中国组织工程研究与临床康复2011，Vol.15Issue(1)：1-6,6.DOI:10.3969/j.issn.1673-8225.2011.01.001
核转染对成体骨髓源性干细胞的影响
Nucleofection effects on adult bone marrow-derived stem cells
摘要
Abstract
BACKGROUND: Adult bone marrow-derived stem cells are promising cell source in cell therapy. Tracking of adult bone marrow-derived stem cells is crucial to demonstrate the mechanism of stem cell migration and differentiation,and develop novel strategy for functional regeneration and stem cell therapy.OBJECTIVE: To explore effects of transfected adult bone marrow clonogenic stem cells (AMCSCs) on cell phenotype, proliferation and cardiac differentiation potential.METHODS: Plasmid-encoded reporter gene maxGFP was used for nucleofection of AMCSCs with U-23 program.Growth curves of AMCSCs before and after nucleofection were compared based on results of MTT assay. AMCSCs before and after nucleofection were treated with 3 μmol/L 5-azacytidine for inducing cardiac differentiation. The cardiac differentiation specific markers, GATA4 and MLC-2v, were applied to confirm cardiac differentiation by RT-PCR. The maxGFP transfected AMCSCs were conducted the intramyocardium injection into an adult Sprague-Dawley rat left anterior descending coronary artery (LAD)ligation model to trace the in vivo expression of transfected maxGFP gene. Fluorescence images of the injected heart were analyzed on days 2 and 7 postinjection.RESULTS AND CONCLUSION: At 24 hours following nucleofection, the transfection efficiencies of AMCSCs at passages 47 and 119 were 49.4％ and 43.1 ％ . On 5 hours, green fluorescence positive cells were observed. Following nucleofection,AMCSCs maintained round shape, and could adhere and show clone-shape growth. MTT assay results demonstrated that the passages 47 and 119 of AMCSCs exhibited similar growth curves before and after nucleofection. Mean population doubling time was 8.57 and 10.28 hours in passages 47 and 119 of AMCSCs prior to nucleofection, and 9.42 and 10.42 hours following nucleofection(P=0.551 , P=0.774). RT-PCR results showed that AMCSCs expressed GATA4 before and after 5-azacitidine treatment prior to nucleofection, and strongly expressed MLC-2v strip after treatment. AMCSCs expressed GATA4 prior to and following 5-azacitidine treatment after nucleofection, and strongly expressed MLC-2v after treatment. No significant difference was determined in above-mentioned indexes prior to and following nucleofection. In vivo experiment results demonstrared that a few green fluorescence positive cells were apparent in injected myocardium on days 2 and 7 following transfected AMCSCs injection.Results indicated that nucleofection is an effective and fast method for transfection of exogenous DNA into cell. The AMCSCs which are experienced with nucleofection are able to maintain their morphology, proliferation and cardiac differentiation potential.However, only a few transfected AMCSCs express the transferred gene, GFP, after intramyocardium injection.关键词
成体骨髓呈克隆样干细胞/核转染/增殖/分化/示踪分类
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李震,沈晓涛,曹亮,毛颖玉,陈斯韵,郑馨,蔡冬青..核转染对成体骨髓源性干细胞的影响[J].中国组织工程研究与临床康复,2011,15(1):1-6,6.基金项目
国家"863"项目(2007AA02Z105),课题名称:针对缺血心脏血管改变靶向子的鉴定及其效应的研究.国家自然科学基金项目(30973158),课题名称:与心脏衰老相关表型的功能研究.国家自然科学基金项目(30770886),课题名称:BDNF-TrkB通路与老年缺血坏死心肌再生相关机制的研究.国家自然科学基金项目(30570369),课题名称:心脏血管特异性靶向短肽H1靶向介导缺血心肌血管新生的研究.国家自然科学基金项目(30340038),课题名称:新筛选能特异性靶向心脏血管短肽的作用研究.广东省自然科学基金重点项目(04105826),课题名称:新筛选心脏血管特异性短肽的靶向作用研究.广东省科技攻关项目(2004B30601007),课题名称:老年心脏血管新生功能下降的蛋白组学研究.广东省国际合作项目.中央高校基本科研业务费专项资金. （2007AA02Z105）
