摘要目的 分离和培养成人脂肪来源干细胞(hADSC),并观察非接触式细胞体外共培养条件下,hADSC对人胰岛细胞形态、存活率及功能的影响及机制.方法 采用胶原酶消化法分离并原代培养获得hADSC,形态学、免疫荧光及成骨、成脂诱导分化鉴定hADSC.采用Liberase酶消化及Ficoll 400液分离、纯化成人胰岛细胞,并将胰岛细胞分为共同培养组和单独培养组;倒置相差显微镜观察两组胰岛细胞的生长形态,比较两组体外培养3、7和14d时的胰岛细胞存活率、胰岛素分泌量、胰岛素刺激指数及培养14 d时上清液中肝细胞生长因子(HGF)、转化生长因子-β(TGF-β)、血管内皮细胞生长因子(VEGF)及碱性成纤维细胞生长因子(b-FGF)浓度.结果 培养第3代hADSC呈均一的长梭形纤维细胞样形态,免疫荧光检测显示CD44和CD49d为阳性,CD31、CD34和CD106均为阴性;具有成骨、成脂等多向分化潜能.培养14d时,共同培养组胰岛细胞的形态较单独培养组完整,共同培养组胰岛细胞存活率为(82.83±2.32)％,较单独培养组的(53.00±2.82)％明显提高(P＜0.01);共同培养组、单独培养组高糖培养时的胰岛素分泌量分别为(23.66±2.11) mIU/L和(7.82±1.09)mIU/L,低糖培养时分别为(13.22±0.77) mIU/L和(6.40±0.44) mIU/L,两组比较,差异均有统汁学意义(P＜0.01);共同培养组胰岛细胞胰岛素刺激指数由培养3d时的1.90±0.03降为培养14d时的1.77±0.13,降低不明显;单独培养组由培养3d时的1.67±0.10降为培养14 d时的1.22±0.12,显著降低(P＜0.01).培养14d时,共同培养组上清液HGF、TGF-β、VEGF、b-FGF浓度均显著高于单独培养组(P＜0.05).结论 从成人脂肪组织中能够成功分离及培养hADSC;在体外与人胰岛细胞共同培养时,hADSC可以通过分泌HGF、TGF-β、HGF、b-FGF来提高胰岛细胞的存活率,改善胰岛细胞的功能.

abstractsObjective To isolate and culture human adipose-derived stem cells (hADSCs),investigate the influence of hADSCs on the cellular morphology,survival rate,and function of human islet cells under the in vitro non-contact co-culture conditions,and explore its mechanism.Method hADSCs were isolated by collagenase digestion method,then cultured,and identified by morphology,immunofluorescence and multi-directional differentiation.Adult islet cells were separated and purified by Liberase enzyme and Ficoll 400,then divided into co-culture group and individual group.The cellular growth morphology of islet cells was observed by inverted phase contrast microscope.The survival rate of islet cells,insulin secretory volume,insulin stimulation index and concentration of growth factor in the supernatant were compared between the two groups.Result hADSCs of the third generation showed uniform long spindle fibrocyte-like morphology,and had multi-directional differentiational potentials of osteogenesis and adipogenesis.Immunofluorescence test of surface antigens on hADSCs revealed CD44 + and CD49d +,CD31-,CD34-and CD106-.After 14-day culture,the islet cellular morphology in co-culture group was more intact than that in individual group.The survival rate of islet cells in co-culture group was (82.83 + 2.32) ％,and that in individual group was (53.00 + 2.82) ％ (P＜0.01).Insulin secretory volumes were (23.66 + 2.11) and (7.82 +1.09) mU/L respectively in co-culture group and individual group under high glucose concentration,and 13.22 + 0.77 and 6.40 + 0.44 mU/L respectively under low glucose concentration (P＜0.01 for all).Insulin stimulation index was decreased from 1.67 + 0.10 (at 3rd day) to 1.77 + 0.13 (at 14th day) in co-culture group,and from (1.67 + 0.10) (at 3rd day) to (1.77 + 0.13) (at 14th day) in individual group (P＜0.01).After 14-day culture,the concentrations of HGF,TGF-β,VEGF and bFGF in the supernatant were higher in co-culture group than in individual group (P＜0.01).Conclusion hADSCs were isolated and cultured successfully from adult adipose tissue.They could increase the survival rate and improve the function of islet cells when co-culture with the adult islet cells in vitro through secreting HGF,TGF-β,VEGF and b-FGF.