microRNA-9在大鼠脑损伤后的表达变化及意义

发布时间：2018-06-23 06:59

本文选题：创伤性脑损伤 + miRNA　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:观察创伤性脑损伤(traumatic brain injury,TBI)后大鼠伤灶周围脑皮质区miRNA-9的表达变化规律并在细胞水平上探讨其在颅脑损伤后发挥的作用及分子机制。方法:对已发表的创伤性脑损伤后micro RNA芯片数据进行整合并重新分析,筛选出差异表达的基因miRNA-9,并应用生物信息学技术预测出其下游靶基因PTCH1;实验一:选取成年雄性SD大鼠60只建立控制性皮质撞击损伤模型(controlled cortical impact,CCI),并收集TBI后6 h,1、3、7、14、28d各时间点伤灶周围脑组织,应用Q-PCR法检测miRNA-9和PTCH1基因表达变化及应用Western blot法检测PTCH1蛋白及hedgehog信号通路蛋白SHH、Gli1蛋白的表达情况。实验二:提取并培养原代大鼠脑微血管内皮细胞,使用不同浓度的含依托泊苷的培养基进行处理,根据CCK-8结果选定最佳处理浓度;然后将内皮细胞依次分为对照组、损伤模型组[用依托泊苷(etoposide,ETO)损伤]、miRNA-9过表达损伤模型组、空转染损伤模型组,分别应用Q-PCR法验证miRNA-9转染效果、CCK-8法检测各组细胞活力及Western blot法检测各组细胞中B细胞淋巴瘤/白血病-2蛋白(B-cell lymphoma-2,Bcl-2)、B细胞淋巴瘤/白血病-2相关X蛋白(Bcl-2Associated X,Bax)、活化半胱氨酸天冬氨酸特异性蛋白酶-3蛋白(cleaved cysteinyl aspartate specific proteinase 3,cl-caspase-3)表达水平。实验三:提取并培养原代大鼠脑微血管内皮细胞,将内皮细胞依次分为对照组、miRNA-9抑制组(inhibitor组)和miRNA-9过表达组(mimic组),分别应用Western Blot法和明胶酶谱法检测各分组内皮细胞培养基中基质金属蛋白酶9(matrix metalloprotein,MMP-9)的表达量及生物活性,并应用划痕实验和Matrigel血管生成实验检测各组细胞迁移能力和成管能力。实验四:提取并培养原代大鼠脑微血管内皮细胞,先将内皮细胞依次分为对照组、miRNA-9过表达组(mimic)、损伤模型组(ETO)和miRNA-9过表达损伤模型组(ETO+mimic),检测不同组间PTCH1蛋白的表达变化;后将内皮细胞依次分为对照组、miRNA-9过表达组(mimic组)、miRNA-9过表达NF-κB干预组(mimic+QNZ)、NF-κB干预组(QNZ),检测各组细胞Gli1、NF-κB、MMP-9及VEGF的表达。结果:实验一:(1)Q-PCR结果显示:从伤后7d起,伤灶周围脑组织中miRNA-9表达开始增加(P0.05),并于伤后14d达高峰(P0.01),后表达降低且趋于正常水平;PTCH1 m RNA从伤后6h即开始大量表达(P0.01),其表达量随着时间点的迁移逐渐降低,7d时已恢复正常水平;(2)Western Blot结果显示:PTCH1蛋白在伤后6h即开始大量表达(P0.05),但在伤后3d时已趋于正常水平,其结果和Q-PCR结果较吻合;SHH蛋白在伤后随时间点迁移其表达趋势逐渐增加,于14d达高峰(P0.05),后表达趋于正常水平;Gli1蛋白表达趋势和SHH蛋白相似,在伤后14d表达量达高峰(P0.05),后逐渐降低并趋于正常水平。实验二:(1)CCK-8结果显示:最佳药物处理浓度为20μmol/L;(2)内皮细胞建模转染后,Q-PCR结果提示损伤模型组较对照组miRNA-9表达显著降低(P0.01),但miRNA-9过表达损伤模型组miRNA-9表达显著高于损伤模型组(P0.05);(3)CCK-8结果同样显示:miRNA-9过表达损伤模型组细胞活力明显高于损伤模型组(P0.05);(4)Western Blot结果显示:相比于损伤模型组,miRNA-9过表达损伤模型组内皮细胞Bcl-2蛋白表达增加(P0.05),Bcl-2/Bax值增加(P0.05),但Bax蛋白、活化caspase-3蛋白表达降低(P0.05)。实验三:(1)Western Blot结果显示:miRNA-9过表达组内皮细胞培养基中MMP-9蛋白表达较对照组显著增加(P0.05);(2)明胶酶谱实验结果显示:miRNA-9过表达组内皮细胞培养基中MMP-9活性较对照组显著增加(P0.05);(3)划痕实验结果显示:miRNA-9过表达组细胞迁移距离较对照组明显增加(P0.05);(4)细胞成管实验结果同样显示:miRNA-9过表达组细胞成管数目较对照组明显增加(P0.05)。实验四:(1)Western Blot结果显示:miRNA-9过表达组较对照组PTCH1蛋白表达显著降低(P0.01),而损伤模型组与对照组相比PTCH1表达明显增高(P0.05),但在损伤模型基础上过表达miRNA-9可显著降低PTCH1的表达(P0.05);(2)Western Blot结果显示:与对照组相比,miRNA-9过表达组内皮细胞核内NF-κB蛋白、胞质内Gli1蛋白、MMP-9蛋白和VEGF蛋白表达显著升高(P0.05),但给予NF-κB抑制剂QNZ之后可以逆转miRNA-9过表达的效应,促使NF-κB、MMP-9及VEGF蛋白表达显著降低(P0.05)。结论:创伤性脑损伤恢复期伤灶周围脑组织中miRNA-9表达增多,且过表达miRNA-9可通过激活Hedgehog信号通路上调核内NF-κb蛋白表达水平促进MMP-9及VEGF的表达提高内皮细胞活力、促进内皮细胞迁移和管腔形成,提示TBI后miRNA-9的表达增多有助于脑血管重塑的发生,促进神经功能恢复。
[Abstract]:Objective: To observe the changes in the expression of miRNA-9 in the cerebral cortex surrounding the injured traumatic brain injury (TBI) and to explore the role and molecular mechanism of the miRNA-9 after the traumatic brain injury. Methods: the micro RNA chip data of the published traumatic brain injury were integrated and re analyzed and screened. The differentially expressed gene miRNA-9 was produced and its downstream target gene PTCH1 was predicted by bioinformatics. Experiment 1: 60 adult male SD rats were selected to establish a controlled cortical impact damage model (controlled cortical impact, CCI), and 6 h after TBI, and 1,3,7,14,28d was injured around the brain tissue at each time point. Q-PCR method was used to detect miRNA-. 9 and PTCH1 gene expression changes and the use of Western blot method to detect the expression of PTCH1 protein and hedgehog signaling pathway protein SHH, Gli1 protein. Experiment two: extract and culture the primary rat brain microvascular endothelial cells, use different concentrations of etoposide containing culture medium, select the best treatment concentration according to the results of CCK-8; then, and then select the best treatment concentration according to the result of CCK-8; The endothelial cells were divided into the control group, the damage model group [using etoposide (etoposide, ETO) damage], the miRNA-9 overexpression damage model group and the empty transfection damage model group, the miRNA-9 transfection effect was verified by the Q-PCR method. CCK-8 method was used to detect the cell viability and the Western blot method to detect the B cell lymphoma / leukemia -2 protein in each group. (B-cell lymphoma-2, Bcl-2), B cell lymphoma / leukemia -2 related X protein (Bcl-2Associated X, Bax), activated cysteine aspartate specific protease -3 protein (cleaved cysteinyl) expression level. Experiment three: extraction and culture of the primary rat brain microvascular endothelial cells, the endothelial cells will be fine endothelium. The cells were divided into the control group, the miRNA-9 inhibition group (inhibitor group) and the miRNA-9 overexpression group (mimic group). The expression of matrix metalloproteinase 9 (matrix metalloprotein, MMP-9) and the biological activity of the matrix metalloproteinase (matrix metalloprotein, MMP-9) were detected by Western Blot method and gelatinase spectrum method respectively, and the scratch test and Matrigel angiogenesis were used. Test four: experimental four: extraction and cultivation of primary rat brain microvascular endothelial cells, the endothelial cells were first divided into control group, miRNA-9 overexpression group (mimic), injury model group (ETO) and miRNA-9 overexpression damage model group (ETO+ mimic), and the expression of PTCH1 protein in different groups was detected; after that, the expression of PTCH1 protein in different groups was detected. The skin cells were divided into control group, miRNA-9 overexpression group (Group mimic), miRNA-9 overexpression of NF- kappa B intervention group (mimic+QNZ) and NF- kappa B intervention group (QNZ). The expression of Gli1, NF- kappa B, and the expression of NF- kappa B were detected. Results: (1) the results showed that the expression began to increase in the brain tissue around the wound and after injury. 14d reached the peak (P0.01), then decreased and tended to normal level; PTCH1 m RNA began to express a large number of expressions from 6h after injury (P0.01), and its expression gradually decreased with the migration of time, and 7d had been restored to normal level. (2) Western Blot results showed that PTCH1 protein began to be expressed in large quantities after injury, but it had tended to normal water after injury. The results were in good agreement with the results of Q-PCR, and the expression trend of SHH protein was gradually increased with time point after injury, at the peak of 14d (P0.05) and then to the normal level. The expression trend of Gli1 protein was similar to that of SHH protein. The expression of 14d reached the peak after injury (P0.05), and then gradually decreased and tended to normal level. Experiment two: (1) CCK-8 results showed as follows: The optimal concentration of drug treatment was 20 mol/L; (2) after transfection of endothelial cells, the results of Q-PCR showed that the expression of miRNA-9 in the damage model group was significantly lower than that of the control group (P0.01), but the expression of miRNA-9 in the miRNA-9 overexpressed model group was significantly higher than that in the injury model group (P0.05). (3) the result of CCK-8 also showed that miRNA-9 overexpressed the cell viability of the damage model group. Significantly higher than the damage model group (P0.05), (4) the results of Western Blot showed that compared to the damage model group, the expression of Bcl-2 protein in the endothelial cells of the miRNA-9 overexpressed model group increased (P0.05), the Bcl-2/Bax value increased (P0.05), but the Bax protein, the activated caspase-3 protein table was reduced (P0.05). Experiment three: (1) Western results showed the over expression group. The expression of MMP-9 protein in the endothelial cell medium was significantly higher than that in the control group (P0.05). (2) the results of gelatinase assay showed that the activity of MMP-9 in the endothelial cell culture medium of miRNA-9 overexpression group was significantly higher than that of the control group (P0.05); (3) the results of scratch test showed that the migration distance of miRNA-9 overexpressed group was significantly increased (P0.05), (4) cells (4). The results of the tube formation showed that the number of cells in the miRNA-9 overexpressed group was significantly higher than that in the control group (P0.05). Experiment four: (1) the results of Western Blot showed that the expression of PTCH1 protein in the miRNA-9 overexpressed group was significantly lower than that of the control group (P0.01), but the damage model group was significantly higher than the control group (P0.05), but in the damage model basis. The expression of miRNA-9 significantly decreased the expression of PTCH1 (P0.05); (2) Western Blot results showed that the expression of NF- kappa B protein in the endothelial nuclei of the miRNA-9 overexpressed group was significantly higher than that in the control group, and the expression of Gli1 protein, MMP-9 protein and VEGF protein in the cytoplasm increased significantly (P0.05). The expression of NF- kappa B, MMP-9 and VEGF protein was significantly reduced (P0.05). Conclusion: the expression of miRNA-9 in the brain tissue around the traumatic brain injury is increased, and the overexpression of miRNA-9 can increase the expression of NF- kappa B protein by activating Hedgehog signaling pathway to promote the expression of MMP-9 and VEGF to increase the vitality of endothelial cells and promote the migration of endothelial cells. And the formation of lumen, suggesting that the increase of miRNA-9 expression after TBI may contribute to the occurrence of cerebral vascular remodeling and promote the recovery of neurological function.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15

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