人脂肪间充质干细胞与脐静脉内皮细胞共培养与丝素蛋白支架联合构建组织工程脂肪的体外研究  

Experimental study of tissue-engineered adipose constructed by co-culture system of hADSCs and HUVEC with silk fibroin scaffolds in vitro

作　　者：刘刚强[1] 张诚[1] 刘毅[1] 亢婷[1] 王刚[1] 

机构地区：[1]兰州军区兰州总医院全军烧伤整形中心,甘肃兰州730050

出　　处：《兰州大学学报（医学版）》2015年第4期1-9,共9页Journal of Lanzhou University（Medical Sciences）

基　　金：全军医学科研"十二五"重点项目(BWS11C061)

摘　　要：目的探讨人脂肪间充质干细胞(1aADSCs)与人脐静脉内皮细胞(}tUVEC)共培养后,共培养体系与丝索蛋白支架联合构建组织工程脂肪的可行性。方法从人体正常脂肪组织中提取hADCSs,培养并扩增,行成脂及成骨诱导鉴定hADCSs的多向分化特性;流式细胞术检测hADCSs表面分子CD34、CD44、CD45、CD90,检测HUVEC表面分子CD31及CD34;测定hADCSs与HUVEC增殖曲线,探讨共培养比例;将共培养体系种植于丝素蛋白支架后行电镜扫描;成脂诱导14d后,反转录一聚合酶链反应(RT-PCR)鉴定成脂基因PPAR叫-2的表达。结果 hADSCs可以向脂肪细胞及成骨细胞分化;流式细胞术检测hADSCs结果显示CD34(+)、CD44(十)、CD45(-)、CD90(-),检测mJVEC结果显示CD31(+)、CD34弱阳性;m5VEC增殖速度快于hADSCs,以HUVEC:hADSCs为1:4比例接种于培养皿培养3 d后,细胞融合时两者比例约为1:1;共培养体系接种于丝素蛋白支架成脂诱导14 d后,通过RT-PCR成功测定出PPAR叫-2基因的表达。结论 hADSCs与HI_YVEC共培养,在体外可成功构建出组织工程脂肪。Objective To investigate the feasibility of constructing the engineering adipose tissue by co-culture system of human adipose mesenchymal stem cells （hADSCs） and human umbilical vein endothelial cells （HUVEC） with silk fibroin scaffold. Methods hADSCs was extracted from human normal adipose tissue, cultured and amplified. The multi-directional differentiation characteristics of hADSCs were identified by adipogenic and osteogenic induction. Flow cytometry was used to detect the expressions of surface molecule CD34, CD44, CD45, CD90. The surface molecules of HUVEC, CD31 and CD34, were detected by flow cytometry. The proliferation curve of hADSCs and HUVEC were measured to explore the co-culture ratio. Co-cultured cells were observed under scanning electron microscope after being seeded on silk fibroin scaffold. After adip ogenic induction of the cell-scaffold complexe in adipogenic medium for 14 days the expression of specific gene PPARy-2 was tested by RT-PCR. Results hADSCs can differentiated toward adipocyte and osteoblast with molecular characteristics of CD34 （＋）, CD44 （＋）, CD45 （-） and CD90 （-） testified by flow cytometry. Flow cytometry results for HUVEC indicated CD31 （＋） and CD34 weakly positive. The proliferation speed of HUVEC was faster than that ofhADSCs, therefore, the numbers ofhADSC and HUVEC were equal when the cells come to be confluent 3 days after co-culture of HUVEC and hADSCs with the ratio of 1 ： 4. PPAR-2 gene expression was successfully determined by RT-PCR 14 days after adipogenic induction when co-culturing hADSCs and HUVEC with silk fibroin scaffolds. Conclusion Co-culture of hADSCs and HUVEC with silk fibroin can successfully construct engineered adipose.

关 键 词：人脂肪间充质干细胞 人脐静脉内皮细胞 丝素蛋白 支架 共培养体系 组织工程脂肪 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]