| Objective1. To observe the cell morphology and biological characteristics of rat bone marrow mesenchymal stem cells(BMMSCs) with different passage time in vitro. To establish a stable and efficient isolation culture system to acquire the cell source with sufficient quantity and good quality for cell therapy.2. To establish the best method and optimal concentration for labeling bone marrow mesenchymal stem cells with Chloromethyl-benzamidodialkylcarbocyanine(CM-Dil) and explore its impact on biological characteristics and proliferation of labeled stem cells. To establish optimal labeling condition and an effective and safe in vivo tracing technique for cell therapy.3. To assess the effect of transplanted rat bone marrow mesenchymal stem cells on wound healing in a full-thickness incisional cutaneous wound model and investigate the mechanisms of this therapy.Methods1. BMMSCs isolated from the rat bone marrow(2-3week) by whole bone marrow culture method, density gradient centrifugation method and adherence with different speed method, were purified, cultured and amplified in vitro. The configuration and morphology of the stem cells was observed under inverted microscope and a stable isolation culture system of rat bone marrow mesenchymal stem cells was established. Expression of BMMSCs at the fourth passage isolated and cultured by this system were detected by flow cytometer. The proliferative ability of these cells were accessed by MTT test and a growth curve was drawn.2. The fourth passage SD rat BMMSCs were labeled by diverse concentration, the fluorescence intensity, labeling rate, viability and proliferation of labeled cell with different conditions were analyzed.3. The forth passage SD rat bone marrow mesenchymal stem cells cultivated by the whole bone marrow culture method were labeled with 5μM fluorescent dye (CM-Dil) at 24 hours before transplantation, suspended in phosphate-buffered saline and prepared for transplantation using to treat full-thickness incisional wounds in rat skin. Research one:15 SD Rats were randomized into 3 groups, namely systemic transplantation group(n=9) with one full-thickness dorsal incisional wound, local implantation group(n=3) with two paralleled full-thickness dorsal incisional wounds and PBS control group(n=3) (the same model with systemic transplantation group as control). Every three rats in systemic transplantation group random mixed were administered 5×106,1×107,1.5×107 labeled stem cells by intravenous injection respectively. The local implantation group were administered 5 X 106 labeled stem cells. The PBS control group were administered PBS (injected volume were all lml). The biological activity of the transplanted labeled stem cells was monitored by dynamic cell tracking technique in vivo and made optical imaging at different time points after transplantation. Research two:The fifth passage F344 rats bone marrow mesenchymal stem cells proliferated culture in vitro were labeled with 5μM fluorescent dye (CM-Dil) at 24 hours before transplantation, suspended in phosphate-buffered saline and repaired for transplantation using to treat full-thickness incisional wounds in rat skin.30 F344 rats were randomized into 3 groups, namely systemic transplantation group(n=10), local implantation group(n=10) and PBS control group(n=10). F344 rats in systemic transplantation group were all administered 1.5×107 stem cells by intravenous injection. The local implantation group were administered 5×106 stem cells. The PBS control group were administered PBS (injected volume were all lml). Wound appearance were observed generally. Then the healing conditions of deriving tissue in every group at same time point measured by the imaging processing of the histological insection and semiquantitative analysis. Finally, the data of research one and two were analyzed by statistical software SPSS13.0.Results1. The adhering fibro-like colonies formed at 3th-5th day from primary culture of BMMSCs obtained and purified via different methods. From 5th-10th day, BMMSCs grew faster and got together to form bigger colonies. After 14 days from primary culture, BMMSCs fused in monolayer, then were subcultured by trypsine. The passage BMMSCs proliferated fast, and could be subcultured about every 3-5 day. BMMSCs at the fourth passage isolated and cultured by this system were positive on the expression of CD29, CD90, while negative of CD45. The average proportion of these cells was approximately 97%. They appeared to great potential for self-renewal. The doubling time was 38 hours.2. The labeled stem cells with CM-Dil displayed red fluorescence. The fluorescence intensity and labeling rate changed concentration-dependently. There was no statistically significant differences between labeled stem cells with optimal concentration and unlabeled cells in viability and proliferation.3. The aggregation of labeled BMMSCs were observed from 40h to 14d in systemic transplantation group and from 24h to 52d in local implantation group. More CD68+cells and CD163+cells were recruited; CD31+cells increased in formation stage of granulation tissue and made great contribution for collagen secretion and deposition. There were significant differences between transplantation groups and controls.ConclusionBMMSCs can be obtained by different isolation methods in vitro. A stable isolation culture system is investigated and established. The P4 BMMSCs isolated and cultured via this system have a good biological characteristics and could been purified primarily. They can be used as a powerful tool for this research on stem cell transplantation. The optimal labeling concentration is 5μM. CM-Dil is an optimal alteratives for tracing stem cells for the researches on the stem cell therapy. Rat bone marrow mesenchymal stem cell therapy is a viable approach to significantly affect the course of tissue wound repair and promote wound healing by modulate inflammation and make great contribution for formation stage of granulation tissue, and collagen secretion or deposition.
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