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目的研究蛋白激酶C(PKC)主要亚型在人骨肉瘤143B细胞中的表达情况及其与143B细胞耐药的关系。方法采用Western blot法检测PKC主要亚型α、β和γ磷酸化形式p-PKCα、p-PKCβ、p-PKCγ和多药耐药蛋白-1(mutidrugresistance protein-1,MDR-1)在人骨肉瘤143B/WT野生型细胞和143B/ADM耐药细胞中的表达水平,通过PKC特异性抑制剂staurosporine抑制PKC对MDR-1蛋白表达的影响;CCK法检测药物敏感性;荧光分光光度计法检测细胞内阿霉素浓度,流式细胞术检测细胞凋亡水平和细胞外排罗丹明水平。结果相对于143B/WT野生型细胞,143B/ADM耐药细胞中p-PKCα表达显著增加,而p-PKCβ和p-PKCγ表达则无明显差异,同时143B/ADM耐药细胞中MDR-1表达也明显上调;143B/ADM耐药细胞对阿霉素的耐药指数为15.7,而加入staurosporine抑制143B/ADM细胞PKC表达后,细胞耐药指数下降为5.5(P<0.01);143B/ADM耐药细胞接受ADM处理后,细胞凋亡率为(28.41±4.33)%,而其PKC表达被抑制后,细胞内阿霉素浓度增加的同时(P<0.01),细胞凋亡率增加为(70.63±4.80)%(P<0.01),外排罗丹明减少,同时MDR-1蛋白表达明显下调。结论人骨肉瘤耐药细胞中过表达的PKCα可通过调控MDR-1参与骨肉瘤耐药。
Objective To study the expression of major protein kinase C (PKC) subtype in human osteosarcoma 143B cells and its relationship with 143B cell resistance. Methods Western blot was used to detect the expression of phosphorylated forms of p-PKCα, p-PKCβ, p-PKCγ and mutidrug resistance protein-1 (MDR-1) 143B / WT wild-type cells and 143B / ADM resistant cells by PKC-specific inhibitor staurosporine inhibition of PKC MDR-1 protein expression; CCK test drug sensitivity; fluorescence spectrophotometer detection of cells Endogenous doxorubicin concentration, flow cytometry to detect apoptosis levels and extracellular efflux rhodamine levels. Results Compared with 143B / WT wild-type cells, the expression of p-PKCα in 143B / ADM resistant cells was significantly increased, while the expression of p-PKCβ and p-PKCγ was not significantly different, while the expression of MDR-1 in 143B / ADM resistant cells (P <0.01). The resistance index of 143B / ADM cells to doxorubicin was 15.7, while the inhibitory index of PKC of 143B / ADM cells was decreased to 5.5 after adding staurosporine (P <0.01) After treated with ADM, the rate of apoptosis was (28.41 ± 4.33)%, while the expression of PKC was inhibited (P <0.01). The apoptosis rate increased to (70.63 ± 4.80)% (P <0.01). The efflux of rhodamine decreased and the expression of MDR-1 protein was significantly down-regulated. Conclusion Overexpression of PKCα in human osteosarcoma drug-resistant cells may be involved in the drug resistance of osteosarcoma by regulating MDR-1.