原代培养大鼠脑星形胶质细胞液压冲击损伤后蛋白差异性表达的研究

发布时间：2018-01-17 17:00

本文关键词：原代培养大鼠脑星形胶质细胞液压冲击损伤后蛋白差异性表达的研究　出处：《河北医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:建立原代培养星形胶质细胞液压冲击损伤模型,利用双向电泳结合质谱技术观察脑皮质星形胶质细胞液压冲击损伤后蛋白质组的表达变化,探寻脑损伤的特异性生物标志蛋白。方法:取出生后24h内SD大鼠(军事医学科学院实验动物中心提供)大脑皮质组织,于含有10%胎牛血清的DMEM-F12培养基中进行星形胶质细胞体外分离培养和纯化,并以胶质纤维酸性蛋白(GFAP)免疫细胞化学染色进行纯度鉴定。随机将细胞分为对照组和损伤后4h、8h、12h、24h、48h组,复制Scott's细胞液压冲击损伤模型(0.2MPa)。用台盼蓝染色观察星形胶质细胞损伤前后形态学变化,测培养液中LDH活力反映细胞的损伤程度,PI/Hoechst33342染色进行凋亡分析。提取细胞总蛋白,经双向电泳和MALDL-TOF质谱分析鉴定差异蛋白质。结果:1星形胶质细胞原代培养和纯化:经2-3次摇床传代纯化培养后的星形胶质细胞形态均一,胞体较大而扁、胞质较丰富、形状不规则;胞突丰富、分支多呈放射状,一级胞突较粗,常有二级分支;胞核圆形或椭圆形,常偏于胞体一侧。培养至24-26d,细胞生长状态良好,铺满皿底,形成大致均一的扁平细胞层。 2星形胶质细胞的纯度鉴定:采用GFAP免疫细胞化学染色(SP法),计数结果显示培养的星形胶质细胞纯度可达94.9%±1.9%。 3液压冲击损伤后星形胶质细胞形态学的改变:光镜下观察,细胞随时间发生间隙增大、肿胀、变圆、恢复,部分细胞发生皱缩、胞膜破裂、脱壁等显著的形态学改变;PI/Hoechst33342(1:1)荧光双染可见凋亡细胞。 4液压冲击损伤后细胞培养液中LDH的变化:与正常对照组细胞培养液相比,损伤后各组细胞培养液中LDH活力均降低,4h组降至最低,与对照组相比有统计学差异(P0.05)。然后其它组逐渐上升,48h组与对照组相比无统计学差异(P0.05)。 5液压冲击损伤后星形胶质细胞双向电泳图谱分析:双向电泳图谱经PDquest软件分析检测到的各组总蛋白点数:对照组1369±56,4h组1325±94,8h组1136±81,12h组1197±112,24h组1257±63,48h组1176±73。蛋白点多分布于pI5.2-6.6、MW30kDa-70kDa的范围;以对照组为参考胶,平均图谱匹配率达82%以上。损伤后共有265个蛋白质点的相对强度与正常对照组相比有显著性差异(P0.05),其中38个蛋白质点在损伤后表达升高,111个蛋白质点在损伤后表达下降,45个蛋白质点为损伤后新表达,有71个蛋白质点在损伤后各组均未表达。 6差异蛋白质的质谱鉴定:对9个差异蛋白点行MALDI-TOF质谱鉴定,其中8个点得分大于61分(P0.05),分别为60S酸性核糖体蛋白P2(60S acidic ribosomal protein P2)、细胞视黄醇结合蛋白(cellular retinol binding protein, Crbp)、脑脂肪酸结合蛋白7(brain fatty acid binding protein 7, FABP7)、S-100钙结合蛋白A11(S100 calcium binding protein A11, S100A11 )、假设蛋白LOC685814 ( hypothetical protein LOC685814)、真核生物翻译起始因子1A(Eukaryotic translation initiation factor 1A, eIF-1A)、乳腺癌扩增序列2同源体( Breast carcinoma amplified sequence 2 homolog, BCAS2)、钙结合蛋白3(calponin 3)。其中对FABP7行Western-blot验证,结果显示:FABP7在星形胶质细胞中存在表达,而且该蛋白在星形胶质细胞液压冲击损伤后的变化趋势与双向电泳图像经PDQuest软件分析后得到的蛋白质变化趋势相一致。结论:1液压冲击损伤可引起星形胶质细胞形态学发生改变,且有一定的时间依从性。 2液压冲击损伤能导致星形胶质细胞蛋白质组表达发生变化,损伤后共有265个蛋白点的相对强度与正常对照组相比有显著性差异,对其深入研究,有可能探寻到脑损伤的生物标志蛋白。 3经MALDL-TOF质谱分析初步确定的8种蛋白大致分为代谢调节、信号转导调节、翻译起始相关类及其它等四类,这些蛋白表达的变化表明它们在参与星形胶质细胞损伤后早期反应、促进细胞功能恢复、抑制细胞继发性损伤、参与细胞骨架组织重组及改善蛋白质合成等环节起重要作用,对其进行进一步研究,有望深入了解脑损伤的分子机制。
[Abstract]:Objective: to establish a primary hydraulic shock injury model of astrocytes, and to observe the proteome expression of astrocytes after hydraulic impact injury by two-dimensional electrophoresis and mass spectrometry, and to explore the specific biomarker of brain injury.
Methods: after the birth of 24h SD rats (provided by experimental animal center of Military Medical Science Academy of the PLA) in the cerebral cortex, containing 10% fetal bovine serum DMEM-F12 culture were cultured and purified astrocytes in vitro medium, and glial fibrillary acidic protein (GFAP) immunohistochemical staining for purity. The cells were divided into random the control group and the injury after 4h, 8h, 12h, 24h, 48h group, Scott's replication cell model of fluid percussion injury (0.2MPa). To observe the morphologic changes by trypan blue staining before and after astrocyte injury, measured the activity of LDH in liquid culture reflect the damage of cells, PI/Hoechst33342 staining for apoptosis analysis. The total proteins were extracted. The electrophoresis and mass spectrometry identification of differential protein MALDL-TOF.
Results: 1 astrocytes cultured and purified by 2-3 times table purified uniform morphology of astrocytes cultured, large cell body and flat, had abundant cytoplasm, irregular cytoplasmic processes; rich, multi branch radial, a cytoplasmic process is thick, often have two branches nucleus; round or oval, often partial on the cell body side. Cultured 24-26d, the cells were in good condition with the dish bottom, forming a layer of flat cells substantially uniform.
2 the purity identification of astrocytes: GFAP immunocytochemical staining (SP). The count results show that the purity of the cultured astrocytes can reach 94.9% + 1.9%.
3 hydraulic shock morphological changes of astrocytes after injury: under light microscope, cell gap increases over time, swelling, rounding, recovery, partial collapse, rupture of membranes, and the wall morphology changed significantly; PI/Hoechst33342 (1:1) double fluorescent staining showed the apoptosis of cells.
4 changes in liquid hydraulic impact damage of LDH cells: compared with normal control group, cell culture medium after injury were compared to cell culture LDH activity was decreased to the lowest, 4H group compared with the control group, there was significant difference (P0.05). Then the other group gradually increased, no statistically significant difference compared with the control group 48h group (P0.05).
Analysis of astrocytes electrophoresis after injury 5 hydraulic shock: two dimensional electrophoresis analyzed by PDquest software to detect the total protein of each group of points: 1369 + 1325 + 56,4h group 94,8h group 81,12h group 1136 + 1197 + 1257 + 112,24h group 63,48h group 1176 + 73. protein spots distributed in pI5.2-6.6, the range of MW30kDa-70kDa; in the control group as a reference gel, the average map matching rate was 82%. After the injury of a total of 265 protein spots in relative intensity compared with the normal control group had significant difference (P0.05), the expression of 38 protein spots increased after injury, expression of 111 protein spots decreased after injury, 45 protein spots as a new expression after injury, 71 protein spots in the injury groups were not expressed.
MS identification of 6 differentially expressed proteins of 9 different protein spots by MALDI-TOF mass spectrometry, of which 8 points score more than 61 points (P0.05), respectively, 60S acidic ribosomal protein P2 (60S acidic ribosomal protein P2), cellular retinol binding protein (cellular retinol binding protein, Crbp), brain fatty acid binding protein 7 (brain fatty acid binding protein 7, FABP7), S-100 calcium binding protein A11 (S100 calcium binding protein A11, S100A11), LOC685814 (hypothetical protein hypothetical protein LOC685814), eukaryotes translation initiation factor 1A (Eukaryotic translation initiation factor 1A, eIF-1A), breast carcinoma amplified sequence 2 homolog (Breast carcinoma amplified sequence 2 homolog, BCAS2), calcium binding protein 3 (calponin 3). The FABP7 Western-blot verification, the results show that FABP7 is expressed in astrocytes, and the The change trend of protein after the hydraulic impact damage of astrocytes is in accordance with the change trend of the two dimensional electrophoresis images after the PDQuest software analysis.
Conclusion: 1 the damage of hydraulic shock can cause the morphological changes of astrocytes and have a certain time dependence.
2, hydraulic impact injury can lead to changes in proteome expression of astrocytes. After injury, there are significant differences between the 265 protein spots relative to the normal control group. Further study of them may lead to the discovery of biomarkers of brain injury.
8 kinds of protein 3 by MALDL-TOF mass spectrometry analysis identified can be divided into metabolic regulation, signal transduction, translation initiation related and other four categories, these changes in protein expression showed that they participate in early response of astrocytes after injury, promote cell function, inhibit secondary injury, involved in cytoskeletal reorganization and improve the protein synthesis and other aspects play an important role for further research on it, is expected to further understand the molecular mechanism of brain injury.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

【参考文献】