| Stem cells are special kind of cells that has a unique capacity to renew itself and to give rise to specialized cell types. For the past few years, adult stem cells have received more attention and have became the front edge of modern life science. Mesenchymal stem cells (MSCs) in the bone marrow of adult animal are the most studied type of adult stem cells. MSCs have fundamental feature of stem cells, which can differential into several kind of mesenchymal tissue under certain conditions. The multi- differential potency of MSCs is showed not only in the tendency to mesenchymal tissue, but also non- mesenchymal tissue like nerve cells. MSCs have a abundant source and are easily cultured and cultured in vitro. We can also take advantage of the facility of introduction and exogenous of gene expression of MSCs. For this reason, MSCs will be used for cell therapy and gene therapy, even tissue engineering.In case of peripheral nerve defect, the district dominated by defective peripheral nerve is emerging to be sensory disability andfunctional disturbance, which severely influence the quality of patient's life, even psycho- health of the victim. Though autologous nerve graft is the golden standard in clinic for the therapy of peripheral nerve defects, this method is to be present the shortage of donator, sense dysfunction in donor, misdirection regeneration of nerve fiber, and even formation of nerve tumor. Tissue engineering has accelerated its development as a newly rising science, and peripheral nerve tissue engineering is hoped to solve the impairment of autologous nerve graft. Schwann cells (SCs ) have been the seed cells of peripheral nerve tissue engineering for a long time. Nevertheless, the issues existing in SCs impede the application of tissue engineering peripheral nerve in clinic. Autologous MSCs have a lot of superiorities against SCs and will turned out to be a kind of perspective seed cells for TEPN.In this research, method of tissue engineering was applying to construct the TEPN for repair of peripheral nerve defect. We have studied the culture of MSCs and induced them to differentiate into neuroglia-like cells. Differentiated autologous MSCs and Poly (ε-caprolactone-block-D,L-lactide) were used to form the TEPN in repair progress, and it is expected to be a better method for repair peripheral nerve defects.There are four parts in this research as follows: Part 1:Culture of seed cells in vitro:Aim: The attempt was to isolate and culture MSCs and SCs for peripheral nerve engineering. Method: Autologous MSCs of adult rats was isolated by Percoll and cultured in vitro. Growth curve of cultured cells was drawn. Result showed that MSCs of fibroblast shape weremore homogeneous in morphous or array after several times of passage, which hinted that it was effective in purification of MSCs for passage progress. Evaluation of growth curve displayed the great adaptability and fast multiplication of MSCs, by which could conclude the huge potentiality for amplification of MSCs; At the same time, Sciatic nerve of 7d old SD rats and newborn dog were digested by trypsin and collagenase for several times. Cytosine arabinoside and geneticin were used to eliminate fibroblast, and forskolin can promote the proliferation of SCs. The purified cells were identified with rabbit anti-S100 protein antibody by SABC immunohistochemistry methods. All result shows that the method used in this experiment is a optimal means to get the purified SCs for TEPN.Part 2: Induced differnetiation of bone marrow mesenchymal stem cell into glial-like cell in vitro:Aim: To observe induced differentiation of bone marrow mesenchymal stem cell (MSCs) into glial-like cells in vitro, and investigate the feasibility of MSCs being the seed cells of tissue engineered peripheral nerve (TEPN). Method: MSCs from adult rats were isolated and harvested. In chemical-inducing group, MSCs were exposed to Beta-Mercaptoethanol(BME), Butylated Hydroxyanisole (BHA),dimethyl sulfoxide(DMSO) All-Trans-Retinoic Acid (ATRA), Forskolin and bFGF ,ect. In co-culture group,Snap-on culture dish named Millicell was used to construct a co-culture environment to simulates the nerve regeneration cabinet,in which MSCs and Schwann cells shared same medium.The undifferentiated control group was set up.The morphology,immunocytochemistry, western blot and RT-PCRwere used to identify cells type. Results: Morphous of glial cells were observed in both inducing groups but control groups. MSCs in co-culture group remained acceleration and could be passaged yet after induction process. The expressing of protein and mRNA for nestin, glail fibrillary acidic protein (GFAP) and S-100 were similar to the morphological results. Conclusion: In this study, We founded a new method of inducing MSCs into glial-like cells, which simulated the environment of body fluid after cells transplantation. The method not only gains the same effect of traditional inducing method but also conserves the better vigor of induced MSCs. We conclude that MSCs would be the ideal cells source of TEPN. Part 3: Construction of tissue engineering peripheral nerve:The aim is to prepare the TEPN for repair of peripheral nerve defect and examine the biocompatibilities of different scaffold materials including acellular blood vessel for TEPN. The method is to combine the co-culture differentiated MSCs or SCs with membrane of different biomaterial. Biocompatibilities of these materials were determined by MTT method. The result showed that cells in Poly (e-caprolactone-block-D, L-lactide) (PLCA) group grew better proliferation capacity than ones in other groups, especially acellular blood vessel group. (P<0.01); In construction of TEPN, prepared gel with cell was injected into the PLCA conduit, the compound cloted under 37℃, 5% CO2 in 30 min and was cultured in DMEM. Through the investigation in vitro, MSCs could grow and proliferate as normal in the constructed TEPN. Conclusion: As a kind of reshaping material of PLGA, PLCA possess good biocompatibility. Acellular blood vessel in...
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