利用活体生物光学分子成像示踪不同途径移植人脐带间充质干细胞在脑缺血大鼠体内的功能及通心络的影响

A lot of animal experiments and some human clinical trials have proved that the transplant of HUCMSCs to treat the ischemic stroke is effective and safe. However, the evaluation of the study about tracting the distribution, survive, migration and differentiation of the stem cells in the living body is obviously less advanced than the observational study and the study of the mechanism. At the same time, it becomes the bottleneck and hotspot of the study about the stem cells. The early studies have showed that tongxinluo capsule can accelerate the proliferation and oriented differentiation. Based on that theory, if we can use the In vivo optical molencular imaging technology tracer technique to compare the distribution, survive, migration and differentiation of the HUCBMCs after the cerebral ischemia of the mice with HUCMSCs transplant through the veins, arteries, ventricles，parenchymal and LP，and the influence of the tonxinluo capsule and do the test in the different timepoints combining the vivo tracer tenique ,then maybe we can figure out the mechanism of the proliferation, differentiation and migration of the HUCBMCs transplant after the cerebral ischemia. It is really meaningful to observe the effect and mechanism of tongxinluo capsule after the cerebral ischemia thereby to testify the "collateral disease" theory. It is of great importance to study the mechanism of tongxinluo capsule and will do good to the communication of the western medicine and traditional Chinese medicine.

动物实验和小规模人体临床试验已证实，人脐带间充质干细胞（HUCMSCs ）移植治疗缺血性卒中是安全有效的，然而评价HUCMSCs不同途径移植后在动物活体内存活、分布、迁移、增殖及分化的差异及机制研究明显滞后于功能改善观察和机理的研究，成为干细胞治疗机制探讨的瓶颈因素。我们前期研究表明通心络对大鼠脑缺血后内源性NSCs的增殖与定向分化有明显的促进作用，有助于脑梗死患者移植HUCMSCs后功能改善。在此基础上运用活体生物光学分子成像技术示踪比较大鼠脑缺血后经静脉、动脉、脑室、脑实质内、腰穿移植HUCMSCs后不同时间点大鼠体内HUCMSCs分布、存活、迁移情况的差异及通心络干预后的影响,并在各时间点行组织学检测，与活体示踪法相结合，明确移植入的 HUCMSCs增殖、分化、迁移的机制，实时、动态观察通心络促进脑缺血损伤后HUCMSCs移植的疗效及机制，探讨中医药在干细胞移植治疗脑缺血中的作用。

本研究观察中药通心络对MCAO小鼠经颈动脉、静脉、脑实质内、侧脑室、腰穿五种途径移植Lenti-Fire Luciferase-eGFP-HUCMSCs后不同时间点在小鼠脑组织内的存活增殖分化的影响及机制，取得以下成果：.1. 经颈动脉途径移植HUCMSCs可明显减轻脑梗死体积，优于其它四种途径效果，通心络干预后可减轻各移植组的梗死体积；并探索了MCAO小鼠模型步态变异与基因表达的关系。.2.采用IVIS Spectrum system连续动态检测，发现总体经颈动脉移植组缺血侧HUCMSCs的荧光信号强度高于其它移植途径组。各组HUCMSCs荧光信号强度随时间延长而渐衰减，其中在第24h、3天时脑实质移植组荧光信号最强，第7天时经动脉组信号最强，后时点渐衰减，荧光信号强度依次为脑实质组、静脉组、侧脑室及腰穿组；中药通心络干预后各移植组HUCMSCs的荧光信号强度均高于移植组；.3.荧光免疫双标成像发现在缺血灶周边及SVZ、SGZ区，eGFP标记的HUCMSCs于各时间点的增殖分化能力不同，在第24h、3天时脑实质移植组eGFP+细胞增殖最强，于7天时颈动脉移植组增殖最强，后渐减弱，依次为脑实质组、静脉组、侧脑室及腰穿组；在各区分化以GFAP+细胞为主，少量Olig+细胞，30天始有少量NeuN+细胞表达。经通心络干预后各时间段HUCMSCs的增殖分化均高于细胞移植组，但无统计学差异。.4.HUCMSCs不同途径移植组脑缺血相关炎性因子的蛋白表达均明显减少，促进神经修复及血管再生的蛋白表达均明显增加，中药通心络有调节作用。.总结：总体趋势经颈动脉移植的HUCMSCs存活、增殖分化能力较强，对MCAO小鼠疗效最好；通心络干预后可增强其存活增殖分化能力，其作用机制与调控炎性因子表达及促进神经修复和血管再生有关。