BrdU标记胎兔骨髓间充质干细胞及对细胞生物学行为影响的体外研究  

IN VIRTO BROMODEOXYURIDINE LABELING OF BONE MARROW MESENCHYMAL STEM CELLS FROM BREPHO-RABBIT

作　　者：李德绘[1] 黎洪棉[1,2] 梁自乾[1] 柳大烈[1,3] 

机构地区：[1]广西医科大学第一附属医院烧伤整形外科,南宁530021 [2]中山大学附属中山医院博士后科研工作站,整形美容外科,中山528403 [3]南方医科大学附属珠江医院整形外科,广州510282

出　　处：《广西医科大学学报》2011年第4期506-511,共6页Journal of Guangxi Medical University

基　　金：广西自然科学基金资助项目(No.桂科自0991112)

摘　　要：目的:通过用5-溴脱氧尿苷(5-bromodeoxyuridine,BrdU)标记胎兔骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs),探讨BrdU作为干细胞标记示踪方法的可行性。方法:取胎兔骨髓组织密度梯度离心法分离培养BMSCs。取P3代细胞以终浓度分别为5,10,15,20mg/L的BrdU进行标记,分别记为A,B,C,D组;另1孔不含BrdU,作为空白对照(E组)。标记12,24,48,72h后,采用免疫组织化学检测各组细胞阳性率,苔盼蓝排染法细胞计数观察标记后细胞活性。结果:细胞培养观察:原代培养的细胞形态主要为宽大扁平短梭形细胞;传代后细胞形态为长梭形;第3代BMSCs经诱导均能向成骨细胞和脂肪细胞分化。免疫组织化学观察:第3代BMSCs经BrdU标记后,在荧光显微镜下胞核呈绿色荧光。孵育12h,A,B,C,D组细胞标记阳性率逐渐增高,分别为(33.2±4.3)%、(34.5±3.9)%、(47.9±4.8)%、(52.8±5.1)%,对照组细胞标记阳性率为0,C,D组与A组比较,差异有统计学意义(P<0.01);24h,A,B,C,D组标记率分别为(48.1±4.0)%、(83.4±5.3)%、(91.7±4.9)%、(92.6±4.2)%,对照组细胞标记阳性率为0。48h和72h,A,B,C,D,E组标记情况与24h相似。孵育24,48,72h,10,15,20mg/L组细胞阳性率与5mg/L组比较,差异有统计学意义(P<0.01)。细胞计数:孵育12h,各组细胞活性均在90%以上,A、B、C、D组与对照组(E)比较,差异无统计学意义(P>0.05);孵育24,48,72h,各组细胞活性情况与12h情况相似,A,B,C,D组与对照组(E)比较,差异无统计学意义(P>0.05)。随着标记时间的延长和BrdU剂量的增加,标记率逐渐增高,BrdU终浓度为10mg/L以上,且标记48h后细胞标记率>90%,活细胞数>90%。结论:BrdU标记BMSCs的最佳时间为48h,最佳浓度为10mg/L;BrdU对细胞毒性小,标记率及安全性高。Objective.We explored the optimal dosage, timing and cytotoxicity for bromodeoxyurident （Br- dU） labeling of brepho-rabbit bone marrow mesenchymal stem cells （BMSCs） in vitro, in order to confirm its feasibility for stem cells labeling and tracer means. Methods：Brepho-rabbit bone marrow was isolated and cultured the BMSCs using the density gradient centrifugation method in vitro. The three passages of BMSCs were incubated with BrdU at different concentrations （5,10,15 and 20 mg/L ） for incubating time （12,24, 48 and 72 h）, to identify the optimal BrdU concentration and incubating time for cell labeling. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labeling index （positive rate） and the cells activity for different time after BrdU labeling. Results：The observation of bmSCs＇ culture： the main appearance of primary BMSCs is wide, applanate and short fusiform shape, and long fusiform shape is the maim appearance of passage 3 BMSCs. The passage 3 of BMSCs can be differentiated into osteoblast and adipocyte under correspoonding inductive medium. The observation of immunohistochemistry ： the BMSCs＇ nucleus showed green fluor under fluorescence microscope after labeled by the BrdU. The labeling ratio increasingly elevated of Groups A, B,C and D after the BMSCs were incubated 12 h,their mean labeling ratio was （33.2±4.3）%,（34.5±3.9）%,（47.9±4.8）% and （52.8±5.1）%, respee tively, and the labeling ratio of group E is zero, which were significantly difference among the groups A,B andD （P〈0.01）. The labeling ratio of groups A,B,C and D was （48.1±4.0）%,（83.4±5.3）%,（91.7 ±4.9）% and （92.6±4.2）%,respectively after the BMSCs were incubated 24 h, and the labeling ratio of group E is zero. The results of all groups after incubating 48 h,72 h were similar to those of the 24 h. The rate of label increased gradually with time and concentration, more than 90% BMSCs were labelled after 48 h and when the concentration of

关 键 词：骨髓间充质干细胞 组织工程 尿嘧啶核苷酸类 细胞标记 细胞毒性 

分 类 号：Q2-33[生物学—细胞生物学]