| Treatment for chronic wound healing complicated by pathological conditions, such as diabetes, radiotherapy or large area defect of skin and so on, has been a tough challenge in clinical practice. Transplantation of skin, usually used for the treatment of wound of large area, is restrained by some disadvantages. Normal wound healing is deteriorated by multiple factors, including decreased inflammatory cell infiltration, reduced secretion of growth factors and collagen synthesis etc. Bone marrow-derived mesenchymal stem cells (BMSCs) have been the focused target of cell and/or gene therapy for inherited and acquired diseases because of their great plasticity. Moreover, a large quantity of BMSCs can be achieved conveniently by in vitro amplification. Recently, researchers have successfully associated BMSCs with chronic wound healing therapy. Platelet-derived growth factor (PDGF), stored in theαgranules of platelet and released firstly in the process of wound healing, contributes to wound repair. Open chronic wound provides a good nutrient condition for microorganism growth and the prevention of infection will benefit to both local wound healing and prevention of systemic side effects. Beta defensin 2 (BD2) is a kind of natural antimicrobial polypeptide synthesized by cells in skin and mucosa in mammals. Its different antimicrobial mechanisms from traditional antibiotics render it with relatively low frequency of resistance in bacteria.After the different gene modified, The biol-effects of the BMSCs may be changed, this changes should be analyzed according to the concrete gene transfecting, so as to make sure the BMSC`s safety and validity as a kind of seed cell. When this gene modified MSCs has been transplanted to the skin incised wound, it can promote the healing, but how it works?what other functional cells the MSCs maybe differentiate into? it worths attentions and investigation. Additionally, microRNAs ,as a endogenous post-transcriptional control pathway, can maintain the stem cell, regulate its` proliferation,differentiation,apoptosis, control the development of the living being , organ formation and the neoplasia, et al. So combining the microRNAs with the stem cell may be a new hint for stem cell research.The aim of this experiment is to use the constructed recombinant adenovirus vector expressing human PDGF-A (hPDGF-A) and human BD2 (hBD2) simultaneously, then topically transplant recombinant adenovirus-transfected BMSCs into skin incised wound and observe the wound healing. The main methods, techniques and results are summerized as follows:1. We used the successfully constructed recombinant adenovirus vector Adv-hPDGF-A-IRES-hBD2 that expressed hPDGF-A/hBD2 at the same time, infected BMSCs , observed the biological effects of the MSCs transfected with recombinant adenovirus vector. By using liposome transfection technique, recombinant adenovirus vector into 293 cells for virus packaging and amplification. BMSCs were isolated, cultured and infected by adenovirus-containing supernatant. The exogenous gene-modified BMSCs were comprehensively studied on their biological features, in terms of morphology, cell growth curve, cell cycle, and adipogenic, osteogenic and myogenic differentiation ability. The result shows that hPDGF-A-IRES-hBD2 gene-modified BMSCs did not show obvious changes in cell viability, proliferation, cell cycle distribution, cell differentiation。2. The effect of topical transplantation of BMSCs transfected with recombinant adenovirus vector on skin incised wound. Firstly, we constructed a mouse skin incised wound model. Then transplanted the hPDGF-A-IRES-hBD2 gene-modified BMSCs to the wound surface. Animals were randomly divided into two groups: group C (20 rats) which accepted equal volume of normal saline, group T (20 rats) which accepted transplantation of recombinant adenovirus-infected BMSC. HE staining used to reflect wound histopathological changes during wound healing, has been done on day 7, 14 posttransplantation. The new born granulation tissue was fixed in 4%PFA, paraffin imbedded, sliced, and applied the immunofluorescence histochemistry test(FITC marked the transplanted MSCs, TRITC marked the angiogenesis) . After transplantation, the result of HE staining showed that the condition of wound healing in group T was superior to that of group C ;the BMSCs survived and expressed the the exogenous genes in wound for at least two weeks indicated by EGFP expression; the transplanted MSCs can help the formation of hair follicle and differentiated into the fibroblasts,endothelial cells that speed up the process of wound healing.3. This part aimed at the effects of the miR-21 in MSCs and wound healing. For the cell model,CCK(cell counting kit)was applied for checking out the miR-21 to cell proliferation. 10T1/2 mesencheymal cell planting in 96 well plate was divided into six group:10T1/2cell; 10T1/2+TGF-β; 10T1/2+LNA-sc; 10T1/2+LNA-miR-21; 10T1/2+LNA-sc+TGF-β; 10T1/2+LNA-miR-21+ TGF-β. When RNA extraction from the 10T1/2 cell after TGF-βinduced was finished, the expression changes of the miR-21 related gene: Bcl-2,PTEN,TMP1 by RT-PCR. were analyzed, the alteration of miR-21 by northern blot was observed. Besides, in the incised wound model, we extracted the RNA from the new born granulation tissue on day 4,7 after incised injury. The miR-21 changes was confirmed by northern blot and gene microarray analysis. The result demonstrated : proliferation ability of 10T1/2 mesencheymal cell increased after TGF-βinducing as compared to that without TGF-β. But when both were induced by TGF-β, the group of miR-21 knocked down has low proliferation than the TGF-βgroup. the RT-PCR shows that the tumor suppressor gene PTEN and TMP1 were downregulated, but tumor gene Bcl-2 was upregulated. The northern blot indicated that :the miR-21 expression enhanced both in MSCs within TGF-βand in the new born granulation tissue after injury.Conclusions:1. BMSCs were not only good carriers for exogenous hPDGF-A and hBD2 genes but also satisfied seed cells for cell therapy even after hPDGF-A/hBD2 modification.2.Topical transplantation of gene-modified BMSCs promoted wound healing, the transplanted MSCs can help the hair follicle formation, and differentiated into the fibroblasts,endothelial cells that speed up the wound healing.3. The expression of miR-21 increased not only in 10T1/2 mesencheymal cell after TGF-βinduced, but also new born granulation tissue in the wound surface. It indicated that miR-21 may play the most important part in MSCs promoting wound healing.
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