高糖对高钾刺激引起大鼠星形胶质细胞外液谷氨酸含量的影响

发布时间：2018-05-15 11:17

本文选题：星形胶质细胞 + 高糖　；参考：《辽宁医学院》2012年硕士论文

【摘要】：目的探讨高糖对高钾刺激引起大鼠星形胶质细胞外液谷氨酸含量的影响。方法从新生(1d~3d)的SD大鼠的大脑皮质上提取星形胶质细胞（Astrocytes，AST），传代培养3~4代，并以胶质酸性纤维蛋白（Glial fibrillary acidic protein，GFAP）免疫荧光法对所获的细胞进行鉴定。然后随机分组：分别用不同浓度的葡萄糖细胞外液孵育胶质细胞，其浓度分别为5、10、20和40mmol·L~(-1)；然后在此基础上，加入氯化钾75mmol·L~(-1)，而葡萄糖的浓度没有变化，，和之前的保持一致，于0、1、2、3、4和5min后分别取细胞培养液测定谷氨酸（Glutamate，Glu）的含量。采用倒置显微镜观察胶质细胞的形态学变化；噻唑蓝（MTT）法测定胶质细胞的存活率；利用柱前邻苯二甲醛（OPA）和N-异丁酰基-D-半胱氨酸(IBDC)衍生高效液相色谱法测定细胞培养液中Glu的含量。结果经过免疫组化鉴定本试验培养的星形胶质细胞（AST）的纯度为95%以上。对培养的AST的细胞外液中的Glu含量进行了测定。并对Glu含量的测定方法进行了方法学研究，Glu浓度在12.5～200μg·L~(-1)范围内线性关系良好（r=0.9959）；峰面积的相对标准差为0.79%；平均回收率为98.5%，表明此Glu检测方法可靠。利用该方法测定了细胞外培养液中的Glu含量，结果发现用含有不同浓度葡萄糖的细胞外液孵育胶质细胞0，1和3h后，细胞培养液中Glu含量无明显变化，同时用MTT法测定了孵育3h后细胞的存活率，也无明显变化；高浓度的氯化钾（75mmol·L~(-1)）孵育1、2、3、4、5min后，含10、20和40mmol·L~(-1)葡萄糖的细胞培养液内Glu含量比含5mmol·L~(-1)葡萄糖均明显升高（P0.01）；但各组反应并不一致。甘露醇组Glu含量变化与5mmol·L~(-1)葡萄糖组相似。结论高糖可能通过促进星形胶质细胞(AST)谷氨酸释放及抑制吸收来影响细胞外液中的谷氨酸含量。从而提高细胞外液内的谷氨酸的含量。
[Abstract]:Purpose To investigate the effect of high glucose on the content of glutamate in extracellular fluid of astrocytes induced by high potassium stimulation in rats. Method Astrocytes were isolated from the cerebral cortex of SD rats and cultured for 3 ~ 4 generations. The cells were identified by immunofluorescence method. Then, the glial cells were incubated with different concentrations of glucose extracellular solution at concentrations of 5 ~ 10 ~ (10) ~ (20) and 40mmol ~ (-1) ~ (-1) respectively. Then, potassium chloride (75mmol) was added to the solution, and glucose concentration was not changed, which was the same as before. The content of glutamate Glutamate Glu4 was measured in the culture medium after 0 ~ 1 and 2 ~ (2) ~ (2) ~ (2) ~ (3) ~ (3) ~ (3) and 5min, respectively. The morphological changes of glial cells were observed by inverted microscope, and the survival rate of glial cells was determined by thiazolyl methacrylate (MTT) method. The content of Glu in cell culture medium was determined by high performance liquid chromatography with precolumn phthalaldehyde (OPA) and N- isobutylol-D- cysteine (IBDC) derivatives. Result The purity of cultured astrocytes was over 95% by immunohistochemistry. The content of Glu in extracellular fluid of cultured AST was determined. The method for the determination of Glu content was studied. The linear range of Glu concentration was 12.5 渭 g / L ~ (-1). The relative standard deviation of peak area was 0.79 and the average recovery was 98.5%, which indicated that the method was reliable for the determination of Glu in the range of 12.5 渭 g / L ~ (-1), and the relative standard deviation of peak area was 0.79% and the average recovery was 98.5 渭 g / L ~ (-1), respectively. The Glu content in the extracellular culture medium was determined by this method. The results showed that the Glu content in the cell culture medium did not change significantly after incubating glial cells with different concentrations of glucose for 0 and 3 hours. At the same time, MTT assay was used to determine the survival rate of the cells incubated for 3 h, and the survival rate of the cells was not significantly changed after incubation with high concentration of KCl (75mmol / L) for 5 minutes. The content of Glu in the cell culture medium containing 10 ~ (20) and 40mmol L ~ (1) glucose was significantly higher than that in the medium containing 5mmol L ~ (1), but the response was not consistent among the three groups. The change of Glu content in mannitol group was similar to that in 5mmol Ln-1) glucose group. Conclusion High glucose may affect the content of glutamate in extracellular fluid by promoting the release of glutamate from astrocytes and inhibiting its absorption. In order to increase the content of glutamate in extracellular fluid.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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