本文選題：顱腦損傷 + 冷誘導(dǎo)��； 參考：《天津醫(yī)科大學(xué)》2016年博士論文

【摘要】：第一部分:顱腦損傷大鼠亞低溫治療作用下CIRP表達(dá)的研究目的:亞低溫療法是現(xiàn)階段重型顱腦損傷急性期重要的治療措施之一,但亞低溫對顱腦損傷患者神經(jīng)保護(hù)作用調(diào)控機(jī)制尚未明確。研究顯示CIRP可能是亞低溫保護(hù)作用的溫度依賴性關(guān)鍵調(diào)控蛋白,本次研究以CIRP為切入點(diǎn),觀察腦皮層、海馬、下丘腦部位腦細(xì)胞凋亡與CIRP表達(dá)情況,在不同時(shí)間點(diǎn),檢測下丘腦腦組織內(nèi)CIRP蛋白表達(dá)與ERK通路激活情況,從而討論CIRP表達(dá)在顱腦損傷大鼠亞低溫治療過程中的作用機(jī)制。方法:將大鼠隨機(jī)分為對照組(Group 1),空白AD5-GFP轉(zhuǎn)染組(Group 2)及AD5-GFP-CIRP-Si RNA轉(zhuǎn)染組(Group 3),每組再分為4個(gè)亞組分別為Sham組、TBI組、MH組及TBI+MH組。病毒轉(zhuǎn)染Group 2,3組,檢測病毒轉(zhuǎn)染情況,造模后檢測各組皮層、海馬、下丘腦部位腦組織細(xì)胞凋亡情況,應(yīng)用RT-PCR法檢測三個(gè)部位腦組織CIRP m RNA表達(dá)情況,應(yīng)用Western blot法檢測下丘腦部位不同時(shí)間段CIRP、MEK、p-MEK、ERK-1/2、p-ERK-1/2蛋白表達(dá)情況。結(jié)果:病毒載體有效轉(zhuǎn)染入Group2,3組對應(yīng)部位腦組織內(nèi)。細(xì)胞凋亡檢測顯示,亞低溫治療可顯著減輕皮層、海馬、下丘腦部位腦組織凋亡(p0.05),其中抗凋亡作用在下丘腦部位較其他部位更加顯著(p0.05),經(jīng)過CIRP靜默表達(dá)后亞低溫抗凋亡作用消失。RT-PCR法檢測顯示,亞低溫治療可以使皮層、海馬、下丘腦部位CIRP表達(dá)升高(p0.05),同時(shí),下丘腦部位較皮層、海馬區(qū)表達(dá)顯著升高(p0.05)。Western blot檢測顯示,亞低溫治療使下丘腦CIRP蛋白表達(dá)從6h開始顯著升高(p0.05),并于48 h達(dá)到高峰,于72 h呈下降趨勢。MEK激活度檢測顯示,TBI、MH、TBI+MH三組MEK激活度均表現(xiàn)出進(jìn)行性升高,并于24 h達(dá)到峰值,隨后進(jìn)行性下降,MEK激活程度與CIRP靜默表達(dá)無相關(guān)性,說明CIRP蛋白表達(dá)對MEK激活無影響。ERK-1/2激活檢測顯示,亞低溫治療可導(dǎo)致ERK-1/2激活度峰值前移,并迅速降低其激活度,當(dāng)CIRP靜默表達(dá)時(shí)此作用消失。結(jié)論:在亞低溫治療顱腦損傷過程中,CIRP因低溫作用而在皮層、海馬、下丘腦部位過表達(dá),其中下丘腦部位更加明顯,CIRP作用機(jī)制可能通過直接調(diào)控ERK-1/2激活,使其激活度隨時(shí)間推移迅速降低,減少對應(yīng)部位腦組織細(xì)胞凋亡,從而發(fā)揮神經(jīng)保護(hù)作用。第二部分:參附注射液對顱腦損傷大鼠亞低溫期CIRP表達(dá)的影響目的:從CIRP表達(dá)情況推測參附注射液輔助亞低溫治療顱腦損傷患者的作用機(jī)制。方法:將大鼠隨機(jī)分為對照組(Group 1),空白AD5-GFP轉(zhuǎn)染組(Group 2)及AD5-GFP-CIRP-Si RNA轉(zhuǎn)染組(Group 3),再將Group 1,2,3各分為三個(gè)亞組,分別為創(chuàng)傷亞低溫對照組(Control)、低劑量組(Low dose)、高劑量(High dose)藥物治療組,均首先給予顱腦損傷致傷及亞低溫治療造模,造模成功后進(jìn)行分組處理。取大鼠腦皮層、海馬、下丘腦部位腦組織,檢測腦細(xì)胞凋亡、CIRP、MEK、ERK表達(dá)。結(jié)果:不同部位腦細(xì)胞凋亡檢測顯示,在Group 1,2中低劑量、高劑量組中皮層、海馬、下丘腦部位細(xì)胞凋亡指數(shù)均較對照組顯著降低(p0.05),通過藥物治療顯著降低這三個(gè)部位細(xì)胞凋亡,經(jīng)CIRP靜默表達(dá)后,各組凋亡指數(shù)差異無顯著性(p0.05),亞低溫及藥物對腦細(xì)胞凋亡抑制作用消失。RT-PCR檢測顯示,在Group1,2中低劑量組、高劑量組較對照組CIRP m RNA表達(dá)均顯著升高(p0.05),在Group 3中,各組各部位CIRP m RNA表達(dá)均無顯著差異(p0.05),經(jīng)CIRP靜默表達(dá)后,CIRP m RNA不受亞低溫及藥物影響。Western blot檢測顯示,腦皮層、海馬、下丘腦三個(gè)部位,在Group 1,2中低劑量組、高劑量組較對照組CIRP蛋白表達(dá)均顯著升高(p0.05);MEK激活在Group 1,2,3中各組表達(dá)無顯著差異(p0.05);在Group 1,Group 2中ERK激活在低劑量組、高劑量組較對照組表達(dá)均顯著降低(p0.05),在Group 3中,各組及各部位ERK激活表達(dá)均無顯著差異(p0.05)。結(jié)論:應(yīng)用參附注射液可能通過促進(jìn)CIRP過表達(dá),直接作用于ERK,降低ERK表達(dá),抑制繼發(fā)的轉(zhuǎn)錄因子磷酸化的啟動信號轉(zhuǎn)導(dǎo),從而減少神經(jīng)細(xì)胞凋亡,發(fā)揮其輔助亞低溫治療的抗凋亡作用。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R651.15

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