Study On Rats Mesenchymal Stem Cells Transplant To Repair The Injured Gastric Mucosa

Background:Bone marrow mesenchymal stem cells(BMMSCs)are non-hematogenic stem cells isolated from marrow.They have been paid extensive attention in medical field due to their multi-directional differentiation potential, low immunogenicity and can be easily isolated and cultured in vitro. Transplantation of MSCs has already been used in treatment of ischemic and injury diseases.Therefore,stem cell replacement therapy is expected to become a new therapeutic means.Objective:To study the effect and mechanism of cultured mesenchymal stem cells(MSCs)on the repair of injured gastric mucosa.Methods:(1)Density gradient centrifugation and differential attachment methods were used to isolate of MSCs from adult Wistar rats and the proliferation ability of the cells was analyzed by MTT examination.The surface antigens of MSCs were detected by flow cytometry.(2)CFDA SE was used to label the MSCs in vitro,then the growth state of MSCs was observed by fluorescence microscope. In situ hybridization technique was used to trace the transplanted cells by detecting the sex-determining gene(SRY)of rats.(3)Fifty-four healthy Wistar rats of 8 weeks old(200g-250g,half of them were female)were randomly divided into three groups:the control group and two gastric mucosa injury groups.Each group had 18 rats.Gastric mucosa injury was induced by hypodermical injection of indomethacinand injected amount was 30mg/Kg.The control group received hypodermical injection of NaHCO₃ with 10ml/Kg.Two hours after injection,one group of rats with gastric mucosa injury and control group were received MSCs(1×10⁷/rat)by intravenous injection,while another group anamals were received PBS as the same way.The gastric tissues were dissected respectively 24-h,48-h, 72-h after transplanting of MSCs respectively.The degree of gastric injury was evaluated by the ulcer index(UI).The patho-histological changes of gastric tissues corresponding to different time points were investigated through biological microscope.The difference expression of VEGF and EGFR in the experimental group and control group were detected by immunohistochemistry.Results:(1)MSCs have the characteristics of adherent growth,The morphology of cultured MSCs was fibroblast-like form.MSCs possessed intensive self-renewal potential and integrated in single layer in 12-14 days after planting.The growth ability of subcultured MSCs was greater than primary cultured MSCs.Nearly all of subcultured MSCs were adhesive in 24 hours. Compare with density gradient centrifugation,the growth rate of MSCs is slower in differential attachment method group.But there was no significant difference. Examination of MSCs revealed that in passage 3 of initial culture almost all adherent cells took on typical morphologies of fibroblastoid cells in vitro and maintained similar morphology with passages.They were positive for CD90, CD44 and were negative for CD31,CD45.(2)The cell multiplication of MSCs had not been affected after labelling with CFDA SE in vitro.The green fluorescence can be handed down to off-spring with cell division.On the 48-h and 72-h after MSCs transplantation,the CFDA SE labeled cells were found scattered in the gastric of the rats that underwent MSCs transplantation,while these were not detected in the control group at the corresponding points.Moreover,the SRY gene was detected in the injuried gastric mucosa of rats that underwent the transplantation of MSCs derived from male rats 2 weeks after transplantation,while it was not detected in the control group.(3)The UI was significantly decreased at 72-h after transplanting of MSCs(P＜0.05).(4)The immunohistochemistry staining of VEGF and EGFR demonstrated that the positive expression of cells were increased in the injuried gastric mucosa of the rats underwent MSCs transplantion than that of control group,it was significantly on 72-h after transplantion. Conclusion:(1)MSCs can grow stablely in vitro and nonadherent hematopoietic cells were removed through replacing medium.Compared with density gradient centrifugation,the differential attachment method is simple,easy to operate and grasp.(2)The technique of CFDA SE labelling is applicable to be used to trace the transplanted cells in vivo.In present study,it revealed that MSCs could be located in the injured gastri mucosa through intravenous injection. (3)The MSCs of rats may accelerate gastric wound healing course and the mechanism maybe that MSCs could not only differentiate into gastric epithelium but also secreted some biological factors.