乳酸堆积对肝星状细胞增殖、分化、凋亡的影响  

Effect of lactic acid accumulation on proliferation,differentiation and apoptosis of hepatic stellate cells

作　　者：孙辉[1] 马凌波[1] 董飞[1] 胡光荣[1] 何永捍 邓颖[1] SUN Hui;MA Lingbo;DONG Fei;HU Guangrong;HE Yonghan;DENG Ying(Department of Emergency,the Second Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang 150081,China;Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming,Yunnan 650223,China)

机构地区：[1]哈尔滨医科大学附属第二医院急诊科,150081 [2]中国科学院昆明动物研究所,昆明650223

出　　处：《重庆医学》2019年第16期2743-2746,共4页Chongqing medicine

基　　金：黑龙江省教育厅科学技术研究项目(12541437)

摘　　要：目的探讨乳酸堆积对肝星状细胞(HSCs)增殖、分化、凋亡的影响。方法体外培养人HSCs,在HSCs中加入终浓度分别为0(对照组)、0.5、1.0、2.0mmol/L的乳酸,共同培养72h,留取样本。采用四甲基偶氮唑蓝(MTT)法测定乳酸对HSCs增殖的影响;流式细胞仪检测乳酸对HSCs凋亡和细胞周期的影响;real-timePCR检测α-SMA和mRNA凋亡基因FaslmRNA的表达;免疫荧光法检测caspase-3的活性。结果MTT实验结果表明,低浓度乳酸(0.5、1.0、2.0mmol/L)对HSCs增殖具有明显促进作用(P<0.05),之后随着乳酸浓度的增加,乳酸对HSCs毒性增加,对HSCs增殖具有明显的抑制作用(P<0.05)。流式细胞仪检测结果显示,与对照组比较,在乳酸浓度为0.5、1.0、2.0mmol/L时HSCs凋亡减少(P<0.05);低浓度乳酸作用72h可调节细胞周期,G0/G1期细胞减少(P<0.05),S期细胞增加(P<0.05)。real-timePCR检测结果显示,与对照组比较,低浓度乳酸(0.5、1.0、2.0mmol/L)可明显提高α-SMAmRNA的表达(P<0.05),减少FaslmRNA的表达(P<0.05);免疫荧光法检测结果显示与对照组比较,低浓度乳酸可降低caspase-3活性。结论低浓度乳酸可促进HSCs增殖,抑制HSCs凋亡,促进细胞外基质合成,使HSCs向成纤维细胞分化,增加肝纤维化风险。Objective To investigate the effects of lactic acid accumulation on the proliferation,differentiation and apoptosis of hepatic stellate cells(HSCs).Methods The HSCs was cultured in vitro.Lactic acid with the final concentration of 0 mmol/L(control group),0.5 mmol/L,1.0 mmol/L,and 2.0 mmol/L were cultured with HSCs for 72 h.The specimens were collected for the following tests,the effect of lactic acid on HSCs proliferation was determined by MTT assay;Flow cytometry was used to detect the effect of lactic acid on apoptosis rate and cell cycle in HSCs;Real-time PCR was used to detect the expression ofα-SMA mRNA and Fasl mRNA;The activity of caspase-3 was detected by immunofluorescence assay.Results MTT assay showed that low concentration of lactic acid(0.5,1.0,2.0 mmol/L)significantly promoted the proliferation of HSCs(P<0.05).Then,with the increase of the concentration of lactic acid,the toxicity of lactic acid on HSCs increased,and it had obvious inhibitory effect on HSCs(P<0.05).The results of flow cytometry showed that,compared with the control group,the apoptosis of HSCs was decreased when the lactic acid concentration was 0.5 mmol/L,1.0 mmol/L and 2.0 mmol/L respectively(P<0.05);the cell cycle was regulated by low concentration of lactic acid,the cells in G 0/G 1 phase were decreased(P<0.05),and the cells in S phase were increased(P<0.05).The results of real-time PCR showed that low concentration of lactic acid(0.5 mmol/L,1.0 mmol/L,2.0 mmol/L)could significantly stimulate the expression of theα-SMA mRNA(P<0.05),and reduce the expression of FasL mRNA(P<0.05)when compared with control group.Immunofluorescence assay showed that low concentration of lactic acid(0.5,1.0,2.0 mmol/L)decreased the activity of caspase-3 when compared with control group.Conclusion Low concentration of lactate can promote the proliferation of HSCs and the synthesis of extracellular matrix,inhibit the apoptosis of HSCs,differentiate HSCs into fibroblasts,and increase the risk of liver fibrosis.

关 键 词：乳酸 肝星状细胞 细胞增殖 细胞分化 细胞凋亡 

分 类 号：R575.29[医药卫生—消化系统]