本文選題：創(chuàng)傷性腦損傷 + miRNA��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:觀察創(chuàng)傷性腦損傷(traumatic brain injury,TBI)后大鼠傷灶周圍腦皮質(zhì)區(qū)miRNA-9的表達(dá)變化規(guī)律并在細(xì)胞水平上探討其在顱腦損傷后發(fā)揮的作用及分子機(jī)制。方法:對已發(fā)表的創(chuàng)傷性腦損傷后micro RNA芯片數(shù)據(jù)進(jìn)行整合并重新分析,篩選出差異表達(dá)的基因miRNA-9,并應(yīng)用生物信息學(xué)技術(shù)預(yù)測出其下游靶基因PTCH1;實(shí)驗(yàn)一:選取成年雄性SD大鼠60只建立控制性皮質(zhì)撞擊損傷模型(controlled cortical impact,CCI),并收集TBI后6 h,1、3、7、14、28d各時(shí)間點(diǎn)傷灶周圍腦組織,應(yīng)用Q-PCR法檢測miRNA-9和PTCH1基因表達(dá)變化及應(yīng)用Western blot法檢測PTCH1蛋白及hedgehog信號通路蛋白SHH、Gli1蛋白的表達(dá)情況。實(shí)驗(yàn)二:提取并培養(yǎng)原代大鼠腦微血管內(nèi)皮細(xì)胞,使用不同濃度的含依托泊苷的培養(yǎng)基進(jìn)行處理,根據(jù)CCK-8結(jié)果選定最佳處理濃度;然后將內(nèi)皮細(xì)胞依次分為對照組、損傷模型組[用依托泊苷(etoposide,ETO)損傷]、miRNA-9過表達(dá)損傷模型組、空轉(zhuǎn)染損傷模型組,分別應(yīng)用Q-PCR法驗(yàn)證miRNA-9轉(zhuǎn)染效果、CCK-8法檢測各組細(xì)胞活力及Western blot法檢測各組細(xì)胞中B細(xì)胞淋巴瘤/白血病-2蛋白(B-cell lymphoma-2,Bcl-2)、B細(xì)胞淋巴瘤/白血病-2相關(guān)X蛋白(Bcl-2Associated X,Bax)、活化半胱氨酸天冬氨酸特異性蛋白酶-3蛋白(cleaved cysteinyl aspartate specific proteinase 3,cl-caspase-3)表達(dá)水平。實(shí)驗(yàn)三:提取并培養(yǎng)原代大鼠腦微血管內(nèi)皮細(xì)胞,將內(nèi)皮細(xì)胞依次分為對照組、miRNA-9抑制組(inhibitor組)和miRNA-9過表達(dá)組(mimic組),分別應(yīng)用Western Blot法和明膠酶譜法檢測各分組內(nèi)皮細(xì)胞培養(yǎng)基中基質(zhì)金屬蛋白酶9(matrix metalloprotein,MMP-9)的表達(dá)量及生物活性,并應(yīng)用劃痕實(shí)驗(yàn)和Matrigel血管生成實(shí)驗(yàn)檢測各組細(xì)胞遷移能力和成管能力。實(shí)驗(yàn)四:提取并培養(yǎng)原代大鼠腦微血管內(nèi)皮細(xì)胞,先將內(nèi)皮細(xì)胞依次分為對照組、miRNA-9過表達(dá)組(mimic)、損傷模型組(ETO)和miRNA-9過表達(dá)損傷模型組(ETO+mimic),檢測不同組間PTCH1蛋白的表達(dá)變化;后將內(nèi)皮細(xì)胞依次分為對照組、miRNA-9過表達(dá)組(mimic組)、miRNA-9過表達(dá)NF-κB干預(yù)組(mimic+QNZ)、NF-κB干預(yù)組(QNZ),檢測各組細(xì)胞Gli1、NF-κB、MMP-9及VEGF的表達(dá)。結(jié)果:實(shí)驗(yàn)一:(1)Q-PCR結(jié)果顯示:從傷后7d起,傷灶周圍腦組織中miRNA-9表達(dá)開始增加(P0.05),并于傷后14d達(dá)高峰(P0.01),后表達(dá)降低且趨于正常水平;PTCH1 m RNA從傷后6h即開始大量表達(dá)(P0.01),其表達(dá)量隨著時(shí)間點(diǎn)的遷移逐漸降低,7d時(shí)已恢復(fù)正常水平;(2)Western Blot結(jié)果顯示:PTCH1蛋白在傷后6h即開始大量表達(dá)(P0.05),但在傷后3d時(shí)已趨于正常水平,其結(jié)果和Q-PCR結(jié)果較吻合;SHH蛋白在傷后隨時(shí)間點(diǎn)遷移其表達(dá)趨勢逐漸增加,于14d達(dá)高峰(P0.05),后表達(dá)趨于正常水平;Gli1蛋白表達(dá)趨勢和SHH蛋白相似,在傷后14d表達(dá)量達(dá)高峰(P0.05),后逐漸降低并趨于正常水平。實(shí)驗(yàn)二:(1)CCK-8結(jié)果顯示:最佳藥物處理濃度為20μmol/L;(2)內(nèi)皮細(xì)胞建模轉(zhuǎn)染后,Q-PCR結(jié)果提示損傷模型組較對照組miRNA-9表達(dá)顯著降低(P0.01),但miRNA-9過表達(dá)損傷模型組miRNA-9表達(dá)顯著高于損傷模型組(P0.05);(3)CCK-8結(jié)果同樣顯示:miRNA-9過表達(dá)損傷模型組細(xì)胞活力明顯高于損傷模型組(P0.05);(4)Western Blot結(jié)果顯示:相比于損傷模型組,miRNA-9過表達(dá)損傷模型組內(nèi)皮細(xì)胞Bcl-2蛋白表達(dá)增加(P0.05),Bcl-2/Bax值增加(P0.05),但Bax蛋白、活化caspase-3蛋白表達(dá)降低(P0.05)。實(shí)驗(yàn)三:(1)Western Blot結(jié)果顯示:miRNA-9過表達(dá)組內(nèi)皮細(xì)胞培養(yǎng)基中MMP-9蛋白表達(dá)較對照組顯著增加(P0.05);(2)明膠酶譜實(shí)驗(yàn)結(jié)果顯示:miRNA-9過表達(dá)組內(nèi)皮細(xì)胞培養(yǎng)基中MMP-9活性較對照組顯著增加(P0.05);(3)劃痕實(shí)驗(yàn)結(jié)果顯示:miRNA-9過表達(dá)組細(xì)胞遷移距離較對照組明顯增加(P0.05);(4)細(xì)胞成管實(shí)驗(yàn)結(jié)果同樣顯示:miRNA-9過表達(dá)組細(xì)胞成管數(shù)目較對照組明顯增加(P0.05)。實(shí)驗(yàn)四:(1)Western Blot結(jié)果顯示:miRNA-9過表達(dá)組較對照組PTCH1蛋白表達(dá)顯著降低(P0.01),而損傷模型組與對照組相比PTCH1表達(dá)明顯增高(P0.05),但在損傷模型基礎(chǔ)上過表達(dá)miRNA-9可顯著降低PTCH1的表達(dá)(P0.05);(2)Western Blot結(jié)果顯示:與對照組相比,miRNA-9過表達(dá)組內(nèi)皮細(xì)胞核內(nèi)NF-κB蛋白、胞質(zhì)內(nèi)Gli1蛋白、MMP-9蛋白和VEGF蛋白表達(dá)顯著升高(P0.05),但給予NF-κB抑制劑QNZ之后可以逆轉(zhuǎn)miRNA-9過表達(dá)的效應(yīng),促使NF-κB、MMP-9及VEGF蛋白表達(dá)顯著降低(P0.05)。結(jié)論:創(chuàng)傷性腦損傷恢復(fù)期傷灶周圍腦組織中miRNA-9表達(dá)增多,且過表達(dá)miRNA-9可通過激活Hedgehog信號通路上調(diào)核內(nèi)NF-κb蛋白表達(dá)水平促進(jìn)MMP-9及VEGF的表達(dá)提高內(nèi)皮細(xì)胞活力、促進(jìn)內(nèi)皮細(xì)胞遷移和管腔形成,提示TBI后miRNA-9的表達(dá)增多有助于腦血管重塑的發(fā)生,促進(jìn)神經(jīng)功能恢復(fù)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.15

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