创伤失血模型中miRNA对中性粒细胞的凋亡调控及对肝损伤的影响
发布时间：2018-08-24 14:35
【摘要】：背景与目的：创伤失血是临床常见的病理过程,机体受损后,损伤部位的细胞会大量释放炎症介质、组织中性粒细胞(PMN)、内皮细胞(EC)等细胞被快速激活,被激活的PMN及EC释放氧自由基、毒性产物、蛋白酶及炎症因子,并相互作用,炎症反应逐级放大,形成恶性循环,导致PMN游离出血管外、血管通透性增大、渗出增多和组织水肿等一系列病理变化。重度创伤失血后,肝脏组织往往会表现出剧烈的反应,该过程涉及巨噬细胞、中性粒细胞、树突状细胞等的归巢,肝细胞炎性因子分泌量升高,肝脏组织病变等一系列生理生化反应。在MOF、SIRS模型中肝、肺、肠等器官常可见大量PMN浸润,抑制PMN过度激活及炎症反应强度为防治重度创伤后脏器损伤的可行思路之一。已有研究表明,miRNA参与了胚胎发育、造血过程、糖脂类代谢、细胞凋亡、癌症发生等许多生物学过程中。因此,如何有效的阐明创伤失血的发病机制,减缓(甚至阻止)创伤失血的病程和后遗症发生,成为迫切需要解决的议题。本研究通过：一、以SD大鼠为研究对象,采用“骨折损伤合并失血”的方法制备大鼠创伤失血模型,经过免疫组织化学的方法考察伤后大鼠不同时间点的肝组织病变情况,流式细胞术分析肝组织细胞中PMNs比例,采用IL-1β、TNF-α趋化因子等为检测指标,考察了创伤失血大鼠炎性细胞因子的分泌;二、通过生物信息学方法筛选与大鼠肝脏中性粒细胞凋亡相关的候选miRNA,然后采用实时定量PCR方法验证候选miRNA;三、采用大鼠创伤失血16h后的PMNs为研究对象,考察候选miRNA对肝脏中性粒细胞细胞凋亡的影响;四、提前给于大鼠miRNA静脉注射,然后建立大鼠创伤失血模型,通过蛋白表达水平分析和组织病理切片分析,考察miRNA对创伤失血大鼠肝脏损伤的防护作用。方法：第一部分,通过HE染色分析和血清ALT、AST检测,比较建模不同时间点大鼠肝脏损伤情况;采用ELISA方法检测肝组织匀浆液中炎性细胞因子的表达量,流式分析建模不同时间点肝脏中CD45+细胞数量;第二部分,通过生物信息学预测了大鼠PMNs凋亡相关的niRNAs,采用实时定量PCR的方法对预测得到的miRNAs进行进一步筛选验证;第三部分,以大鼠伤后16h的PMNs为研究对象,采用Western Blot方法考察了目标miRNA对中性粒细胞的L-选择素,CD18, CDllb等分子表达的影响,经流式细胞术分析目标miRNA干预后中性粒细胞凋亡率的变化;第四部分,经免疫组织化学、ELISA、流式细胞术等方法考察miRNA对创伤失血大鼠肝脏损伤的保护作用。结果：第一部分：建模16h时,血清ALT、AST含量在伤后16h时达到峰值,结合肝组织HE染色结果提示伤后16h时肝脏的损伤情况最为严重,肝脏中多种炎性细胞因子的表达峰值出现在损伤4h到16h之间,流式细胞术提示损伤后16-24hCD45+细胞数量最高,反映了肝脏中激烈的免疫反应。第二部分：通过大量的生物信息学预测和随后实验研究,我们筛选到与中性粒细胞凋亡高度相关的miRNA: miR-126a-5p。第三部分：体外细胞实验证实miR-126a-5p能抑制中性粒细胞中L-选择素、CD18、CDllb等分子的表达,并促进受损大鼠的中性粒细胞的凋亡;第四部分：给予大鼠尾静脉注射miR-126a-5p进行干预,大鼠肝脏由创伤失血引起的病理变化,miRNA干预组明显轻于模型对照组,比较两组动物肝脏炎性细胞因子及肝脏损伤标志物转氨酶的表达水平,miRNA对照组明显低于模型对照组。结论：成功建立了创伤失血肝损伤模型并确定了创伤失血后大鼠肝脏损伤最严重的时间节点;首次筛选到了一个与大鼠骨折创伤失血后中性粒细胞凋亡高度相关的miRNA:miR-126a-5p,研究证实该]miRNA可减少中性粒细胞和内皮细胞粘附,促中性粒细胞凋亡,保护肝功能、降低创伤失血后肝脏炎症因子表达水平,对创伤失血引起的大鼠肝脏损伤有一定的保护作用。
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641;R-332
本文编号：2201118
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641;R-332
【参考文献】
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1 George P.COBB1,Todd A.ANDERSON;Identification and characterization of new plant microRNAs using EST analysis[J];Cell Research;2005年05期
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