第10号染色体缺失的磷酸酶及张力蛋白同源物基因／雷帕霉素靶蛋白调节细胞骨架构建在神经细胞分化过程中的变化及意义

Construction of cytoskeleton modulated by phosphatase and tensin homolog deleted on chromosome ten/mammalian target of rapamycin during the differentiation of bone marrow stem cell-derived nerve cells

目的观察在体外诱导成人骨髓基质干细胞向神经细胞分化过程中，第10号染色体缺失的磷酸酶及张力蛋白同源物基因（PTEN）／雷帕霉素靶蛋白（roTOR）调节细胞骨架构建的变化特征，并探讨其意义。方法从成人骨髓中分离基质干细胞，在体外经细胞因子诱导分化2周后成为神经样细胞；应用PTEN抑制剂BPV作用于该细胞，半定量逆转录-聚合酶链反应（RT-PCR）方法研究PTEN下游信号分子mTOR的表达变化；应用结合有荧光剂的鬼笔环肽染色细胞骨架，在荧光显微镜下观察细胞突起内细胞骨架的变化特征及测量细胞突起长度。结果成人骨髓来源的基质干细胞经诱导分化后，神经细胞特征性标志分子神经元特异性烯醇化酶（NSE）和B-Ⅲ微管蛋白（tubulin）表达水平增加，而神经干细胞的标志物巢蛋白表达先增加后减少（P〈0．05）；经BPV作用后mTOR的表达水平高于作用前（P〈0．05）；细胞突起生长的长度从（4．58±1．21）μm增加到（9．09±2．68）μm（P〈0．05）。结论BMSCs来源的神经细胞分化成熟时，PTEN／mTOR具有调节神经细胞突起内细胞骨架构建的能力。

Objective To observe the construction of cytoskeleton modulated by phosphatase and tensin homolog deleted on chromosome ten （ PTEN ）/mammalian target of rapamycin （mTOR） during the nerve cell differentiation by human bone marrow stem cells in vitro, and to analyze the implication. Meth- ods Adult bone marrow cells were isolated and induced into nerve-like cells by some cytokines in vitro af- ter 2 weeks. The mRNA expression of mTOR which is the downstream molecule of PTEN was compared be- tween un-treated and treated by the BPV （a type of PTEN inhibitor） using semi-quantitative reverse tran- scriptase-polymerase chain reaction （RT-PCR）. The construction patterns of cytoskeleton were indicated by Phalloidin-fluoresceine isothiocyanate （FITC）, and observed by the fluorescence microscopy. Results Neuron specific enolase （NSE） and β-Ⅲ tubulin were expressed in these cells after the induction by adult human bone marrow stem cells, nestin, the marker of neural progenitors was expressed increasingly at the beginning, and decreasingly subsequently. The mRNA expression level of roTOR was increased and the lenth of the neurites was from （4. 58 ± 1.21 ） μm to （9. 09 ±2. 68） μm after the cells were treated by BPV （P 〈 0. 05 ）. Conclusion When the nerve cells derived from bone marrow stern cells obtain the mature differentiation, PTEN/mTOR may possess the the ability of cytoskeleton construction modulation in the neuritis of nerve cells in vitro.