發(fā)布時(shí)間：2018-06-23 07:18

  本文選題：創(chuàng)傷失血性休克 + miRNA　； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2016年博士論文

【摘要】：目的創(chuàng)傷失血性休克(Traumatic hemorrhagic shock, THS)作為臨床常見(jiàn)疾病之一,以其發(fā)病機(jī)制復(fù)雜、病程長(zhǎng)及死亡率高等特點(diǎn)已被廣泛關(guān)注,多種細(xì)胞炎癥因子參與該過(guò)程的調(diào)控,但具體機(jī)制尚未完全闡明,特別是miRNA參與該過(guò)程調(diào)控的具體機(jī)制尚需進(jìn)一步闡明。因此,本文擬采用急性機(jī)械性損傷方法建立大鼠THS模型,結(jié)合血清細(xì)胞因子和心肌巨噬細(xì)胞標(biāo)志蛋白檢測(cè)確定最佳檢測(cè)時(shí)間點(diǎn),給予miRNA二代測(cè)序和生物信息學(xué)分析得到THS大鼠血清和心肌表達(dá)差異miRNAs,結(jié)合miRNAs生物學(xué)功能驗(yàn)證以期獲得其參與THS調(diào)控的具體機(jī)制,為從miRNA角度詮釋THS的預(yù)防及治療提供新的手段和潛在靶標(biāo)。方法首先,基于急性機(jī)械性損傷法建立大鼠THS模型,并于造模后0h、1h、2h、 4h、8h、16h、24h、48h收集大鼠血清和心肌組織樣本,采用酶聯(lián)免疫吸附測(cè)定法檢測(cè)血清腫瘤壞死因子-a(Tumor necrosis factor-alpha,TNF-α)、白細(xì)胞介素-2(Interleukin-2, IL-2)、白細(xì)胞介素-6(Interleukin-6, IL-6)、白細(xì)胞介素-10(Interleukin-10, IL-10)等細(xì)胞炎癥因子表達(dá)水平以確定血清miRNA樣本最佳檢測(cè)時(shí)間點(diǎn)：針對(duì)心肌巨噬細(xì)胞MO、M1和M2型分化標(biāo)志蛋白CD68,誘導(dǎo)性一氧化氮合酶(inducible nitric oxide synthase, iNOS)和精氨酸酶-1(Arginase-1, ARG1)利用免疫組織化學(xué)(Immunohistochemistry, IHC);去檢測(cè)以確定心肌miRNA樣本最佳檢測(cè)時(shí)間點(diǎn)。然后,針對(duì)THS大鼠血清采用Illumina HiSeq4000系統(tǒng)進(jìn)行小RNA(small RNA, sRNA)深度測(cè)序,通過(guò)生物信息學(xué)分析獲得血清差異性表達(dá)miRNAs及聚類(lèi),結(jié)合Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)富集獲得rniRNA調(diào)控的靶基因生物學(xué)功能及信號(hào)通路。針對(duì)血清樣本中顯著上調(diào)的5個(gè)miRNAs和顯著下調(diào)的5個(gè)miRNAs,采用實(shí)時(shí)定量PCR(qRT-PCR)驗(yàn)證,選擇已確證存在顯著差異的miR-92a-1-3p和細(xì)胞因子進(jìn)行相關(guān)性分析。最后,針對(duì)THS大鼠心肌組織采用Illumina HiSeq4000系統(tǒng)進(jìn)行sRNA深度測(cè)序,通過(guò)生物信息學(xué)分析獲得血清差異性表達(dá)miRNAs及聚類(lèi),結(jié)合Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)富集獲得獲得miRNA調(diào)控的靶基因生物學(xué)功能及信號(hào)通路。針對(duì)與Toll樣受體-4(Toll-like receptors, TLR-4)調(diào)控相關(guān)的5個(gè)心肌細(xì)胞miRNAs,采用qRT-PCR驗(yàn)證,選擇已確證存在顯著差異的miR-155采用雙熒光素酶報(bào)告基因系統(tǒng)驗(yàn)證其對(duì)靶基因TLR4的調(diào)控,進(jìn)而研究心肌細(xì)胞因子TNF-α轉(zhuǎn)化生長(zhǎng)因子-β(transforming growth factor-β, TGF-β)、IL-6、IL-10的表達(dá)變化。同時(shí),針對(duì)]miR-155 mimic進(jìn)行體外合成,尾靜脈注射后結(jié)合蘇木精-伊紅(Hematoxylin-eosin, HE)染色和IHC檢測(cè)心肌組織病理學(xué)變化及巨噬細(xì)胞M1和M2型標(biāo)志蛋白。結(jié)果1)利用急性機(jī)械性損傷法成功建立了大鼠THS模型,炎性因子TNF-α和IL-6的表達(dá)水平隨著造模時(shí)間的增加顯著降低,4h達(dá)谷值并緩慢升高至穩(wěn)定期,抗炎因子IL-2和IL-10的表達(dá)水平隨造模時(shí)間的增加顯著增加,4h達(dá)峰值并緩慢降低至穩(wěn)定期,說(shuō)明4h是血清的最佳變化時(shí)間點(diǎn)。同樣地,大鼠心肌組織巨噬細(xì)胞MO型、M1型和M2型極化標(biāo)志蛋白CD68、iNOS和ARG1在造模后16h變化最明顯,說(shuō)明16h是心肌細(xì)胞最佳變化時(shí)間點(diǎn)。2)血清測(cè)序結(jié)果顯示：共獲得86個(gè)顯著差異miRNAs,包括68個(gè)已知miRNAs和18個(gè)新miRNAs。其中,miR-92a-1-3p的表達(dá)水平與高通量測(cè)序分析結(jié)果一致,并且與細(xì)胞因子IL-6和CRP負(fù)相關(guān),與細(xì)胞因子IL-10正相關(guān)。3)心肌組織測(cè)序結(jié)果顯示：共發(fā)現(xiàn)744個(gè)miRNAs,包括729個(gè)已知miRNAs和15個(gè)新miRNAs。其中,miR-155的表達(dá)水平與高通量測(cè)序分析結(jié)果一致,可通過(guò)調(diào)控TLR4的3'-UTR去調(diào)控其轉(zhuǎn)錄和翻譯。外源miR-155 mimic可顯著抑制巨噬細(xì)胞M1型極化標(biāo)志蛋白iNOS及其分泌的炎性細(xì)胞因子TNF-α和IL-6的表達(dá),促進(jìn)巨噬細(xì)胞M2型極化標(biāo)志蛋白ARG1及其分泌的炎性細(xì)胞因子IL-10和MCL1的表達(dá)。結(jié)論本研究通過(guò)高通量測(cè)序、生物信息學(xué)分析及分子生物學(xué)驗(yàn)證獲得了血清及心肌組織特異性差異表達(dá)的miRNA,為T(mén)HS相關(guān)疾病的治療提供了新的方案和靶標(biāo)。同時(shí),從miRNA角度解釋其可通過(guò)干預(yù)TLR4影響巨噬細(xì)胞M1型和M2型極化狀態(tài),為T(mén)HS致心肌細(xì)胞損傷作用機(jī)理研究提供了重要參考,具有一定的臨床應(yīng)用價(jià)值。
[Abstract]:Objective Traumatic hemorrhagic shock (THS) is one of the common clinical diseases. It has been widely concerned with its complicated pathogenesis, long course of disease and high mortality, and many cytokines are involved in the regulation of the process, but the specific mechanism has not been fully elucidated, especially the specific mechanism of miRNA involved in the regulation of the process. The system still needs to be further clarified. Therefore, this paper uses acute mechanical damage method to establish the rat THS model, combined with serum cytokine and myocardial macrophage marker protein detection to determine the best detection time point, and give miRNA two generation sequencing and bioinformatics analysis to obtain the difference of serum and myocardial expression between THS rats and miRNAs, combined with miRNAs birth. In order to obtain the specific mechanism to participate in the regulation of THS, it provides new means and potential targets for the prevention and treatment of THS from the miRNA point of view. Method first, based on the acute mechanical damage method, the rat THS model was established. After the model, 0h, 1H, 2h, 4h, 8h, 16h, 24h, 48h rats serum and myocardial tissue samples were collected and enzymes were used. The level of serum tumor necrosis factor -a (Tumor necrosis factor-alpha, TNF- alpha), interleukin -2 (Interleukin-2, IL-2), interleukin -6 (Interleukin-6, IL-6), interleukin -10 (IL-2) and other cytokines were detected to determine the optimal detection time point of serum samples: needle MO, M1 and M2 type differentiation marker protein CD68, inducible nitric oxide synthase (inducible nitric oxide synthase, iNOS) and arginase -1 (Arginase-1, ARG1) were used to determine the optimum detection time point of the cardiac muscle samples. The Illumina HiSeq4000 system was used to sequence the small RNA (small RNA, sRNA) depth, and the serum differential expression miRNAs and clustering were obtained through bioinformatics analysis. The biological function and signal pathway of the target gene were enriched with Gene Ontology (GO) and Kyoto Encyclopedia. 5 miRNAs and 5 miRNAs, which were significantly up-regulated, were verified by real-time quantitative PCR (qRT-PCR), and the correlation analysis of miR-92a-1-3p and cytokine, which had proved significant differences, was selected. Finally, the Illumina HiSeq4000 system was used for THS rat myocardial tissue to be sequenced by sRNA depth, and the blood was obtained by bioinformatics analysis. Differentially expressed miRNAs and clustering, combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment to obtain the biological functions and signaling pathways of the target genes regulated by miRNA. The selection was confirmed for 5 cardiac cells. MiR-155 with a significant difference was used to verify the regulation of the target gene TLR4 by the dual luciferase reporter gene system, and then study the expression changes of TNF- alpha transforming growth factor - beta (transforming growth factor- beta, TGF- beta), IL-6, IL-10. At the same time, it was synthesized in vitro for]miR-155 mimic, combined with the tail vein injection. Hematoxylin eosin (Hematoxylin-eosin, HE) staining and IHC detection of myocardial histopathological changes and macrophage M1 and M2 type marker proteins. Results 1) rat THS model was successfully established by acute mechanical injury. The expression level of inflammatory factors TNF- A and IL-6 decreased significantly with the increase of model time, and the value of 4H reached the Valley and increased slowly. The expression level of anti inflammatory factors IL-2 and IL-10 increased significantly with the increase of model time, and 4H reached the peak and slowed down to the stable period, indicating that 4H was the best time point for the change of serum. Similarly, the MO, M1 and M2 polarization markers of macrophage in rat myocardium were marked by egg white CD68, iNOS and ARG1 were most obvious after the model was made. 16h was the best time point.2 of cardiac myocytes.) serum sequencing results showed that 86 significant differences were obtained, including 68 known miRNAs and 18 new miRNAs., the expression level of miR-92a-1-3p was consistent with high throughput sequencing analysis, and negative correlation with cytokine IL-6 and CRP, and the positive correlation with cytokine IL-10.3) myocardium group The results of sequencing showed that 744 miRNAs, including 729 known miRNAs and 15 new miRNAs. were found. The expression level of miR-155 was consistent with the high throughput sequencing analysis, and the transcription and translation could be regulated by regulating the 3'-UTR of TLR4. Exogenous miR-155 mimic could significantly inhibit the M1 polarization marker protein iNOS and its secretion of macrophage cells. The expression of inflammatory cytokines TNF- alpha and IL-6 promotes the expression of macrophage M2 polarization marker protein ARG1 and its secreted inflammatory cytokines, IL-10 and MCL1. Conclusion this study obtained the miRNA of specific differential expression of serum and myocardial tissue by high throughput sequencing, bioinformatics analysis and molecular biological verification, which is a related disease of THS. The treatment of disease provides a new scheme and target. At the same time, it can be explained from the miRNA point of view that it can affect the M1 and M2 type polarization state of macrophages by interfering with TLR4. It provides an important reference for the study of the mechanism of myocardial injury induced by THS, and has a certain clinical value.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R605.971

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