1、人脂肪干細(xì)胞向內(nèi)皮分化最佳誘導(dǎo)體系的建立         10-09-07 16:07:00     編輯：studa20                       作者：楊旭芳何 旭 何 牮 張慕蕊 張麗紅 李玉林 【摘要】  目的 探

2、討人脂肪干細(xì)胞(human Adiposederived stem cells，hADSCs)體外分化為內(nèi)皮細(xì)胞的最佳誘導(dǎo)體系。方法 利用膠原酶消化法和貼壁篩選法從人脂肪組織中分離、培養(yǎng)及擴(kuò)增hADSCs，分3組分別進(jìn)行內(nèi)皮誘導(dǎo)：即纖維連接蛋白(fibronectin,FN)包被組、明膠包被組和未鋪組;同時(shí)誘導(dǎo)液分為兩組：單純內(nèi)皮細(xì)胞生長培養(yǎng)基(EGM2MV)組及內(nèi)皮細(xì)胞生長培養(yǎng)基(EGM2MV)+50 ng/ml血管內(nèi)皮細(xì)胞生長因子(VEGF165)組。誘導(dǎo)15 d后，通過流式細(xì)胞術(shù)檢測各組內(nèi)皮細(xì)胞特異性表面抗原CD34的表達(dá);通過相差顯微鏡觀察誘導(dǎo)前后細(xì)胞的一般形態(tài)學(xué)特征。結(jié)果 體外分離、

3、培養(yǎng)出高度同源性的hADSCs，CD29、CD44、CD90、CD105及CD166呈陽性表達(dá)，而CD34、CD45及HLADR呈陰性表達(dá);誘導(dǎo)后細(xì)胞流式結(jié)果顯示單純EGM2MV誘導(dǎo)的3組細(xì)胞內(nèi)皮細(xì)胞特異性標(biāo)志CD34陰性表達(dá)，而EGM2MV+50 ng/ml VEGF165誘導(dǎo)的細(xì)胞,其中FN包被組CD34表達(dá)率為8.4%，明膠包被組為5.4%，未鋪組為4.2%;相差顯微鏡下可見誘導(dǎo)后細(xì)胞呈三角形或多邊形，融合處呈鋪路石樣生長。結(jié)論 FN作為細(xì)胞外基質(zhì)與EGM2MV+50 ng/ml VEGF165組成的誘導(dǎo)液協(xié)同作用，構(gòu)成了hADSCs體外分化為內(nèi)皮細(xì)胞的最佳誘導(dǎo)體系，可為臨床上缺血性疾病

4、的自體細(xì)胞移植治療提供大量的種子細(xì)胞來源。 【關(guān)鍵詞】  人脂肪干細(xì)胞;流式細(xì)胞術(shù);內(nèi)皮分化;誘導(dǎo)體系【Abstract】 Objective To discuss the best method for endothelial differentiation of human adiposederived stem cells (hADSCs). Methods hADSCs were isolated from human adipose tissue by collagenase digestion and adherence to flasks, divided into t

5、hree groups and induced into endothelia respectively, including Fibronectin (FN)coated, gelatincoated group and noncoated group. At the same time, inducing medium was divided into pure endothelial cell growth medium (EGM2MV) and endothelial cell growth medium (EGM2MV) +50 ng/ml vascular endothelial

6、growth factor (VEGF165) groups. After induced 15 d, CD34，which was endothelial cellspecific surface antigen, was detected by flow cytometry. General morphological characteristics of hADSCs and induced hADSCs were observed by phasecontrast microscope. Results A high degree of homology and the CD34 ne

7、gative hADSCs were succeed to isolate and cultivate. At the end of differentiation, flow results of the three groups induced by simple EGM2MV all showed CD34 negative expression, but the other three groups induced by EGM2MV plus 50 ng/ml VEGF165 showed that CD34 expression rate of FNcoated group was

8、 8.4%, gelatincoated group was 5.6%, notcoated group was 4.2%; Induced hADSCs were triangular or polygonal, paving stonelike growth was observed at the place of local integration. Conclusions Combination of FNcoated and EGM2MV +50 ng/ml VEGF165 is the best method for endothelial differentiation of h

9、ADSCs. The results will offer abundant of seeding cells for autologous cell transplantation therapy of ischemic disease.【Key words】 Human adiposederived stem cells (hADSCs); Flow cytometry; Endothelial differentiation; Induction system 缺血性疾病是當(dāng)今世界的常見病之一，尤以動(dòng)脈粥樣硬化性血管疾病為主要代表。目前常用的藥物療法不能徹底解決已經(jīng)發(fā)生狹窄的血管解剖結(jié)構(gòu)

10、，而動(dòng)脈搭橋術(shù)又面臨著遠(yuǎn)期通暢率不夠理想等問題，因此尋找新的治療方法和移植材料已成為晚期缺血性疾病研究的重要課題。近年來隨著干細(xì)胞研究的進(jìn)展，將干細(xì)胞作為種子細(xì)胞為缺血組織血管新生(vasculogenesis)提供了新機(jī)遇1，2，但如何便捷有效地獲取種子細(xì)胞,是影響組織工程血管應(yīng)用于臨床的一個(gè)重要問題。本實(shí)驗(yàn)旨在探討人脂肪干細(xì)胞(human Adiposederived stem cells，hADSCs)向內(nèi)皮分化的最佳誘導(dǎo)體系，為臨床上各種晚期缺血性疾病患者提供取材方便、適于體外大量培養(yǎng)增殖、無免疫原性的可供自體移植的種子細(xì)胞。1 材料與方法1.1 主要試劑 LGDMEM、10%優(yōu)質(zhì)胎牛

11、血清(Hyclone,USA)，0.25%胰酶、膠原酶(Invitrogen)，小鼠抗人單克隆抗體CD44、CD90及CD166(BD,USA)，小鼠抗人單克隆抗體CD29、CD105、CD34、CD45、HLADR(BioLegend,USA)，EGM2MV(Lonza,USA)，VEGF165(Peprotech，USA)。1.2 hADSCs的分離與培養(yǎng)及鑒定DMEM、10%FBS、100 U/ml penicillin,100 g/ml streptomycin)重懸5，37、5% CO2、飽和濕度的孵箱內(nèi)培養(yǎng)，48 h后首次換液，以后每隔3 d更換1次培養(yǎng)基，待細(xì)胞長到培養(yǎng)皿底面積8

12、0%時(shí)，用0.25%胰蛋白酶消化傳代，以下實(shí)驗(yàn)均取自P2代hADSCs。1.3 hADSCs內(nèi)皮誘導(dǎo)體系的探索 利用細(xì)胞因子及細(xì)胞外基質(zhì)等因素誘導(dǎo)hADSCs向內(nèi)皮細(xì)胞分化。為了探討細(xì)胞因子及細(xì)胞外基質(zhì)對(duì)hADSCs向內(nèi)皮細(xì)胞分化的影響，實(shí)驗(yàn)共分6組，分別為纖維連接蛋白(FN)包被與單純內(nèi)皮細(xì)胞生長培養(yǎng)基(EGM2MV)誘導(dǎo)組;FN包被與EGM2MV+50 ng/ml血管內(nèi)皮細(xì)胞生長因子(VEGF165)誘導(dǎo)組;明膠包被與單純EGM2MV誘導(dǎo)組;明膠包被與EGM2MV+50 ng/ml VEGF165誘導(dǎo)組;無包被與單純EGM2MV誘導(dǎo)組;無包被與EGM2MV+50 ng/ml VEGF165誘導(dǎo)組。hADSCs按照2×103個(gè)/cm2細(xì)胞密度接種，每隔3 d換液，誘導(dǎo)15 d。1.4 hADSCs內(nèi)皮誘導(dǎo)后表型鑒定 內(nèi)皮誘導(dǎo)結(jié)束后，消化收集細(xì)胞，分別與小鼠抗人單克隆抗體CD34(1100稀釋)室溫孵育30 min，與 FITC標(biāo)記的二抗(150稀釋)4避光孵育20 min，用300 l PBS重懸，流式細(xì)胞儀檢測。1.5