间充质干细胞对脓毒症及其肾损伤的保护作用研究

发布时间：2018-05-09 19:30

本文选题：间充质干细胞 + 脓毒症肾损伤　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：研究背景：脓毒症(Sepsis)是世界范围内最主要的死亡原因之一，尤其是在经济欠发达国家，给家庭及国家带了沉重的经济负担。近年来，尽管危重病医学及血液净化医学得到了极大的发展，，但是脓毒症病人的死亡率仍然居高不下，尤其是合并脓毒症肾损伤的患者，死亡率更是高达50%以上。脓毒症肾损伤已经成为脓毒症的独立死亡因素，合并肾损伤的重度脓毒症增加了治疗的难度及费用，因此如何避免及干预脓毒症及脓毒症肾损伤就成为医学界的研究热点。近年来，具有来源丰富、简单；自我分化、更新能力强；低免疫源性等特性的骨髓间充质干细胞在基础研究及临床工作中具有广泛的应用前景。 IL-17是一种重要的前炎症因子，是连接固有免疫和适应性免疫的桥梁。已有研究证明IL-17参与了脓毒症的发病，但是IL-17对脓毒症肾损伤的发病及MSC对脓毒症IL-17的影响尚不清楚，因此本文即通过盲肠结扎穿孔术(CLP)建立小鼠脓毒症模型，观察间充质干细胞对脓毒症及其肾损伤的干预作用，并初步探讨IL-17在MSC发挥对脓毒症导致的急性肾损伤保护机制中的可能作用。方法：第一部分，建立小鼠CLP诱导的脓毒症模型，经尾静脉注射106个C57/BL6小鼠骨髓来源的MSC，（1）观察造模后一般状态及72小时生存率；（2）血清送检我院生化科，检测血清肌酐、尿素氮、谷丙转氨酶、谷草转氨酶（n=10）；（3）评价脓毒症肝脏及肺脏病理损伤程度，评价脓毒症肾脏病理损伤程度及肾小管坏死评分（n=5）；（4）ELISA检测循环中炎症因子IL-6、IL-17、IL-10、TNF-α、INF-γ的表达；(5)双光子显微镜技术追踪红色荧光蛋白标记的MSC在脓毒症小鼠各器官的分布情况（n=3）。第二部分，给IL-17基因敲除鼠建立CLP诱导的脓毒症模型，（1）观察造模后一般状态及72小时生存率；（2）血清送检我院生化科，检测血清肌酐、尿素氮、谷丙转氨酶、谷草转氨酶（n=10）；（3）PAS染色评价脓毒症肾脏病理损伤程度及肾小管坏死评分（n=5）；（4）免疫荧光技术检测浸润肾脏的中性粒细胞（Ly-6G）的数量变化（n=5）；（5）QT-PCR检测肾脏组织炎症因子IL-1β、IL-6、IL-17、IL-10、TNF-α、INF-γ以及趋化因子CXCL1、CXCL2、CXCL5的mRNA表达；(6)检测脓毒症肾脏局部的TUNEL阳性细胞；（7）Western Blot检测肾脏局部的Cleavedcaspase3表达。第三部分，（1）免疫荧光技术检测浸润至肝脏、肺脏及肾脏的中性粒细胞（Ly-6G）及巨噬细胞（F4/80）的数量变化（n=5）；（2）流式细胞技术检测脓毒症小鼠脾脏中分泌IL-17的γδT细胞和αβT细胞的比例（n=5）；（3）取脓毒症小鼠肾脏组织提取RNA，QT-PCR检测炎症因子IL-6、IL-1β、IL-17、IL-10、TNF-α、INF-γ以及趋化因子的mRNA表达；（4）流式细胞技术检测脓毒症小鼠肾脏中分泌IL-17的γδT细胞的比例（n=5）。结果：在本研究第一部分中，我们成功建立了盲肠结扎穿孔导致的脓毒症损伤模型。造模后3h经尾静脉注射106个MSC可以改善脓毒症一般状态及生存率，血清肌酐、尿素氮、谷丙转氨酶、谷草转氨酶水平下降，各组织的病理改变减轻。MSC注射至脓毒症小鼠体内后可显著减少循环中炎症因子的表达，尤其以IL-17改变比较明显，同时增加IL-10的表达。MSC尾静脉注射至脓毒症小鼠体内后，始终未在肝脏、肾脏和心脏定植，而3h即可出现在肺脏、脾脏及腹腔淋巴结。在本研究第二部分中，我们应用IL-17基因敲除鼠建立脓毒症模型，发现基因敲除鼠的一般状态及生存率明显好于野生型小鼠的脓毒症模型，生化指标及肾脏病理改变均较野生型比也显著好转。肾脏局部炎症因子、趋化因子的mRNA表达及中性粒细胞的浸润亦较野生型改善。同时肾脏局部的TUNEL阳性细胞及Cleaved caspase3的表达均明显下降，说明敲除IL-17可以明显改善脓毒症及脓毒症肾损伤。在本研究的第三部分，MSC可减少浸润至肝脏、肺脏及肾脏的中性粒细胞数量和巨噬细胞数量。脓毒症损伤可以使脾脏分泌IL-17的αβT细胞及γδT细胞比例增加，且以γδT细胞增加为主。MSC可减少脾脏分泌IL-17的γδT细胞的比例。同时，我们发现脓毒症小鼠肾脏组织的炎症因子及趋化因子的mRNA水平均明显升高。而MSC治疗可以降低IL-6、IL-1β、IL-17、TNF-α、INF-γ及趋化因子的mRNA水平并进一步升高IL-10的mRNA水平。流式细胞检测发现脓毒症损伤可以使小鼠肾脏组织IL-17分泌明显增加，且主要来自γδT细胞。MSC可减少浸润至肾脏局部的分泌IL-17的γδT细胞。结论：（1）MSC可改善脓毒症及其肾损伤；（2）MSC可以改善脓毒症小鼠的炎症状态；（3）在脓毒症小鼠IL-17主要来自γδT细胞；（4）MSC可以抑制脾脏及肾脏局部分泌IL-17的γδT细胞；（5）敲除IL-17后可以起到保护脓毒症肾损伤的作用。抑制IL-17可能是MSC发挥保护脓毒症及其肾损伤的重要机制之一。
[Abstract]:Background: sepsis (Sepsis) is one of the most important causes of death in the world, especially in economically underdeveloped countries, with a heavy economic burden on families and countries. In recent years, despite the great development of critical medicine and blood purification medicine, the mortality of patients with sepsis remains high, especially in the case of sepsis. The mortality rate of patients with sepsis renal injury is more than 50%. Sepsis renal injury has become an independent death factor of sepsis. Severe sepsis with renal injury has increased the difficulty and cost of treatment. Therefore, how to avoid and interfere with sepsis and sepsis kidney damage has become a research hotspot in the medical field. The bone marrow mesenchymal stem cells (MSCs) have a wide range of application prospects in basic research and clinical work, with rich and simple sources, self differentiation, strong renewal ability and low immunogenic characteristics.
IL-17 is an important pro-inflammatory factor, a bridge linking inherent immunity and adaptive immunity. Research has shown that IL-17 is involved in the pathogenesis of sepsis. But the effect of IL-17 on the pathogenesis of sepsis and the effect of MSC on sepsis IL-17 is not clear. Therefore, this paper is to establish a mouse sepsis model by blind intestinal ligation and perforation (CLP). To observe the effect of mesenchymal stem cells on sepsis and renal injury, and to explore the possible role of IL-17 in the protection mechanism of acute renal injury caused by sepsis in MSC.
Methods: in the first part, a model of CLP induced sepsis in mice was established, MSC of 106 C57/BL6 mice was injected into the tail vein, and (1) the general state and 72 hour survival rate were observed. (2) serum serum creatinine, urea nitrogen, alanine aminotransferase, and glutamic aminotransferase (n=10) were detected by serum, and (3) to evaluate the liver of sepsis and (3) to evaluate the liver of sepsis and to evaluate the liver of sepsis. The degree of lung pathological damage, the degree of renal pathological injury and renal tubular necrosis score (n=5), and (4) the expression of inflammatory factors IL-6, IL-17, IL-10, TNF- a, INF- gamma in the ELISA detection cycle; (5) the distribution of MSC in the sepsis mice was traced by two photon microscopy (n=3).
The second part was to establish a CLP induced sepsis model for IL-17 knockout mice, (1) to observe the general state and 72 hour survival rate of the model, and (2) serum serum creatinine, urea nitrogen, alanine aminotransferase, n=10, and (3) PAS staining was used to evaluate the degree of renal pathological injury and renal tubular necrosis score (3). (4) (4) immunofluorescence technique was used to detect the number of neutrophils (Ly-6G) in the kidneys (n=5), and (5) QT-PCR was used to detect the inflammatory factors IL-1 beta, IL-6, IL-17, IL-10, TNF- alpha, INF- gamma, and chemokine CXCL1, CXCL2, and (6) detection of nephrotic positive cells in the kidney of sepsis; (7) detecting kidney Cleavedcaspase3 expression in the dirty local.
The third part, (1) immunofluorescence technique to detect the number change of neutrophils (Ly-6G) and macrophage (F4/80) in the liver, lungs and kidneys (n=5); (2) flow cytometry was used to detect the ratio of IL-17 to T cells and alpha beta T cells secreted in the spleen of sepsis mice (n=5); (3) RNA, QT-PCR in sepsis mice kidney tissues. Detection of inflammatory factors IL-6, IL-1 beta, IL-17, IL-10, TNF- alpha, INF- gamma, and chemokine mRNA expression; (4) flow cytometry was used to detect the proportion of IL-17 in the kidneys of sepsis mice (n=5).
Results: in the first part of this study, we successfully established a model of sepsis induced by cecal ligation and perforation. After the model, 106 MSC injected through the tail vein of 3H can improve the general state and survival rate of sepsis. Serum creatinine, urea nitrogen, glutamic aminotransferase, glutamic aminotransferase, and cereal transaminase level decrease, and the pathological changes of each tissue reduce.MSC injection. In mice with sepsis, the expression of inflammatory factors in the circulation could be reduced significantly, especially with IL-17 change, while the expression of IL-10 was increased by.MSC tail vein injection to sepsis mice. The liver, kidney and heart were not colonized, and 3H could appear in the lungs, spleen and peritoneal lymph nodes.
In the second part of this study, we used IL-17 gene knockout mice to establish a sepsis model, and found that the general state and survival rate of the gene knockout mice were better than the sepsis model in the wild type mice. The biochemical indexes and renal pathological changes were significantly better than those of the wild type. The mRNA expression of local inflammatory factors and chemokines in the kidneys and the expression of chemokines were in the middle of the study. The infiltration of granulocytes was also better than that in the wild type. The expression of TUNEL positive cells and Cleaved Caspase3 in the kidney was significantly decreased, indicating that the knockout of IL-17 could significantly improve the renal injury of sepsis and sepsis.
In the third part of this study, MSC can reduce the number of neutrophils and macrophages infiltrating to the liver, lungs and kidneys. Sepsis damage can increase the proportion of IL-17's alpha beta T cells and gamma delta T cells in the spleen, and.MSC can reduce the proportion of IL-17 in the spleen secreted by the spleen. At the same time, we hair MRNA levels of inflammatory factors and chemokines in renal tissues of mice with sepsis were significantly increased. MSC therapy could reduce the mRNA level of IL-6, IL-1 beta, IL-17, TNF- a, INF- gamma and chemokine, and further increase mRNA levels of IL-10. Flow cytometry found that sepsis injury could increase the IL-17 secretion of renal tissue in mice. And mainly from the gamma delta T cell.MSC can reduce the infiltration of IL-17 delta gamma cells T into the kidney.
Conclusions: (1) MSC can improve sepsis and renal injury; (2) MSC can improve the inflammatory state of sepsis mice; (3) IL-17 in septic mice mainly comes from gamma delta T cells; (4) MSC can inhibit the partial secretion of IL-17 in the spleen and kidneys of the gamma delta T cells; (5) after knockout IL-17, it can protect the renal injury of sepsis. MSC can play an important role in protecting sepsis and renal injury.

【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】：2014
【分类号】：R459.7

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