【作者】 房林；

【导师】 赵振民； 王佳琦； 宋维铭；

【作者基本信息】 中国协和医科大学 ， 外科学， 2010， 博士

【摘要】 研究目的1.观察大鼠骨髓间充质干细胞(BMMSCs)的生长情况和生物学特性,建立大鼠BMMSCs稳定的扩增体系。2.建立荧光染料CM-Dil标记BMMSCs的最适方法和最佳浓度,探讨该染料对细胞活性及增殖能力的影响。3.以一期切口为模型,观察BMMSCs在皮肤创伤处的存活情况及对创伤愈合的影响,并对作用机制进行初步探讨。方法1.通过全骨髓培养法、密度梯度离心法、差速贴壁法从幼年大鼠骨髓中分离BMMSCs,进行纯化、扩增。通过对培养细胞形态及生物学特性的观察,确立稳定的扩增体系,并鉴定P4代细胞,绘制生长曲线。2.用不同浓度CM-Dil分别标记P4代SD大鼠BMMSCs,观察不同浓度和方法标记细胞的成活情况,对标记后细胞的荧光强度、标记率、活性和增殖能力进行比较。3.建立大鼠背部皮肤一期切口愈合模型,动物随机分为三组：静脉组,对照组,局注组。实验1：15只SD大鼠,随机分组：静脉组(n=9,尾静脉移植细胞量分为5×106,1×107,1.5×107,每注射剂量各3只)、局注组(n=3,创缘移植细胞量为5×106)、对照组(n=3,尾静脉注入等量不含细胞的PBS液lml)。将标记后P4代BMMSCs(SD品系)分别以相应移植方式、剂量注入大鼠体内,于不同时段动态示踪BMMSCs体内分布情况。对30只F344大鼠随机分组：静脉组(n=3,尾静脉移植细胞1.5×107),局注组(n=3,创缘移植细胞5×106)、对照组(n=3注入PBS)。P2代BMMSCs (F344品系)体外扩增后移植,观察愈合不同时段的切口外观及组织切片。结果1. BMMSCs约3-5日形成成纤维样细胞集落；5-10日生长迅速；12-14日融合成单层。传代细胞增殖速度快,约3-5日传1代。扩增体系下获得P4代细胞CD29+、CD90+、CD45-,纯度约97%,倍增时间为38小时。2.荧光显微镜下CM-Dil标记大鼠BMMSCs发红色荧光；最佳浓度为5μM且标记后细胞与未标记细胞在活性和增殖能力上差异无统计学意义。3.静脉移植后40小时-14日、局部移植后24小时-52日均发现BMMSCs聚集于皮肤创伤处；移植组炎症期CD68+细胞及CD163+细胞增多,肉芽形成期CD31+细胞增多,观察后期胶原分泌量增加,与对照组比较差异有统计学意义。结论利用大鼠BMMSCs扩增体系获得的细胞纯度及活性较满意。以5μM浓度CM-Dil标记的BMMSCs活性及增殖力正常,示踪效果理想。移植的BMMSCs通过调节早期炎症反应,快速启动修复过程；以不同方式提高血管内皮细胞数量,加速肉芽组织形成；促进胞外胶原分泌及成熟。BMMSCs在修复的不同阶段均有促愈合作用。各种因素造成的皮肤创伤不仅毁损外形而且影响功能,如眼睑皮肤创伤后不仅影响面容,而且会影响视力。因此,促进皮肤创伤愈合的研究一直是整形外科领域的研究重点。干细胞生物技术的发展为皮肤创伤愈合的研究提供了新的思路。本文将有关细胞生物学的基础研究与创伤愈合的临床研究相结合,探讨BMMSCs对皮肤创伤愈合的促进作用。其创新之处为：首次将染料CM-Dil应用于活体动态示踪技术中,为有关皮肤创伤愈合的细胞治疗研究提供了可行的标记方法及示踪手段；比较详细的对干细胞在活体内生存及归巢情况进行示踪；在细胞水平上,对创伤愈合的不同阶段均进行了详尽的观察,发现BMMSCs可调节炎症反应,以不同方式增加内皮细胞数量,促进胶原分泌和成熟。它促创伤愈合的作用贯穿于修复的全过程。更多还原

【Abstract】 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.更多还原