Mesenchymal Stem Cells Over-expressing Hepatocyte Growth Factor Improve Liver Regeneration In A Rat Small-for-size Liver Transplantation Model

Partâ… Construction of hepatocyte growth factor expression vector systemand stable expression in rat bone marrow mesenchymal stem cellsObjectiveTo construct the pDC316-HGF-IRES-EGFP adenoviral expressionvector system containing human full-length HGF cDNA, and to producethe virus containing hHGF, using 293 cells, then to study the transducingefficiency and the expression ofhHGF in MSCs.MethodsThe HGF cDNA encoding the full-length human HGF (hHGF) genewas amplificated from the expression plasmid pCMV-HGF by PCR,using primers containing Ageâ… and Nheâ… restriction enzyme sequence(HGF-Ageâ… -f : 5'-TAAACTATACCGGTATGTGGGTGACCAAACTC-3', and HGF-Nheâ… -r:5'-TAAACTATGCTAGC CTATGACTGTGGTACCTT-3'), then digestedwith Ageâ… and Nheâ… , and subcloned into the same site of the plasmidpDC316-IRES-EGFP vector. The recombinants were named pDC316-HGF-IRES-EGFP, and identificated with restriction enzymedigestion and sequencing. Subsequently, we produce virus Ad-HGF byhomologous recombination in 293 package cell. BM-MSCs wereharvested and cultured, and then were transduced with Ad-HGF. In thisway we generated HGF-producing MSCs, named HGF/MSCs. Theefficiency of Ad-HGF transduction was assessed by FACS analysis usingGFP gene expression. The HGF concentrations in supernatants ofHGF/MSCs were determined by ELISA using anti-human HGFmonoclonal antibody.ResultsThe adenoviral expression vector system pDC316-HGF-IRES-EGFPwas digested with restriction enzyme, and agarose gel electrophoresisshowed there were fragments of 2.3kb (hHGF) and 5.2kb(pDC316-IRES-EGFP). The DNA sequencing of HGF was identical tothe report in Genebank and did not reveal any mutation. Forty-eighthours after transduction, 98.26% of HGF/MSCs were GFP positive.Peak concentration levels of hHGF (103ng/mL) in the culturedsupernatants were detected on day 2 post-transduction.ConclusionsOur data demonstrate that the adenoviral expression vector systempDC316-HGF-IRES-EGFP containing human full-length HGF cDNAhave been constructed sucessfully, and their effective expressions also have been obtained in MSCs. It will provide material basis for the nextstudy on liver regeneration after small-for-size liver transplantation.Partâ…¡Mesenchymal Stem Cells Over-expressing Hepatocyte Growth FactorImprove Liver Regeneration in a Rat Small-for-size LiverTransplantation ModelObjectiveIschemia-reperfusion (I/R) injuries associated with liver transplantationimpair liver graft regeneration. MSCs have the capability under specificconditions, of differentiating into hepatocytes. HGF has potentanti-apoptotic and mitogenic effects on hepatocytes during liver injury,and has been utilized for many applications and clinical applications.The present study was designed to test whether HGF-expressing MSCswill protect small-for-size liver grafts after liver transplantation.MethodsIn this study, we implanted HGF-expressing MSCs into liver grafts viathe portal vein, using a 30% small-for-size rat liver transplantation model.HGF, c-met expression, hepatic injury and liver regeneration wereassessed after liver transplantation.ResultsOur study demonstrated that HGF-expressing MSCs reduced mortality in rats after small-for-size liver transplantation. These animals alsoexhibited improved liver function and liver weight recovery during theearly post-transplantation period. Using GFP gene as a marker, wedemonstrated that during the later post-transplantation period, theengrafted cells and their progeny incorporated into remnant livers andproduced albumin, and played an instrumental role in promoting thepreservation of normal liver architecture and increasing the survival ratein the later post-transplantation period.ConclusionsThese findings suggest that MSCs genetically modified toover-expressing HGF and implanted in the liver graft, may offer a novelapproach to promoting liver regeneration after small-for-sizetransplantation.