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健脾化瘀法含药血清对肝癌细胞Smad3蛋白及EMT的影响

Impact of Jianpi Huayu Therapy on Phosphorylation of Smad3 Protein and Epithelial Mesenchymal Transition of Hepatocellular Carcinoma Cell
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摘要 目的:探讨健脾化瘀法含药血清对肝癌细胞Smad3蛋白磷酸化及上皮间质转移标志蛋白E-cadherin/Ncadherin表达水平及侵袭转移能力的影响。方法:分别以不同浓度(10%和20%)健脾化瘀法中药含药血清及10%小牛血清对照组培养肝癌细胞MHCC97-H,实时定量PCR检测细胞Smad3和Snail mRNA水平,Western blot检测Smad3、p-Smad3、Snail、E-cadherin和N-cadherin蛋白表达水平。划痕法和Transwell小室检测肝癌细胞侵袭/迁移能力。结果:实时定量PCR结果显示高浓度组(20%含药血清组)、低浓度组(10%含药血清组)和对照组(10%小牛血清对照)Smad3的mRNA水平分别为0.87±0.24、0.97±0.28和1.18±0.34(P>0.05);高浓度组Snail的mRNA水平低于对照组(0.49±0.05 vs.1.10±0.21,P=0.034)。与对照组相比,高浓度含药血清组48h的p-Smad3、Snail和N-cadherin明显降低,E-cadherin明显升高(P<0.05)。与对照组相比,低浓度含药血清组48h的E-cadherin升高,N-cadherin降低(P<0.05);而Smad3、p-Smad3及snail的结果与对照组无明显差异(P>0.05)。划痕法结果显示,与对照组比较,20%含药血清组及10%含药血清组肝癌细胞的划痕愈合较慢,差异均有统计学意义(P<0.05)。Transwell显示与对照组相比,20%含药血清组及10%含药血清组MHCC97-H细胞的迁移受抑制,差异有统计学意义(P<0.05)。结论:健脾化瘀法中药能抑制肝癌细胞侵袭转移,可能与抑制Smad3蛋白磷酸化及上皮间质转化有关。 Objective :To investigate the impact of Jianpi Huayu Recipes on phosphol^clation of Smad3 protein and epithelial mesenchymal transition (EMT) essential protein ( E - cadherin/N - cadherin) of hepatocellular carcinoma cell. Methods : Hepato- cellular carcinoma cell line MHCC97 - H was cultured in different concentrations ( 10% and 20% ) of Jianpi Huayu Therapy medicated serum and 10% blank serum,respectively. Real -time PCR (qPCR) and western blot were used to detect the mRNAlevels of Smad3 and Snail and protein levels of Smad3, p - Smad3, Snail, E - cadherin and N - eadherin, respectively. Wound healing assays and Transwell chamber were respectively used to detect the invasion and migration ability of MttCC97 - H cell. Resuits:The qPCR results reveals that mRNA level of Smad3 of high -concentration group (20% ,Jianpi Huayu Therapy medicated serum) ,low -concentration group ( 10% Jianpi Httayu Therapy medicated serum) and control group ( 10% blank serum ) was 0. 87 ± 0. 24,0.97 ± 0.28 and 1.18±0.34, respectively ( P 〉 0. 05 ). The mRN A level of Snail of high - cnneemration group was lower than that of control group( 0.49 ± 0. 05 vs. 1. 10 ± 0. 21 , P = 0. 034 ). After cultured for 48 hours, protein levels of p- Stand3, Snail and N - eadherin of high - concentration group were lower than those of control group, respeetivel ( P 〈 0. 05 ). The protein level of E - cadherin of high - concentration gronp was higher than that of control group ( P 〈 0. 05 ). Difference of protein level of N - cadherin and E - cadherin between low - concentration group and control group was significant, respectively ( P 〈 0. 05 ) while the difference of Smad3, p - Smad3 and Snail was not significant, respectively ( P 〉 0. 05 ). Wouhl healing assays re- vealed that the speed of wouhl healing of high - concentration group and low - concentration group was slower than that of control group, respectively ( P 〈 0. 05 ). Consistent with the results of would healing assays,Transwell assay showed that the migration of MHCC97 -H cell of high -concentration group and low -concentration group was inhibited than that of control group, respectively (P 〈0. 05 ). Conclusion :Jianpi Huayu therapy inhibited the invasion and migration of HCC. The potential mechanism was inhibiting the phosphorylation of Smad3 and epithelial mesenchymal transition of HCC.
出处 《辽宁中医杂志》 CAS 北大核心 2016年第11期2344-2347,共4页 Liaoning Journal of Traditional Chinese Medicine
基金 国家自然科学青年基金项目(81403397) 广东省自然科学基金项目(2014A030313408) 广东省科技计划项目(2012B061700032) 2016年广东省科技计划项目(2016A020226052)
关键词 肝细胞癌 健脾化瘀法 SMAD3蛋白 磷酸化 上皮间质转化 hepatocellular carcinoma Jianpi Httay, therapy Smad3 protein phosphnrylation epithelial rnesenchymal transi- tion
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