脂联素基因转染的内皮祖细胞自体移植对缺血性脑损伤的保护作用

Vascular endothelial dysfunction is the initial and central change in ischemic brain. Vascular endothelial progenitor cells (EPC) can target the damaged vascular endothelia and repair it, also help vascular regeneration, while its level is insufficient. Our previous research demonstrated that autologous transplanted EPC after in vitro amplification could increase the vascular regeneration and improve the prognosis of brain ischemia, however, the application of EPC is restricted because of its limited resources and difficulty to culture, also its function will degenerate in pathological condition. It is reported that adiponectin can regulate and enhance the proliferation, differentiation, mobilization, homing of EPC, and regeneration of vessels. Moreover, APN itself can improve cells metabolism, resist inflammation and protect brain function after brain ischemia, while its reduced level in aging and brains with underlying disorders could not maintain the function of EPC. Based on these results, we prospect that the function of EPC and APN will be enhanced both if APN gene is transferred into EPC and expresses. Therefore, this study is designed to use lentiviral vector to construct the EPC lines with APN highly and stably expressed, then increase the proliferation activity of EPC by using stem cells differentiation technique, and transplant these EPCs to rats with brain ischemia. Finally evaluate the result and affecting factors of this method, and the mechanism as well, which will contribute the treatment of brain ischemia eventually.

血管内皮受损是缺血性脑损伤的始动因素和中心环节，而血管内皮祖细胞（EPC）具有靶向内皮修复和血管新生作用，但其体内含量不足。本课题组前期研究表明：将体外扩增后的EPC自体移植治疗脑缺血，虽能促进血管增生，改善预后，但EPC来源少、培养困难、病理环境下功能易退化，故应用受限。研究发现，脂联素（APN）能调控和强化EPC增殖、分化、动员、归巢及血管新生能力；另外，APN本身具有改善细胞代谢、抗炎症损伤及保护缺血性脑组织的功能，但APN随年龄增长和基础疾病影响而下降，不足以维持EPCs的功能。我们设想：将APN基因转入EPC并得以表达，二者的生物学功能将同时得以提升。为此，本研究拟利用"慢病毒载体"构建稳定高效表达APN的EPC系，采取干细胞去分化技术加强EPC增殖活性，然后自体移植EPC干预缺血性脑损伤大鼠，评估其功效和影响因素，同时探讨作用机制，为脑缺血损伤探索切实有效的治疗方法。

脑血管内皮受损是缺血性脑损伤的始动因素和中心环节，而血管内皮祖细胞（EPCs）具有靶向内皮修复和血管新生作用，脂联素（APN）能调控和强化EPC增殖、分化、动员、归巢及血管新生能力；但高龄、高脂血症、糖尿病、高血压等病理环境下，体内EPCs、APN含量双双下降、功能受损。如何大量的获得具有良好功能活性的EPCs、将其注入到不同的缺血性脑损伤大鼠，探索其神经保护效果及机制，是本实验的目的。为此我们体外分离培养EPC，正常培养8天、在MOI为100:1的条件下成功构建载有人脂联素基因的慢病毒载体感染EPC系（LV-APN-EPCs），有效表达了APN；以此种LV-APN-EPCs大鼠尾静脉注射干预不同；其中，在急性MCAO的实验中观察到干预组：第7天转杆实验改善、第14天测得的脑梗塞体积缩小、微血管密度增加，脑梗死交界区细胞凋亡减轻。在雌性大鼠急慢性缺血模型中观察到：14天时两细胞治疗组转杆实验结果无差别，其他结果类似前述研究，并且SRY基因原位杂交表明缺血病灶内APN-EPC组异体来源细胞较EPC组丰丰富；在糖尿病MCAO模型上的研究显示：LV-APN-EPCs干预的7天后，mNSS评分明显高于其他对照组，14天后MRI观察到的梗死体积明显小于对照组，CD31免疫荧光染色缺血脑组织周围皮质，观察LV-APN-EPCs组较LV-EPCs组，EPCs组和PBS组CD31+微血管密度大、促炎因子和促凋亡因子明显降低。本研究表明“慢病毒载体”构建的LV-APN-EPCs细胞能稳定高效表达高活性APN及高性能EPCs，移植干预缺血性脑损伤大鼠后，可能通过EPCs的内皮修复、血管新生作用和\或APN的抗炎作用来实现缺血性脑损伤的保护作用。