小鼠骨髓基质细胞参与缺血组织血管再生及时相关系  

Mouse bone marrow-derived stroma cells for neovascularization in ischemic tissues and the phase relationship

作　　者：张坡[1] 黄岚[1] 蒋世忠[1] 武晓静[1] 宋明宝[1] 于世勇[1] 

机构地区：[1]解放军第三军医大学新桥医院全军心血管内科中心,重庆市400037

出　　处：《中国临床康复》2005年第3期143-145,F003,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30270568)~~

摘　　要：目的:探讨体外培养的骨髓基质细胞体内移植后在缺血区新血管生成中的作用及其时相关系。方法:实验选用健康BALB/C小鼠,体质量18～22g,5～6周龄,雌雄各半。分离小鼠胫骨、股骨,用预冷的DMEM/F12培养基冲洗出骨髓,经密度梯度离心分离出骨髓单个核细胞体外培养。体外诱导分化鉴定分离的细胞,建立下肢缺血模型,荧光标记的体外扩增的骨髓基质细胞被移植入左侧缺血组织作为移植组,右侧注射等量培养介质作为对照组,分为移植后第7,14,28天3个观察期(n=6),冷冻切片荧光显微镜观察,毗邻切片HE及vWF染色,检查荧光阳性细胞与染色阳性细胞的时空关系,并计算移植前后毛细血管密度的变化。结果:体外分离培养的骨髓基质细胞在成内皮细胞供及物存在条件下表达vWF,并分泌一氧化氮。移植后第7天检测到大量幼稚的荧光阳性细胞存活,第14天荧光阳性细胞呈与骨骼肌直径相似的网状分布;移植后28d,移植组毛细胞血管数量犤(57±18)/mm2犦明显高于对照组犤(36±12)/mm2犦(t=3.618,P<0.05)。结论:体外培养的骨髓基质细胞在体外能够定向分化为血管内皮细胞,移植入体内缺血区能促进缺血区新血管生成,可望用作组织缺血细胞治疗的种子细胞。AIM:To study the function of bone marrow stroma cells (BMSCs)cultured in vitro and their time relationship after inter-transplantation in postnatal neovascularization under ischemic repair. METHODS:Healthy BALB/C mice, weighing 18-22 g, aged 5-6 weeks, half males and females, were selected. After tibias and femurs dissected, the bone marrow was flushed out with ice-cold DMEM/F12 medium. Mononuclear cells of the marrow were obtained with density gradient centrifugation and cultured in vitro, after that, they were identified with induction differentiation. Lower limb ischemia model was established, BMSCs amplified in vitro were labeled with fluorescence labeling and transplanted into left ischemic tissue, the same volume into the right ischemic tissue as control group.Three observation periods were 7, 14 and 28 days after transplantation respectively(n=6).Frozen slices were observed under microscope, and other slices were HE and vWF stained and observed to study time relationship between fluorescent positive cells and staining positive cells. Capillary density was calculated before and after transplantation. RESULTS:In the presence of endothelial cell growth supplement, the cultured MSCs in vitro expressed vWF and released NO.After 7 days of transplantation, large amount of infant fluorescence positive cells were detected; After 14 days, fluorescence positive cells presented net distribution similar to skeletal muscle diameter; After 28 days, hair cell number in the transplantation group [(57±18) / mm2 ]was significantly higher than that in the control group [(36±12) / mm2] (t=3.618, P< 0.05). CONCLUSION:BMSCs cultured in vitro can differentiate into endothelial cells.Transplantation of BMSCs into ischemic area can promote neovascularization and be used as therapeutic seed cells for tissue ischemia.

关 键 词：骨髓细胞 细胞 培养的 新生血管化 

分 类 号：R551.3[医药卫生—血液循环系统疾病]