摘要
背景:一些研究已显示,重组脑源性神经营养因子基因修饰的骨髓间质干细胞具有向神经样细胞分化的潜能,且能提高脑缺血模型鼠的神经缺失功能。目的:拟进一步观察脑源性神经营养因子基因修饰的骨髓间质干细胞移植对阿尔茨海默病鼠学习记忆能力的改善。设计、时间及地点:随机对照动物观察,于2006-01/2007-06在中山大学一附院神经病学实验室完成。材料:清洁级8周龄雄性SD大鼠48只,随机分为正常对照组、损伤模型组、基因修饰细胞组、未修饰细胞组,12只/组。另取出生3d的清洁级SD大鼠10只,用于骨髓间质干细胞的分离培养。方法:取传至第6代的骨髓间质干细胞,双酶切后亚克隆至pcDNA3质粒,行增强型绿色荧光蛋白基因转染,采用电穿孔法得到重组腺病毒Ad-BDNF,转染293细胞进行病毒包装。除正常对照组外,其余3组大鼠均建立阿尔茨海默病模型。造模后12d,基因修饰细胞组取重组腺病毒脑源性神经营养因子基因修饰的不含血清骨髓间质干细胞悬液,于伤侧侧脑室坐标前囟后1.6mm、外侧1.5mm、腹侧4.0mm注入8μL;未修饰细胞组移植等量的单纯骨髓间质干细胞悬液。主要观察指标:Western blotting法鉴定重组病毒在骨髓间质干细胞中表达,流式细胞仪检测基因感染效率。细胞移植后2周采用Morris水迷宫检测大鼠学习记忆能力。结果:Ad-BDNF感染的骨髓间质干细胞存在Mr14000的脑源性神经营养因子蛋白条带,2d后约34%骨髓间质干细胞被感染。基因修饰细胞组、未修饰细胞组大鼠平均逃避潜伏期均明显低于损伤模型组(P<0.01),且前者基本达到正常水平。与损伤模型组寻找平台的运动策略比较,基因修饰细胞组与未修饰细胞组趋向式、直线式显著增多(P<0.01),边缘式、随机式明显减少(P<0.01),且前者基本达到正常水平。结论:移植的骨髓间质干细胞经脑源性神经营养因子基因修饰后,可明显改善阿尔茨海默病模型鼠的记忆能力。
BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） modified by recombinant brain-derived neurotrophic factor （BDNF） gene has a ability to differentiate into nerve-like cells, and promotes nerve deletion of rat models of cerebral ischemia. OBJECTIVE： To observe the memory amelioration of Alzheimer＇s disease model rats transplanted with BMSCs modified by BDNF gene. DESIGN, TIME AND SETTING： The randomized control animal observation was performed at the Neurologia Laboratory of First Affiliated Hospital of Sun Yat-sen University from January 2006 to June 2007. MATERIALS： A total of 48 clean male Sprague Dawley rats aged 8 weeks were randomly assigned into a normal control group, an trauma group, a modified group and a unmodified group, 12 in each group. An additional ten clean Sprague Dawley rats aged 3 days were obtained for isolation and culture of BMSCs. METHODS： At the sixth passage, BMSCs cloned to pcDNA3 plasmid following double enzyme digestion and received enhanced green fluorescent protein gene transfection. Recombinant adenovirus Ad-BDNF was obtained by electroporation. Transfection 293 cells received virus package. Except the normal control group, rat models of Alzheimer＇s disease were established in other three groups. At 12 days after modeling, 8 μ L serum-flee BMSC suspension modified by Ad-BDNF gene was infused into 1.6 mm posterior to the anterior fontanel, 1.5 mm from lateral side and 4.0 mm from ventricle side in the injured lateral ventricle of rat models in the modified group. The same volume of BMSC suspension was infused into rats in the unmodified group. MAIN OUTCOME MEASURES： Recombinant virus expression in BMSCs was detected by Western blotting. Gene efficiency of infection was measured by flow cytometry. At 2 weeks after transplantation, rat learning memory ability was examined by Morris water maze. RESULTS： Ad-BDNF infected BMSCs had Mr14 000 BDNF protein strap. About 34% BMSCs were infected at 2 days. Mean escaping latency of the modified group and unmodified group was shorter than that of the trauma group （P 〈 0.01 ）, and the modified group mostly reached a normal level. Compared to the motion strategy of trauma group, trend and orthoscopic movement increased remarkably in the modified group and unmodified group （P 〈 0.01 ）, and marginal and random strategies decreased significantly （P 〈 0.01）, and the modified group mostly reached a normal level. CONCLUSION： The memory amelioration of Alzheimer＇s disease model rat was obtained by transplanting BDNF-gene modified BMSCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第38期7431-7434,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
贵州省优秀科技人才省长基金(黔省专合字(2005)231号)~~
