PTX3通过促进M2型巨噬细胞的分化减轻糖尿病肾病的肾损伤

发布时间：2018-05-27 14:33

本文选题：PTX3 + 足细胞　；参考：《山东大学》2017年博士论文

【摘要】：研究背景及目的糖尿病是一种全球性疾病,国际糖尿病联盟数据显示,糖尿病的患病率在全世界范围呈上升趋势,目前患病率是8.8%,中国糖尿病患者人数为1.09亿人,居全球首位,并且增长速度非常快。大约有三分之一的病人发展成糖尿病肾病(diabetic nephropathy, DN ),DN属于一种微血管病变,是造成患者死亡、残疾的原因之一,也是终末期肾病的主要原因。因而进一步研究糖尿病肾病的发生发展机制,寻求新的治疗靶点和治疗策略显得尤为重要。DN的发病机制尚不明确,高血糖、高血脂、高血压等多种因素参与糖尿病肾病的发生与发展,这些因素诱导多种炎性介质分泌,促进炎性细胞聚集导致DN出现免疫损伤,因此免疫系统激活和微炎症状态是目前较一致认可的DN发病机制。正五聚蛋白3 (PTX3)是一种与C-反应蛋白(CRP)和血清淀粉样蛋白A(SAP)同属于正五聚蛋白超家族的急性炎症蛋白,由多种炎症免疫细胞如中性粒细胞、巨噬细胞、树突样细胞和内皮细胞等分泌或合成。CRP和SAP属于短链正五聚蛋白,而PTX3属于长链正五聚蛋白,在多种炎症相关性疾病如微生物感染、自身免疫性疾病、动脉粥样硬化等发挥着重要作用,它能够通过模式识别、激活补体等机制发挥调理素和调节炎症的功能。在急慢性感染性疾病中,PTX3和CRP可以作为标志物在血液中很快升高,在一些无菌性炎症中,它的水平可以更直接的反映血管炎症状态。最近,一些临床研究表明,升高的血浆PTX3水平与心血管疾病和慢性肾脏病(CKD)有关。PTX3水平与体重指数(BMI)呈负相关表明PTX3可能在肥胖和代谢综合征中起到一定作用。最近,AbuSemanN等研究表明,血浆PTX3水平的降低与2型糖尿病和糖尿病肾病有关。然而,目前的研究只集中在内源性PTX-3水平表达的升高或是沉默与其他疾病的关系,对于PTX3治疗作用的研究却不多,只有零星的研究发现,PTX3能有效预防巨细胞病毒感染,减少肺部曲霉菌感染,降低脓毒血症小鼠模型的病死率等。在急性心肌梗动物模型中具有保护心脏的作用,可能与PTX3有抗动脉粥样硬化有关。Lech M等研究发现,内源性或外源性PTX3能够减轻小鼠缺血再灌注后的急慢性肾损伤。基于上述背景,本研究第一部分探讨PTX3是否具有减轻糖尿病肾病的肾损伤作用。材料与方法1.糖尿病肾病(DN)模型的建立24只8-12周龄雄性C57BL/6小鼠,6只正常饲养小鼠为正常对照组(NC组)。18只小鼠腹腔注射链脲佐菌素STZ (50mg/kg)连续5天,定期检测血糖和尿蛋白。STZ注射4周后经化验证实DN小鼠模型建模成功,进行干预分组,分为三组,PTX3组(n=6),DN小鼠建模成功后腹腔注射Recombinant PTX3 (0.5 mg/kg),每日一次,共注射4周;Anti-PTX3组(n=6),为中和内源性PTX3,建模成功后腹腔注射抗鼠PTX3单克隆抗体(anti-mouse PTX3 mAb,0.2 mg/kg)每日一次,共注射4周;Control组(n=6)为糖尿病肾病小鼠对照组,腹腔注射同等剂量的缓冲液;干预4周(即STZ给药8周)后处死所有小鼠,药物干预前(STZ给药4周)和干预后(STZ给药8周)分别留取血样检测血肌酐和24小时尿液,测定小鼠尿微量白蛋白的水平。Western blot检测正常对照组(NC组)和DN小鼠Control组肾组织内足细胞标记物Desmin和Nephrin的蛋白表达水平。2. PTX3能够减轻糖尿病肾病的肾损伤18 只 DN 小鼠分为三组,PTX3 组(n=6), Anti-PTX3 组(n=6)和 Control组(n=6 ),用免疫组化法检测Desmin在三组小鼠肾组织中的表达,Western blot检测三组肾组织Nephrin和WT-1的蛋白水平。应用流式细胞术(FCM)检测三组肾组织中炎性细胞包括CD4+ T细胞、CD8+ T细胞、Ly6G+中性粒细胞和CD11b+巨噬细胞的数目。Western blot检测三组肾组织炎症因子包括IFN-γ、TNF-α、IL-4和IL-13的表达水平。结果1. STZ注射4周后糖尿病肾病模型组(Control组)血糖和尿微量白蛋白水平明显高于正常对照组(NC组)(P0.05),Control组足细胞损伤标记物Desmin蛋白表达水平显著高于NC组,而正常足细胞标记物Nephrin的蛋白水平显著低于NC组(P0.05),说明糖尿病肾病小鼠模型成功建立。2.通过对DN小鼠三组(PTX3组、Anti-PTX3组、Control组)实验结果的比较发现,PTX3干预糖尿病肾病小鼠4周后,尿微量白蛋白定量明显低于干预前(P0.05),在同期对照比较中,PTX3组尿微量白蛋白定量明显低于Control组(P0.05); Desmin在PTX3组小鼠肾组织中的表达减少,Nephrin和WT-1的蛋白水平显著高于Control组(P0.05),而Anti-PTX3组则显示出相反的结果,加重了 DN的肾损伤。3. PTX3组肾组织中浸润的炎性细胞包括CD4+ T细胞、CD8+ T细胞、Ly6G+中性粒细胞和CD11b+巨噬细胞的数目明显少于Control组(P0.05),PTX3能够上调抑炎性因子IL-4和IL-13,下调促炎性因子IFN-γ、TNF-α的表达(P0.05),说明PTX3能够抑制促炎性免疫反应。结论1. PTX3可以降低STZ诱导的糖尿病肾病小鼠尿微量白蛋白定量,具有稳定足细胞结构、减少足细胞损伤的作用,从而减轻糖尿病肾病的肾损伤。2.PTX3能够减少糖尿病肾病小鼠肾组织中炎性细胞浸润,上调抑炎性因子IL-4和IL-13,下调促炎性细胞因子IFN-γ、TNF-α的表达,减轻肾组织的炎性损伤。研究背景及目的糖尿病肾病的发病机制尚不明确,高血糖、高血脂、高血压等多种因素参与糖尿病肾病的发生与发展,这些因素诱导多种炎性介质分泌,促进炎性细胞聚集导致DN出现免疫损伤。因此多种炎症因子和免疫细胞参与DN的免疫损伤,这是目前一致认可的DN发病机制。DN早期,巨噬细胞作为最主要的炎性细胞浸润在肾小球和肾间质中,由于具有极强的异质性,能够在不同环境诱导下分化为不同表型的亚群,从而发挥不同的功能。Th1细胞因子IFN-γ、TNF-α可以刺激巨噬细胞向M1型分化(经典活化型),M1主要表达iNOS、CD16/32、IL-12及TNF-a等促炎因子,主要发挥抗原提呈和免疫炎症作用。Th2分泌IL-4和IL-13可以诱导巨噬细胞向M2型分化(替代活化型),M2高表达CD206、Arg-1及IL-10等分子,表现为抗炎活性,有很强的组织修复能力。临床及动物实验均显示在糖尿病组织损伤部位存在以M1型巨噬细胞为主。在一定条件下,M1和M2亚型可相互转换。我们第一部分实验结果显示PTX3能够减少活性炎性细胞的浸润,下调促炎性因子IFN-γ、TNF-α和上调抑炎性因子IL-4和IL-13的表达,从而减少肾脏促炎性免疫反应,减轻糖尿病肾病的肾损伤。而IL-4和1L-13可以诱导巨噬细胞向M2型分化,推测PTX3可能具有调节巨噬细胞M1/M2动态平衡,促进巨噬细胞向M2型分化,减少炎症损伤的功能。因此本研究第二部分通过体内和体外实验研究PTX3是否能够抑制M1型巨噬细胞表达,促进巨噬细胞向M2型转化,进一步说明PTX3通过促进M2型巨噬细胞的分化减轻糖尿病肾病的肾损伤。材料与方法1.为研究PTX3是否影响巨噬细胞的极化作用,分别从DN小鼠模型和体外RAW264.7细胞培养进行研究。利用本研究第一部分中三组DN小鼠(Control组、PTX3组和Anti-PTX3组)的肾脏取材标本,Western blot检测M1型巨噬细胞表型标记物iNOS、CD16/32和M2型巨噬细胞表型标记物CD206、Arg-1蛋白水平的变化,流式细胞术(Flow Cytometry,FCM)检测M1型和M2型巨噬细胞的数目。2.小鼠单核巨噬细胞株RAW264.7用RPMI 1640培养基培养,含10%FBS、青霉素100u/mL和链霉素100u/mL。采用高糖(右旋葡萄糖)浓度依赖性(11.1mM,20mM,30mM,35mM)以及时间依赖性(0h,6h,12h,24h,48h)剌激RAW264.7细胞株。用酶活性法检测巨噬细胞活化表型标志物iNOS的活性。Recombinant PTX3 (200ng/ml)干预高糖(30mM)组 RAW264.7 细胞,预孵育 12小时,Trizol法提取细胞RNA,采用RT-PCR技术检测M1型巨噬细胞表型标记物iNOS、CD16/32和M2型巨噬细胞表型标记物CD206、Arg-1 mRNA的变化。结果1.与DN小鼠Control组相比,M1型巨噬细胞表型标记物iNOS、CD16/32的蛋白水平在PTX3组中表达明显增多而M2型标记物CD206、Arg-1蛋白水平则显著减少(P0.05),通过流式细胞术检测M1型和M2型巨噬细胞的数目发现,PTX3组M2型巨噬细胞的数目明显多于M1型(P0.05),而Anti-PTX3组则显示出相反的结果,说明PTX3可以促使DN小鼠肾组织的巨噬细胞由M1型向M2型转化。2.高糖环境下能够刺激RAW264.7的iNOS活性增强,呈浓度依赖性上升。其中,30mM高糖组刺激RAW264.7表达iNOS活性达最大值(48.1±6.6 U/mgprot,P0.05)。高糖时间依赖性刺激RAW264.7,高糖组从0至12h产生iNOS活性随时间延长表达逐渐增加,呈时间依赖性。高糖刺激RAW264.7达12h时iNOS活性达最大值(44.6±10.7U/mgrot,P0.05)。高糖(30mM,121h)刺激M1型标记物iNOS、CD16/32 mRNA 表达增加(P0.05),M2 型标记物 CD206、Arg-1 mRNA表达下降(P0.05)。高糖(30mM)+ PTX3干预后,M1型标记物iNOS、CD16/32 mRNA 表达下降(P0.05),M2 型标记物 CD206、Arg-1 mRNA 表达增加(P0.05),说明高糖环境可以促使巨噬细胞向M1型活化,PTX3可以促使高糖环境下的RAW264.7细胞由M1型向M2型转化。结论1. PTX3在体内和体外实验中均促使巨噬细胞由M1型向M2型转化,达到减轻糖尿病肾病的肾损伤作用。2.高糖促进RAW264.7细胞以时间和浓度依赖性表达iNOS活力,促使巨噬细胞向M1型转化。
[Abstract]:Background and objective diabetes is a global disease. The International Diabetes Association data shows that the prevalence of diabetes is on the rise around the world, the prevalence rate is 8.8%, the number of people with diabetes in China is 109 million, the world is the first, and the rate of growth is very fast. About 1/3 of the patients develop into diabetes. Diabetic nephropathy (DN), DN is a kind of microvascular disease, which is one of the causes of death and disability, and is also the main cause of end-stage renal disease. Therefore, further research on the pathogenesis and development mechanism of diabetic nephropathy and the search for new therapeutic targets and treatment strategies are particularly important for the pathogenesis of.DN, which is not clear and high. Many factors such as blood sugar, hyperlipidemia and hypertension are involved in the development and development of diabetic nephropathy. These factors induce the secretion of various inflammatory mediators, promote inflammatory cell aggregation and lead to DN immune damage. Therefore, the activation of the immune system and the state of microinflammation are the consistent recognized pathogenesis of DN. Positive five polyprotein 3 (PTX3) is a kind of anti - C- CRP and serum amyloid A (SAP) belong to the acute inflammatory proteins belonging to the positive five polyprotein superfamily, which are secreted or synthesized by a variety of inflammatory immune cells such as neutrophils, macrophages, dendritic cells, and endothelial cells..CRP and SAP belong to the short chain positive five egg white, and PTX3 belongs to the long chain positive five protein, in a variety of inflammatory phases. Related diseases, such as microbial infection, autoimmune diseases, and atherosclerosis, play an important role. It can play a role of modulating hormone and regulating inflammation through pattern recognition, activating complement and other mechanisms. In acute and chronic infectious diseases, PTX3 and CRP can be used as a marker to increase rapidly in the blood and in some aseptic inflammation. Recently, some clinical studies have shown that elevated plasma PTX3 levels have a negative correlation with body mass index (BMI) related to cardiovascular disease and chronic renal disease (CKD), suggesting that PTX3 may play a role in obesity and metabolic syndrome. Recent studies have shown that AbuSemanN, and so on. The decrease of plasma PTX3 level is associated with type 2 diabetes and diabetic nephropathy. However, the current study is focused only on the elevation of endogenous PTX-3 levels or the relationship between silence and other diseases. There are few studies on the role of PTX3 therapy. Only sporadic studies have found that PTX3 can effectively prevent cytomegalovirus infection and reduce lung music. Fungal infection, lower mortality of mice model of sepsis. In acute myocardial infarction model, the role of protecting the heart, and the possible anti atherosclerotic related.Lech M studies in PTX3 have found that endogenous or exogenous PTX3 can reduce acute and chronic renal injury after ischemia reperfusion in mice. Based on the above background, this study The first part is to investigate whether PTX3 has the effect of reducing renal injury in diabetic nephropathy. Materials and methods 1. diabetic nephropathy (DN) model, 24 8-12 weeks male C57BL/6 mice were established, 6 normal mice were fed as normal control group (group NC) and.18 mice were injected with streptozotocin STZ (50mg/kg) for 5 days. Blood glucose and urine eggs were regularly detected. After 4 weeks of white.STZ injection, the model of DN mice was successfully modeled and divided into three groups, group PTX3 (n=6), DN mice were injected Recombinant PTX3 (0.5 mg/kg) after successful modeling, once a day for 4 weeks; Anti-PTX3 group (n=6) was a neutralization endogenous PTX3, and the anti rat PTX3 monoclonal antibody was injected intraperitoneally after the modeling was successful. Use PTX3 mAb, 0.2 mg/kg) was injected once a day for 4 weeks; group Control (n=6) was the control group of diabetic nephropathy mice, and the same dose of buffer solution was intraperitoneally injected. All mice were killed after 4 weeks (i.e. 8 weeks of STZ administration). The serum creatinine and 24 hours urine were measured before the intervention (STZ administration 4 weeks) and the dry prognosis (STZ for 8 weeks). The level of.Western blot in urine microalbuminuria was determined in the normal control group (group NC) and the protein expression level of Desmin and Nephrin in the renal tissue of Control group of Control group of DN mice,.2. PTX3 could reduce the renal injury of diabetic nephropathy, 18 DN mice were divided into three groups, PTX3 group (n), Immunohistochemistry was used to detect the expression of Desmin in the renal tissue of three groups of mice. Western blot was used to detect the protein levels of Nephrin and WT-1 in the three groups of renal tissues. The flow cytometry (FCM) was used to detect the inflammatory cells in the three groups of renal tissues including CD4+ T cells, CD8+ T cells, Ly6G+ neutrophils and CD11b+ macrophages, and three groups of kidneys were detected. Tissue inflammatory factors included the expression level of IFN- gamma, TNF- alpha, IL-4 and IL-13. Results after 4 weeks of 1. STZ injection, the levels of blood glucose and urine microalbuminuria in the diabetic nephropathy model group (group Control) were significantly higher than those in the normal control group (NC group) (P0.05). The expression of Desmin protein in the Control group of foot cell damage markers was significantly higher than that in the NC group, and the normal podocyte markers were significantly higher than those in the group Control group. The protein level of Nephrin was significantly lower than that of group NC (P0.05), indicating that the mice model of diabetic nephropathy successfully established.2. through the comparison of the experimental results of three groups of DN mice (group PTX3, Anti-PTX3 group, Control group), and it was found that the amount of urine albumin in urine was significantly lower than that before intervention (P0.05) after 4 weeks in diabetic nephropathy mice (P0.05). The amount of urine microalbuminuria in group PTX3 was significantly lower than that in group Control (P0.05), and the expression of Desmin in the renal tissue of PTX3 mice decreased, and the protein level of Nephrin and WT-1 was significantly higher than that in Control group (P0.05), while the Anti-PTX3 group showed the opposite result, which aggravated the DN renal injury. The number of cells, CD8+ T cells, Ly6G+ neutrophils and CD11b+ macrophages was significantly less than that of group Control (P0.05). PTX3 could up regulate the anti inflammatory factors IL-4 and IL-13, down regulated the proinflammatory factor IFN- gamma, TNF- alpha expression (P0.05), indicating that it could inhibit the anti inflammatory immune response. Conclusion 1. can reduce the induced diabetic nephropathy mice. Urine microalbuminuria, which can stabilize the podocyte structure, reduce the effect of foot cell injury, and reduce the renal injury of diabetic nephropathy,.2.PTX3 can reduce inflammatory cell infiltration in renal tissue of diabetic nephropathy mice, increase anti inflammatory factors IL-4 and IL-13, down regulate the expression of proinflammatory cytokines IFN- gamma, TNF- alpha, and reduce inflammation of kidney tissue. The pathogenesis of diabetic nephropathy is not clear. Many factors such as hyperglycemia, hyperlipidemia, hypertension and other factors are involved in the development and development of diabetic nephropathy. These factors induce various inflammatory mediators to secrete and promote inflammatory cell aggregation to lead to DN immune injury. So a variety of inflammatory factors and immune cells are involved in DN The immune injury, which is the unanimous DN pathogenesis in the early stage of.DN, is the most important inflammatory cell infiltrating in the glomeruli and the renal interstitium. Because of its strong heterogeneity, it can differentiate into subgroups of different phenotypes in different environments, thus causing different functional.Th1 cytokines, IFN- gamma, and TNF- alpha. The macrophages were stimulated to differentiate into M1 type (classical activation type), and M1 mainly expressed iNOS, CD16/32, IL-12 and TNF-a, which mainly played the antigen presentation and immune inflammation,.Th2 secreted IL-4 and IL-13, which could induce macrophage to differentiate into M2 type (instead of activating type). The strong tissue repair ability. Clinical and animal experiments showed that M1 type macrophages were dominant in the site of diabetic tissue injury. Under certain conditions, M1 and M2 subtypes could be converted to each other. Our first experimental results showed that PTX3 could reduce the infiltration of active inflammatory cells, the lower modulation of inflammatory factors IFN- gamma, TNF- alpha and up regulation of anti inflammatory properties. The expression of factor IL-4 and IL-13 reduces renal proinflammatory response and reduces renal damage in diabetic nephropathy. IL-4 and 1L-13 can induce macrophage to differentiate into M2 type. It is presumed that PTX3 may have the function of regulating the dynamic balance of M1/M2 in macrophages, promoting the differentiation of macrophages into M2 type, and reducing the function of inflammatory damage. Therefore, second parts of this study were studied. Whether PTX3 can inhibit the expression of M1 type macrophages and promote the transformation of macrophage to M2 type through the experiment in vitro and in vitro, further illustrates that PTX3 can reduce the renal damage of diabetic nephropathy by promoting the differentiation of M2 type macrophages. Material and method 1. is to study whether PTX3 affects the polarization of macrophage cells, from the DN mouse model, respectively. In the first part of this study, three groups of DN mice (group Control, PTX3 and Anti-PTX3) were used in the study. Western blot was used to detect the phenotypic markers of M1 type macrophages, iNOS, CD16/32 and M2 type macrophages, the changes in protein level, and flow cytometry. Etry, FCM) detection of the number of M1 and M2 macrophages in the.2. mouse mononuclear macrophage strain RAW264.7 with RPMI 1640 culture medium, containing 10%FBS, penicillin 100u/mL and streptomycin 100u/mL. using high glucose concentration dependent (11.1mM, 20mM, disposable) stimulant cell strain. Activity method detected active.Recombinant PTX3 (200ng/ml) of macrophage activation phenotypic marker iNOS (200ng/ml) to interfere with high glucose (30mM) group RAW264.7 cells, incubated for 12 hours, Trizol method was used to extract cell RNA, and RT-PCR technique was used to detect phenotypic markers of M1 macrophage, CD16/32 and phenotype markers of macrophages were changed. Results 1. compared with the Control group of DN mice, the phenotype markers of M1 macrophages were iNOS, the protein level of CD16/32 increased significantly in the PTX3 group and the M2 type marker CD206, and the Arg-1 protein level decreased significantly (P0.05). The number of M1 and M2 type macrophages was detected by flow cytometry. More than type M1 (P0.05), and Anti-PTX3 group showed the opposite results, indicating that PTX3 could promote the increase of iNOS activity of RAW264.7 in DN mouse kidney tissue from M1 to M2 type.2. high sugar environment, which increased in a concentration dependent manner. The maximum value of 30mM high glucose group stimulated RAW264.7 expression to reach the maximum value (48.1 + 6.6). Rot, P0.05). High glucose time dependent stimulation of RAW264.7, the activity of iNOS in high glucose group increased from 0 to 12h and increased with time. The activity of high glucose was time dependent. The activity of iNOS was maximum (44.6 + 10.7U/mgrot, P0.05) when high glucose stimulated RAW264.7 (12h). CD206, Arg-1 mRNA expression decreased (P0.05). High sugar (30mM) + PTX3 dry prognosis, M1 type iNOS, CD16/32 mRNA expression decreased (P0.05). Conclusion 1. PTX3 in both in vivo and in vitro stimulated the transformation of macrophages from M1 type to M2 type to alleviate the renal injury of diabetic nephropathy, and.2. high glucose could promote RAW264.7 cells to express iNOS activity in time and concentration dependent manner, and promote the transformation of macrophages into M1 type.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

【参考文献】