摘要
目的检测多能成体祖细胞(MAPC)的培养条件对猴骨髓间充质细胞(BMMSCs)和人脂肪干细胞(hADSCs)生长的影响,旨在获得更适合治疗视网膜变性疾病的供体细胞。方法通过细胞形态观察、MTT实验、克隆形成率、PCR检测、以及成脂、成骨、成软骨分化潜能检测等,研究MAPC培养条件下猴BMMSCs和hADSCs的特征,并用DMEM/LG和MAPC培养条件培养的hADSCs进行RCS大鼠视网膜下腔移植,通过视网膜电图(ERG)和TUNEL检测,判断细胞移植治疗对视功能及视网膜细胞凋亡的影响。结果与常规培养基相比,MAPC培养条件能促进猴BMMSCs增殖,细胞变小,但传2代后,细胞变得宽大扁平,出现衰老征象;然而,MAPC培养条件下的hADSCs细胞增殖能力及克隆形成率均增强,形成的克隆较大可稳定传10代以上,且具有成脂、成骨、成软骨的多向分化潜能,细胞表面标记物及细胞因子出现差异表达:CD140b、CD90、CD47、HGF和PEDF显著上调,CD73、CD105和IL-6显著下调。与对照组相比,移植DMEM/LG和MAPC培养条件培养的hADSCs(P4)3周后,RCS大鼠的B波波幅明显升高,外核层细胞凋亡明显减少。结论 MAPC培养条件培养的hADSCs显示出更好的视网膜神经保护作用,适合用于治疗视网膜退行性疾病。
Objective To investigate the effects of multipotent adult progenitor cell （MAPC） conditions on biological characteristics and therapeutic effects of monkey bone marrow mesenchymal stem cells （BMMSCs） and human adipose-derived stem cells （hADSCs）, thus to obtain optimal donor cells for treatment of retinal degeneration （RD）. Methods The cell morphology, MTT assay, clone formation, adipogenic, osteogenic, chondrogenic differentiation potential and qRT-PCR technique were used to detect biological characteristics of monkey BMMSCs and hADSCs. The hADSCs were cultured under MAPC condition medium and transplanted into the subretinal space of RCS rats. The retinal functions were evaluated by electroretingram （ERG） recording, histological examination and TUNEL assay. Results Compared with conventional medium, MAPC conditions promoted the growth of monkey BMMSCs with smaller cell size. However, two passages later by subculture, monkey BMMSCs changed into larger and flatter morphology, showing senescence characteristics. MAPC culture conditions, compared with conventional hADSCs culture medium, maintained hADSC’s morphology, high proliferative capacity and multi-lineage differentiation potentials in long-term culture （beyond 10 passages）, and fewer cells displayed senescence. Gene expression profiles for cell surface markers and cytokines showed a differential expression patterns：Under MAPC culture conditions, CD140b,CD90, CD47, HGF and PEDF were significantly up-regulated, whereas CD73, CD105 and IL-6 were significantly down-regulated compared with those under conventional culture medium. In comparison with untreated RCS rats, DMEM/LG-hADSCsand MAPC-hADSC transplanted RCS rats showed significantly improved b-wave amplitudes in ERG examination in 3 weeks after the transplantation, and significant reduction of apoptotic cells in the outer nuclear layer. Conclusions hADSCs treated with MAPC culture conditions were more effective in protect retinal neurons and hold great promise for clinical use in
出处
《中华细胞与干细胞杂志（电子版）》
2014年第1期21-28,共8页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
基金
国家重点基础研究发展计划(2013CB967501)
国家自然科学基金(C120111)
高等学校博士学科点专项科研基金(新教师基金:20100072120051)
关键词
全能干细胞
骨髓间充质干细胞
视网膜变性
细胞移植
Totipotent stem cell： Bone mesenchymal stem cells： Retinaldegeneration： Cell transplantation
