不同来源成体干细胞BMP-2基因转染并微囊化后细胞活性及分泌量观察  

Observation of viability and BMP-2 secretion of three different microcapsulated adult stem cells transfected with BMP-2 gene

作　　者：沈阳[1] 张书红[1] 汤亭亭[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院骨科/上海市骨科内植物重点实验室,200011

出　　处：《中国医药生物技术》2011年第2期96-100,共5页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30772183);上海教委重点学科建设基金(J50206)

摘　　要：目的比较不同来源成体干细胞BMP-2基因转染并微囊化后的存活情况,并检测分泌至微囊外的BMP-2蛋白数量。方法从大鼠的骨髓、脂肪和滑膜组织内分离培养不同类型的成体干细胞:骨髓间充质干细胞(BMSCs)、脂肪干细胞(ADSCs)和滑膜干细胞(SMSCs)。利用高压静电装置制备海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊,在荧光显微镜下观察绿色荧光蛋白(GFP)标记细胞在微囊内的存活情况。利用含有BMP-2基因的重组腺病毒感染不同来源的成体干细胞后微囊化包裹,用酶联免疫吸附法(ELISA)检测分泌到微囊外的BMP-2蛋白量。结果包裹4周后荧光显微镜观察发现,BMSCs、ADSCs和SMSCs可在微囊内长期存活。ELISA检测发现,3种转基因细胞在微囊化后都可以持续分泌BMP-2蛋白,其中BMSCs分泌最多,在第2、3、4周和其他2种细胞相比有明显差异(P<0.01)。结论 3种来源的成体干细胞都可以作为BMP-2基因给药促进骨再生研究的候选细胞,其中以骨髓来源的间充质干细胞为首选。Objective To test the viability and BMP-2 secretion from microcapsulated BMP-2 gene transfected adult stem cells derived from different tissue. Methods The bone marrow mesenchymal stem cells （BMSCs）, adipose-derived stem cells （ADSCs）, synovium-derived mesenchymal stem cells （SMSCs） from rat tissue were isolated and encapsulated by the alginate-poly-L-lysine-alginate （APA） microcapsules produced by high voltage electrostatic generator. The viability of GFP labeled cells in APA microcapsules were observed by fluorescence microscope at 1 and 4 weeks after encapsulation. Then the BMSCs, ADSCs and SMSCs were infected by the adenovirus containing BMP-2 gene and encapsulated in APA microcapsules. The ELISA method was used to determine the BMP-2 secretion in supematant of cultured microcapsules. Results The viability of encapsulated GFP labeled stem cell could be demonstrated by the microscope at 1 and 4 weeks after encapsulation. All encapsulated BMP-2 gene-transfected stem cells were capable of constitutive secrete BMP-2 proteins. BMSCs could secret most BMP-2 proteins which was significant higher than ADSCs or SMSCs at 2, 3 and 4 weeks （P 〈 0.01） after BMP-2 gene transfection and cells encapsulation. Conclusion All three kinds of adult stem cells could be used to promote the bone regeneration by the strategy of microcapsule based BMP-2 gene medicine, among which BMSCs is the first choice.

关 键 词：成体干细胞 骨形态发生蛋白质类 基因转移技术 

分 类 号：R683[医药卫生—骨科学]