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目的研究多药耐药肿瘤细胞PKC活性、PKC亚型的表达和亚细胞分布与KBV200细胞多药耐药的关系。方法MTT法检测敏感株KB和耐药株KBV200细胞的耐药性;32P掺入法测定PKC的活性;Westernblot法检测KBV200及其亲本KB细胞株PKC亚型的表达和亚细胞分布。结果长春新碱(VCR)和阿霉素(ADR)对KBV200细胞的IC50值分别高于KB细胞(P<0.01);耐药指数(RI)分别为65.03和19.8。KBV200细胞的膜组分、浆组分的PKC活性均较KB细胞高,KBV200细胞的总PKC活性是KB细胞的2.12倍。Westernblot结果发现KBV200和KB细胞膜组分及浆组分均有PKCα的表达,且KBV200细胞的表达较KB明显增强。PMA可升高KBV200细胞的PKC总活性和膜组分PKC活性,降低浆组分PKC活性、表达(P<0.01);PMA可升高VCR、ADR对KBV200细胞的IC50值(P<0.01)。SP可降低PKC活性;SP预孵育使PKCα膜组分和浆组分的表达均降低,可降低VCR、ADR对KBV200细胞的IC50值(P<0.01)。结论KBV200细胞的PKC活性、表达、亚细胞分布与KB细胞有明显差别,这种差别可能与KBV200耐药性的变化密切相关,在PKC亚型中,PKCα的表达明显高于其他亚型,且高于亲本株,提示KBV200细胞中PKCα与多药耐药相关。
Objective To study the relationship between the PKC activity, the expression of PKC isoforms and the subcellular distribution of multidrug-resistant tumor cells and multidrug resistance in KBV200 cells. Methods MTT assay was used to detect the drug resistance of KB and resistant KBV200 cells. The 32P incorporation method was used to determine the activity of PKC. The expression and subcellular distribution of PKC subtypes of KBV200 and its parent KB cells were detected by Western blot. Results The IC50 values of vincristine (VCR) and doxorubicin (ADR) in KBV200 cells were higher than those in KB cells (P <0.01). The drug resistance index (RI) was 65.03 and 19.8, respectively. The membrane fraction and pulp fraction of KBV200 cells had higher PKC activity than KB cells, and the total PKC activity of KBV200 cells was 2.12 times that of KB cells. The result of Western blot showed that the expression of PKCα in KBV200 and KB cell membrane fractions and plasma components was significantly higher than that in KB cells. PMA could increase PKC activity and PKC activity of KBV200 cells, decrease PKC activity and expression of plasma components (P <0.01). PMA increased IC50 of VCR and ADR on KBV200 cells (P <0.01). SP decreased the activity of PKC200. The preincubation of SP decreased the expression of PKCα membrane fraction and plasma fraction, and decreased the IC50 of VCR and ADR on KBV200 cells (P <0.01). Conclusion PKC activity, expression and subcellular distribution of KBV200 cells are significantly different from those of KB cells. This difference may be closely related to the change of KBV200 resistance. In PKC subtypes, the expression of PKCα is significantly higher than that of other subtypes Higher than the parent strain, suggesting that KBV200 cells PKCα and multidrug resistance related.