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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 investigate the effect of recombinant adenovirus mediated bone morphogenetic protein-9 (Adv-BMP-9) on the differentiation of mouse embryonic liver stem cells into mature hepatocytes. Methods The constructed recombinant adenovirus expressing BMP-9, hepatocyte growth factor (HGF) and green fluorescent protein (GFP) were respectively infected into mouse embryonic hepatic stem cells and induced to differentiate. Semi-quantitative PCR and Real-time PCR, immunofluorescence and luciferase reporter assay were used to detect the expression of hepatocyte markers such as AFP, ALB and CK18. Results The mRNA expression of AFP in BMP-9 and HGF groups after induction for 7 days were (0.027 ± 0.003) and (0.023 ± 0.002), respectively, which was significantly higher than that of GFP control group ± 0.018). The △△ Ct values of ALB mRNA were (0.041 ± 0.005) and (0.031 ± 0.001) respectively, which were significantly higher than those of GFP control group (0.013 ± 0.002) 0.094 ± 0.003), (0.083 ± 0.007), respectively, which was significantly higher than that of GFP control group (0.040 ± 0.008) (P <0.05). Immunofluorescence staining showed that hepatocyte- CK18 and other proteins in the control group were almost no expression; Luciferase reporter gene detection, 1,4,7 d-induced BMP-9 group, HGF group ALB expression of luciferase A values were (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 ± 1868.03)] compared with GFP control group (4 732.49 ± 68.22 ), (9 407.71 ± 487.84) and (20 894.83 ± 1 100.54), respectively (P <0.05). Conclusion BMP-9 induces the differentiation of mouse embryonic liver stem cells into mature hepatocyte-like cells, and BMP-9 induces the differentiation of mouse embryonic stem cells even compared with the traditional hepatocyte-derived differentiation factor HGF.