TrKA基因调控对BMSCs体内分化的影响及相关机制研究

Our previous study demonstrated that BMSCs could survived and differentiated into Schwann-like cells in vivo in monkey radial-nerve model. TrKA is receptor of NGF,but we haven't find the relation between TrKA and the BMSCs' differentiation in vivo. Study in-vitro implied that TrKA make the neuron cell differentiate and grow, MEK/ERK/MAPK pathway is related to the regulation of differentiation of cell, Bcl-2 also have effects on the neuronal differentiation.Therefore, we attempt to establish BMSCs with stable overexpression of TrKA and to emxamine how TrKA impact the differentiation of BMSCs in-vivo.As to further investigate the mechanism of TrKA's impact on the differentiation of BMScs in-vivo, we also attempt to establish BMSCs with transfection of Bcl-2 gene or MAPK gene, a combined method with MTT，flow cytometry，TUNEL, ELISA assays, RT-PCR，western blot，zymography, immunohistochemistry and luciferase will be applied to vetify how TrKA modulates the differentiation of BMScs in-vivo. We hope our work may clarify TrKA's impact and its mechanism on the differentiation of BMSCs in-vivo and then provide new therapeutic method for peripheral nerve defect after transplantation of tissue-engineered nerve seeded with BMSCs．

我们已证明，自体BMSCs植入体内可分化为类雪旺细胞，并促进神经再生。TrkA与神经元存活、分化有关。那么，TrKA对BMSCs体内分化的影响及相关机制是什么？体外实验证明，MEK/ERK/MAPK通路参与调控细胞分化，Bcl-2可影响细胞神经样分化。由此，我们首次提出TrKA及其通路对BMSCs体内分化的调控机制：NGF与TrKA结合，由MEK/ERK/MAPK通路调控Bcl-2基因表达，从而影响细胞分化。以此为基础，本课题拟：分别建立以TRKA稳定高表达BMSCs、Bcl-2稳定高表达BMSCs和MAPK稳定高表达BMSCs为种子细胞的鼠神经缺损模型，验证TrKA通过激活MEK/ERK/MAPK通路完成调控Bcl-2基因表达功能。本项目将阐明TrKA及其信号通路对BMSCs体内分化的调控机制，为确立BMSCs作为高效组织工程化神经种子细胞提供充分的科学依据。

【背景】周围神经缺损的修复及功能恢复一直是神经学科的前沿问题之一，组织工程化神经为周围神经修复带来了新希望。BMSCs作为组织工程化神经种子细胞得到广泛应用。.申请人前期研究表明：自体BMSCs植入猕猴周围神经缺损后，可在体内诱导分化为类雪旺细胞并促进神经再生。TrkA被公认为NGF的受体，此受体与NGF结合后, 可依次引起级联反应维持神经元存活和生长。体外实验表明，Ras /MEK/ERK/MAPK信号通路是NGF诱导细胞分化及存活的重要信号转导途径；Bcl-2家族蛋白是在神经细胞分化及信号传导过程中起重要作用的一类蛋白质。申请人前期研究结果提示，自体BMSCs植入体内后内NGF及TrKA表达水平显著提高。.【研究内容】本课题分别建立以TRKA稳定高表达BMSCs、Bcl-2稳定高表达BMSCs和MAPK稳定高表达BMSCs为种子细胞的鼠神经缺损模型，验证TrKA通过ERK/MAPK/Bcl-2通路调控并促进BMSCs体内分化为类雪旺细胞。.【重要结果，结论及意义】（1）TrkA表达上调可以明显促进BMSCs体内分化为类雪旺细胞，TrkA表达下调可以显著抑制BMSCs体内分化为类雪旺细胞。（2）TrkA表达上调可以明显减少BMSCs在体内移植物内死亡，TrkA表达下调可以显著增加BMSCs在移植物内死亡。（3）TrKA表达上调的BMSCs促进神经纤维再生及神经功能恢复； TrKA表达下调的BMSCs抑制神经纤维再生及神经功能恢复。（4）TrkA可通过Erk1/2途径激活靶分子Bcl-2等的表达从而促进BMSCs体内分化为类雪旺细胞。.本项目研究表明，TrKA通过ERK/BCL-2通路调控、促进BMSCs体内分化为类雪旺式细胞并促进神经再生，结果有助于理解BMSCs体内分化为类雪旺细胞的调控机制。