不同血清对成人骨髓基质干细胞成骨诱导分化的影响  

Effects of different serums on osteogenic induction of human bone marrow mesenchymal stromal cells in vitro

作　　者：李方国[1] 王磊[1] 张鑫鑫[1] 姜晓锐[2] 金丹[1] 江汕[1] 赵培冉[1] 裴国献[3] 

机构地区：[1]南方医科大学南方医院创伤骨科,广州510515 [2]山东省烟台毓璜顶医院手足骨科 [3]第四军医大学西京医院骨科

出　　处：《中国矫形外科杂志》2011年第5期406-410,共5页Orthopedic Journal of China

基　　金：国家重点基础研究发展计划(973)资助项目(2009CB930003);国家自然科学基金项目(No.31070866)

摘　　要：[目的]探讨三种不同来源血清对体外培养成人骨髓基质干细胞(hBMSCs)向成骨诱导分化的作用。[方法]将体外第3代hBMSCs向成骨诱导分化培养,分为胎牛血清组(对照组)、AB血清组和自体血清组。对比观察细胞碱性磷酸酶(ALP)染色、钙结节染色。诱导培养后4、7、14 d和21 d,各血清组分别进行钙黄绿素法荧光显微镜动态观察矿盐沉积,检测ALP活性,实时荧光定量PCR(RT-qPCR)法检测成骨基因(ALP)、骨桥蛋白(OPN)和骨钙素(OCN)的表达。[结果]ALP染色和钙结节染色结果显示,与自体血清(AS)组、胎牛血清(FBS)组相比,AB血清(ABS)组明显提高了hBMSCs的染色阳性率。荧光显微镜下观察诱导21 d的hBMSCs,ABS组较FBS组、AS组呈现更多的钙盐沉积。ALP活性结果显示,同一时间点ABS组的ALP活性均明显高于FBS组和AS组,差异有统计意义(P<0.05)。RT-qPCR结果显示,在7、14 d和21d,ABS组ALP、OPN和OCN成骨基因表达均明显高于FBS组、AS组,其中,ALP基因表达在7 d出现峰值,OPN基因表达在14 d出现峰值,OCN基因表达在21 d出现峰值,差异均有统计学意义(P<0.05)。[结论]ABS对hBMSCs成骨分化作用较FBS、AS明显增强。ABS有望替代FBS建立符合骨组织工程临床应用要求的体外hBMSCs培养体系。[Objective]To investigate the effects of different serums on osteogenic induction of human bone mesenchymal stromal cells（hBMSCs）. [Methods]The third passage of hBMSCs were cultured in osteogenic medium containing either fetal bovine serum（FBS）,pooled human AB serum（ABS）,or autologous serum（AS）.Alkaline phosphatase（ALP）and the calcium staining were used to evaluate the effect of different serums on osteogenic differntiation of hBMSCs.At 4,7,14 and 21 days after culture,fluorescence analysis of calcein incorporated into mineralized bone matrices and ALP activity was measured in all serum groups.The specific osteogenic genes [ALP,osteopontin（OPN）,osteocalcin（OCN）] expression were also measured by real-time quantitative PCR analysis during these time points.[Results]ABS showed higher ALP and calcium staining than FBS or AS.A great quantity of mineralized bone matrices were produced in ABS which was higher than that in FBS or AS through fluorescence microscope at 21 days after culture.ALP activity was higher in ABS than that in FBS or AS at each time point（P0.05）.The gene expressions of ALP,OPN and OCN were higher in ABS than that in FBS or AS at 7,14 and 21 days after culture（P0.05）.ALP gene expression reached a peak at 7 days,OPN gene expression reached a peak at 14 days,OCN gene expression reached a peak at 21 days.[Conclusion]ABS has been found to have a higher osteogenic capability than FBS and AS so as to establish a new and safer culture model in clinical cytotherapy of bone repair.

关 键 词：骨髓基质干细胞 AB血清 自体血清 成骨分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]