本文選題：創(chuàng)傷性腦損傷 + miR-21-5p��； 參考：《天津醫(yī)科大學(xué)》2015年博士論文

【摘要】：研究目的:創(chuàng)傷性顱腦損傷(TBI)致殘、致死率高,其所致的繼發(fā)性腦損傷與TBI預(yù)后密切相關(guān)。神經(jīng)-血管單元(NVU)是由神經(jīng)元、神經(jīng)膠質(zhì)細(xì)胞、血管內(nèi)皮細(xì)胞及細(xì)胞外基質(zhì)等構(gòu)成的功能單元,通過其中各種成分的相互作用維持腦組織內(nèi)環(huán)境穩(wěn)態(tài)。促進(jìn)TBI后NVU損傷修復(fù)是治療繼發(fā)性腦損傷、救治TBI的關(guān)鍵。mi RNAs在TBI領(lǐng)域中的研究報(bào)道尚少。我所在課題組的前期研究發(fā)現(xiàn),TBI大鼠創(chuàng)傷側(cè)大腦皮層mi R-21-5p表達(dá)量較傷前明顯升高。此外,初步證實(shí)mi R-21-5p表達(dá)水平的升高可以減輕腦組織細(xì)胞凋亡,并改善TBI大鼠的神經(jīng)功能預(yù)后。本實(shí)驗(yàn)將通過動(dòng)物實(shí)驗(yàn),進(jìn)一步闡明mi R-21-5p在NVU損傷修復(fù)中的作用及機(jī)制,為探索TBI后mi RNAs治療的新策略提供理論依據(jù)。方法:1.應(yīng)用q RT-PCR及mi RNA原位雜交+免疫熒光雙染技術(shù),檢測(cè)TBI后mi R-21-5p在創(chuàng)傷灶腦組織的表達(dá)變化,及其在神經(jīng)元、星形膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞及腦微血管內(nèi)皮細(xì)胞中的原位表達(dá)變化。2.通過改進(jìn)的神經(jīng)功能評(píng)分及水迷宮實(shí)驗(yàn),評(píng)價(jià)TBI大鼠神經(jīng)功能;應(yīng)用TUNEL法細(xì)胞凋亡檢測(cè),評(píng)價(jià)mi R-21-5p對(duì)腦組織細(xì)胞凋亡的影響;應(yīng)用免疫熒光法檢測(cè)腦微血管密度,評(píng)價(jià)mi R-21-5p對(duì)腦組織血管生成修復(fù)的影響;通過腦組織干濕重檢測(cè)、血腦屏障(BBB)Evans Blue滲漏量檢測(cè)及緊密連接蛋白(Occludin及Claudin-5)定量檢測(cè),評(píng)價(jià)mi R-21-5p對(duì)BBB通透性的影響。3.應(yīng)用Western Blot和q RT-PCR技術(shù),檢測(cè)mi R-21-5p可能的作用靶點(diǎn)PTEN、AKT信號(hào)通路及其下游蛋白(凋亡相關(guān)蛋白Bax、Bcl-2、Caspase-3;血管生成及穩(wěn)定性相關(guān)因子Ang-1、Tie-2)的表達(dá)變化,探討mi R-21-5p影響細(xì)胞凋亡、血管生成修復(fù)及BBB通透性的相關(guān)機(jī)制。結(jié)果:1.(1)TBI后,創(chuàng)傷灶腦組織mi R-21-5p表達(dá)量自傷后6h起逐漸升高,于傷后3d達(dá)峰,隨后逐漸下降,于傷后14d降低至假手術(shù)組大鼠水平;且mi R-21-5p在神經(jīng)元、星形膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞、腦微血管內(nèi)皮細(xì)胞(BMVECs)中的原位表達(dá)水平均有不同程度的升高。(2)在TBI前后,mi R-21-5p在腦神經(jīng)組織中均主要表達(dá)于神經(jīng)元及星形膠質(zhì)細(xì)胞。(3)通過側(cè)腦室注射脂質(zhì)體攜帶的mi R-21-5p agomir/antagomir,可以上調(diào)/下調(diào)創(chuàng)傷灶腦組織于傷后6h、1d及3d的mi R-21-5p表達(dá)水平,且同時(shí)上調(diào)/下調(diào)mi R-21-5p在以上細(xì)胞中的原位表達(dá)水平。2.(1)TBI后,腦組織mi R-21-5p表達(dá)水平的升高可以改善神經(jīng)功能預(yù)后。(2)mi R-21-5p的表達(dá)可以抑制腦組織細(xì)胞凋亡。(3)mi R-21-5p的表達(dá)可以促進(jìn)腦組織血管生成修復(fù)。(4)mi R-21-5p的表達(dá)可以減輕BBB滲漏。3.(1)TBI后,腦組織mi R-21-5p的表達(dá)量可以影響凋亡相關(guān)蛋白的表達(dá)水平。(2)mi R-21-5p可以在轉(zhuǎn)錄后水平靶向抑制腦組織PTEN基因的表達(dá),并促進(jìn)Akt磷酸化。(3)mi R-21-5p的表達(dá)可以促進(jìn)腦組織血管生成及穩(wěn)定性相關(guān)因子Ang-1及Tie-2的表達(dá)。結(jié)論:1.TBI后,創(chuàng)傷灶腦組織mi R-21-5p表達(dá)水平升高,且在神經(jīng)元、星形膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞、腦微血管內(nèi)皮細(xì)胞中的原位表達(dá)水平均有不同程度的升高。2.TBI后,mi R-21-5p通過在轉(zhuǎn)錄后水平抑制靶基因PTEN的表達(dá),激活A(yù)kt通路,從而調(diào)控下游凋亡相關(guān)蛋白的表達(dá),產(chǎn)生抑制腦組織細(xì)胞凋亡的作用。3.TBI后,BMVECs表達(dá)的mi R-21-5p通過激活A(yù)ng-1/Tie-2通路,產(chǎn)生促進(jìn)腦組織血管生成修復(fù)、減輕BBB滲漏的作用。4.TBI后,mi R-21-5p可以通過上述作用,保護(hù)神經(jīng)-血管單元,減輕繼發(fā)性腦損傷,從而改善TBI預(yù)后。5.mi R-21-5p是TBI后繼發(fā)性腦損傷的潛在治療靶點(diǎn)。
[Abstract]:Objective: traumatic brain injury (TBI) is disabled and has a high mortality rate. The secondary brain damage caused by it is closely related to the prognosis of TBI. The neurovascular unit (NVU) is a functional unit consisting of neurons, glial cells, vascular endothelial cells and extracellular matrix, and maintains the internal environment of the brain through the interaction of various components. NVU damage repair after TBI is the treatment of secondary brain injury, and the key.Mi RNAs in the treatment of TBI is rarely reported in the field of TBI. The earlier study in my group found that the R-21-5p expression of MI in the cerebral cortex of the TBI rats was significantly higher than that before the injury. Furthermore, it was preliminarily confirmed that the increase in the expression level of MI R-21-5p could be reduced. The apoptosis of brain tissue and the prognosis of neural function in TBI rats will be improved. Through animal experiments, this experiment will further elucidate the role and mechanism of MI R-21-5p in the repair of NVU damage, and provide a theoretical basis for exploring new strategies for MI RNAs treatment after TBI. Methods: 1., Q RT-PCR and Mi RNA in situ hybridization + immunofluorescence double staining technique was used to detect TBI after TBI. The changes in the expression of MI R-21-5p in the brain tissue of the trauma, and in situ expression changes in neurons, astrocytes, microglia and cerebral microvascular endothelial cells..2. was evaluated by improved neural function score and water maze test to evaluate the neural function of TBI rats. The TUNEL method of apoptosis was used to evaluate the Mi R-21-5p on the brain tissue. The effect of apoptosis, detection of cerebral microvascular density by immunofluorescence, and evaluation of the effect of MI R-21-5p on angiogenesis and repair of brain tissue, quantitative detection of blood brain barrier (BBB) Evans Blue leakage and quantitative detection of close connexin (Occludin and Claudin-5) by brain tissue dry and wet weight, and to evaluate the.3. application of MI R-21-5p to BBB permeability Western Blot and Q RT-PCR techniques are used to detect the possible targets of MI R-21-5p, PTEN, AKT signaling pathway and its downstream proteins (Bax, Bcl-2, Caspase-3, angiogenesis and stability related factors). After 1. (1) TBI, the expression of MI R-21-5p in the traumatic brain tissue increased gradually after the injury, and the 3D reached the peak after injury, and then decreased gradually. The 14d decreased to the level of the rat in the sham operation group after injury, and the expression level of MI R-21-5p in the neurons, astrocytes, microglia and intracerebral microvascular skin cells (BMVECs) were in varying degrees. (2) before and after TBI, MI R-21-5p was mainly expressed in neurons and astrocytes in the brain nerve tissue. (3) the MI R-21-5p agomir/antagomir carried by liposomes was injected into the lateral ventricle. The MI R-21-5p expression of 6h, 1D and 3D in the traumatic brain tissue could be up-regulated and downregulated at the same time. After the expression level of.2. (1) TBI in the cell, the elevation of the expression level of MI R-21-5p in the brain can improve the prognosis of neural function. (2) the expression of MI R-21-5p can inhibit the apoptosis of the brain tissue. (3) the expression of MI R-21-5p can promote the angiogenesis and repair of the brain tissue. (4) the expression of MI R-21-5p can reduce BBB leakage.3. (1) The expression of 21-5p can affect the expression level of apoptosis related proteins. (2) mi R-21-5p can inhibit the expression of PTEN gene in the brain tissue at post transcriptional level and promote Akt phosphorylation. (3) the expression of MI R-21-5p can promote the expression of Ang-1 and Tie-2 in the angiogenesis and stability related factors of brain tissue. Conclusion: after 1.TBI, the brain tissue of the traumatic brain tissue The expression level of MI R-21-5p increased and the expression level of in situ expression in neurons, astrocytes, microglia and cerebral microvascular endothelial cells increased to varying degrees of.2.TBI. Mi R-21-5p inhibited the expression of target gene PTEN and activated the Akt pathway through the post transcriptional level, thus regulating the expression of downstream apoptosis related proteins and producing inhibition of the expression of apoptosis related proteins. After the effect of.3.TBI on the apoptosis of brain tissue cells, the MI R-21-5p expressed by BMVECs activates the Ang-1/Tie-2 pathway to promote the angiogenesis and repair of the brain tissue, and reduces the effect of BBB leakage on.4.TBI. Mi R-21-5p can protect the nerve and vascular units and reduce the subsequent brain damage. Thus, the.5.mi R-21-5p is the prognosis of TBI. The.5.mi R-21-5p is the prognosis of TBI. Potential therapeutic targets for secondary secondary brain injury.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.15

【共引文獻(xiàn)】

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9 徐t，

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