Pax6和Ngn2基因重编程星形胶质细胞为神经元治疗小鼠脑梗死的研究

Ischemic infarction causes neuronal deficits. Thus, close attention has been paid to neuronal replacement and regeneration after ischemic infarction. Lineage reprogramming, which can be defined as direct conversion of specific differentiated cell type into another lineage, has been a promising approach for producing functional cells. In our previous study, we found out that reactive glial cells induced by ischemic infarction retained the ability to resume proliferation and acquired stem cell properties both in the peri-infarction area and the remote region. However, whether reactive glial cells can be reprogrammed into functional neurons in the environment of ischemic infarction has not been reported. Therefore, we plan to apply a mouse model of ischemic infarction, and use direct reprogramming of reactive glial cells into neurons by force expression of Pax6 and Ngn2, which are two intrinsic neurogenic determinants of astrocyte precursor cells. The identity of reactive glial cells following retroviral transduction will be determined by time-lapse video microscopy. Immunofluescence staining will be performed to track whether reprogrammed neurons express neuronal markers. To confirm the neuronal identity of the reprogrammed neurons, patch clamp recordings will be performed. By investigating the underlying molecular mechanisms, our study may provide an experimental basis for a promising new approach to initiate brain repair cell replacement and regeneration after ischemic infarction.

脑梗死后神经元损伤和死亡导致功能缺失，如何运用神经元替代和再生进行修复是目前研究的热点。谱系重编程是直接将成熟细胞转分化为其他类型的功能细胞。本课题组前期研究发现，小鼠脑梗死后皮层病灶周围和丘脑远隔部位大量增生的应激性星形胶质细胞表现出神经干细胞的特性，但能否通过重编程将梗死后应激性星形胶质细胞诱导为功能性神经元目前尚不清楚。本项目拟采用脑梗死小鼠模型，应用调控神经胶质前体细胞分化的两个关键因子Pax6和Ngn2，将应激性星形胶质细胞重编程为神经元，经细胞示踪技术动态观察细胞转化过程及其是否表达成熟神经元表面标记分子；通过膜片钳检测这些神经元是否整合入局部神经网络并具备活性功能，进而探讨其可能的分子机制，为脑梗死后细胞替代和再生治疗提供实验依据。

脑卒中是全世界范围内三大主要的致死致残性原因之一，具有发病率、致残率及死亡率均高的特点。脑梗死后导致神经元损伤和死亡从而使功能缺失。因此我们的研究目的主要是有效地促进脑梗死后神经元再生以治疗脑梗死及功能恢复。我们采用内皮素皮层注射法制作脑梗死小鼠模型，应用调控神经胶质前体细胞分化的两个关键因子Pax6和Ngn2，通过病毒载体将应激性星形胶质细胞重编程为神经元。我们将小鼠分为实验组、对照组、手术组和假手术组，分别对其进行Nissl染色、GFAP免疫荧光染色、行为学测试。手术组与假手术组脑片Nissl染色以及行为学测试结果表明我们所采用的皮层内皮素注射法能够成功建立脑梗死模型，并产生相应的运动功能行为学改变，与临床上的脑梗死症状相一致。与假手术组比较，手术组的手术皮层区可看到高表达的GFAP绿色荧光，提示脑梗死后有大量的星形胶质细胞增生，为本研究的星形胶质细胞重编程为神经元提供了基础支持。本研究的体外实验是将转录因子Pax6和Ngn2通过慢病毒感染离体培养的星形胶质细胞，在荧光显微镜下观察细胞的形态变化，发现有神经元生成，提示转录因子Pax6和Ngn2能够将星形胶质细胞重编程为神经元。