Objective:To construct the adenoviral expression vector system containing human hepatocyte growth factor(hHGF)cDNA,and to further study the transduction efficiency and the expression of HGF in mesenchymal stem cells(MSCs).Methods:The HGF cDNA was amplificated from the expression plasmid pCMV-HGF,and was subcloned into the adenovirus shuttle plasmid pDC316-IRES-EGFP vector containing a green fluorescence protein(GFP)reporter gene.Virus Ad-HGF was produced by homologous recombination in HEK293 package cells.Bone marrow derived MSCs were harvested and cultured,and then were transduced with Ad-HGF.The efficiency of Ad-HGF transduction was assessed by FACS analysis using GFP gene expression.And HGF/MSCs were generated.The HGF concentrations in supernatants of HGF/MSCs were determined by ELISA using anti-human HGF monoclonal antibody.Results:The recombinant,named pDC316-HGF-IRES-eGFP,was digested with restriction enzyme,and the DNA sequencing of HGF was identical to the report in Genebank and did not reveal any mutation.GFP expression could be observed on the second day after packing of the linearized pAd-HGF in HEK293 cells and 7.15×1010pfu/ml titer of Ad-HGF was obtained.Forty-eight hours after transduction,96.89% of HGF/MSCs were GFP positive.Peak concentration levels of hHGF(103ng/mL)in the cultured supernatants were detected on day 2 post-transduction,and the adenovirus-mediated expression of HGF by MSCs was maintained for at least 2 weeks in vivo.Conclusion:Our data demonstrated that the adenovirus expression vector system pDC316-HGF-IRES-EGFP has been constructed successfully,and their effective expressions also have been obtained in MSCs.This will provide material basis for the next study on liver regeneration after small-for-size liver transplantation.
Yue Yu Feng Zhang Ling Lü Ye Fan Guoqiang Li Xuehao Wang
目的探讨缺血预处理(IPC)对减体积肝移植大鼠缺血再灌注损伤的保护作用及其机制。方法将36只成年雄性SD大鼠行50%减体积肝移植,随机分为对照组(Control)组和IPC组,检测术后2、6、24 h血清丙氨酸转氨酶(ALT)水平变化。检测术后24 h肝组织形态学变化、丙二醛(MDA)和超氧化物歧化酶(SOD)含量;检测髓性过氧化物酶(MPO)活性以反映中性粒细胞浸润情况;ELISA法检测肝组织中肿瘤坏死因子α(TNF-α)水平。结果与Control组比较,IPC组术后6、24 h ALT水平显著下降(P<0.01);组织病理学显示,Control组肝细胞明显空泡样变性伴局部坏死,小叶结构破坏,门脉周围水肿、充血,炎症细胞浸润明显,而IPC组损伤减轻;与Control组比较,IPC组MDA水平显著下降而SOD含量则明显增加(P<0.01),肝组织中TNF-α和MPO亦显著降低(P<0.01)。结论IPC明显减轻减体积肝移植术后再灌注损伤,其机制部分与增强抗氧化、抑制脂质过氧化、减轻炎症反应密切相关。
