【作者】 李芳；

【导师】 李小峰； 张奉春；

【作者基本信息】 山西医科大学 ， 内科风湿病学， 2009， 博士

【摘要】 【背景】类风湿关节炎(Rheumatoid arthritis,RA)是一种高度致残性自身免疫性疾病,在我国RA的发病率约0.34%,患病总人数达500万,每年直接用于RA的治疗费用和因劳动力丧失带来的间接经济损失很大。近年的研究发现间充质干细胞(Mesenchymal Stem Cells,MSCs)具有免疫调节作用,同时MSCs可迁移到损伤和炎症部位,在局部微环境作用下分化为外胚层、中胚层细胞,甚至内胚层细胞,参与组织重建和修复,这些为RA患者的免疫抑制治疗,以及受损关节的修复带来了新的希望。【目的】通过对RA动物模型胶原诱导关节炎(CIA)大鼠的实验研究,观察异基因MSCs移植对(早期、晚期)CIA大鼠免疫细胞和分子的免疫学作用,并探讨其在体内发挥免疫调节作用的机制;观察异基因MSCs移植在CIA大鼠免疫器官和关节炎症部位的归巢和分布,并探讨其修复损伤关节可能的机制。【方法】第一部分:采用密度梯度离心结合贴壁培养法体外分离、培养大鼠MSCs,细胞表型鉴定。第二部分:(1)弗氏完全佐剂+Ⅱ型胶原初次免疫雌性Wistar大鼠,14d弗氏完全佐剂+Ⅱ型胶原第2次免疫动物,建立CIA大鼠模型。(2)大鼠分为早期治疗组(佐剂初次诱导时治疗组)和晚期治疗组(再次诱导时治疗组),同时设立正常组和早、晚期CIA对照组,每组8只。(3)MSCs移植均采用尾静脉注射,每只大鼠移植的细胞数为1×10~7个/Kg;对照组以同样方法给予等容量的生理盐水。(4)观察42天不同分组大鼠的关节症状及进行关节炎指数和肿胀度测定。(5)MSCs移植后第42天全部处死动物取脾,通过CCK-8测定T细胞、B细胞的增殖;RT-PCR测定Foxp3 mRNA的表达水平;流式细胞术测定CD4~+CD25~+调节性T细胞的变化,ELISA测定外周血TGF-β、IL-17和TNF-α,收集数据并做统计分析。第三部分:(1) PKH-26和Brdu标记MSCs。(2)将64只大鼠随即分成正常组和CIA组,均给予MSCs移植。(3)移植后第3、11、30、42天分别处死8只动物,取不同分组大鼠的胸腺、脾和关节组织制作病理切片,共聚焦显微镜联合免疫组化技术,观察MSCs移植后不同时点在不同脏器的迁移和分布。【结果】1.大鼠MSCs为均一的梭形的成纤维细胞样生长,贴壁及增殖能力强,生长曲线呈S型;流式细胞术鉴定为间充质干细胞。2.早晚期MSCs治疗组AI、后肢足肿胀度均较CIA对照组明显改善(P＜0.05),早期治疗组与晚期治疗组比较,关节炎指数较低,差异有统计学意义(P＜0.05)。3.早晚期CIA对照组Treg水平低于正常组和治疗组,差异有统计学意义(P＜0.05);早期治疗组Treg比晚期治疗组有所升高,差异有统计学意义(P＜0.05)。4.早晚期CIA对照组Foxp3 mRNA的表达水平比正常组和治疗组低,差异有统计学意义(P＜0.05);早期治疗组与晚期治疗组比较差异无统计学意义(P＞0.05),Foxp3 mRNA的表达水平与正常组接近。5.早晚期CIA对照组T、Bcell增殖水平明显高于正常组和治疗组,差异有统计学意义(P＜0.05);早期MSCs治疗组Tcell水平低于晚期治疗组(P＜0.05),早晚期治疗组Bcell水平接近(P＞0.05)。7.早晚期CIA对照组TGF-β1水平较正常组和治疗组低(P＜0.05),早期治疗组TGF-β1水平高于晚期治疗组,差异有统计学意义(P＜0.05)。早晚期CIA对照组血清IL-17和TNF-α水平较正常组和治疗组升高(P＜0.05),其中早期治疗组MSCs对IL-17的抑制优于晚期治疗组,差异有统计学意义(P＜0.05);早晚期MSCs治疗组TNF-α被抑制的程度接近,差异无统计学意义(P＞0.05)。8.异基因骨髓MSCs移植在CIA大鼠的脾脏、胸腺、关节炎症部位可较长期(42天)的存在,归巢的MSCs细胞数量明显高于正常组。【结论】1.体外分离、培养大鼠骨髓MSCs呈现均一的成纤维细胞样生长,贴壁及增殖能力强。2.异基因MSCs移植对(早期、晚期)CIA大鼠的关节炎症状、滑膜病理都有所改善,并通过上调CIA大鼠体内Treg水平,促进Foxp3 mRNA的表达及TGF-β的分泌,抑制T、B淋巴细胞的增殖,减少血清中TNF-α、IL-17的含量,而发挥其在体内的免疫调节作用;早期治疗组的疗效优于晚期治疗组。3.异基因骨髓MSCs移植在CIA大鼠的脾脏、胸腺、关节炎症部位可较长期(42天)的存在;移植的MSCs优先定位于受损伤的组织和器官而发挥其治疗作用。更多还原

【Abstract】 BackgroundRheumatoid arthritis is a highly disabled autoimmune disease.Disease incidence is about 0.34%in China and the total patients are 5 millions.There is a big indirect economic loss because of treatment and loss of work ability.Recent study showed that mesenchymal stem cells have immuno-regulation effect,at the same time MSCs can move to injury and inflammatory area in microenvironment.It can differentiate to dermal layer,mesoblast and endoderm,participating in tissue rebuilding and restoration.All of these bring a hope for RA patients.ObjectiveTo observe the immunologic effect of transplantation of MSCs through studying early and later period of Collagen-induced-arthritis;to observe homing of MSCs in immune organs and inflammatory joints in CIA rats.In this view we discuss the mechanisms of underlying immunomodulating properties and potential applications.MethodsIn the first part,rats MSCs were isolated and expanded from bone marrow cells by density gradient cent rifugation and adhering to the culture plastic,and identified with cell morphology, and the phenotypes were assessed by flow cytometry.In the second part,we established collagen-induced-arthritis rats model immuned by Freund’s complete adjuvant and typeⅡcollagen for two times.40 rats were randomly divided into 5 groups,including:early MSCs treatment group(MSCs were transplanted by the time the rats were induced for the first time), later MSCs treatment group(MSCs were transplanted by the time the rats were induced for the second time),normal control group,early CIA control group and later CIA control group.MSCs were injected through tail Venus.Treatment effects were assessed clinically and histologically before sacrificed.All of rats were sacrificed at 42 days after transplantation of MSCs.We detected the proliferation of lymphocyte to ConA and LPS with CCK-8,observed the expression of Foxp3 mRNA using RT-PCR,assayed the level of CD4~+CD25~+ T cell by means of flow cytometry.In the third part,MSCs were labled by PKH-26 and Brdu.64 rats were randomly divided into normal group and CIA group.Every 8 rats were sacrificed at 3,11,30,42 days after transplantation of MSCs.At the end of the experiment,the specimens of thymus gland,spleen, ankle joints were exposed,fixed,decalcified,wrapped and cut into slices.We use confocal laser scanning microscope and immuno-histochemical methed to observe migration and distribution of MSCs in different organs.Results1.Uniform spindle-shaped in appearance and showed active proliferative capacity and had S shape of growth curve.2.The improvement of arthritis index of early and later treatment groups was higher than CIA control group,which showed statistically significant difference(P＜0.05).Arthritis index of early treatment group was lower than later treatment group,which showed statistically significant difference(P＜0.05).3.The percentage of Treg in early and later CIA groups was lower than normal control group and treatment groups,which showed statistically significant difference(P＜0.05).The level of Treg in early MSCs treatment group was higher than the later MSNs treatment group,which showed statistically significant difference(P＜0.05).4.Compared to the normal group and treatment groups,the expression level of Foxp3 mRNA in early and later CIA groups was decreased markedly.While the early MSNs treatment group versus the later treatment group showed no statistically significant difference(P＞0.05).The intensity of Foxp3 mRNA in treatment groups was close to normal group.5.The proliferation level of T cell and B cell was higher in CIA control groups than normal and treatment groups,which showed statistically significant difference(P＜0.05).Compared to the later treatment group,the percentage of T cell was decreased markedly.While the level of B cell in early and later MSNs treatment groups showed no statistically significant difference(P＞0.05).6.The level of TGF-β1 was lower in early and later CIA control groups than normal and treatment groups(P＜0.05).Rats serum TGF-β1 level of the early treatment group was higher than that of the later treatment group,which showed statistically significant difference(P＜0.05). The levels of TNF-αand IL-17,which were increased in CIA control groups,higher than normal and treatment groups(P＜0.05).Compared to the later treatment group,IL-17 in the early treatment group was increased markedly(P＜0.05).TNF-αof treatment groups showed no statistically significant difference(P＞0.05).7.It was found that allogenic MSCs could stay in spleen,thymus gland and joints of CIA rats for a relatively long period(42 days).The quantity of MSCs was higher in CIA group than normal group. Conclusions1.Rats MSCs were isolated and expanded from bone marrow cells.MSCs were uniform spindle-shaped in appearance and showed actively proliferative capacity,with S shape of growth curve. After MSCs were labeled by PKH-26 and Brdu,biological properties were not changed.2.Our results proved that allogenie MSCs transplantation can relieve inflammatory symptoms of joints and pathology of synovial membrane.Moreover,in our experiments,MSCs showed significant immunomodulatory effects,to upregulate the level of Treg in CIA rats,accelerate the expression of Foxp3 mRNA,inhibit the proliferation of T cell and B cell,stimulate secretion of TGF-β,decrease the content of TNF-αand IL-17 in serum.The early treatment group was more effective than the later treatment group.3.It was found that allogenic MSCs can stay in spleen,thymus gland and joints of CIA rats, whatever later or early groups,for a relatively long period(42 days).MSCs stayed in injured tissue and organs preferentially.更多还原