谷氨酸对人骨髓间充质干细胞中Par-4蛋白表达的影响

Effects of Glutamate on Expression of Par-4 Protein in Human Bone Mesenchymal Stem Cells

目的探讨谷氨酸对人骨髓间充质干细胞(BMSCs)中Par-4蛋白表达的影响及意义。方法用BMSCs分离液和密度梯度离心分离和培养人BMSCs,取第3～5代培养的细胞,分别加入小鼠抗人CD29-FITC、CD44-FITC、CD105-FITC、CD45-FITC、CD14-FITC、CD34-FITC单克隆抗体,流式细胞术鉴定其免疫表型。采用0.1nmol/L、1.0nmol/L、10.0nmol/L等不同剂量谷氨酸处理人BMSCs24h,并设正常对照组。Westernblot测定各组Par-4与突触素1(Synapsin-1)的蛋白表达量,并进行2种蛋白表达的相关性分析。结果在显微镜下,BMSCs呈星形或梭形。流式细胞术鉴定发现培养的细胞中CD29、CD44、CD105表达阳性,而CD45、CD14、CD34表达阴性,符合BMSCs特点。正常对照组Par-4蛋白相对表达量为17.43±5.9,0.1nmol/L、1.0nmol/L、10.0nmol/L谷氨酸诱导人BMSCs中Par-4蛋白表达依次升高,分别为39.33±8.2、49.45±11.5、86.41±14.9(Pa<0.01)。谷氨酸处理导致BMSCs中Synapsin-1表达下调,并呈剂量依赖性(F=49.1P<0.01)。10.0nmol/L谷氨酸处理24h后,Par-4蛋白的表达与Synapsin-1的表达量呈显著负相关(r=-0.643P<0.01)。结论谷氨酸诱导人BMSCs中Par-4表达升高,并可能使植入到缺血微环境中的BMSCs发挥神经细胞替代的作用受到负面影响。

Objective To investigate the effects and significance of glutamate on expression of Par - 4 protein in human bone mesenchyreal stem cells （BMSCs）. Methods Human BMSCs were incubated and were divided into normal group. With monoclonal antibody CD29 - FITC, CD44 - FITC, CD105 - FITC, CD45 - FITC, CD14 - FITC and CD34 - FITC, immunophenotype of the cells were detected by Flow cytometry. 0.1 nmol/L, 1.0 nmol/L, 10.0 nmol/L glutamate treated groups and normal control group were set up. Western blot was used to determine the protein levels of Par - 4 and Synapsin - 1. Correlation analysis of Par - 4 and Synapsin - 1 expressions was done. Results BMSCs was fusiform or star - shaped, which showed positive immunophenotype of CD29, CD44 and CD105. But CD45, CD14 and CD34 were negative. Glutamate induced an increase of Par - 4 protein level in a dose - depended way （ F = 49. 1 P 〈 0.01 ）. The relative expression level of Par -4 protein in normal control group was 17.43 ± 5.9. In 0.1 mnol/L, 1.0 nmol/L and 10.0 nmol/L glutamate - treated groups, Par -4 protein was respectively enhanced to 39.33 ± 8.2, 49.45 ± 11.5 and 86.41 ± 14.9 （ Pa 〈 0. 01 ）. The expression of Synapsin - 1 protein in human BMSCs with exposure of glutamate was significantly decreased （ F = 49. 1 P 〈 0. 01 ）. After human BMSCs were exposed to 10.0 nmoL/L glutamate for 24 h, the expression level of Par-4 protein was significantly inversely correlated with Synapsin- 1 protein level （ r = - 0. 643 P 〈 0.01 ）. Conclusions Glutamate induced the increase of Par - 4 in human BMSCs, which could impart the effect of BMSCs for nerve cell replacement therapy in isehemic microenvironment.