羊水来源胎儿间充质干细胞向成骨细胞的诱导分化  

Differentiation of amniotic fluid-derived fetal mesenchymal stem cells into osteoblasts

作　　者：沈海波[1] 朱哲[1] 潘骏[2] 陈方[1] 

机构地区：[1]上海交通大学医学院附属新华医院泌尿外科,上海市200092 [2]上海儿科医学研究所,上海市200092

出　　处：《中国组织工程研究与临床康复》2009年第27期5314-5318,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：上海市科学技术委员会资金资助项目(05jc14040)~~

摘　　要：背景:羊水细胞已广泛应用于与基因突变有关疾病的产前诊断,但目前关于羊水来源胎儿间充质干细胞的分离培养、细胞表面特征鉴定、细胞分化及其应用前景的文献并不多见。目的:体外诱导孕中期羊水来源的胎儿间质干细胞向成骨细胞方向分化。设计、时间及地点:细胞学体外观察,于2005-08/2006-05在新华医院实验中心完成。材料:10例羊水标本取自孕18~22周行产前诊断或流产的孕妇,取前均征得孕妇本人同意。方法:采用机械手段挑取孕中期羊水细胞培养体系中的胎儿间充质干细胞集落,体外培养扩增。取第3代胎儿间充质干细胞,以100nmol/L地塞米松、10mmol/Lβ-甘油磷酸和50mg/L维生素C持续诱导14d。主要观察指标:免疫组化染色检测碱性磷酸酶和Ⅰ型胶原的表达,Western Blot蛋白印记法检测Ⅰ型胶原蛋白的表达,Von Kossa染色法检测钙结节形成能力,激光共聚焦显微镜检测细胞骨架蛋白形成。结果:分离出的胎儿间充质干细胞为CD44和HLA-ABC阳性,CD34、CD45、HLA-DR阴性,符合间充质干细胞特点。成骨诱导14d后,细胞碱性磷酸酶染色阳性率达91%,Ⅰ型胶原阳性率达87%;表达Ⅰ型胶原蛋白;随诱导时间的延长钙结节逐渐增多、增大;镜下可见细胞胞质中的微丝呈绿色荧光,细胞周围的微丝形成致密的周围束。结论:羊水来源的胎儿间充质干细胞可以分化为成骨细胞,且与典型的成骨细胞具有相似的形态学和生物学特征。BACKGROUND： Amniotic fluid cells have been widely used in antenatal diagnosis for gene mutation-related diseases. However, there are few reports concerning isolation, culture, surface character identification, differentiation and application perspective of fetal mesenchymal stem cells （MSCs） derived from amniotic fluid. OBJECTIVE： To investigate the differentiation of fetal mesenchymal stem cells （MSCs） derived from second-trimester amniotic fluid into osteoblasts in vitro. DESIGN, TIME AND SETTING： The cytological in vitro study was conducted at the Experimental Center of Xinhua Hospital from August 2005 to May 2006. MATERIALS： Ten amniotic fluid samples were obtained from pregnant women （18-22 weeks after conception） or aborted women The informed consents were obtained from pregnant women. METHODS： Fetal MSCs were separated mechanically from amniocyte culture system and expanded in medium in vitro. At passage 3, fetal MSCs were induced in 100 nmol/L dexamethasone, 10 mmol/L [^-glycerophosphodc acid and 50 mg/L vitamin C for 14 days. MAIN OUTCOME MEASURES： Collagen I and alkaline phosphatase expression was detected by immunohistochemistry. Collagen I protein expression was determined by Western Blot analysis. Calcium tuberoses were measured by Von Kossa staining. The cytoskeletal protein was detected by laser confocal microscopy. RESULTS： The isolated fetal MSCs were uniformly positive for CD44 and HLA-ABC, negative for CD34, CD45 and HLA-DR. After being induced with osteogenic medium for 14 days, 91% cells were positive for alkaline phosphatase, and 87% cells for collagen I. Cells expressed collagen I protein, Number of calcium tuberoses was increased and became big over time. Cytoplasm microfilament presented green fluorescence and the microfilament surrounding cells formed dense bundle. CONCLUSION： Fetal MSCs derived from amniotic fluid could be induced into osteoblasts and displayed a typical osteoblastic morphology and biological characteristic.

关 键 词：间充质干细胞 羊水 成骨细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R681[医药卫生—基础医学]