体外培养大鼠星形胶质细胞缺营养损伤模型的制作

The Formation of Innutritive Astrocyte Model of Rat in Vitro

目的制作理想的大鼠星形胶质细胞的损伤模型，观察形态变化。方法使用牛后3d内的SD乳鼠大脑皮质制成细胞悬液后，采用含15％的胎牛血清的DMEM／F12培养基培养，通过差速贴肇和不同速度的摇床及逐渐传代纯化星形胶质细胞。采用PBS缓冲液代替培养基模拟缺营养模型，缺营养3h后恢复营养，观察细胞缺营养前后形态变化。同时使用RT-PCR检测损伤后晕形胶质细胞GFAP mRNA表达的变化来明确缺营养损伤模型中晕形胶质细胞对缺营养损伤的反应。结果缺营养3h后部分细胞胞体变小，胞突变短，个别细胞呈网形，复营养后大多数细胞胞体变大，胞突变长并相互交错，基本恢复缺营养前形态，仍然可见少数细胞死亡漂浮在贴肇细胞层面上。RT-PCR检洲发现损伤后星形胶质细胞GFAPmRNA表达较正常组明显增强，且有统计学意义。结论通过上述方法培养后可获得高纯度的星形胶质细胞，原代星形胶质细胞可耐受一定的缺营养损伤．恢复营养后可基本恢复至损伤前的形态。

Objective To establish the nulli-nutrition model of rat astrocytes and observe the morphological change.Methods The astrocytes were isolated from cerebral cortex of neonatal rats which are born within 3 days, inoculated in the 0.01% poly-L-lysine-coated 75cm2 bottle by 10^7 - 10^9/L,placed in the incubator （5% CO2,3 7 ℃）, cultivated by DMEM/F12 media added with 15% fetal bovine serum for 8 days,and depurated by differential velocity adherent technique, swinging and passaging.Making nulli-nutrition model by PBS buffer instead of the medium for 3h to observe cell morphological changes before and after lacking of nutrition.Using the RT-PCR detection the expression of GFAP mRNA response to nulli-nutrition in injuried astrocytes.Results After lacking of nutrition for 3h,part of cells＇ body become smaller,cytoplasmic process become shorter,few of cells become round,after re- nutrition,it was similar with the state before lacking ＆nutrition,the majority of cell body become larger, the cytoplasmic process become longer and intertwined,few of cells dead.RT-PCR shows the expression ofGFAP mRNA significantly increased in injured astrocyte compared with control group,and there is statistical significance.Conclusions Shaking and differential velocity adherent technique can obtain high-purity astrocytes,primary astrocytes may tolerante a certain lack of nutritional damage,the restoration of nutrition can be restored to normal form.