APOE在脊髓损伤干细胞移植治疗中的机理和应用研究

spinal cord injury has high incidence and great hazardous.Its treatment and Rehabilitation process is difficulty and complex,moreover it has a serious threat to human health. Recently experimental and clinical studies find that stem cell transfusion therapy has a certain effect on spinal cord injury,so it becomes one of the hot spots of the treatment of spinal cord injury research. Pre-through the DIGE-2DE more cases of umbilical cord blood stem cells implanted in the cerebrospinal fluid of patients before and after the protein expression profile differences,we found that 28 protein with anti-inflammatory of the anti-apoptotic and promote nerve growth and repairing. The expression levels of APOE in the umbilical cord blood stem cells after implantation continued elevated and the content of APOE positively correlated with the degree of recovery of patients. We speculate that the the ApoE may play an important role in the directed differentiation of umbilical cord blood stem cells into neurons. This subject is to be in the original experiment based on vitro experiments, animal models and clinical trials, explore the participating role and molecular mechanism of ApoE umbilical cord blood stem cells differentiate into nerve cell ; preliminary clarify the differences of ApoE in varying degrees of spinal cord injury patients and infact on never repairing; explore the feasibility of ApoE as indicator molecules and gene therapy on spinal cord injury.study the mechanism of this protein and to establish a set of criteria as indicators of the degree of spinal cord injury nerve repairing in accordance with the ApoE expression amount.

脊髓损伤发生率高、危害性大、治疗困难、严重威胁人类健康。近期的实验和临床研究发现脐血干细胞移植治疗对脊髓损伤具有一定的疗效，是目前治疗脊髓损伤修复的研究热点之一。前期通过DIGE-2DE比较多例脐血干细胞植入前、后患者脑脊液蛋白质表达谱的差异，发现了28个与抗氧化、抗细胞凋亡和促进神经生长和修复相关的蛋白质，其中APOE（Apolipoprotein E ）在植入脐血干细胞后表达水平持续升高，且APOE的含量，与患者的恢复程度正相关。因此，我们推测ApoE可能在脐血干细胞向神经细胞定向分化中发挥重要作用。本课题拟在原有实验基础上，通过体外实验、动物模型和临床实验，探索ApoE对脐血干细胞分化为神经细胞过程中参与的作用和分子机理；初步阐明ApoE在不同程度的脊髓损伤患者中的差异及其对神经修复的影响；探索ApoE作为神经修复指标分子和脊髓损伤基因治疗的可行性。

脊髓损伤发生率高、危害性大、治疗困难、严重威胁人类健康。近期的实验和临床研究发现脐血干细胞移植治疗对脊髓损伤具有一定的疗效，是目前治疗脊髓损伤修复的研究热点之一。本课题拟在原有实验基础上，通过体外实验、动物模型和临床实验，探索ApoE对脐血干细胞分化为神经细胞过程中参与的作用和分子机理。收集50例脊髓损伤患者，对患者进行APOE基因型测定，对入选患者进行综合康复治疗训练，患者分别于入院时、治疗1个月后进行综合评估，观察其ASIA脊髓损伤程度、轻触觉、针刺觉评分，运动评分及日常生活活动能为（FIM）等方面的变化，分析APOE基因多态性对脊髓损伤康复治疗效果的影响。本研究采用脑立体定位仪自制脊髓打击器制作小鼠脊髓损伤动物模型，探究ApoE在脊髓损伤后的修复作用及其可能的作用机制。建模后每天行BBB功能评分直至实验结束，记录脊髓损伤后小鼠运动功能恢复状态。对脊髓损伤处行H&E染色，观察损伤节段细胞水肿及炎症反应；体外试验：将PC12转染含人ApoE3基因的pEGFP质粒及空载体pEGFP质粒，经持续筛选建立起稳定表达人ApoE3基因和质粒pEGFP的PC12的细胞系，用H2O2诱导4小时，通过CCK-8检测细胞存活率，Hochest33258、流式检测细胞凋亡率。通过本研究发现脊髓损伤患者神经功能的恢复与APOE基因多态性相关，携带APOE4的脊髓损伤患者运动功能和日常生活能力恢复情况较差，在轻触觉和针刺觉的恢复方面APOE4携带者和非携带者没有显著差异 。脐血干细胞移植治疗后，患者感觉和运动评分均有不同程度的增加，但是均未能引起ASIA残损分级的变化；3名患者脐血干细胞移植治疗之前、移植后1月、3月的均有明显改善，表现为Barthel指数评分明显提高； 脊髓损伤后小鼠体内ApoE表达含量随着病程进展呈进行性增加，说明ApoE与脊髓损伤后修复有重要关系；通过两种小鼠脊髓损伤后BBB评分和损伤节段H&E染色结果对比，从主客观两方面表明，ApoE缺乏的情况下，小鼠后肢运动功能恢复进程较缓慢，炎症反应加重，提示ApoE对脊髓损伤后的神经修复有正向作用。ApoE3基因高表达对H2O2所致的PC12细胞氧化损伤有很好的保护作用