【摘要】 目的 观察血管内皮生长因子C(vascular endothelial growth factor-C,VEGF-C)在顺铂诱导的人肾小管上皮细胞(HK-2)毒性损伤模型中mRNA的表达变化,探究VEGF-C对顺铂诱导人肾小管上皮细胞毒性损伤的作用和介导其作用的相关分子机制。方法 体外培养人近端肾小管上皮细胞系,用不同浓度的顺铂刺激HK-2细胞24 h后,利用实时荧光定量RT-PCR法检测HK-2细胞VEGF-C mRNA的表达变化,CCK8法检测不同浓度的顺铂对HK-2细胞存活率的影响。分别用0、50、100、200 ng/rr的人重组VEGF-C因子预培养HK-2细胞4 h后,再加入50μmol/L的顺铂和一定浓度的VEGF-C继续培养HK-2细胞24 h,利用CCK8法检测细胞存活率的变化,明确VEGF-C对顺铂诱导的肾小管上皮细胞毒性损伤的作用。用不同浓度的VEGF-C刺激HK-2细胞30 min后,Western blot方法检测细胞p-Akt、Akt、p-Erk、Erk、p-p38及p38蛋白水平的变化。结果 不同浓度的顺铂刺激HK-2细胞后细胞VEGF-C mRNA水平的表达呈现先上升后下降的趋势。顺铂能引起HK-2细胞存活率减低,并呈现浓度依赖性。而外源性加入VEGF-C能减轻顺铂所导致的HK-2细胞毒性损伤,提高细胞的存活率,并且VEGF-C浓度为200 ng/ml时对HK-2细胞的保护作用最明显。不同浓度的VEGF-C刺激HK-2细胞30 min,随着VEGF-C浓度的增加,HK-2细胞p-Akt、pErk的表达也逐渐增加,而Akt、Erk、p-p38及p38表达不变。结论 VEGF-C能减轻顺铂诱导的肾小管上皮细胞毒性损伤,提高细胞的存活率,其作用机制可能与激活细胞内PI3K/Akt及MAPK/Erk信号通路有关。更多还原

【Abstract】 Objective To identify the mRNA expression of VEGF-C on HK-2 cells induced by differrent concentrations of cisplatin and the impact of VEGF-C on the cisplatin-induced cytotoxicity of HK-2cells,and to explore the possible mechanisms.Methods HK-2 cells(human kidney tubular epithelial cell line) were treated with cisplatin at different concentrations for 24 h,and the mRNA expression of VEGF-C was detected by realtime fluorescence quantitative PCR.HK-2 cells were stimulated by cisplatin at different concentrations for 24 h and the cell viability was evaluated by cell counting kit-8(CCK-8).Cells were incubated with different recombinant wild-type VEGF-C for 4 h,then 50 μmol/L cisplatin was added for another24 h.Cell viability was measured by CCK-8 using microplate reader.Serum-starved HK-2 cells were treated with VEGF-C at different concentrations for 30 min and the effects of VEGF-C on Akt,Erk,and p38 phosphorylation in HK-2 cells were examined by Western blotting.Results The expression of VEGF-C was increased in a dose-dependent manner after treatment with cisplatia VEGF-C could inhibit cisplatin-induced cell viability loss in a dose-dependent manner with the most protective concentration being 200 ng/ml.VEGF-C could increase Akt(Ser473) and Erk phosphorylation in a dose-dependent manner,but had no significant influence on the expression of total Akt and total Erk,VEGF-C could not increase the p38 phosphorylation Conclusions VEGF-C could reduce the cytotoxicity of cispaltin on human kidney tubular epithelial cells via both autocrine and paracrine mechanisms.Its role may be associated with activation of PDK/Akt and MAPK/Erk signaling pathway.更多还原