间充质干细胞通过活化M2型巨噬细胞减轻急性肾损伤的机制研究

发布时间：2018-07-11 17:27

本文选题：间充质干细胞 + 横纹肌溶解　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：研究背景横纹肌溶解(rhabdomyolysis，RM)指任何原因引起的广泛的横纹肌细胞坏死,肌细胞内容物外漏至细胞外液及血液循环中，导致急性肾损伤(acute kidney injury, AKI)、电解质紊乱等一系列并发症,重症患者预后极差。与肌红蛋白尿相关的AKI是创伤和非创伤性横纹肌溶解最严重的并发症，现有治疗无法根本改善其预后。如何提高RM后肾小管上皮细胞坏死的修复再生、降低AKI死亡率，一直是医学界研究的重大课题。近年来骨髓间充质干细胞（mesenchymal stem cells, MSCs）在急慢性肾脏病中的应用日益受到关注，给AKI的治疗指明新的方向。多项研究均证实，MSCs治疗可明显改善肾小管损伤并有利于肾功能的恢复。巨噬细胞是天然免疫系统中重要的调节细胞,可分为经典活化的M1型巨噬细胞和替代性活化的M2型巨噬细胞两种极性状态。M2型巨噬细胞具有抑制炎症反应，促进组织重塑和再生修复的功能。体外研究中MSCs可与巨噬细胞相互作用，促进M2型巨噬细胞的产生。那么，诱导M2型巨噬细胞的生成是否是MSCs减轻AKI的机制？目前尚无相关报道。本研究旨在建立RM所致AKI的小鼠动物模型，探讨MSCs是否通过诱导M2型巨噬细胞的生成来减轻AKI，为阐明MSCs治疗减轻AKI的机制提供理论依据。方法第一部分C57BL/6小鼠经双下肢肌肉注射50%甘油8ml/kg建立RM所致AKI模型，观察第6h、12h、24h、48h、72h、96h、120h和168h时间点血尿素氮(BUN)、肌酐(Cr)和磷酸肌酸激酶(CK)的变化趋势，以及肾脏、肌肉和肺脏的病理改变。将C57BL/6小鼠随机分为Sham+生理盐水(NS)组、Sham+MSCs组、RM+NS组和RM+MSCs组。6小时后，MSCs组给予1×106个红色荧光蛋白（RFP）标记的C57BL/6小鼠骨髓来源的MSCs尾静脉注射, NS组给予等体积的生理盐水尾静脉注射；（1）生化检测血BUN、Cr和CK水平（n=10）；（2）酶联免疫吸附测定法（ELISA）检测血清细胞因子IL-6、TNF-α和IL-10水平（n=5）；（3）光镜观察肌肉和肾脏病理损伤程度，计算肾小管坏死评分（n=4）；（4）PCNA免疫组化评价肾小管上皮细胞损伤后增殖情况（n=5）；（5）双光子显微镜检测RFP标记的MSCs在RM模型小鼠各器官的定植情况（n=3）。第二部分C57BL/6小鼠经50%甘油8ml/kg肌肉注射建立RM模型，随机分为RM+NS组和RM+MSCs组，n=5。取第24h、48h和72h的肾脏组织，免疫荧光技术检测巨噬细胞（F4/80）浸润数量的变化和M2型巨噬细胞（CD206）浸润数量的变化；Western Blot检测横纹肌溶解后不同时间点的肾脏组织M2型巨噬细胞标志物CD206的表达水平。第24小时，Western Blot检测sham+NS组、sham+MSCs组、RM+NS组和RM+MSCs组的M2型巨噬细胞标志物CD206表达情况。在损伤后第24小时，以MSCs治疗的横纹肌溶解AKI小鼠为研究对象，使用氯屈膦酸二钠脂质体清除其体内的巨噬细胞，空白脂质体作为对照，观察两组小鼠在第48h和72h血BUN、Cr和肾脏病理的改变。第三部分将小鼠巨噬细胞系RAW264.7细胞分为三组：常规培养的细胞为M0组，脂多糖（LPS）2.5ug/ml刺激2h的细胞为M1组，LPS刺激后并与MSCs共培养72小时的细胞为M2组。使用细胞免疫荧光检测三组细胞的巨噬细胞表面标志物F4/80和M2型巨噬细胞表面标志物CD206的表达，流式细胞技术检测各组的CD206和IL-10表达，ELISA检测不同时间点培养液上清IL-6、TNF-α和IL-10的水平（n=5）。使用氯膦酸二钠脂质体清除小鼠体内巨噬细胞，然后建立横纹肌溶解AKI模型，并随即尾静脉注射1×107个M0组、M1组和M2组的RAW264.7细胞（n=5）。在AKI的第72h检测血BUN、Cr和肾脏病理。结果本研究第一部分，成功建立了C57BL/6小鼠RM所致AKI模型，损伤后血BUN、Cr和CK进行性升高，并观察到CK于第24小时达峰值，BUN和Cr于第72小时达峰值的趋势。第6h经尾静脉注射1×106个MSCs或等体积的NS对照，发现MSCs治疗使RM小鼠血BUN、Cr和CK水平明显下降(P0.01)，，血促炎细胞因子IL-6和TNF-α的水平明显下降(P0.01)，血抑炎细胞因子IL-10的水平明显升高(P0.01)。肾组织PAS染色显示RM+MSCs组的肾小管损伤减轻，肾小管坏死评分下降。PCNA免疫组化见RM+MSCs组的肾小管上皮细胞明显增生。双光子显微镜活体检测MSCs主要定植在肺脏和肌肉，肾脏未发现RFP标记的MSCs，并用组织免疫荧光证实。本研究第二部分，组织免疫荧光发现RM发生后，肾脏的巨噬细胞浸润逐渐增加，RM+MSCs组肾组织CD206阳性的M2型巨噬细胞提前出现。WesternBlot证实RM+MSCs组肾脏M2型巨噬细胞标志物CD206的表达水平显著高于RM+NS组(P0.01)，且表达在损伤后呈上升趋势。不同组肾脏CD206表达分析发现：损伤后第24小时，仅RM+MSCs组出现CD206的明显表达(P0.01)。MSCs治疗AKI小鼠的巨噬细胞清除实验证实，在第24小时清除M2型巨噬细胞使第48h和72h小鼠的血BUN和Cr再次升高，伴肾脏损伤病理加重。本研究第三部分，细胞免疫荧光检测M0组、M1组及M2组的RAW264.7细胞均表达F4/80，仅M2组高表达CD206。流式细胞技术检测发现M2组的RAW264.7细胞高表达CD206和IL-10。ELISA测定RAW264.7培养液上清细胞因子浓度，LPS使IL-6和TNF-α的水平升高，MSCs使IL-6和TNF-α的水平降低、IL-10的水平升高(P0.01)。氯屈膦酸二钠脂质体清除小鼠体内的巨噬细胞后建立横纹肌溶解AKI模型，空白脂质体作为对照组，给予不同组巨噬细胞过继转移，损伤后第72小时发现接受M2组RAW264.7细胞小鼠的血清BUN、Cr和病理损伤均较对照组、M0组和M1组明显减低(P0.01)。结论（1）MSCs治疗可调节体内炎症反应，减轻RM所致AKI；（2）MSCs不通过直接定植于肾脏发挥保护作用；（3）MSCs治疗促进肾脏M2型巨噬细胞浸润的数量增加，清除巨噬细胞使已减轻的肾损伤再次加重（4）MSCs可在体外诱导巨噬细胞向M2型的转换。（5）过继转移M2型巨噬细胞可改善RM所致的AKI。
[Abstract]:Background rhabdomyolysis (rhabdomyolysis, RM) refers to a wide range of rhabdomyocyte necrosis caused by any cause, muscle cell contents leaking into extracellular fluid and blood circulation, causing acute renal injury (acute kidney injury, AKI), electrolyte disorder and a series of disorders. The prognosis of severe patients is very poor. The AKI related to myoglobin urine is very poor. It is the most serious complication of traumatic and non traumatic rhabdomyolysis. Existing treatment can not improve its prognosis. How to improve the repair and regeneration of tubular necrosis of renal tubular cells after RM and reduce the mortality of AKI has been a major issue in the medical field. In recent years, bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) are in acute and chronic kidney. A number of studies have confirmed that MSCs therapy can obviously improve renal tubular injury and benefit the recovery of renal function. Macrophages are important regulatory cells in the natural immune system, which can be divided into classical activated M1 type macrophages and alternative activated M2 type megagagi. .M2 type macrophages of two polar states have the function of inhibiting inflammatory response and promoting tissue remodeling and regeneration. In vitro, MSCs can interact with macrophages to promote the production of M2 type macrophages. Then, is the mechanism of inducing M2 type macrophages to be a mechanism for MSCs to reduce AKI? There is no related report. This study aims at this study. In the establishment of a mouse model of AKI induced by RM, it is discussed whether or not MSCs reduces AKI by inducing the formation of M2 type macrophages, providing a theoretical basis for clarifying the mechanism of MSCs therapy to alleviate AKI.
Methods in part 1, C57BL/6 mice were injected with 50% glycerol 8ml/kg to establish a RM induced AKI model, and the changes in blood urea nitrogen, creatinine and creatine kinase were observed at 6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h time, and the pathological changes of the kidney, muscle and lungs. After.6 hours in group NS, group Sham+MSCs, group RM+NS and RM+MSCs, group MSCs was given MSCs tail vein of bone marrow of C57BL/6 mice with 1 x 106 red fluorescent protein (RFP) labeled C57BL/6 mice, and NS group was given equal volume of saline tail vein, and (1) biochemical test of BUN, Cr and levels; (2) enzyme linked immunosorbent assay Test serum cytokine IL-6, TNF- alpha and IL-10 level (n=5); (3) observe pathological damage of muscle and kidney and calculate renal tubular necrosis score (n=4); (4) PCNA immunohistochemistry to evaluate the proliferation of renal tubular epithelial cells (n=5); (5) double light microscopy detection of MSCs in RFP markers in each organ of RM model mice Condition (n=3).
The second part of C57BL/6 mice were injected with 50% glycerol and 8ml/kg to establish RM model. They were randomly divided into group RM+NS and RM+MSCs group. N=5. took the renal tissue of 24h, 48h and 72h. The changes in the number of macrophage (F4/80) infiltration and the quantity of M2 macrophage (CD206) were detected by immunofluorescence. The expression level of M2 type macrophage marker CD206 of the kidney tissue at the same time. Twenty-fourth hours, Western Blot was used to detect the expression of CD206 expression of M2 type macrophage markers in group sham+NS, sham+MSCs, RM+NS and RM+MSCs. Twenty-fourth hours after the injury, AKI mice were dissolved in the rhabdomydric acid two with MSCs treatment, and chlordric acid two was used. Sodium liposomes scavenged macrophages in vivo and blank liposomes as controls. The changes of BUN, Cr and renal pathology in 48h and 72h blood of two groups of mice were observed.
In the third part, the mouse macrophage RAW264.7 cells were divided into three groups: the conventional cultured cells were group M0, the lipopolysaccharide (LPS) 2.5ug/ml stimulated 2H cells in the M1 group, and the LPS stimulated and the MSCs co cultured for 72 hours was the M2 group. The macrophage surface markers, F4/80 and M2 macrophages, were detected by cell immunofluorescence. The expression of surface marker CD206, CD206 and IL-10 expression in each group were detected by flow cytometry. ELISA was used to detect the level of IL-6, TNF- A and IL-10 (n=5) at different time points. Chlorphosphonic acid two sodium liposomes were used to remove macrophages in mice, and then a rhabdomyolysis AKI model was established, and then 1 x 107 M0 groups were injected into the tail vein, M1. M1 RAW264.7 cells (n=5) in group M2 and group AKI were detected in BUN, Cr and renal pathology at 72h of AKI.
Results in the first part of this study, the AKI model induced by RM in C57BL/6 mice was successfully established. The blood BUN, Cr and CK were increased after injury, and the peak value of CK at twenty-fourth hours was observed, and the peak value of BUN and Cr at seventy-second hours was reached. 1 * 106 MSCs or equal volume NS controls were injected through the tail vein. Significantly decreased (P0.01), blood proinflammatory cytokines IL-6 and TNF- alpha significantly decreased (P0.01), the level of blood suppressor cell factor IL-10 increased significantly (P0.01). Renal tissue PAS staining showed that renal tubule injury in group RM+MSCs was reduced, renal tubular necrosis score decreased in.PCNA immunization, and the renal tubular epithelial cells in the RM+MSCs group were obviously proliferated. In vivo, MSCs was mainly colonized in lungs and muscles, and no RFP labeled MSCs was found in the kidneys. The results were confirmed by tissue immunofluorescence.
In the second part of this study, tissue immunofluorescence found that the infiltration of macrophages in the kidneys increased gradually, and the CD206 positive M2 macrophages in the RM+MSCs group showed.WesternBlot in group RM+MSCs, which showed that the expression level of CD206 of the M2 type macrophage marker in the RM+MSCs group was significantly higher than that in the RM+NS group (P0.01), and the expression increased after the injury. Trend. CD206 expression analysis in different groups of kidneys found that twenty-fourth hours after injury, only RM+MSCs group showed CD206 obvious expression (P0.01).MSCs treatment of AKI mice macrophage clearance experiment confirmed that the clearance of M2 type macrophages in twenty-fourth hours made 48h and 72h mice blood BUN and Cr increased again, with the pathological aggravation of renal injury.
The third part of this study, cell immunofluorescence test M0 group, M1 group and M2 group RAW264.7 cells all express F4/80, only M2 group high expression CD206. flow cytometry detection found that RAW264.7 cells in M2 group high expression CD206 and IL-10.ELISA determine the concentration of cell factor in the RAW264.7 culture liquid supernatant. The level of F- alpha and the level of IL-10 increased (P0.01). After scavenging of chlordronic acid two liposomes, the rhabdomyolysis AKI model was established. The blank liposomes were used as the control group, and the macrophages were adoptive and transferred in different groups. The serum BUN, Cr and pathological lesion of the M2 group RAW264.7 cell mice were found after seventy-second hours of injury. The injury was significantly lower in the M0 group and the M1 group than in the control group (P0.01).
Conclusion (1) MSCs therapy can regulate the inflammatory response in the body and reduce the AKI caused by RM; (2) MSCs does not use the direct colonization of the kidney to protect the kidney; (3) MSCs therapy promotes the increase in the number of M2 macrophage infiltration in the kidney, and clears the macrophages to aggravate the reduced renal injury (4) MSCs can induce the conversion of macrophage to M2 type in vitro. 5) adoptive transfer of M2 macrophages can improve the AKI. induced by RM.
【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】：R692

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