamori方法提取、纯化耐药逆转前后及KB细胞的N GSLs ,以高效薄层层析 (HPTLC)分析不同细胞亚株N GSLs的表达。采用MTT法分析糖脂合成抑制剂DL PPMP对肿瘤细胞多药耐药性 (MDR)的逆转作用。结果pHβApr 1neo/ 5mR3及pHβApr 1neo可以在KB、KBv2 0 0 细胞中稳定表达 ,mdr1 核酶可以特异性地切割mdr1,导致KBv2 0 0 / 5mR3的mdr1mRNA含量下降 ,P gp表达减低。KB和KBv2 0 0 的N GSLs表达有差异 ,KBv2 0 0 的单乙糖神经鞘氨醇 (monohexosylceramide ,CMH)、二乙糖神经鞘氨醇 (dihexosylceramide ,CDH)表达较KB强 ,核酶逆转MDR后 ,KBv2 0 0 / 5mR3的CMH、CDH表达降低 ,以CMH为著。DL PPMP可通过抑制CMH的合成 ,逆转KBv2 0 0 对长春新碱 (VCR)的耐药。结论 肿瘤细胞mdr1与其细胞表面N GSLs表达相关 ,CMH为耐药相关N GSLs,抑制耐药细胞CMH的合成可能?

Objective To study the relationship between MDR and expression of Nglycosphingolipids (HGSLs) in tumor cells. Methods Ribozyme with specific catalytic activity, capable of cleaving mdr1 mRNA, was transfected into KBv 200 cells. RNA dot blot was used to detect the expression of ribozyme in the transfected cells. Northern blot and immunocytochemistry were employed to examine the expression of mdr1 mRNA and Pglycoprotein. Cellular NGSLs was isolated and purified with a modified Hakamori′s method and analysed by high performance TLC. The effect of DLPPMP, a glycolipids sythase inhibitor, on the reversion of MDR was assayed by MTT method. Results The ribozyme stably expressed in KBv 200/5mR3 cells decreased the level of mdr1 mRNA expression by 819% and inhibited the formation of Pglycoprotein. The levels of monohexosylceramide (CMH) and dihexosylceramide (CDH) in KBv 200 cells were increased as compared to those in KB cells. When MDR was reversed by the ribozyme, the KBv 200/5mR3 showed a sharply decreased level of CMH. PPMP could reverse MDR by inhibiting synthesis of CMH. Conclusion[WT5”BZ] CMH is a kind of MDRrelated glycolipid. Inhibition of its expression in tumor cells may be a new approach to reverse MDR.