PTEN表达下调促进体外活化大鼠肝星状细胞黏附  

Down-regulation of PTEN expression promotes the adhesion in activated rat hepatic stellate cells in vitro

作　　者：郝礼森 张家琪 刘博 章广玲 靳丽敏 张明婷 张朋垒 

机构地区：[1]华北理工大学附属医院消化内科,河北唐山063000 [2]华北理工大学基础医学院,河北唐山063000

出　　处：《基础医学与临床》2017年第3期364-368,共5页Basic and Clinical Medicine

基　　金：河北省自然科学基金(H2013209327);中国肝炎防治基金会天晴肝病研究基金(CFHPC20132078)

摘　　要：目的探讨腺病毒介导的短发夹RNA(shRNA)下调第10号染色体缺失的磷酸酶张力蛋白同源物PTEN基因的表达对体外培养的活化肝星状细胞(HSC)黏附的影响及其信号传导机制。方法体外培养活化大鼠肝星状细胞系HSC-T6,以腺病毒为载体将靶向PTEN的RNA干扰(shRNA)瞬时转染HSC;实验分为3组:1)对照组,在腺病毒转染时以DMEM代替病毒液;2)Ad-GFP组,转染仅表达绿色荧光蛋白(GFP)的空病毒Ad-GFP;3)Ad-shRNA/PTEN组,转染携带靶向PTEN的shRNA并表达GFP的重组腺病毒Ad-shRNA/PTEN。用实时荧光定量PCR法检测HSC的PTEN mRNA表达;Western blot检测HSC的PTEN、黏着斑激酶(FAK)、磷酸化FAK[p-FAK(Tyr397)]蛋白表达;甲苯胺蓝染色法及四甲基偶氮唑盐(MTT)法测定HSC黏附能力。结果腺病毒感染HSC 48 h,Ad-shRNA/PTEN组PTEN蛋白及mRNA表达明显低于对照组及Ad-GFP组(P<0.05);Ad-shRNA/PTEN组HSC的p-FAK(Tyr397)表达较对照组及Ad-GFP组显著升高(P<0.05);Ad-shRNA/PTEN组HSC黏附细胞数及黏附率较对照组及Ad-GFP组明显增加(P<0.05)。结论 PTEN表达下调可通过上调FAK信号传导活性促进体外活化HSC的黏附。Objective To investigate the down regulation of phosphatase and tensin homolog deleted on chromosome 10 （PTEN） gene by adenovirus mediated short hairpin RNA （shRNA） on the adhesion in activated hepatic stellate cells （ HSC ） in vitro and the related signal transduetion mechanism. Methods The recombinant adenovirus （ Ad- shRNA/PTEN） with shRNA targeting PTEN and expressing green fluorescent protein （GFP） were transient trans- fected into the cultural activated HSC in vitro. The experimental group as follows： 1 ）Control group, viral medium was replaced by DMEM at virus transfeetion step. 2）Ad-GFP group, HSC were infected with adenovirus expressing GFP alone. 3 ）Ad-shRNA/PTEN group, HSC were infected with adenovirus both taking shRNA targeting PTEN andexpressing GFP. PTEN mRNA expression was detected by RT-qPCR, and western blot was used for detecting pro- tein expressions of PTEN, focal adhesion kinase （FAK） and phosphorylated FAK （Thr397） [ p-FAK（Tyr397） ] in HSC. The toluidine blue stain method and MTT colorimetric method were used to determine the adhesion ability of HSC. Results When HSC were infected by adenovirus for 48 hours, PTEN protein and mRNA expressions in Ad- shRNA/PTEN group significantly decreased （P 〈 0. 05 ）, compared to control group and Ad-GFP group, and the expressions of p-FAK （Tyr397） in Ad-shRNA/PTEN group were significantly higher than those in control group and Ad-GFP group （ P 〈 0. 05 ）. The adhesion cell counting and the adhesion rate of HSC in Ad-shRNA/PTEN group significantly increased as compared with control group and Ad-GFP group （ P 〈 0. 05 ）. Conclusions The down-regulation of PTEN expression can promote the adhesion by increasing the activation of FAK signaling trans- duction in activated HSC in vitro.

关 键 词：肝星状细胞 PTEN RNA干扰 细胞黏附 

分 类 号：R575.2[医药卫生—消化系统]