rEPO联合MSCs移植治疗早产儿脑白质损伤的疗效及其机制研究

WWith the increasing survival rate in critically ill preterm neonates, brain white matter damage(WMD) steps forward and manifests as a major cause of cerebral palsy. Currently,the effects of rEPO and MSCs transplantation for brain damage diseases are affirmative in adults, while the therapy have not been confirmed and used for WMD in premature neonates. Earlier we used rEPO and MSCs to intervene the animal model of premature WMD.With the BrdU cell tracer and Micro-MRI,we discovered homing to the damage area. Using protein and molecule technics and neurobehavioral tests,we also proved that MSCs can promote nerve growth and repair on physical and functional level.The propose: observe the regulatory effect of rEPO to SDF-1 / CXCR4 predominant in homing .With the following technics: Erythropoietin nanoparticles structuring, genetic engineering for CXCR4 up/down-expression,SDF-1 blocking,Micro-MRI,neurobehavioral tests, we will try to observe, in vivo, if the homing effect of SDF-1/CXCR-4 exists in premature brain and to explain and evaluate the role of rEPO in the homing of MSCs, mainly its effect on SDF-1/CXCR-4 . From the perspective of prenatal and postnatal care, this preclinical research meet requirement of the National Natural Science Foundation of China.

随着危重早产儿存活增加，早产儿WMD突出，易致脑瘫。目前rEPO、MSCs治疗成人脑损伤性疾病疗效是肯定的，但尚未有用于早产儿WMD治疗和研究。前期课题组运用rEPO和MSCs干预早产儿WMD模型，通过BrdU标记追踪、Micro-MRI发现MSCs定向归巢到未成熟脑损伤部位；蛋白和分子水平、神经行为评估证实有明显促进神经生长和修复现象。本次拟：探讨rEPO对早产儿WMD模型MSCs归巢中起核心作用基质细胞衍生因子1及其受体对未成熟脑功能性趋化与调控效应的影响机制；构建rEPO纳米粒子，利用在体观察、基因转染检测CXCR4过/沉默表达、SDF-1受体阻断技术正负检测；Micro-MRI、神经行为近远期评估SDF-1/CXCR-4在rEPO作用下对未成熟脑损伤应用MSCs的归巢影响，揭示rEPO对MSCs在早产儿WMD中神经修复机制。本研究是国家基金鼓励优生优育创新性的基础研究。

目的：探讨EPO对早产儿WMD模型MSC归巢中起核心作用基质细胞衍生因子1及其受体对未成熟脑功能性趋化与调控效应的影响机制，构建EPO纳米粒子，利用在体观察、基因转染检测CXCR4过/默表达，SDF-1受体阻断技术正负检测，Micro-MRI、神经行为近远期评估SDF-1/次、CXCR4在EPO作用下对未成熟脑损伤应用MSC的归巢影响，揭示EPO对MSC在早产儿WMD中神经修复机制。结果：本研究成功以全骨髓贴壁法提取干细胞并完成鉴定，建立早产儿脑损伤模型，并以Brdu标记干细胞，发现干细胞有少量可通过血脑屏障进入脑实质，使模型鼠脑病理改变明显减轻，EPO对模型鼠进行干预，发现具有脑保护作用；初步证实BMSCs在未成熟WMD中的修复作用；大鼠骨髓间充质干细胞对脑白质损伤模型幼鼠脑内炎症环境有改善作用；完成了体外慢病毒感染BMSCs，沉默其CXCR-4的表达，并且探索CXCR-4低表达的BMSCs移植入脑白质损伤模型后，体外证实CXCR-4低表达的BMSCs迁移能力下降。意义：本研究源头创新，为早产儿脑白质损伤的临床治疗提供可靠的实验基础，为进一步探讨EPO及MSC联合治疗脑损伤提供新思路。