重组腺病毒介导的骨形态发生蛋白-9对小鼠胚胎肝脏干细胞诱导分化的影响  

Adenovirus-mediated mouse bone morphogenetic proteins-9 induces mouse fetal liver stem cells differentiation

作　　者：陈聪[1] 罗庆[1] 毕扬[1] 陈肯[1] 田雯[1] 迭小红[1] 曹光彪[1] 周建武[1] 康权[1] 

机构地区：[1]重庆医科大学附属儿童医院儿童发育疾病研究省部共建教育部重点实验室,儿科学重庆市重点实验室,重庆市(儿童发育重大疾病诊治与预防)国际科技合作基地,重庆400014

出　　处：《第三军医大学学报》2012年第15期1481-1486,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金(30973062);重庆市自然科学基金(CSTC2008BB5390;CSTC2009BB5408);重庆医科大学重点课题(XBZD201015)~~

摘　　要：目的探讨重组腺病毒介导的骨形态发生蛋白-9(Adv-BMP-9)诱导小鼠胚胎肝干细胞向成熟肝细胞定向分化的影响。方法将已构建的表达BMP-9、肝细胞生长因子(HGF)和绿色荧光蛋白(GFP)的重组腺病毒,分别感染小鼠胚胎肝干细胞,诱导其分化,然后采用半定量PCR、Real-time PCR、细胞免疫荧光、荧光素酶报告基因检测等方法检测甲胎蛋白(AFP)、白蛋白(ALB)和细胞角蛋白18(CK18)等肝细胞标志物的表达。结果半定量及Real-time PCR检测,诱导7 d后的BMP-9组、HGF组AFP的mRNA表达△△Ct值分别为(0.027±0.003)、(0.023±0.002),较GFP对照组(0.103±0.018)显著下降,ALB的mRNA表达△△Ct值分别(0.041±0.005)、(0.031±0.001),较GFP对照组(0.013±0.002)明显升高,CK18的mRNA表达△△Ct值分别(0.094±0.003)、(0.083±0.007),较GFP对照组(0.040±0.008)明显升高(P<0.05);细胞免疫荧光染色,诱导7 d后的细胞胞浆内表达肝细胞特有的ALB、CK18等蛋白,而对照组几乎无表达;荧光素酶报告基因检测,诱导1、4、7 d的BMP-9组、HGF组的ALB表达荧光素酶A值分别[(5 509.35±213.08),(33 198.14±666.39),(50 815.03±1 730.75)]、[(5 539.25±132.62),(31 400.71±2 622.39),(49 088.28±1 868.03)],较GFP对照组(4 732.49±68.22),(9 407.71±487.84),(20 894.83±1 100.54)明显升高(P<0.05)。结论 BMP-9有诱导小鼠胚胎肝脏干细胞向成熟肝细胞样细胞分化的作用,并且BMP-9对小鼠胚胎肝干细胞的诱导分化作用甚至和传统的肝干细胞诱导分化因子HGF相当。Objective To determine the effect of recombinant adenovirus-mediated mouse bone morphogenetic proteins-9（Adv-mBMP-9） on the differential regulation of mouse fetal liver stem cells to mature hepatocytes.Methods The mouse fetal liver stem cells were infected respectively by the constructed recombinant adenoviruses expressing BMP-9,hepatocyte growth factor（HGF）,and green fluorescent protein（GFP）.Expression of alpha fetoprotein（AFP）,albumin（ALB） and cell keratin 18（CK18） were tested by half quantitative PCR,real-time PCR,cellular immune fluorescence staining and fluorescent element enzyme gene report to investigate the regulation of stem cells differentiation.Results In 7 d after transfection,the △△Ct for mRNA expression of AFP were 0.027±0.003 and 0.023±0.002 respectively in the cells induced with BMP-9 and HGF,obviously lower when compared to the cells induced by GFP（0.103±0.018）.For ALB,the △△Ct were 0.041±0.005 and 0.031±0.001 for the cells induced with BMP-9 and HGF,significantly higher than the GFP-induced cells（0.013±0.002）.And for CK18,the △△Ct were 0.094±0.003 and 0.083±0.007 for the cells after BMP-9 and HGF transfection,significantly higher than the GFP-induced cells（0.040±0.008）.Cellular immune fluorescence assay showed that the specific markers of mature hepatocyte,ALB and CK18 were strongly expressed in 7 d after induction,but no expression was observed in the GFP-induced cells.Luciferase assay indicated that the expression of ALB in 1,4 and 7 d were 5 509.35±213.08,33 198.14±666.39 and 50 815.03±1 730.75 respectively after BMP-9 induction,and 5 539.25±132.62,31 400.71±2 622.39,and 49 088.28±1 868.03 respectively after HGF induction,all were significantly higher when correspondingly compared to the cells after GFP induction（4 732.49±68.22,9 407.71±487.84,20 894.83±1 100.54,all P〈0.05）.Conclusion BMP-9 induces the mouse fetal liver stem cells to different into mature hepatocytes.This effect is even similar to that of HGF,a convention

关 键 词：骨形态发生蛋白-9 小鼠胚胎肝干细胞 分化 重组腺病毒 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学] R394.2[医药卫生—基础医学]