人羊水来源干细胞与生物可降解材料体内外复合培养构建组织工程膀胱平滑肌  

Construction of tissue-engineered bladder smooth muscle tissue from human amniotic fluid colony-derived stem cells and biodegradable materials in vitro and in vivo

作　　者：高同斌[1] 马晓荣[1] 张胜利[1] 谢华[1] 周君梅[1] 陈方[1] 

机构地区：[1]上海交通大学附属儿童医院小儿泌尿外科,200092

出　　处：《中华小儿外科杂志》2011年第7期543-549,共7页Chinese Journal of Pediatric Surgery

基　　金：上海市科学技术委员会基金（编号：07XD14024;08jc1416000）;上海市教育委员会科研创新项目（编号：09YZ90）

摘　　要：目的探讨体外高效地诱导人羊水来源干细胞向平滑肌样细胞的分化，评估细胞与聚羟基乙酸（PGA）支架复合物的生物相容性，由此探讨该复合物用于组织工程膀胱平滑肌重建的可行性。方法孕中期B超引导下穿刺抽取羊水，体外分离挑选原代人羊水来源的干细胞（hAFCSCs），取第三代细胞用肌源性生长因子（PDGF-BB）和转化生长因子β1（TGF-β1）和左旋维生素C（LAA）在体外分别诱导分化3周、4周和5周，然后光镜下观察诱导后细胞生长的形态变化，免疫荧光细胞染色和RT-PCR检测平滑肌细胞的标志物。取第四代人羊水来源干细胞种植到非编织PGA材料上，体外诱导培养4周后移植到裸鼠背部皮下，4周后取材进行体内外种植效果的评价。结果hAFC-SCs体外增殖能力强，RT-PCR、免疫荧光细胞染色显示经诱导的hAFCSCs表达平滑肌细胞的标志物平滑肌肌动蛋白（a—SMA），肌钙蛋白（CALP），平滑肌22a（SM22a）和肌球蛋白重链（MHC）。PGA支架上的hAFCSCs在种植4h后开始正常贴附、生长和增殖，细胞形态良好。扫描电镜显示hAFC—SCs紧密贴附到PGA支架材料上并分泌细胞外基质，冰冻切片免疫荧光染色显示组织块中表达平滑肌细胞的标志物。结论hAFCSCs能在肌源性生长因子（PDGF-BB和TGF-β1）和左旋维生素C（L-AA）的诱导作用下高效地分化为平滑肌样细胞。复合hAFCSCs的PGA支架体外具有良好的生物相容性及体内较好的组织相容性，表明以hAFCSCs为种子细胞、PGA为支架构建组织工程膀胱平滑肌具有可行性。Objective To investigate the potential of human amniotic fluid colony-derived stem cells （hAFCSCs） efficiently differentiating into smooth rnusle cells,and evaluate the biocompatibility of cell-PGA composite scaffolds and the fesibility of reconstraction of bladder smooth muscle tissue in vitro and in vivo . Methods hAFCSCs were obtained from second trimester amniotic fluid by ultra- sound-guided arnniocentesis performed on pregnant women. After expended in vitro, the 3rd genera- tion hAFCSCs were induced in myogenic differentiation medium supplemented by myogenic growth factors （PDGF-BB and TGF-β1 ） and L-Ascorbic acid （L-AA） for 3 weeks, 4 weeks and 5 weeks, and the differentiated cells were identified morphologically and the myogenic phenotype were detected by gross observation,RT-PCR and immunofluorescence staining. The 4th generation hAFCSCs cells were seeded omo polyglycolie acid （PGA） unwoven fiber scaffolds and cultured for 4 weeks in vitro in myo- genic differentiation medium, then PGA-cell composite scaffolds and pure scaffolds were separately im- planted inlo subcutaneous pockets of nude mice. After 4weeks of implantation, the specimens were harvesled and examined. Results The hAFCSCs expended rapidly in vitro. After induction with rnyo- genie growth factors and L-AA, RT-PCR and immunofluorescence staining demonstrated that a-smoothmuscle actln（α-SMA）, calponin（CALP ） smooth muscle 22α （SM22α） and myosin heavy chain（MHC） were expressed. The adhesion and proliferation of hAFCSCs on PGA fiber scaffolds were observed after 4 h.. As polymer degradation degrad- ed, the hAFCSCs kept proliferation and converged, Microscopic and scanning e- lectron mfcroscope （SEM） observationsshowed that hAFCSCs spreaded over the PGA fibers with isecreted extracellular matrices filling the space among the fibers.. Conclusions hAFCSCs derived from second trimester amniotic fluid can be efficiently induced into smooth muscle cells by myogenic growth factors （PDGF-BB and TGF-β1 ） and

关 键 词：羊水 干细胞 组织工程 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]