BrdU, CFSE和GFP标记大鼠间充质干细胞的比较

1、BrdU, CFSE和GFP标记大鼠间充质干细胞的比较         08-10-11 14:02:00     编辑：studa20           作者：付霞霏，何援利，杨芳，彭冬先，刘木彪   【摘要】  目的: 研究BrdU, CFSE和GFP作为大鼠间充质干细胞标记物的可行性. 方法: 分别用上述三种标记物标记大鼠间充质干细胞，检测

2、即时、15和30 d的标记率，通过检测细胞周期、凋亡率和描绘生长曲线，来明确标记物对体外培养大鼠间充质干细胞生长特性的影响；通过流式细胞仪检测标记物对细胞表面标志（CD29, CD34, CD44, CD45）表达的影响. 结果: BrdU, CFSE, GFP的即时标记率为77.0%, 99.4%, 89.7%，1 mo后分别下降至51.9%, 77.5%, 82.9%. 三者对体外培养的大鼠间充质干细胞的生长特性和表面标志的表达均无明显影响. 结论: GFP, BrdU和CFSE均可以作为大鼠间充质干细胞的标记物. 【关键词】  绿色荧光蛋白；溴脱氧尿嘧啶核苷；荧光染料CFSE；

3、间充质干细胞【Abstract】 AIM: To  investigate the feasibility of labeling of BrdU, CFSE and GFP as labels of rat mesenchymal stem cells (MSCs) and to investigate the influence of the 3 markers on the growth characteristics and expressions of surface markers of rat MSCs. METHODS: Wistar rats bone marrow

4、 derived MSCs were isolated and purified in vitro by Percoll density gradient centrifugation combined with adherent method and were labeled with BrdU, CFSE and GFP, respectively. The immediate, 15day and 30day labeling efficiency of the 3 markers was quantified. The labeling efficiency of BrdU was d

5、etected with immunocytochemical method, and that of CFSE and GFP was detected via flow cytometry (FCM). The labeled MSCs were observed under microscope. Cell cycle and apoptosis rate of labeled MSCs were revealed and the growth curves were delineated, so as to identify whether or not the markers cas

6、ted an influence on the cell growth characteristics in vitro. Furthermore, the surface markers (CD29, CD34, CD44, CD45)  of the labeled MSCs were detected through flow cytometry and were compared with those of nonlabeled MSCs. RESULTS: The immediate labeling efficiency of BrdU, CFSE, and GFP wa

7、s 77.0%, 99.4%,  89.7% respectively, and the efficiency decreased to 51.9%, 77.5%, and 82.9% respectively after 30 d. There was no statistical difference in cell cycle and apoptosis ratio between labeled MSCs and nonlabeled MSCs. The growth curves of labeled MSCs were similar to that of nonlabe

8、led MSCs. The 3 markers didnt influence the expressions of surface markers (CD29, CD34, CD44, CD45) of rat MSCs. CONCLUSION: The 3 markers have no effects on the growth characteristics and expressions of surface markers of rat MSCs cultured in vitro. BrdU, CFSE and GFP can serve as labeling markers

9、of rat MSCs. BrdU and CFSE labeling can be used for short time tracing, and GFP labeling for long time tracing.【Keywords】 GFP; BrdU; CFSE; MSCs0引言骨髓间充质干细胞（mesenchymal stem cells, MSCs）是定居于骨髓微环境中的一种成体干细胞. MSCs不仅能分泌多种细胞因子调节造血细胞的增生与分化，还具有自我增殖、多向分化的潜能1. 因其采集方法简单、能在体外大量扩增、可自体移植避免免疫排斥和伦理问题，已成为组织修复的理想种子细胞和

10、基因治疗的靶细胞2. 而寻找合适的标记方法用于追踪观察MSCs在实验研究中是很重要的. 目前常用的标记方法包括：溴脱氧尿嘧啶（5bromodeoxyuridine, BrdU）、荧光染料（如二醋酸盐琥珀酰亚胺酯，5,6carboxyfluorescein diacetate succinimidyl ester，CFSE）和绿色荧光蛋白（green fluorescent protein，GFP）标记. 我们从标记率、MSCs的生物学特性和表面标志的表达等方面对这三种标记方法进行较全面的比较.1材料和方法1.1材料近交系SPF级68周龄Wistar大鼠20只，雌雄不拘，体质量120150 g，

11、由广东省动物中心提供. DMEMF12培养基（Gibco公司），胎牛血清（Hyclone公司），CFSE（Sigma公司），BrdU（Sigma公司），小鼠抗BrdU抗体（Chemicon公司），山羊抗小鼠FITC（Santa Crutz公司），AdGFP（珠江医院妇产科构建），CCK8试剂盒（Dojindo公司），磷酸盐缓冲液（PBS，武汉博士德公司）. CO2培养箱（Shell公司），荧光显微镜（Leica公司），流式细胞仪（Becton Dickinson公司）.1.2方法1.2.1大鼠MSCs的分离、培养及扩增用100 g/L水合氯醛麻醉大鼠后，无菌条件下取出胫骨和股骨，制备单细胞悬液

12、，小心加入预先有密度为1.083的Percoll分离液的离心管内，两种液体体积比为11. 离心后，小心吸取中间界面乳白色的单个核细胞层，用PBS洗2次后加完全培养基（含100 mL/L胎牛血清的DMEM/F12培养基）重悬，接种培养. 待细胞铺满瓶底的80%90%时，按12传代. 本实验中选用生长良好的第3代大鼠MSCs.1.2.2BrdU标记方法及标记率的检测待细胞长至约50%融合时，吸弃培养基，加入BrdU浓度为10 mol/L的培养基，置培养箱中孵育48 h. 弃去培养基，PBS清洗，以40 g/L多聚甲醛固定30 min. 再用PBS清洗3次后，加入10 g/L牛血清白蛋白，甩净余液后

13、，加入小鼠的抗BrdU抗体，于湿盒中37孵育4 h，PBS清洗3次后，避光加入山羊抗小鼠FITC荧光抗体，避光孵育1 h，PBS清洗3次后，荧光显微镜下观察细胞的即时标记情况. 随机选取5个视野，平均标记率，即为BrdU的即时标记率. 同批BrdU标记的大鼠MSCs继续培养、传代，分别于15，30 d后再检测标记率.1.2.3CFSE标记方法和标记率检测待大鼠MSCs长至约铺满瓶底的80%时，加入CFSE，并使其终浓度为5 mol/L，置培养箱中继续孵育40 min. PBS洗3次后，流式细胞仪检测荧光标记率，以未标记的MSCs作为对照. 同批CFSE标记的大鼠MSCs继续培养、传代，分别于15，30 d后再检测标记率.1.2.4AdGFP标记方法和标记率的检测取病毒储存液在HEK293细胞中反复扩增，收集培养上清及细胞，反复冻溶破坏细胞离心取上清、过滤除菌，氯化铯密度梯度离心法进行纯化，空斑实验法测定转染液病毒滴度为2×1013 pfuL. 待大鼠MSCs长至约铺满瓶底的80%时，加入感染复数（multiplicity of infection，MOI）为100的AdGFP，并于12，24，48和72 h，于荧光显微镜下观察标记情况. GFP标记72 h后，以未标记的MSCs做对照，用流式细胞仪进行荧光标记率检测. 同批GFP标记