| The use of stem cells for wound repair mechanism and promote the research has become one of hotspot in research of trauma.The adult stem cells as the ethical restrictions are less,and has obvious multi-directional differentiation potential,so the related research are numerous,and broad application prospect.Adult stem cells can have a variety of sources,including bone marrow,fat,pancreas,skin and nerve tissue.Because of the skin tissue update speed,regeneration ability,and conveniently,is used in the soft tissues such as skin wound repair has the characteristic such as good compatibility,therefore,from a local skin of adult stem cells in the process of wound repair more significant role.With the research results show that there is also a dermal tissue stem cells,which originated in mesenchymal cells,strong ability to this kind of cell proliferation,and can be converted to a variety of cells,such as smooth muscle cells,neurons and glial cells,fibroblasts and fat cells.This type of cell regeneration ability,is a type of pluripotent stem cells,known as the dermal pluripotent stem cells.Previous research results,we found that the wound early wound fluid have clear effect on promoting proliferation of dermal pluripotent stem cells;The whole animal experiment showed that the skin cut injury rat dermal pluripotent stem cells transplantation can shorten wound healing time,the wound repair,including the merger of radioactive damage painless sex is good to promote the healing of the wound.These results suggest that dermal pluripotent stem cells involved in wound repair process,and promote wound healing is one of the most important ingredients,repair cells and dermal pluripotent stem cell proliferation is the important way of promoting wound repairSecretory leukocyte protease inhibitor(secretory leukocyte protease inhibitor,SLPI)is a kind of can fight against all kinds of serine protease activity of the protein,the amino terminal has antifungal,anti-inflammatory and antibacterial activity,and the carboxyl terminal in such aspects as inhibit chymotrypsin activity performance is good.Studies have shown that SLPI also have effect on promoting cell proliferation,was also involved in cell apoptosis and the regulation of immune function,have the function of inhibiting cell apoptosis and regulating immune response.These biological effects in a way this SLPI and has close inner link between the wound repair process.Through previous research we have found that dermal pluripotent stem cells in the early wound wound fluids under the stimulus of multiplication rate increased significantly;In gene chip technology and curb cuts we found with the help of hybrid technology,under the effect of early wound wound fluid,the dermal pluripotent cells express SLPI content significantly increased;And local tissue trauma SLPI also has high expression;Prompt SLPI could play through promoting dermal pluripotent stem cells proliferation role adjusting process of wound repair.In order to study the effects of SLPI on promoting proliferation of dermal multipotent stem cells,we isolated and identified dermal multipotent stem cells,then SLPI overexpression vector and SLPI gene si RNA expression vector were constructed,identified by RT-PCR,Western bolt.SLPI gene recombinant expression plasmid were transfected DMSCs,and the effect of SPLI genes on promoting the proliferation of DMSCs were observed using the CCK-8 assay and flow cytometry testing.The main findings and conclusions are as follows:1.DMSCs in neonatal rats were isolated by means of enzyme digestion,and then purified and amplified for further usages.The isolation and purification experiments of dermal Multipotent stem cells form a more uniform,after repeated passage can still maintain a very strong ability to proliferation;under certain inducing conditions,dermal multipotent stem cells have the ability to differentiate into bone cells,fat cells,and muscle cells.These results suggest that the rat dermal multipotent stem cells were successfully isolated and amplified,we also confirmed there self-renewal and pluripotency.2.Based on molecular biology,the successful construction of SLPI overexpression vector and SLPI gene si RNA vector,and they were picked out according to RT-PCR,Western bolt identification.DMSCs recombinant expression plasmid were transfected DMSCs.These results provided the basis of studies from the positive and negative aspects to confirm SLPI gene function.3.SLPI recombinant expression vector could be successfully transfected dermal Multipotent stem cells.The transfected DMSCs were divided into three groups: overexpressing group,interference group and control group(transfection of naked vectors).The results of CCK-8 assay showed,in 48 h,72h and 96 h after transfection,DMSCs in overexpressing group proliferated faster than those in interference group(P<0.05),in 72 h and 96 h after transfection,DMSCs in overexpressing group were significantly different from those in control group(P<0.05),which suggested that SLPI gene might promote the proliferation of DMSCs.The results of Flow cytometry testing showed that cells of G1 phase in overexpressioning group were significantly different from those in interference group(P<0.05),meanwhile,cells of S phase and G2 phase were significant different when compared with those in control group(P<0.05).The results in our study show SLPI gene plays a role in promoting the proliferation of DMSCs,also facilitates DMSCs cell cycle transformation from G1 to S and G2 phase,which suggests that SLPI might promote DMSCs proliferation via regulating cell cycle. |
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