| Part one: The expression of HGF in the transfected MSCsObjective: To investigate the transfection efficiency of mesenchymal stem cells by adenovirus vector encoding GFP gene (Ad-GFP) and the expression of HGF in MSCs infected with adenovirus vector encoding HGF gene (Ad-HGF).Methods: Ad-HGF and Ad-GFP were amplificated in vitro,and then their titer were evaluated. Rat,s bone marrow-derived mesenchymal stem cells (MSCs) were isolated using Percoll desity gradient method and according to the adherent property of MSCs, and were identified by flow cytometer (FCM) for the expression of CD11b/c, CD34, CD44, CD90 and CD29 which were cell surface marker antigen in MSCs. Then we infected rat's MSC with adenovirus vector encoding green fluorescent protein (GFP ) or HGF. The efficiency of infection were detected by FCM, and expression of HGF were detected by RT-PCR and enzyme-linked immunoadsorption assay (ELISA) respectively.Results: The teter of Ad-HGF, Ad-GFP was 1.6×109 pfu/ml, 8.3x1010 pfu/ml respectively by bacteriophage plaque experiment. Isolated MSCs were homogeneous, long-fusiform-shaped, arranged into a fencelike structure. MSCs were positive for FCM, CD29, CD90, CD44, and were negative for CD34, CD11b. It showed that transfection efficency was increasing along with the increase of multiplicity of infection (MOI). A nearly 100% cells were transfected when the 150 MOI was administrated. MSCs could be transfected efficiently as seed cells to accept target genes by Ad-HGF, and a higher level of expression in vitro, which persisted 16 days at least. Expression of HGF mRNA were detected by RT-PCR, which persisted 11 days at least.Conclusions: MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation and be identificated by FCM. MSC are ideal cells for cell-mediated HGF gene therapy or cell therapy. MSC expression HGF at a high level when being transfected with Ad-HGF. Part two: The effects of mesenchymal stem cells transplantation combining with hepotocyte growth factor gene transfection on renal interstitial fibrosisObjective: To explore the effects of mesenchymal stem cells (MSC) transplantation combining with hepotocyte growth factor gene transfection on unilateral ureteral obstruction (UUO) rats renal interstitial fibrosis.Methods: MSC were isolated from SD rat bone marrow. HGF infected MSC by adenovirus vector. The rat model of UUO was established and rats were randomly divided into sham-operated, UUO and transplantion group.The rats were sacrificed at day 3, 7, 14, 28 including serum creatinine (Scr) and blood urea nitrogen (BUN) was examined. The protein levels of HGF andα-SMA were detected by immunohistochemistry. Sections were stained with hematoxylin, transplantation cells were observed in the obstructed kidney for the frozen sections. The expression of FN andα-SMA mRNA were detected by way of RT - PCR.Results: DAPI-labeled transplantation cells were found in the obstructed kidney of rats at day 3, 7, 14 after transplantion. Compared with sham-operated group, the level of serum BUN and Scr in UUO and transplantion group was obvious increase at day7 after operation(P<0.05), reduced at day14 after operation but still higher than sham-operated group, and not obvious differentia between UUO and transplantion group. The level of Scr in transplantion group was lower than UUO group at day28 after operation. Compared with sham-operated group, the expresstion ofα-SMA increased further in other group.The expresstion of HGF in transplantion group was higher than UUO group. The expresstion ofα-SMA mRNA in transplantion group was lower than UUO group at day14 after operation,and the expresstion ofα-SMA and FN mRNA were still lower than UUO group at day28 after operation.Conclusion: MSC transplantation combining with Ad-HGF are capable of homing to unilateral obstructed kidney following renal artery infusion and counteractes renal interstitial fibrosis by negative regulation tubular epidemic-mesenchymal transdifferentiation.
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