【摘要】：背景與目的：創(chuàng)傷失血是臨床常見的病理過程,機體受損后,損傷部位的細胞會大量釋放炎癥介質、組織中性粒細胞(PMN)、內(nèi)皮細胞(EC)等細胞被快速激活,被激活的PMN及EC釋放氧自由基、毒性產(chǎn)物、蛋白酶及炎癥因子,并相互作用,炎癥反應逐級放大,形成惡性循環(huán),導致PMN游離出血管外、血管通透性增大、滲出增多和組織水腫等一系列病理變化。重度創(chuàng)傷失血后,肝臟組織往往會表現(xiàn)出劇烈的反應,該過程涉及巨噬細胞、中性粒細胞、樹突狀細胞等的歸巢,肝細胞炎性因子分泌量升高,肝臟組織病變等一系列生理生化反應。在MOF、SIRS模型中肝、肺、腸等器官�？梢姶罅縋MN浸潤,抑制PMN過度激活及炎癥反應強度為防治重度創(chuàng)傷后臟器損傷的可行思路之一。已有研究表明,miRNA參與了胚胎發(fā)育、造血過程、糖脂類代謝、細胞凋亡、癌癥發(fā)生等許多生物學過程中。因此,如何有效的闡明創(chuàng)傷失血的發(fā)病機制,減緩(甚至阻止)創(chuàng)傷失血的病程和后遺癥發(fā)生,成為迫切需要解決的議題。本研究通過：一、以SD大鼠為研究對象,采用“骨折損傷合并失血”的方法制備大鼠創(chuàng)傷失血模型,經(jīng)過免疫組織化學的方法考察傷后大鼠不同時間點的肝組織病變情況,流式細胞術分析肝組織細胞中PMNs比例,采用IL-1β、TNF-α趨化因子等為檢測指標,考察了創(chuàng)傷失血大鼠炎性細胞因子的分泌;二、通過生物信息學方法篩選與大鼠肝臟中性粒細胞凋亡相關的候選miRNA,然后采用實時定量PCR方法驗證候選miRNA;三、采用大鼠創(chuàng)傷失血16h后的PMNs為研究對象,考察候選miRNA對肝臟中性粒細胞細胞凋亡的影響;四、提前給于大鼠miRNA靜脈注射,然后建立大鼠創(chuàng)傷失血模型,通過蛋白表達水平分析和組織病理切片分析,考察miRNA對創(chuàng)傷失血大鼠肝臟損傷的防護作用。方法：第一部分,通過HE染色分析和血清ALT、AST檢測,比較建模不同時間點大鼠肝臟損傷情況;采用ELISA方法檢測肝組織勻漿液中炎性細胞因子的表達量,流式分析建模不同時間點肝臟中CD45+細胞數(shù)量;第二部分,通過生物信息學預測了大鼠PMNs凋亡相關的niRNAs,采用實時定量PCR的方法對預測得到的miRNAs進行進一步篩選驗證;第三部分,以大鼠傷后16h的PMNs為研究對象,采用Western Blot方法考察了目標miRNA對中性粒細胞的L-選擇素,CD18, CDllb等分子表達的影響,經(jīng)流式細胞術分析目標miRNA干預后中性粒細胞凋亡率的變化;第四部分,經(jīng)免疫組織化學、ELISA、流式細胞術等方法考察miRNA對創(chuàng)傷失血大鼠肝臟損傷的保護作用。結果：第一部分：建模16h時,血清ALT、AST含量在傷后16h時達到峰值,結合肝組織HE染色結果提示傷后16h時肝臟的損傷情況最為嚴重,肝臟中多種炎性細胞因子的表達峰值出現(xiàn)在損傷4h到16h之間,流式細胞術提示損傷后16-24hCD45+細胞數(shù)量最高,反映了肝臟中激烈的免疫反應。第二部分：通過大量的生物信息學預測和隨后實驗研究,我們篩選到與中性粒細胞凋亡高度相關的miRNA: miR-126a-5p。第三部分：體外細胞實驗證實miR-126a-5p能抑制中性粒細胞中L-選擇素、CD18、CDllb等分子的表達,并促進受損大鼠的中性粒細胞的凋亡;第四部分：給予大鼠尾靜脈注射miR-126a-5p進行干預,大鼠肝臟由創(chuàng)傷失血引起的病理變化,miRNA干預組明顯輕于模型對照組,比較兩組動物肝臟炎性細胞因子及肝臟損傷標志物轉氨酶的表達水平,miRNA對照組明顯低于模型對照組。結論：成功建立了創(chuàng)傷失血肝損傷模型并確定了創(chuàng)傷失血后大鼠肝臟損傷最嚴重的時間節(jié)點;首次篩選到了一個與大鼠骨折創(chuàng)傷失血后中性粒細胞凋亡高度相關的miRNA:miR-126a-5p,研究證實該]miRNA可減少中性粒細胞和內(nèi)皮細胞粘附,促中性粒細胞凋亡,保護肝功能、降低創(chuàng)傷失血后肝臟炎癥因子表達水平,對創(chuàng)傷失血引起的大鼠肝臟損傷有一定的保護作用。
[Abstract]:BACKGROUND & OBJECTIVE: Traumatic hemorrhage is a common pathological process in clinic. After the body is damaged, a large number of inflammatory mediators will be released from the injured cells. PMN, EC and other cells are activated rapidly. The activated PMN and EC release oxygen free radicals, toxic products, proteases and inflammatory factors, and interact with each other. After severe trauma and hemorrhage, liver tissues often exhibit severe reactions, which involve the homing of macrophages, neutrophils, dendritic cells and hepatocyte inflammatory factors. Inhibition of excessive activation of PMN and intensity of inflammation is one of the feasible ways to prevent and treat organ injury after severe trauma. Previous studies have shown that microRNAs are involved in embryonic development, hematopoiesis and glycolipid substitution. Therefore, how to effectively elucidate the pathogenesis of traumatic hemorrhage, slow down (or even prevent) the course of traumatic hemorrhage and the occurrence of sequelae has become an urgent issue to be solved. This study adopted: 1. SD rats as the research object, using the "fracture injury combined with hemorrhage" formula. The rat model of traumatic hemorrhage was established. The pathological changes of liver tissues at different time points after traumatic hemorrhage were observed by immunohistochemical method. The ratio of PMNs in liver tissues was analyzed by flow cytometry. The secretion of inflammatory cytokines in traumatic hemorrhage rats was investigated by using IL-1beta and TNF-alpha chemokines. Methods The candidate microRNAs associated with apoptosis of rat liver neutrophils were screened by informatics method, and then real-time quantitative PCR was used to verify the candidate microRNAs. Thirdly, the effects of candidate microRNAs on apoptosis of rat liver neutrophils were investigated by using PMNs after 16 hours of traumatic hemorrhage. Fourthly, the rat microRNAs were injected intravenously in advance and then established. Methods: In the first part, HE staining and serum ALT and AST were used to compare the liver injury of rats at different time points, and the liver homogenate was detected by ELISA. The expression of inflammatory cytokines in the fluid was analyzed by flow cytometry to model the number of CD45 + cells in the liver at different time points. In the second part, the apoptosis-related niRNAs of rat PMNs were predicted by bioinformatics, and the predicted microRNAs were further screened and validated by real-time quantitative PCR. In the third part, PMNs at 16 h after injury were used as the research object. Subjects: Western Blot method was used to investigate the effects of target microRNAs on the expression of L-selectin, CD18, CDllb and other molecules in neutrophils. Flow cytometry was used to analyze the changes of apoptosis rate of neutrophils after target microRNAs intervention. Part IV, immunohistochemistry, ELISA, flow cytometry and other methods were used to investigate the effects of microRNAs on traumatic hemorrhage rats. RESULTS: Part 1: At 16h after injury, the levels of serum ALT and AST peaked at 16h after injury. Combined with HE staining of liver tissue, it was suggested that the liver injury was most serious at 16h after injury. The expression of inflammatory cytokines peaked between 4H and 16h after injury. Flow cytometry indicated the injury. The highest number of CD45 + cells was found at 16-24 hours, reflecting the intense immune response in the liver. Part II: Through a large number of bioinformatics predictions and subsequent experiments, we screened microRNAs highly associated with apoptosis of neutrophils: microRNAs - 126a - 5p. Part III: Cell experiments in vitro confirmed that microRNAs - 126a - 5p inhibited L - 5p in neutrophils. The expression of selectin, CD18, CDllb and other molecules, and promote the apoptosis of neutrophils in injured rats; Part IV: Mi-126a-5p was injected into tail vein of rats to interfere with the pathological changes of liver caused by trauma and hemorrhage. Mi-RNA intervention group was significantly lighter than the model control group, and the inflammatory cytokines and liver damage of the two groups were compared. Conclusion: The model of traumatic and hemorrhagic liver injury was established successfully and the time node of the most severe liver injury was determined. A microRNAs: microRNAs highly correlated with apoptosis of neutrophils after traumatic and hemorrhagic injury in rats were screened for the first time. - 126a-5p, studies have confirmed that the] microRNAs can reduce the adhesion of neutrophils and endothelial cells, promote the apoptosis of neutrophils, protect liver function, reduce the expression of inflammatory factors in the liver after traumatic hemorrhage, and have a certain protective effect on liver injury induced by traumatic hemorrhage in rats.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R641;R-332

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本文編號：2201118