人羊膜间充质干细胞移植对阿尔茨海默病转基因小鼠的行为学和β-淀粉样蛋白的影响

Effects of human amnion membrane mesenchymal stem cell transplantation on behavior and beta-amyloid protein changes in transgenic mice with Alzheimer's disease

背景:研究发现APP基因与阿尔茨海默病发病密切相关。β-淀粉样蛋白是阿尔茨海默病患者脑内老年斑的主要成分,基因突变和外界环境的影响能破坏β-淀粉样蛋白的动态平衡,从而引发或加速阿尔茨海默病的产生和发展。目的:探讨人羊膜间充质干细胞尾静脉移植对阿尔茨海默病转基因小鼠学习记忆能力及脑组织β-淀粉样蛋白表达的影响。设计、时间及地点:随机对照动物实验,于2008-05/10在郑州大学微生物学与免疫学教研室和河南省中医药治疗研究院完成。材料:健康剖宫产产妇志愿捐献的羊膜,由郑州大学第一附属医院产科提供。APP转基因鼠29只,PCR技术鉴定转APP-基因小鼠9只,作为正常组;转APP+基因小鼠20只,随机分为细胞移植组、对照组,10只/组。方法:无菌条件下体外分离培养人羊膜间充质干细胞,传至第3代将细胞浓度调整为1×109L-1,经尾静脉注入0.5mL至细胞移植组小鼠体内;对照组经尾静脉注入同体积的生理盐水;正常组小鼠不给予任何干预措施。主要观察指标:采用Morris水迷宫测定小鼠逃避潜伏期、穿越平台次数及在平台象限的时间,刚果红染色观察小鼠脑组织内β-淀粉蛋白的表达。结果:定位航行试验中,移植前与正常组比较,细胞移植组、对照组小鼠逃避潜伏期差异均有显著性意义(P<0.05);移植后2周细胞移植组小鼠逃避潜伏期与正常组基本相似(P>0.05),但明显短于对照组(P<0.05)。空间探索试验中,移植前后小鼠穿越平台次数及其在平台象限的时间3组间比较差异均无显著性意义(P>0.05)。移植后1个月,正常组未见或仅见极少量的β-淀粉样蛋白沉积,细胞移植组淀粉样蛋白的沉积明显少于对照组。结论:人羊膜间充质干细胞尾静脉移植能促进阿尔茨海默病转基因小鼠空间定位及学习记忆能力的提高,且减少小鼠脑内β-淀粉样蛋白的沉积。

BACKGROUND： APP gene is closely associated with the onset of Alzheimer＇s disease. β -amyloid is the main ingredient of senile plaque in the brain of Alzheimer＇s disese patients. Gene mutation and environment can destroy the dynamic balance of β -amyloid, resulting in the occurrence and development of Alzheimer＇s disease. OBJECTIVE： To study the effects of human anmiotic membrane derived mesenchymal stem cell transplantation via tail venous injection on the capacity of learning, memory and β -amyloid changes in Alzheimer＇s transgenic mice. DESIGN,TIME AND SETTING： The randomized controlled experiment was performed at the Department of Microbiology and Immunology, Zhengzhou University and Henan Institute of Traditional Chinese Medicine from May to October 2008. MATERIALS： The amnion membrance was harvested from healthy puerperants were supplied by Department of Obstetrics, First Affiliated Hospital, Zhengzhou University. A total of 29 APP trangenic mice and 9 APP- trangenic mice identified by polymerase chain reaction served as normal group. A total of APP＋ trangenic mice were equally and randomly assigned into cell transplantation group and control group. METHODS： Human amnion membrane derived mesenchymal stem cells were sterilely isolated and cultured. The third passages of cells （1×10^9/L） （0.5 mL） were transplanted by tail venous pathway to mice. Mice in the control group were injected with an equal volume of saline, whereas mice in the normal group were left intact. MAIN OUTCOME MEASURES： Morris water maze method was adopted to detect the escaping latency, crossing platform times and platform quadrant. Congo red staining was utilized to observe β -amyloid expression in mouse brain tissues. RESULTS： Before transplantation, in location navigation tests, there were significant differences in escaping latency in the cell transplantation group and control group compared with the normal group （P 〈 0.05）. At 2 weeks following transplantation, the escaping latency was similar in the cell transplantation group and the normal group （P 〉 0.05）, which was significantly shorter than in the control group （P 〈 0.05）. In the exploration experiment, no significant difference was detected in crossing platform times and platform quadrant before and after transplantation （P 〉 0.05）. At 1 month following transplantation, no or few β -amyloid expression was found in the normal group. β -amyloid expression was significantly less in the cell transplantation group than in the control group. CONCLUSION： Human amniotic membrane mesenchymal stem cell transplantation through the tail vein could promote the learning and memory ability in space and reduce β -amyloid expression in the brain in Alzheimer＇s disease mice.