从冷诱导RNA结合蛋白表达探讨亚低温治疗对颅脑损伤大鼠作用

发布时间：2018-06-27 06:18

本文选题：颅脑损伤 + 冷诱导　；参考：《天津医科大学》2016年博士论文

【摘要】：第一部分:颅脑损伤大鼠亚低温治疗作用下CIRP表达的研究目的:亚低温疗法是现阶段重型颅脑损伤急性期重要的治疗措施之一,但亚低温对颅脑损伤患者神经保护作用调控机制尚未明确。研究显示CIRP可能是亚低温保护作用的温度依赖性关键调控蛋白,本次研究以CIRP为切入点,观察脑皮层、海马、下丘脑部位脑细胞凋亡与CIRP表达情况,在不同时间点,检测下丘脑脑组织内CIRP蛋白表达与ERK通路激活情况,从而讨论CIRP表达在颅脑损伤大鼠亚低温治疗过程中的作用机制。方法:将大鼠随机分为对照组(Group 1),空白AD5-GFP转染组(Group 2)及AD5-GFP-CIRP-Si RNA转染组(Group 3),每组再分为4个亚组分别为Sham组、TBI组、MH组及TBI+MH组。病毒转染Group 2,3组,检测病毒转染情况,造模后检测各组皮层、海马、下丘脑部位脑组织细胞凋亡情况,应用RT-PCR法检测三个部位脑组织CIRP m RNA表达情况,应用Western blot法检测下丘脑部位不同时间段CIRP、MEK、p-MEK、ERK-1/2、p-ERK-1/2蛋白表达情况。结果:病毒载体有效转染入Group2,3组对应部位脑组织内。细胞凋亡检测显示,亚低温治疗可显著减轻皮层、海马、下丘脑部位脑组织凋亡(p0.05),其中抗凋亡作用在下丘脑部位较其他部位更加显著(p0.05),经过CIRP静默表达后亚低温抗凋亡作用消失。RT-PCR法检测显示,亚低温治疗可以使皮层、海马、下丘脑部位CIRP表达升高(p0.05),同时,下丘脑部位较皮层、海马区表达显著升高(p0.05)。Western blot检测显示,亚低温治疗使下丘脑CIRP蛋白表达从6h开始显著升高(p0.05),并于48 h达到高峰,于72 h呈下降趋势。MEK激活度检测显示,TBI、MH、TBI+MH三组MEK激活度均表现出进行性升高,并于24 h达到峰值,随后进行性下降,MEK激活程度与CIRP静默表达无相关性,说明CIRP蛋白表达对MEK激活无影响。ERK-1/2激活检测显示,亚低温治疗可导致ERK-1/2激活度峰值前移,并迅速降低其激活度,当CIRP静默表达时此作用消失。结论:在亚低温治疗颅脑损伤过程中,CIRP因低温作用而在皮层、海马、下丘脑部位过表达,其中下丘脑部位更加明显,CIRP作用机制可能通过直接调控ERK-1/2激活,使其激活度随时间推移迅速降低,减少对应部位脑组织细胞凋亡,从而发挥神经保护作用。第二部分:参附注射液对颅脑损伤大鼠亚低温期CIRP表达的影响目的:从CIRP表达情况推测参附注射液辅助亚低温治疗颅脑损伤患者的作用机制。方法:将大鼠随机分为对照组(Group 1),空白AD5-GFP转染组(Group 2)及AD5-GFP-CIRP-Si RNA转染组(Group 3),再将Group 1,2,3各分为三个亚组,分别为创伤亚低温对照组(Control)、低剂量组(Low dose)、高剂量(High dose)药物治疗组,均首先给予颅脑损伤致伤及亚低温治疗造模,造模成功后进行分组处理。取大鼠脑皮层、海马、下丘脑部位脑组织,检测脑细胞凋亡、CIRP、MEK、ERK表达。结果:不同部位脑细胞凋亡检测显示,在Group 1,2中低剂量、高剂量组中皮层、海马、下丘脑部位细胞凋亡指数均较对照组显著降低(p0.05),通过药物治疗显著降低这三个部位细胞凋亡,经CIRP静默表达后,各组凋亡指数差异无显著性(p0.05),亚低温及药物对脑细胞凋亡抑制作用消失。RT-PCR检测显示,在Group1,2中低剂量组、高剂量组较对照组CIRP m RNA表达均显著升高(p0.05),在Group 3中,各组各部位CIRP m RNA表达均无显著差异(p0.05),经CIRP静默表达后,CIRP m RNA不受亚低温及药物影响。Western blot检测显示,脑皮层、海马、下丘脑三个部位,在Group 1,2中低剂量组、高剂量组较对照组CIRP蛋白表达均显著升高(p0.05);MEK激活在Group 1,2,3中各组表达无显著差异(p0.05);在Group 1,Group 2中ERK激活在低剂量组、高剂量组较对照组表达均显著降低(p0.05),在Group 3中,各组及各部位ERK激活表达均无显著差异(p0.05)。结论:应用参附注射液可能通过促进CIRP过表达,直接作用于ERK,降低ERK表达,抑制继发的转录因子磷酸化的启动信号转导,从而减少神经细胞凋亡,发挥其辅助亚低温治疗的抗凋亡作用。
[Abstract]:The first part: Study on the expression of CIRP under mild hypothermia treatment in rats with Craniocerebral Injury Objective: hypothermia therapy is one of the important treatment measures in the acute stage of severe craniocerebral injury, but the mechanism of neuroprotective effect of mild hypothermia on craniocerebral injury is not clear. The study shows that CIRP may be the temperature dependence of hypothermia protection. In this study, the apoptosis and CIRP expression of brain cells in cerebral cortex, hippocampus and hypothalamus were observed with CIRP as the breakthrough point. The expression of CIRP protein and activation of ERK pathway in the hypothalamus brain tissue were detected at different time points, and the mechanism of CIRP expression in the process of mild hypothermia treatment in brain injury rats was discussed. Methods: the rats were randomly divided into control group (Group 1), blank AD5-GFP transfection group (Group 2) and AD5-GFP-CIRP-Si RNA transfection group (Group 3). Each group was divided into 4 subgroups, Sham group, TBI group, MH group and TBI+MH group. The virus transfected to Group 2,3 group, the virus transfection condition was detected, and the brain tissue of each group, hippocampus and hypothalamus part of the brain tissue were detected. The expression of CIRP m RNA in the three parts of the brain was detected by RT-PCR method. The expression of CIRP, MEK, p-MEK, ERK-1/2, p-ERK-1/2 protein in the hypothalamus was detected by Western blot method. Results: the virus vector was transfected into the corresponding part of the brain tissue of the Group2,3 group. The apoptosis detection showed that the subhypothermia was treated with mild hypothermia. The apoptosis of cerebral tissue in cortex, hippocampus and hypothalamus could be significantly reduced (P0.05), in which the anti apoptosis effect was more significant in the hypothalamus than in other parts (P0.05). After CIRP silent expression, the anti apoptotic effect of hypothermia was detected by.RT-PCR method, and hypothermia therapy could increase the expression of CIRP in cortex, hippocampus and hypothalamus (P0.05). In the hypothalamus, the expression of the hypothalamus was significantly higher than that in the cortex and hippocampus (P0.05).Western blot detection showed that hypothalamus increased the expression of CIRP protein in the hypothalamus significantly from 6h (P0.05), and reached the peak at 48 h, and the decreasing trend of.MEK activation at 72 h showed TBI, MH, and three groups of TBI+MH. 24 h reached its peak, followed by progressive decline, MEK activation was not related to the silent expression of CIRP, indicating that the expression of CIRP protein had no effect on the activation of.ERK-1/2 in MEK activation, and the mild hypothermia therapy could lead to the shift of the peak of ERK-1/2 activation and rapidly decrease its activation degree. During the treatment of craniocerebral injury, CIRP is overexpressed in the cortex, hippocampus and hypothalamus in the process of hypothermia, in which the parts of the hypothalamus are more obvious. The mechanism of the action of CIRP may be directly regulated by ERK-1/2 activation, and the activation degree decreases rapidly with time, reducing the apoptosis of the brain tissue in the corresponding part, and thus exerts a neuroprotective effect. Second Part: the effect of Shenfu Injection on subhypothermia CIRP expression in rats with Craniocerebral Injury Objective: to estimate the mechanism of the effect of Shenfu Injection on the treatment of craniocerebral injury patients from CIRP expression. Methods: the rats were randomly divided into the control group (Group 1), the blank AD5-GFP transfection group (Group 2) and the AD5-GFP-CIRP-Si RNA transfection group (Group 3). The Group 1,2,3 was divided into three subgroups, which were traumatic mild hypothermia control group (Control), low dose group (Low dose) and high dose (High dose) drug treatment group. All of them were first given brain injury and mild hypothermia treatment model. After successful modeling, the brain tissue of rat cerebral cortex, hippocampus, hypothalamus and brain cell withering were detected. Results: CIRP, MEK, ERK expression. Results: apoptosis in different parts of the brain cells showed that the apoptosis index in the cortex, hippocampus and hypothalamus of the high dose group decreased significantly (P0.05) in the Group 1, 2 group (P0.05), and the apoptosis of these three sites was significantly reduced by drug therapy. The difference of apoptosis index in each group after CIRP was silent. No significant (P0.05), mild hypothermia and the inhibitory effect of drugs on the apoptosis of brain cells.RT-PCR detection showed that in the low dose group of Group1, 2, the expression of CIRP m RNA in the high dose group was significantly higher than that of the control group (P0.05). In Group 3, there was no significant difference in CIRP m RNA expression in each part of each group (P0.05). The.Western blot detection of low temperature and drug effect showed that the expression of CIRP protein in the cerebral cortex, the hippocampus and the hypothalamus was three in the low dose group of Group 1, 2, and the expression of CIRP protein in the high dose group was significantly higher than that in the control group (P0.05), and there was no significant difference in the expression of MEK activation in Group 1,2,3 (P0.05); in Group 1, the low dose group was activated in the low dose group, and the high dose group was compared. The expression of the control group was significantly decreased (P0.05). In Group 3, there was no significant difference in the activation and expression of ERK in each group and part (P0.05). Conclusion: the application of Shenfu injection may directly act on ERK, reduce the expression of ERK and inhibit the activation of the secondary transcription factor phosphorylation by promoting the overexpression of CIRP, thus reducing the apoptosis of the nerve cells. Its anti apoptosis effect is assisted by subhypothermia therapy.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R651.15

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