子宫内膜再生细胞对肾缺血再灌注损伤的治疗作用

发布时间：2018-04-30 14:14

本文选题：子宫内膜再生细胞 + 缺血再灌注损伤　；参考：《天津医科大学》2016年硕士论文

【摘要】：背景:肾脏缺血再灌注损伤(Renal Ischemia Reperfusion Injury,IRI)是影响临床急性肾损伤的主要病因,经常发生在肾移植、肾脏手术、慢性肾疾病、肾外伤中,在临床上十分常见。目前已进行了大量相关研究,但是发病率、死亡率仍然很高,尚无有效的预防及治疗手段。间充质干细胞(Mesenchymal stem cells,MSCs)是来源于中胚层的具有高度自我更新能力和多向分化潜能的多能干细胞,存在于多种组织,可在体外培养扩增,并可在特定条件下诱导分化为神经细胞、成骨细胞、软骨细胞、心肌细胞、脂肪细胞等,是细胞替代治疗和组织工程的种子细胞,具有广阔的临床应用前景。大量研究表明,MSCs能降低肾损伤,保护肾功能。人子宫内膜再生细胞(ERC)是一种新型来源的MSC,目前研究发现其具有保护缺血坏死心肌、促进烧伤组织修复及减轻溃疡性结肠炎肠道病变,而关于ERC对肾缺血再灌注损伤的影响尚未见报道,以及ERC是否通过何种途径发挥抑炎及免疫调控,进而有效减轻肾缺血再灌注损伤,保护肾功能还有待于进一步研究。目的:利用小鼠肾缺血再灌注损伤模型,探讨子宫内膜再生细胞对肾缺血再灌注损伤的治疗作用及机理。方法:(1)应用密度梯度离心法分离培养人子宫内膜再生细胞,观察细胞形态。(2)选取C57BL/6小鼠,雄性,8周龄,体重约20-25g,随机分为3组,每组6只:假手术组(Sham组)、缺血再灌注损伤组(IRI-untreated组)、ERC治疗组(ERC-treated组),缺血再灌注前2小时经尾静脉输注1×106 ERCs(0.25mlPBS重悬细胞)。建立肾IRI模型,行双侧肾蒂微血管夹夹闭30min,去除微血管夹关腹,再灌注48h。(3)术后48h处死小鼠,留取全血、肾组织、脾脏,保存待检测。全自动生化分析仪检测血清肌酐(Cr)和尿素氮(BUN)水平,评价肾功能;HE染色观察肾脏组织病理改变,评价肾病理损伤程度;免疫组化染色观察肾脏CD3+T细胞和中性粒细胞(Ly6G+)浸润情况;ELISA检测全血细胞因子TNF-α,IFN-γ,IL-4和IL-6水平,以评估炎症反应情况;流式细胞分析检测脾脏调节性T细胞(Tregs)、CD4+和CD8+T细胞;流式检测肾脏CD3+T细胞、F4/80+巨噬细胞、CD206+细胞、髓系抑制细胞(CD11B+Ly6C+和CD11B+Ly6G+),评估ERC对肾脏细胞群的影响。结果:1.应用密度梯度离心法分离培养的ERC,显微镜下呈细胞呈梭形、纺锤形、成纤维细胞样,体积大,贴壁生长,细胞生长速度快,呈集落样。2.iri-untreated组与sham组比较,血清肌酐、尿素氮水平显著升高,肾病理损伤严重,肾结构紊乱、肾小管坏死、间质充血水肿;erc-treated组与iri-untreated组相比,血清肌酐、尿素氮水平明显下降,肾功能显著改善,肾病理损伤明显减轻,肾小管坏死减少,无明显间质充血水肿。3.iri-untreated组与sham组比较,肾脏cd3+t细胞和ly6g+细胞浸润增多;erc-treated组与iri-untreated组相比,cd3+t细胞和ly6g+细胞浸润减少。4.iri-untreated组与sham组比较,流式检测分析示:肾脏cd3+t细胞增多,ki67升高,t细胞增殖明显增加;erc-treated组与iri-untreated组相比,cd3+t细胞显著减少,ki67降低,t细胞增殖减弱,p0.05。5.iri-untreated组与sham组相比,脾脏、肾脏cd4+和cd8+t细胞比例显著上升;erc-treated组与iri-untreated组相比,cd4+和cd8+t细胞比例明显下降,p0.05。6.erc-treated组与iri-untreated组相比,脾脏cd4+cd25+tregs细胞比例显著上调,p0.05;iri-untreated组与sham组相比,cd4+cd25+tregs细胞水平相当,p0.05,差异无统计学意义。7.erc-treated组与iri-untreated组相比,cd11b+ly6c+和cd11b+ly6g+细胞比例显著升高;iri-untreated组与sham组相比,cd11b+ly6c+和cd11b+ly6g+细胞轻度升高。8.流式检测分析肾组织中巨噬细胞,iri-untreated组与sham组相比,f4/80+巨噬细胞浸润增加;erc-treated组与iri-untreated组比较,肾组织中f4/80+巨噬细胞浸润减少,但具有修复作用的cd206+细胞(m2巨噬细胞)比例升高,p0.05差异有统计学意义。9.erc-treated组与iri-untreated组相比,细胞因子tnf-α、ifn-γ和il-6明显下降,而il-4水平上升p0.05;iri-untreated组与sham组相比,tnf-α、ifn-γ和il-6水平升高,p0.05差异有统计学意义。结论:1.成功利用小鼠肾缺血再灌注损伤模型,证实erc能够有效改善肾功能,降低肾病理损伤。2.ercs减轻小鼠肾缺血再灌注损伤与下调肾脏cd3+、cd4+、cd8+t细胞和脾脏cd4+、cd8+t细胞比例有关。3.ercs上调脾脏tregs细胞继而降低损伤程度促进肾损伤修复。4.ERCs保护肾组织与下调肾脏F4/80+巨噬细胞,同时上调CD206+细胞(M2巨噬细胞)相关。5.ERCs减轻肾病理损伤,改善肾功能,与提高肾脏髓系抑制细胞CD11b+Ly6C+和CD11b+Ly6G+比例比例有关。6.ERCs通过影响体内炎症因子(TNF-α、IFN-γ、IL-4和IL-6)水平,降低炎症反应,参与肾损伤修复。
[Abstract]:Background: Renal Ischemia Reperfusion Injury (IRI) is the main cause of clinical acute renal injury. It often occurs in kidney transplantation, kidney surgery, chronic renal disease, and renal trauma, which is very common in clinic. A large number of related studies have been carried out, but the incidence and mortality are still high and there is no effective. The means of prevention and treatment. Mesenchymal stem cells (MSCs) is a multipotent stem cell derived from the mesoderm with high self renewal and pluripotent differentiation potential. It exists in a variety of tissues and can be cultured in vitro, and can be induced into nerve cells, osteoblasts, chondrocytes, and myocardium under specific conditions. Cells and adipocytes, which are the seed cells of cell replacement therapy and tissue engineering, have broad prospects for clinical application. A large number of studies have shown that MSCs can reduce renal damage and protect renal function. Human endometrial regenerative cells (ERC) are a new source of MSC. The research has found that it has the protection of ischemic necrosis myocardium and the promotion of burn tissue. The effect of ERC on renal ischemia-reperfusion injury has not been reported, and the effect of ERC on anti-inflammatory and immune regulation is not yet reported, and the renal ischemia reperfusion injury is effectively alleviated, and the protection of renal function is still to be studied. The therapeutic effect and mechanism of endometrial regenerative cells on renal ischemia reperfusion injury were studied. Methods: (1) the density gradient centrifugation was used to isolate and culture human endometrium regenerative cells and observe cell morphology. (2) C57BL/6 mice were selected, male, 8 weeks old, weight about 20-25g, and were randomly divided into 3 groups: 6 group of sham operation group (group Sham), ischemia. Reperfusion injury group (group IRI-untreated), ERC treatment group (group ERC-treated), 1 x 106 ERCs (0.25mlPBS heavy cell) infusion through tail vein 2 hours before ischemia-reperfusion. Establish renal IRI model, bilateral renal pedicle microvascular clip clipping 30min, remove microvascular clips, and reperfusion after 48h. (3) operation 48h to kill mice, leaving whole blood, kidney tissue, spleen, preserving. The total automatic biochemical analyzer was used to detect serum creatinine (Cr) and urea nitrogen (BUN) levels and evaluate renal function; HE staining was used to observe pathological changes of kidney tissue and to evaluate the degree of renal pathological injury; immunohistochemical staining was used to observe the renal CD3+T cells and neutrophils (Ly6G+) immersion; ELISA was used to detect whole blood cytokines TNF- a, IFN- y, IL-4 and IL-6. Level to assess the inflammatory response; flow cytometry was used to detect spleen regulatory T cells (Tregs), CD4+ and CD8+T cells; flow cytometry was used to detect renal CD3+T cells, F4/80+ macrophages, CD206+ cells, myeloid suppressor cells (CD11B+Ly6C+ and CD11B+Ly6G+), and to evaluate the effect of ERC on renal cell groups. Results: 1. the density gradient centrifugation method was used to separate the cultured cells. Under the microscope, the ERC was spindle shaped, spindle shaped, fibroblast like, large, wall growing, and fast growing. The colony.2.iri-untreated group was compared with the sham group, serum creatinine, urea nitrogen level increased significantly, renal pathological injury was serious, renal structure disorder, renal tubular necrosis, interstitial hyperemia edema, and erc-treated group and iri-u Compared with group ntreated, serum creatinine, urea nitrogen level decreased significantly, renal function improved significantly, renal pathological damage was significantly reduced, renal tubular necrosis was reduced, and no interstitial congestion edema was found in group.3.iri-untreated and sham group, and renal cd3+t cell and ly6g+ cell infiltration increased; erc-treated group and iri-untreated group were compared with cd3+t cells and ly6g+. Compared with the sham group, flow cytometry analysis showed that the number of cd3+t cells in the kidney increased, the Ki67 increased and the proliferation of T cells increased obviously, and the cd3+t cells decreased significantly, Ki67 decreased, and the proliferation of T cells decreased in the erc-treated group compared with the iri-untreated group, and the p0.05.5.iri-untreated group was compared with the sham group, the spleen, kidney and kidney. Compared with the iri-untreated group, the proportion of cd4+ and cd8+t cells decreased significantly in the erc-treated group and the p0.05.6.erc-treated group compared with the iri-untreated group. The proportion of cd4+cd25+tregs cells in the spleen was significantly up, P0.05, and the iri-untreated group was comparable to the sham group, and there was no significant difference in the level of the cd4+ cd25+tregs cells. Compared with group iri-untreated, the proportion of cd11b+ly6c+ and cd11b+ly6g+ cells increased significantly in group.Erc-treated, and in iri-untreated and sham groups, cd11b+ly6c+ and cd11b+ly6g+ cells slightly increased.8. flow cytometry to analyze macrophages in renal tissue. Iri-untreated and sham groups increased the infiltration of f4/80+ macrophages. Compared with the ntreated group, the infiltration of f4/80+ macrophages in the renal tissue decreased, but the proportion of the cd206+ cells (M2 macrophages) with the repair effect increased. The difference in P0.05 was statistically significant between the.9.erc-treated group and the iri-untreated group, and the cytokine tnf- a, ifn- gamma and IL-6 decreased obviously, while the IL-4 level increased. The ratio of tnf- alpha, ifn- gamma and IL-6 increased and the difference of P0.05 was statistically significant. Conclusion: 1. the renal ischemia reperfusion injury model was successfully used in mice. It was proved that ERC could effectively improve renal function and reduce renal pathological injury.2.ercs to reduce renal ischemia-reperfusion injury in mice and down regulation of cd3+, cd4+, cd8+t cells and spleen cd4+, cd8+t cells. .3.ercs up regulation of spleen Tregs cells and reducing the degree of injury, promote renal injury to repair the renal tissue and reduce the renal F4/80+ macrophage, and increase the CD206+ cell (M2 macrophage) related.5.ERCs to alleviate renal pathological damage, improve renal function, and improve the proportion of CD11b+Ly6C+ and CD11b+Ly6G+ ratio of renal myeloid cells. 6.ERCs can reduce the inflammatory reaction and participate in the repair of renal injury by affecting the levels of TNF-, IFN-, IL-4 and IL-6 in vivo.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R692

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