大鼠骨髓间充质干细胞促进肝星状细胞凋亡的机制研究-论文.pdf

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1、中华肝脏病杂志2014年3)1第22卷第3期ChinJHepatol，March2014，Vo1．22，No．3·223·大鼠骨髓间充质干细胞促进肝星状细胞凋亡的机制研究韦柳萍覃山羽姜海行沈妍华孟云超【摘要】目的探讨大鼠骨髓间充质干细胞(BMSC)与肝星状细胞(HSC)共培养体系中BMSC旁分泌肝细胞生长因子(HGF)对HSC凋亡的影响及其机制。方法用半透膜建立上下双层细胞共培养体系，各组培养24、48、72h，倒置相差显微镜观察细胞形态学变化，免疫细胞化学法观察0【一肌动蛋白(仅-SMA)表达情况；四甲基偶氮唑盐法检测Y一27632及PH

2、A665752的最佳干预浓度l流式细胞仪检测HSC凋亡率lWesternblot、实时荧光定量PCR检测肝星状细胞RhoAmRNA及蛋白表达情况；酶联免疫吸附法检测HGF及肝细胞生长因子激活因子(HGFA)浓度。计量资料采用单因素方差分析，尸<0．05为差异有统计学意义。结果随着时间的延长，HSC凋亡率逐渐增高，实验b组凋亡率最低，实验C组凋亡率最高，均以72h最为显著，各组在各时段凋亡率差异具有统计学意义(尸<0．05)；实验c组RhoA蛋白及mRNA表达量较其他各组明显下降，差异有统计学意义(尸<0．05)；各组RhoA蛋白及mRNA表

3、达量随时间的延长而逐渐增加；实验各组HGF浓度随时间延长逐渐降低，实验b、C组较对照组增高，差异有统计学意义<0．05)；实验各组HGFA浓度随时间延长逐渐增高，实验b组升高最为明显，差异有统计学意义<0．01)。结论BMSC通过激活HGF并下调Rho通路促进肝星状细胞凋亡。【关键词】肝细胞生长因子；肝细胞生长因子激活因子；肝星状细胞；骨髓间充质干细胞Mechanismofbonemarrow-derivedmesenehymalstemcell-promotedapoptosisofhepaficstellateceasWeiLiup~Q

4、Shanyu,JiangHaixing,ShenYanhu~MengYunchao．DepartmentofGastroenterology,theFirstAfiliatedHospitalofGuangxiMedicalUniversi~Nanning53002LChinaCorrespondingauthor：PmShanyu,Email：qsyaddress@163．com[Abstract]ObjectiveToexploretheroleoftheRhopathwayinthehepatocytegrowthfactor(HGF

5、)paracrinesignal—mediatedbonemarrow-derivedmesench)malstemcell(BMSC)promotionofapoptosisofhepaticstellatecells(nSCs)．MethodsABMSC-HSCCO·culturesystemwasestablishedusingplateswithtranswellinserts．DynamicchangesinresponsetopretreatmentwiththeC-metblockerPHA665752andtheRhopat

6、hwayinhibitorY-27632wereobservedunderaninve~edphasecontrastmicroscopeat24，48and72hofculture．OptimalinterventionconcentrationsofY-27632andPHA665752weredeterminedbyMTTassay．Expressionofalpha-smoothmuscleactininHSCswasevaluatedbyimmunohistochemistry,andtheapoptosisrateofHSCsw

7、asmeasuredbyArmexin—V-FITC／propidiumiodide．RhoAproteinandmRNAlevelsweremeasuredbywesternblotandquantitativereal-timePCRrespectively．ConcentrationsofHGFandhepatocytegrowthfactoractivator(HGFA)werequantifiedbyenzyme—linkedimmunosorbentassay．Between-groupdifferenceswereevalua

8、tedbyone-wayANOVAwithP<0．05indicatingsignificance．ResuRsTheapoptosisratesofD0I：10．3760／cm