CAJ | 학술논문

目的为解决血管组织工程种子细胞来源不足问题,分离和培养人脂肪干细胞(hADSCs),并在体外定向诱导hADSCs分化为内皮细胞.方法利用胶原酶消化法和贴壁筛选法从人脂肪组织中分离、培养及扩增hADSCs;应用纤维粘连蛋白(FN)与富含多种生长因子的内皮细胞支持液EGM2-MV及高浓度血管内皮细胞生长因子(VEGFi6s)50 ng/ml,协同定向诱导hADSCs分化为内皮细胞,然后对其进行形态、表型及功能鉴定.结果体外分离、培养出高度同源性的hADSCs,hADSCs定向内皮分化后免疫组化及RT-PCR结果显示,内皮特异性标志物CD31、CD34及血管内皮细胞生长因子受体(KDR)阳性表达,透射电镜下观察到内皮特异性结构怀布尔-帕拉德(Weibel-Palade)小体,内皮细胞功能性检测结果显示诱导后hADSCs能够吞噬Dil标记的乙酰化的低密度脂蛋白(Dil-Ac-LDL)及在基质胶(Matrigel)上形成小管样结构,成管数(18.9±1.203)、分支数(31.2±1.304)、结点数(16.9±1.183),与HUVEC组比较差异均无统计学意义(P＞0.05).结论在体外可成功诱导hADSCs分化为内皮细胞.
목적 위해결혈관조직공정충자세포래원불족문제,분리화배양인지방간세포(hADSCs),병재체외정향유도hADSCs분화위내피세포.방법 이용효원매소화법화첩벽사선법종인지방조직중분리、배양급확증hADSCs;응용섬유점련단백(FN)여부함다충생장인자적내피세포지지액EGM2-MV급고농도혈관내피세포생장인자(VEGFi6s)50 ng/ml,협동정향유도hADSCs분화위내피세포,연후대기진행형태、표형급공능감정.결과 체외분리、배양출고도동원성적hADSCs,hADSCs정향내피분화후면역조화급RT-PCR결과현시,내피특이성표지물CD31、CD34급혈관내피세포생장인자수체(KDR)양성표체,투사전경하관찰도내피특이성결구부포이-파랍덕(Weibel-Palade)소체,내피세포공능성검측결과현시유도후hADSCs능구탄서Dil표기적을선화적저밀도지단백(Dil-Ac-LDL)급재기질효(Matrigel)상형성소관양결구,성관수(18.9±1.203)、분지수(31.2±1.304)、결점수(16.9±1.183),여HUVEC조비교차이균무통계학의의(P＞0.05).결론 재체외가성공유도hADSCs분화위내피세포.
Objective To investigate the possibility of directional differentiation of human adipose stem cells (hADSCs) into endothelial cells (EC), so as to provide seed cells for tissue engineered vessels.Methods hADSCs were isolated from human adipose tissue by collagenase digestion, cultured and amplified by adherence to flasks. Then hADSCs were directionally induced to differentiate into EC by a combination of fibronectin ( FN ), endothelial cells support liquid ( EGM2-MV ) containing various growth factors and high concentration of VEGF165 (50 ng/ml). Then, the cells morphology, phenotype and function were identified. Results Highly homologous hADSCs were obtained, and then hADSCs were directionally differentiated into EC. CD31 and CD34, the specific markers for EC, and vascular endothelial growth factor receptor (KDR) were positive by immunohistochemical staining and RT-PCR. In addition,unique Weibel-Palade bodies in EC were observed under transmission electron microscope. Functionally,hADSCs could swallow Dil-Ac-LDL and form tube-like structures in matrigel after endothelial differentiation. Conclusions hADSCs can be successfully induced to differentiate into endothelial cells in vitro.