荧光活性染料DiI标记的大鼠骨髓基质干细胞生长增殖及其成骨分化

背景：目前关于骨髓基质干细胞的标记报道较多,各有其优缺点,寻找一种有效、简便的细胞标记与示踪的方法尤为关键。目的：观察经荧光活性染料DiI标记的大鼠骨髓基质干细胞生长增殖与成骨分化情况。设计、时间及地点：细胞学体外观察,于2008-09/12在南方医科大学组织工程研究中心完成。材料：SD大鼠10只,由南方医科大学实验动物中心提供。DiI应用液为美国Molecular ProbeInc公司产品。方法：全骨髓法体外分离培养大鼠骨髓基质干细胞,取传至第3代细胞,加入无血清的DMEM制成细胞悬液,再加入1×109L-1的DiI应用液5μL,37℃下孵育25min,清洗离心后,加入DMEM高糖完全培养基,荧光显微镜观察细胞荧光强度及形态变化。另取传至第3代细胞,加入含地塞米松、维生素C、β-磷酸甘钠、体积分数为10%胎牛血清的成骨诱导剂进行诱导分化。主要观察指标：DiI标记后细胞形态变化,XTT比色法测定细胞增殖状况,细胞上清液中乳酸脱氢酶活性,成骨诱导后细胞碱性磷酸酶活性及成骨分化能力。结果：锥虫蓝染色见骨髓基质干细胞活力好,仅偶见蓝染细胞,荧光显微镜下DiI标记后全部细胞的胞浆、胞膜均显红色荧光,标记的骨髓基质干细胞呈梭形,胞浆丰富,保持了良好的正常形态,DiI标记阳性率为100%,标记早期细胞形态呈荧光环状,48h后细胞中荧光颗粒增多,荧光增强,细胞核未染荧光。与未标记细胞比较,DiI标记的骨髓基质干细胞增殖吸光度、培养上清液乳酸脱氢酶活性、碱性磷酸酶活性均无明显变化（P〉0.05）。成骨诱导7d后,钙钴法染色两组骨髓基质干细胞均见不同程度的胞质呈棕黑色改变。结论：DiI能有效标记体外培养的骨髓基质干细胞,并在细胞内稳定表达,且标记细胞形态良好,对活体细胞无毒性作用;此外,DiI标记不影响骨髓基质干细胞的生长、增殖及成骨分化能力。

Growth,proliferation,and osteogenic differentiation of rats bone marrow stem cells labeled by fluorescent reactive dye DiI

BACKGROUND：There are plenty of labeling regarding bone marrow mesenchymal stem cells（BMSCs） with own merits and demerits. Therefore,it is a key to find a simple,effective labeled method for BMSCs. OBJECTIVE：To observe the growth,proliferation and osteogenic differentiation of fluorescent reactive dye DiI labeled rat BMSCs. DESIGN,TIME AND SETTING：The in vitro cytology observation was performed at the Center of Tissue Engineering Research of Southern Medical University between September and December 2008. MATERIALS：Ten Sprague Dawley rats were provided by animal center of Southern Medical University. DiI application solution was produce by American Molecular Probe Inc. METHODS：The MSCs were isolated primarily from rat bone marrow,and purified by passage culture. The 3rd passage of BMSCs were prepared for cell suspension with adding serum-free DMEM,consequently,5 μL DiI liquor at the density of 1×109/L was added,and then cells were incubated for 25 minutes at 37 ℃,centrifugalized and washed with PBS. DMEM medium was added before coloration and morphologic change of BMSCs. The other 3rd passage of BMSCs were induced to osteoblast differentiation by osteogenic medium containing hexadecadrol,vitamin C,β-glycerophosphate and 10% fetal bovine serum. MAIN OUTCOME MEASURES：The morphologic changes of DiI labelled BMSCs;cell proliferation,lactic dehydrogenase content in supernatant of BMSCs,and alkaline phosphatase（ALP） activity,as well osteogenic differentiation potential of BMSCs. RESULTS：Trypan blue staining showed BMSCs had good activity,and only few blue-stain cells could be seen. Under fluorescent microscopy,all the cells presented red fluorescence after labeling,and the labeled BMSCs maintained good normal shapes. DiI positive rate was 100%. The cells presented fluorescent ring shape at the early period,which increased and enhanced at 48 hours later with increasing fluorescent granules. No fluorescence was found in cell nucleus. Compared to without labeled cells,DiI labeled BMSCs had no significant differences in the absorbance value,activity of lactic dehydrogenase,as well as ALP content（P 0.05） . The cytoplasm of 2 group displayed brownish-black to calcium cobolt staining at day 7 after osteogenic induction. CONCLUSION：DiI can label BMSCs in vitro effectively with stably expression. The DiI labeled cells have good morphology,which have no toxicity for living cells. In addition,DiI labeling has no influence on cell growth,proliferation,as well as osteogenic differentiation.