骨髓间充质干细胞向神经元样细胞分化前后基因及Ca^(2+)浓度的变化  

Gene expression and calcium ion concentration variation in bone marrow mesenchymal stem cells differentiating into neuron-like cells

作　　者：潘国强 齐凤平 徐洪 王相利 

机构地区：[1]迁安市人民医院,河北省迁安市064400

出　　处：《中国组织工程研究》2013年第27期5080-5086,共7页Chinese Journal of Tissue Engineering Research

摘　　要：背景: 单唾液酸四己糖神经节苷脂(Monosialotetrahexosylganglioside,GM1)在神经细胞的生长发育、分化、再生和细胞内外信息传递等多种生理过程中发挥重要作用。目的: 探讨GM1对骨髓间充质干细胞按照Woodbury经典诱导方案将其诱导分化为神经元样细胞前后基因方面和细胞内游离Ca2+浓度的变化。方法: 分离纯化SD大鼠骨髓间充质干细胞进行传代培养,传至第5代时,待细胞融汇成致密单层后,加入50mmol/L神经节苷脂设为GM1组,预培养24h后按照Woodbury经典诱导方案进行诱导,设置对照组,采用免疫组化和RealtimePCR技术分别检测诱导前后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白的蛋白和mRNA表达的变化,采用激光扫描共聚焦显微镜检测诱导前后细胞内游离钙离子浓度的变化。结果与结论: ①加入GM1组诱导分化后较对照组生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白表达水平增高(P<0.05),GM1能够促骨髓间充质干细胞诱导分化为神经元样细胞。②更换诱导液后,两组细胞内荧光强度逐渐增加,到100s达高峰值,其后逐渐减弱,但20min时细胞荧光强度仍高于诱导前,加入GM1组,细胞内游离Ca2+浓度较对照组有所增加(P<0.05)。说明GM1能够促进细胞内Ca2+浓度增加,游离Ca2+在诱导过程中可能有促进作用。③诱导后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白基因表达改变不显著,说明Woodbury经典诱导方案可能为转录后水平即蛋白水平的调控。METHODS： The mesenchymal stem cells from Sprague Dawley rats were cultured after isolated and purified, After 5 passages in culture, the cell integrated into a dense monolayer,,and treated with 50 mmol/l_ monosialotetrahexosylganglioside for 24 hours as the monosialotetrahexosylganglioside group; then the mesenchymal stem cells were induced into neuron-like cells with the methods of Woodbury after pre-cultured for24 hours, and set the control group. The protein and mRNA expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were detected by immunocytochemistry and real-time PCR, respectively. The fluorescence intensity of intracellular free calcium ion before and after inducing was detected by laser scan confocal microscope. RESULTS AND CONCLUSION： After induction, the expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin of the mesenchymal stem cells in the monosialotetrahexosylganglioside group were higher than those in the control group （P 〈 0.05）, demonstrating that monosialotetrahexosylganglioside could promote the differentiation of mesenchymal stem cells into neuron-like cells The fluorescence intensity in mesenchymal stem cells was increased gradually in two groups after the medium was replaced by the induction medium, attained its peak value at 100 seconds and then decreased gradually, but the fluorescence intensity was still higher than that before the induction at 20 minutes. The fluorescence intensity of intracellular free Ca2. was increased significantly in the monosialotetrahexosylganglioside group when compared with the control group （P 〈 0.05）, suggesting that monosialotetrahexosylganglioside could increase the concentration of intraceilular free Ca2＋, and intracellular free Ca2~ may be useful in the course of induction. The changes of protein expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were not significant after induction, indicati

关 键 词：干细胞骨髓干细胞神经节苷脂骨髓间充质干细胞神经元样细胞诱导分化免疫组化聚合酶链反应激光扫描共聚焦显微镜细胞内游离钙离子生长相关蛋白43神经元特异性烯醇化酶神经丝蛋白 神经巢蛋白干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]