人脂肪组织来源的干细胞体外诱导为角膜上皮样细胞的分化潜能  

Differential potential of human adipose-derived stem cells induced into corneal epithelial-like cells in vitro

作　　者：钟刘学颖 刘小伟[1] 戴荣平[1] 秦梅[1] 董方田[1] 李莹[1] 

机构地区：[1]中国医学科学院,北京协和医学院,北京协和医院眼科,北京市100730 [2]中山大学中山眼科中心

出　　处：《眼科新进展》2010年第1期14-19,共6页Recent Advances in Ophthalmology

基　　金：北京协和医院青年科研基金资助~~

摘　　要：目的初步探讨人脂肪组织来源的干细胞(human adipose-derived stem cells,hADSC)体外诱导为角膜上皮样细胞的分化潜能。方法分离、纯化、体外扩增hADSC,流式细胞术鉴定所获得细胞的CD29+、CD34-、CD49d+、CD105+、CD106-的表达情况。成脂、成骨诱导鉴定其多向分化潜能。在DMEM/F12体系、KM体系及上述二者等体积混合的KM/DMEM/F12体系内添加不同梯度浓度的生长因子对hADSC进行体外诱导:0μg·L-1、10μg·L-1、20μg·L-1、30μg·L-1、40μg·L-1、50μg·L-1的表皮生长因子(epidermalgrowthfactor,EGF),及50μg·L-1EGF+10μg·L-1碱性成纤维细胞生长因子(basicfibro blast growth factor,bFGF)和50μg·L-1EGF+20μg·L-1bFGF。连续诱导21d,观察hADSC形态学的改变及第21天时角膜特异性蛋白AE5(CK3/CK12)的免疫化学表达情况,比较不同培养体系及不同浓度生长因子对hADSC诱导作用的差异。结果流式细胞仪测定传3-5代的细胞CD34-、CD106-、CD29+、CD49d+、CD105+。成脂、成骨体外诱导14d后,油红O、碱性磷酸酶染色阳性率分别为74.6%和29.3%。KM体系中加入终浓度为0～50μg·L-1EGF诱导21d后,hADSCAE5阳性细胞率均占90%以上。其中,40μg·L-1EGF诱导下的AE5阳性细胞率为98.7%,50μg·L-1EGF可达到100%。而加入bFGF的hADSC则为AE5弱阳性。而另2个体系对各浓度EGF、bFGF诱导的hADSC的作用均为阴性。结论人吸脂术废弃液中可获得大量高纯度脂肪组织来源的干细胞,经体外条件诱导具备向角膜上皮样细胞分化潜能。添加EGF的角质细胞培养液有利于其分化,并具有浓度依赖性,bFGF则对分化有抑制性作用。Objective To investigate the differential potential of human adiopose-derived stem cells（hADSC） induced into corneal epithelial-like cells in vitro.Methods The hADSC were isolated from processed lipoaspirate tissues,purifed and amplificated in vitro.Phenotypes including CD29＋,CD34-,CD49d＋,CD105＋ and CD106-were identified by flow cytometry.Adipogenesis and osteogenesis inductions were used to identify multi-lineage differentiatial potential abilities of hADSC.The hADSC were induced in vitro by Keratinocyte medium（KM）,Dulbecco’s Modified Eagle Medium/F12（DMEM/F12） and KM/DMEM/F12 with isochoric mixture,which stimulated with different concentrations of growth factors,0 μg·L^-1,10 μg·L^-1,20 μg·L^-1,30 μg·L^-1,40 μg·L^-1,50 μg·L^-1 epidermal growth factor （EGF）,50 μg·L^-1 EGF combined with 10 μg·L^-1 basic fibroblast growth factor （bFGF） and 50 μg·L-1 EGF combined with 20 μg·L^-1 bFGF.After continuously induced for 21 days,the morphological changes and immunohistochemical expression of AE5（CK3/CK12） at the 21st day were observed.The differential potential of hADSC between different mediums and different concentrations of growth factors were compared.Results The phenotypes of cells at passage three to passage five like CD34^-,CD106^-,CD29^＋,CD49d^＋ and CD105^＋ were measured by flow cytometry.After adipogenic and osteogenic induction in vitro for 2 weeks,positive staining of the oil red O was 74.6% and alkaline phosphatase staining was 29.3%.The AE5 positive expression rate of hADSC cultured with 0 μg·L^-1 to 50 μg·L^-1 EGF in KM system for 21 days was over 90%,which increased to 98.7% with 40 μg·L^-1 EGF and 100% with 50 μg·L^-1 EGF.While most of hADSC added with bFGF just expressed weakly.The hADSC cultured in the other two systems with different concentrations of EGF and bFGF were negative.Conclusions A large quantity of purified hADSC can be gained from liposuction liquid,evidenced with differential potential abilities into corneal epithelial-like ce

关 键 词：吸脂术 人脂肪组织来源的干细胞 角膜上皮样细胞 表皮生长因子 碱性成纤维细胞生长因子 体外诱导 

分 类 号：R779.62[医药卫生—眼科]