胃癌耐药细胞系传统型蛋白激酶C的表达及活性.docVIP

胃癌耐药细胞系传统型蛋白激酶C的表达及活性 作者：韩英　时永全　郑悦　张宏博　樊代明 【关键词】 蛋白激酶 　　关键词: 蛋白激酶C;同工酶;胃癌;多药耐药 　　摘 要:目的 观察传统型蛋白激酶C（cPKCs）在胃癌耐药细胞系SGC7901/VCR1.0、耐药亚系SGC7901/VCR0.3，SGC7901/VCR0.7及药敏细胞系SGC7901表达及活性的变化，以及对细胞内药物浓度的影响. 方法 用免疫荧光化学及蛋白印迹方法观察传统型蛋白激酶C在胃癌耐药细胞系及药敏细胞系的表达;竞争蛋白结合法测定PKC的活性;应用流式细胞仪观察细胞内药物浓度的变化. 结果 PKCα，PKCβⅠ ，PKCβ Ⅱ 及PKCγ在胃癌耐药细胞系及药敏细胞系均有表达，PKCα在耐药细胞表达呈强阳性，在药敏细胞表达呈阳性;PKCβⅠ 及PKCβⅡ 在耐药细胞及药敏细胞表达均为阳性;PKCγ在耐药细胞及药敏细胞表达均为强阳性.免疫蛋白印迹实验证实，随耐药指数的增加，PKCα表达呈逐渐增高的趋势，薄层扫描蛋白印迹带的吸光度（A）分别为9584.17，9421.06，8534.64，8088.79，而PKCβⅠ ，PKCβⅡ 及PKCγ在耐药细胞系、耐药亚系及其药敏细胞系表达无明显变化.PKC活性检测结果提示，随耐药指数的增加，PKC活性呈逐渐增高的趋势，以细胞质及细胞核的PKC活性增高为主. 结论 传统型蛋白激酶C在维持胃癌耐药细胞系SGC7901/VCR的多药耐药中起重要作用且具有同工酶特异性. 　　Keywords:protein kinase C;isoenzyme;gastric cancer;mul-tidrug resistance 　　Abstract:AIM To observe the expression and activity of classic protein kinase C in gastric cancer cell SGC7901and its drug-resistant sublines SGC7901/VCR slected by vincristine of different concentration（0.3，0.7and1.0μg#12539;mL-1 ）.To explore the effect of classic protein kinase C on drug concen-tration within gastric cancer cells.METHODS The expres-sion of classic protein kinase C in gastric cancer cells was de-termined by immuno-fluorescent method and Western blot.The PKC activity was determined by competed protein bind-ing method.The drug concentration within cells was deter-mined by FACS.RESULTS Both SGC7901and SGC7901/VCR cells exhibited positive staining of PKCα，PKCβⅠ ，PKCβⅡ and PKCγ.The staining of PKCαin SGC7901/VCR was much stronger than that in SGC7901.There were no dif-ferences in the expression of PKCβⅠ ，PKCβⅡ and PKCγbe-tween SGC7901and SGC7901/VCR.By using Western blot，continuous increasing expression of PKCαin SGC7901/VCR was demonstrated along with the increasing resistant index，as well as no differences between SGC7901and its drug-resis-tant sublines.Results of activity assay showed continuous in-creasing activity of PKC in gastric cancer cells along with in-creasing resistant index.CONCLUSION C