异体骨髓间充质干细胞用于类风湿关节炎治疗的初步探讨
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摘要
目的:
类风湿关节炎(rheumatoid arthritis,RA)是一种慢性、系统性的炎性自身免疫病,通常伴有多关节肿胀与疼痛,若不及时治疗,可导致关节软骨、骨的侵蚀与破坏。尽管发病机制尚不明确,众多证据表明T细胞、B细胞以及多种炎性因子在RA病理过程中均发挥着重要的作用。有效的抑制这些炎性因素,则成为了RA治疗的一个重要目标。近来,以T细胞、B细胞和各种炎性细胞因子为治疗靶向的生物制剂已证实在RA治疗中的具有显著效果,但对于一些难治性RA患者的治疗仍不理想。而有学者认为RA患者自身的干细胞本身就存在功能缺陷,使得目前采用自体干细胞移植来治疗难治性RA的方案也受到质疑。另一方面,RA患者关节局部持续存在的炎症反应,是导致滑膜“肿瘤样”增殖,以及软骨组织最终遭受侵袭破坏的主要原因。尽管RA的治疗在抑制系统性炎症方面已有很大进展,但在炎症因素众多的关节局部如何有效地修复受损的软骨组织,目前还少见报道。
骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)多能分化性以及低免疫原性和免疫抑制特性的发现为解决这些问题带来了希望。以BMSCs为种子细胞构建的组织工程软骨已经在治疗骨关节炎(ostarthritis,OA)患者受损的关节软骨方面取得了满意的效果。另一方面,采用异体BMSCs治疗(graft-versus-host diseases,GVHD)的临床试验也取得成功。此外,BMSCs还被观察到能够缓解实验性脑脊膜炎(experimental allergic encephalomyelitis,EAE)——一种自身免疫病的动物模型的症状。然而,目前利用BMSCs的多能分化性和免疫学特性来治疗RA还少见报道。
因此,本研究旨在BMSCs分离、培养以及体内、外诱导其向软骨细胞/组织分化的基础上:1.研究BMSCs对RA患者II型胶原(type II collagean,CII)特异性T细胞、B细胞的免疫抑制作用,并进一步观察异体BMSCs静脉移植对胶原诱导(collagen induced arthritis,CIA)大鼠模型的治疗作用;2.初步研究BMSCs分化后的软骨细胞的免疫学特性,探讨以BMSCs作为种子细胞构建的组织工程软骨,在炎性因素众多的关节内修复受损软骨同时是否可发挥抑炎作用,从而防止修复后软骨再次遭受炎症破坏的可能性;为临床采用BMSCs治疗RA提供实验依据。
方法(分五部分):
1.建立BMSCs分离、培养、鉴定体系和研究生物特性抽取正常健康供者骨髓3-5 ml,依次采用密度梯度离心法、自然贴壁法分离BMSCs;采用流式细胞术检测表面标记,诱导其向成骨细胞/脂肪细胞分化等方法对其鉴定;观察不同代数细胞形态、生长曲线、细胞周期、衰老染色等各方面的变化以及长期培养的BMSCs的自然分化情况,了解BMSCs的体外生长以及部分生物学特性;通过透射电镜观察BMSCs的微观结构。
2.建立体内、外BMSCs向软骨细胞分化和鉴定体系将BMSCs离心后形成微小细胞团,在TGF-β1、地塞米松(Dex)及维生素C(Vit C)等诱导下向软骨细胞分化。21d后,细胞团经石蜡包埋、切片及HE染色后,进行甲苯胺蓝染色及CoII的免疫组化染色。采用Western blot和RT-PCR,分别检测诱导前后BMSCs中CoII及前CoII mRNA的表达。同时,采用组织工程技术在体外构建BMSCs-CII海绵复合物,在软骨细胞诱导液中培养1周后移植入裸鼠皮下,8 w后观察其大体形态。
3.研究BMSCs及其分化的软骨细胞对CII反应性T细胞的作用收集RA患者外周血(peripheral blood,PB)及关节滑液(synovial fluids,SF),分离单个核细胞(mononuclear cell,PBMC和SFMC)。采用3H-胸腺嘧啶核苷掺入法、流式细胞术、ELISA等方法观察BMSCs及其分化的软骨细胞对RA患者CII特异性T细胞在增殖、CD25/CD29等表面活化标记表达、多种细胞因子分泌(IL-4、IL-10、IL-17A、IFN-γ、TNF-α和TGF-β1)、T细胞亚群变化以及T细胞凋亡等各方面的作用;探讨TGF-β1在BMSCs及其诱导的软骨细胞抑制T细胞的作用机制。
4.研究BMSCs及其分化的软骨细胞对CII反应性B细胞的作用收集RA患者PB及SF,分离PBMC和SFMC。采用BrdU掺入法、流式细胞术、ELISA等方法观察BMSCs及其分化的软细胞对RA患者CII特异性B细胞在增殖、亚群变化、抗CII抗体表达以及B细胞凋亡等各方面的作用;探讨TGF-β1在BMSCs及其诱导的软骨细胞抑制B细胞的作用机制。
5.研究异体BMSCs治疗大鼠CIA采用SD大鼠尾根部皮内注射乳化的鸡CII与完全福氏佐剂(complete Freund’s adjuvant,CFA)混合物的方法,建立CIA大鼠模型;分离、培养健康SD大鼠BMSCs;按静脉给予BMSCs的时间不同,将CIA大鼠分为初次免疫第7 d和21 d两个治疗组。以未注射BMSCs组CIA大鼠作阳性对照,通过CIA模型关节关节炎评分,血清中抗CII抗体水平变化以及关节的组织病理学检查,观察静脉注射异体BMSCs对CIA大鼠治疗的效果。
结果:
1. BMSCs及其生物学特性分离的细胞呈成纤维样的形态,其表面表达多种抗原标志,但并不表达造血细胞系的抗原,且这群细胞具有成功的向成骨细胞和脂肪细胞分化的能力,证实我们所分离出的细胞为BMSCs;传至第2-3代时,无论细胞形态还是其表面抗原的表达均显示BMSCs高度均一,但BMSCs随着传代呈逐渐衰老的趋势;长期培养时,BMSCs可自发的向成骨细胞分化;透射电镜下观察,可见BMSCs具有成熟和幼稚两种细胞形态。
2. BMSC向软骨细胞分化体外诱导分化21d后HE染色显示,细胞呈软骨样细胞形态;胞外基质甲苯胺蓝及CII染色呈阳性;诱导后细胞表达CII蛋白和前CoII的mRNA;此外,8 w后观察到裸鼠皮下植入的BMSCs-CII生物支架复合物已呈透明软骨样组织形态。
3. BMSCs及其分化的软骨细胞对CII反应性T细胞的作用BMSCs及其分化的软骨细胞,不引起RA患者T细胞的增殖与活化,反而能够抑制CII特异性T细胞的增殖、活化抗原(CD25和CD29)的表达,并呈剂量依赖趋势,且抑制效应并不因为BMSCs及其诱导的软骨细胞的延迟加入而改变;BMSCs及其分化的软骨细胞同样能够显著抑制CII特异性T细胞分泌炎性细胞因子如IL-17A、IFN-γ、TNF-α(P<0.05),防止抗炎因子IL-4的降低(P<0.05)以及升高共培养体系中抗炎因子IL-10和TGF-β1的水平(P<0.05);进一步检测发现,BMSCs及其分化的软骨细胞显著抑制T细胞亚群Th1、Th17的升高(P<0.05),防止Th2亚群降低;但对于Treg细胞的上调作用却不显著(P>0.05);此外,BMSCs及其分化的软骨细胞发挥抑制作用的同时并不会造成T细胞凋亡;TGF-β1在BMSCs及其分化的软骨细胞抑制T细胞的作用中扮演重要的角色。
4. BMSCs及其分化的软骨细胞对CII反应性B细胞的作用BMSCs及其分化的软骨细胞,同样不引起RA患者B细胞的增殖,而对CII特异性B细胞的增殖具有抑制作用; BMSCs及其分化的软骨能显著抑制幼稚型CD27-IgG+B细胞比例的降低(P<0.05)和记忆型CD27+IgG+B细胞(non-swiched memory B cells)比例的升高(P<0.05),但对于记忆型CD27+IgD- B细胞(memory B cells)比例的增长,抑制作用却并不显著(P>0.05); BMSCs同样可显著抑制B细胞产生抗CII抗体(P<0.05);BMSCs及其分化的软骨细胞发挥抑制作用时的同样并不会引起B细胞凋亡;TGF-β1在BMSCs及其分化的软骨细胞抑制B细胞的作用中也发挥重要的作用。
5.异体BMSCs治疗大鼠CIA的效果静脉给予CIA大鼠异体BMSCs并不会导致大鼠死亡,反而能够显著缓解CIA的病程发展(即关节肿胀变化过程)(P<0.05)以及显著降低CIA大鼠血清中抗CII抗体的水平(P<0.05);关节组织病理学检查证实,BMSCs能显著缓解CIA大鼠关节滑膜增殖和关节软骨的破坏程度(P<0.05),治疗越早,疗效越明显。
结论:
在成功建立BMSCs培养、鉴定以及诱导向软骨分化的研究体系的基础上,证明异体BMSCs及其诱导的软骨细胞在体外具有抑制RA CII特异性T、B细胞的作用,静脉移植BMSCs能够显著缓解大鼠CIA并减轻关节软骨的破坏。
Objective:
Rheumatoid arthritis (RA) is a chronic and systemic inflammatory auto-immune disease characterized by pain, swelling, and inflammation of joints resulting in progressive destruction of cartilage and bone. Though the mechanisms underlying remain to be clarified, cumulative evidence suggests that T cells, B cells and multiple pro-inflamatory cytokines are all involved in the pathogenesis of RA. Thus, that inhibiting antigen-reactive T-cell/B-cell activities and antagonizing pro-inflammatory cytokines are used as critical methods for RA threatment, which also have been proved effective by the successful using biological agents targeting at T cells, B cells and multiple pro-inflamatory cytokines in RA patients. However, these biological agents have not been got satisfying results in the treatment of some patients with refractory RA, and then autologous bone marrow transplantation (Auto-BMT) has been used as an important substitution. Though arthritis in patients with refractory RA has been notably allevated by auto-BMT, the prostecdtive efficacy of auto-BMT is still unknown. Moreover, RA patients-derived stem cells are recently considered to have some functional defects,which raised some suspection on the role of auto-BMT in RA threatment. On the orther hand, cartilage-repairing in the inflammation-infiltrated RA joints is another aspect for RA therapy, which still has not been well-studied so far.
The multilineage potential of bone mesenchymal stem cells (BMSCs) and their immunosuppressive nature suggest that allogenic BMSCs may be promising candidates for cartilage repairing and immunotherapy in the treatment of RA. BMSCs-based tissue engineering has obtained satisfactory results in repairing the articular cartilage defects in osteoarthritis (OA) models and patients. In addition, allogenic BMSCs also have been successfully used to treat graft-versus-host diseases (GVHD) in patients who received bone marrow transplantation (BMT) and alleviated a T-cell-mediated auto-immune diseas-experimental autoimmune encephalomyelitis (EAE) in an animal model. However, cell therapy for RA using allogenic BMSCs has not been well-studied.
In the present study, we firstly isolated BMSCs from healty adult bone marrow and induced theses cells to differentiate into chondrocyte/cartilage both in vitro and in vivo after expansion in vitro. Then, we explored the therapeutic potentials of BMSCs in RA treatment: the effects of BMSCs on the responses of CII-reactive T cells/ B cells from peripheral blood (PB)/synovial fluid (SF) of RA patients in vitro and on the collagean induced arthritis model by i.v administration were evaluated; we also investigated whether MSCs-differentiated chondrocytes (BMSCs-chondrocytes) possess the same immunological properties as MSCs to explore their cartilage-repairing potentiality in the inflammation-infiltrated RA joints for preventing cartilage re-destruction.
Methods (five parts):
1. Isolation and identification of BMSCs Bone marrow samples (3~5ml) were obtained from healthy adult human donors, and BMSCs were separated by centrifugation in percoll solution followed by adherence to the plastics cultured in vitro; then, the cell surface antigen were detected by flow cytometry (FCM) and their multilineage potential was confirmed through inducing BMSCs to differentiate into osteoblast and adipocytes; in addition, the morphology, cell growth curve, cell cycle and senescence staining were explored in cells from different passages; furthermore, the microstructure of BMSCs were also observed under transmission electron microscope(TEM).
2. Chondrogenic differentiation of BMSCs in vitro and in vivo The micro-cell aggregate was get by centrifugate and was induced by TGF-β1, dexamethasone(Dex) and Vitamin C(Vit C); 21 days later, the micro-cell aggregate were embedded in paraffin, sectioned and stained with HE staining, toluidine blue staining and immunohistochemical staining with anti-type II collagen (CII); the expression of CII in cell plasm were detected by Western blot and The expression of pro CoII mRNA were detected by RT-PCR; in addition, BMSCs-CII scaffod compounds were constructed in vitro followed by cultured in chondrogenesis-inducing medium, and the chondrogenic differentiation of these compounds were observed 8 weeks later after implanting subcutaneously in the athymic mice.
3. Influence of BMSCs and BMSCs-chondrocytes on CII-reactive T cells in vitro Paired peripheral blood mononuclear cells and synovial fluids mononuclear cell were isolated from patients with RA; then the effects of both MSCs and MSCs-chondrocytes on proliferation, activation-antigen expression (CD69 and CD25), and cytokines production (IFN-γ, TNF-α, TGF-β1, IL-17A, IL-10 and IL-4) of CII-reactive T cells and T-cell subsets in RA patients were investigated with the stimulation of CII or not by 3H-thymidine-incorporate detection, flow cytometry and ELISA respectively; CD3/Annexin V staining was used to evaluate T-cell apoptosis in the inhibition by FCM; the role of TGF-β1 underlying the inhibition was also investigated.
4. Influence of BMSCs and BMSCs-chondrocytes on CII-reactive B cells in vitro Paired peripheral blood mononuclear cells and synovial fluids mononuclear cell were isolated from patients with RA; then the effects of both MSCs and MSCs-chondrocytes on proliferation, anti-CII antibody production,B-cell subsets in RA patients were investigated with the stimulation of CII or not by BrdU-incorporate detection, flow cytometry and ELISA respectively; CD19/Annexin V staining was used to evaluate B-cell apoptosis in the inhibition by FCM; the role of TGF-β1 underlying the inhibition was also investigated.
5. Influence of BMSCs by I.V on CIA Rats were isolated and expanded firstly; Collagen-induced arthritis (CIA) model was induced by intradermal injection with chicken CII in complete Freund’s adjuvant (CFA) into SD rats,and a second injection of CII in CFA was administered on 8 day after primary immunization; CIA rats received BMSCs by i.v administration were divided into 2 groups according to the time point they received BMSCs after primary immunization: on 7 day and 21 day, respectively; CIA rats who did not receive BMSCs served as positive control; the effects of allogenic BMSCs on the development of arthritis were investigated by a arthritis scoring system and the titre of anti-CII antibody in serum at different time point, together with a histopathological scoring system in ankle joints after rats being sacrificed.
Results:
1. Isolation, identification of BMSCs and its biological properties The population we isolated showed a fibroblast-like morphology, and expressed multiple cell surface antigens while not expressed cell antigen of hemopoietic stem cell; these cells also retained the capacity to differentiate into osteocytes and adipocytes, which confirmed that the cells we isolated were BMSCs; both morphology and the results of FCM showed that the cells were 90 % and 98 % homogeneous respectively in passage 2 and passage 3; however, BMSCs were found senenscence during sequential passages; in addition, BMSCs were found to differentiate into osteoblast precursors during long-term culture; under TEM,we also found BMSCs were composed of 2 subpopulatons:nave cells and mature cells.
2. BMSCs could be successfully induced to differentiate into chondrocytes in vitro and in vivo 21 days later, the HE result showed that the post-inductive cells exhibited a chondrocyte-like morphology. The cells stained with toluidine blue and Col II were positive in the extracelluar matrix. The result of Western blot and RT-PCR showed that the Col II and its pro Col II mRNA were only expressed in post-inductive cells; 8 weeks later after implantation into athymic mice, BMSCs-CII scaffods compounds showed a cartilage-like morphology.
3. BMSCs and BMSCs-chondrocytes suppressed CII-reactive T cells in vitro Allogenic BMSCs failed to elicit positive responses of CII-reactive T cells, whereas significantly suppressed CII-stimulated T-cell proliferation and activation-antigen expression in a dose-dependent fashion without inducing T-cell apoptosis; the inhibition was still observed even BMSCs were added as late as 3 days after the initiation of stimulation; moreover, BMSCs inhibited T cells in producing IL-17A, IFN-γand TNF-α(P<0.05), while up-regulated the levels of IL-10 and TGF-β1 (P<0.05) and prevented IL-4 from decreasing(P<0.05); furthermore, Th1 and Th17 were found to be significantly suppressed by BMSCs(P<0.05) while Treg were not significantly upregulated (P>0.05); TGF-β1 was confirmed to play a critical role in inhibition. throughout our study, MSCs-chondrocytes shared the similar properties with MSCs.
4. BMSCs and BMSCs-chondrocytes suppressed CII-reactive B cells in vitro Allogenic BMSCs also failed to elicit positive responses of CII-reactive B cells, whereas significantly suppressed CII-stimulated B-cell proliferation and anti-CII antibody prduction without inducing B-cell apoptosis; in addition, BMSCs were also found significantly inhibited the up-regulation of CD27+IgG+non-swiched memory B cells with the stimulation of CII and down-regulation of CD27-IgG+ na?ve B cells(P<0.05); as for CD27+IgD- memory B cells, its elevation in the presence of CII was still suppressed by BMSCs, but insignificantly (P>0.05); TGF-β1 also played a critical role in the inhibition; throughout our study, MSCs-chondrocytes shared the similar properties with MSCs.
5. BMSCs alleviated arthritis in CIA BMSCs administration by i.v. fialed to result in the death of CIA rats for graft-versus-host diseases (GVHD), whereas siginificantly allevated arthritis and prevent the development of joint inflammation in the CIA rats(P<0.05); the titre of anti-CII antibody in CIA serum was also significantly suppressed by BMSCs(P<0.05); furthermore, BMSCs were found to significantly prevented the proliferation of synovium and destruction of cartilage in CIA rat(sP<0.05); in addition, the ealier using BMSCs for therapy ,the better it wil be;
Conclusion:
1. We successfully established the methods in our labaratory for BMSCs isolatation、culture and identification in vitro, and even successffuly induced BMSCs to differentiate into chondrocyte/cartilage both in vitro and in vivo;
2. We found allogenic BMSCs and they differentiated chondrocytes could significantly suppressed the responses of CII-reactive T cells and B cells in vitro;
3. We found allogenic BMSCs significantly alleviated the development of arthritis and destruction of cartilage in CIA rats;
类风湿关节炎(rheumatoid arthritis,RA)是一种慢性、系统性的炎性自身免疫病,通常伴有多关节肿胀与疼痛,若不及时治疗,可导致关节软骨、骨的侵蚀与破坏。尽管发病机制尚不明确,众多证据表明T细胞、B细胞以及多种炎性因子在RA病理过程中均发挥着重要的作用。有效的抑制这些炎性因素,则成为了RA治疗的一个重要目标。近来,以T细胞、B细胞和各种炎性细胞因子为治疗靶向的生物制剂已证实在RA治疗中的具有显著效果,但对于一些难治性RA患者的治疗仍不理想。而有学者认为RA患者自身的干细胞本身就存在功能缺陷,使得目前采用自体干细胞移植来治疗难治性RA的方案也受到质疑。另一方面,RA患者关节局部持续存在的炎症反应,是导致滑膜“肿瘤样”增殖,以及软骨组织最终遭受侵袭破坏的主要原因。尽管RA的治疗在抑制系统性炎症方面已有很大进展,但在炎症因素众多的关节局部如何有效地修复受损的软骨组织,目前还少见报道。
骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)多能分化性以及低免疫原性和免疫抑制特性的发现为解决这些问题带来了希望。以BMSCs为种子细胞构建的组织工程软骨已经在治疗骨关节炎(ostarthritis,OA)患者受损的关节软骨方面取得了满意的效果。另一方面,采用异体BMSCs治疗(graft-versus-host diseases,GVHD)的临床试验也取得成功。此外,BMSCs还被观察到能够缓解实验性脑脊膜炎(experimental allergic encephalomyelitis,EAE)——一种自身免疫病的动物模型的症状。然而,目前利用BMSCs的多能分化性和免疫学特性来治疗RA还少见报道。
因此,本研究旨在BMSCs分离、培养以及体内、外诱导其向软骨细胞/组织分化的基础上:1.研究BMSCs对RA患者II型胶原(type II collagean,CII)特异性T细胞、B细胞的免疫抑制作用,并进一步观察异体BMSCs静脉移植对胶原诱导(collagen induced arthritis,CIA)大鼠模型的治疗作用;2.初步研究BMSCs分化后的软骨细胞的免疫学特性,探讨以BMSCs作为种子细胞构建的组织工程软骨,在炎性因素众多的关节内修复受损软骨同时是否可发挥抑炎作用,从而防止修复后软骨再次遭受炎症破坏的可能性;为临床采用BMSCs治疗RA提供实验依据。
方法(分五部分):
1.建立BMSCs分离、培养、鉴定体系和研究生物特性抽取正常健康供者骨髓3-5 ml,依次采用密度梯度离心法、自然贴壁法分离BMSCs;采用流式细胞术检测表面标记,诱导其向成骨细胞/脂肪细胞分化等方法对其鉴定;观察不同代数细胞形态、生长曲线、细胞周期、衰老染色等各方面的变化以及长期培养的BMSCs的自然分化情况,了解BMSCs的体外生长以及部分生物学特性;通过透射电镜观察BMSCs的微观结构。
2.建立体内、外BMSCs向软骨细胞分化和鉴定体系将BMSCs离心后形成微小细胞团,在TGF-β1、地塞米松(Dex)及维生素C(Vit C)等诱导下向软骨细胞分化。21d后,细胞团经石蜡包埋、切片及HE染色后,进行甲苯胺蓝染色及CoII的免疫组化染色。采用Western blot和RT-PCR,分别检测诱导前后BMSCs中CoII及前CoII mRNA的表达。同时,采用组织工程技术在体外构建BMSCs-CII海绵复合物,在软骨细胞诱导液中培养1周后移植入裸鼠皮下,8 w后观察其大体形态。
3.研究BMSCs及其分化的软骨细胞对CII反应性T细胞的作用收集RA患者外周血(peripheral blood,PB)及关节滑液(synovial fluids,SF),分离单个核细胞(mononuclear cell,PBMC和SFMC)。采用3H-胸腺嘧啶核苷掺入法、流式细胞术、ELISA等方法观察BMSCs及其分化的软骨细胞对RA患者CII特异性T细胞在增殖、CD25/CD29等表面活化标记表达、多种细胞因子分泌(IL-4、IL-10、IL-17A、IFN-γ、TNF-α和TGF-β1)、T细胞亚群变化以及T细胞凋亡等各方面的作用;探讨TGF-β1在BMSCs及其诱导的软骨细胞抑制T细胞的作用机制。
4.研究BMSCs及其分化的软骨细胞对CII反应性B细胞的作用收集RA患者PB及SF,分离PBMC和SFMC。采用BrdU掺入法、流式细胞术、ELISA等方法观察BMSCs及其分化的软细胞对RA患者CII特异性B细胞在增殖、亚群变化、抗CII抗体表达以及B细胞凋亡等各方面的作用;探讨TGF-β1在BMSCs及其诱导的软骨细胞抑制B细胞的作用机制。
5.研究异体BMSCs治疗大鼠CIA采用SD大鼠尾根部皮内注射乳化的鸡CII与完全福氏佐剂(complete Freund’s adjuvant,CFA)混合物的方法,建立CIA大鼠模型;分离、培养健康SD大鼠BMSCs;按静脉给予BMSCs的时间不同,将CIA大鼠分为初次免疫第7 d和21 d两个治疗组。以未注射BMSCs组CIA大鼠作阳性对照,通过CIA模型关节关节炎评分,血清中抗CII抗体水平变化以及关节的组织病理学检查,观察静脉注射异体BMSCs对CIA大鼠治疗的效果。
结果:
1. BMSCs及其生物学特性分离的细胞呈成纤维样的形态,其表面表达多种抗原标志,但并不表达造血细胞系的抗原,且这群细胞具有成功的向成骨细胞和脂肪细胞分化的能力,证实我们所分离出的细胞为BMSCs;传至第2-3代时,无论细胞形态还是其表面抗原的表达均显示BMSCs高度均一,但BMSCs随着传代呈逐渐衰老的趋势;长期培养时,BMSCs可自发的向成骨细胞分化;透射电镜下观察,可见BMSCs具有成熟和幼稚两种细胞形态。
2. BMSC向软骨细胞分化体外诱导分化21d后HE染色显示,细胞呈软骨样细胞形态;胞外基质甲苯胺蓝及CII染色呈阳性;诱导后细胞表达CII蛋白和前CoII的mRNA;此外,8 w后观察到裸鼠皮下植入的BMSCs-CII生物支架复合物已呈透明软骨样组织形态。
3. BMSCs及其分化的软骨细胞对CII反应性T细胞的作用BMSCs及其分化的软骨细胞,不引起RA患者T细胞的增殖与活化,反而能够抑制CII特异性T细胞的增殖、活化抗原(CD25和CD29)的表达,并呈剂量依赖趋势,且抑制效应并不因为BMSCs及其诱导的软骨细胞的延迟加入而改变;BMSCs及其分化的软骨细胞同样能够显著抑制CII特异性T细胞分泌炎性细胞因子如IL-17A、IFN-γ、TNF-α(P<0.05),防止抗炎因子IL-4的降低(P<0.05)以及升高共培养体系中抗炎因子IL-10和TGF-β1的水平(P<0.05);进一步检测发现,BMSCs及其分化的软骨细胞显著抑制T细胞亚群Th1、Th17的升高(P<0.05),防止Th2亚群降低;但对于Treg细胞的上调作用却不显著(P>0.05);此外,BMSCs及其分化的软骨细胞发挥抑制作用的同时并不会造成T细胞凋亡;TGF-β1在BMSCs及其分化的软骨细胞抑制T细胞的作用中扮演重要的角色。
4. BMSCs及其分化的软骨细胞对CII反应性B细胞的作用BMSCs及其分化的软骨细胞,同样不引起RA患者B细胞的增殖,而对CII特异性B细胞的增殖具有抑制作用; BMSCs及其分化的软骨能显著抑制幼稚型CD27-IgG+B细胞比例的降低(P<0.05)和记忆型CD27+IgG+B细胞(non-swiched memory B cells)比例的升高(P<0.05),但对于记忆型CD27+IgD- B细胞(memory B cells)比例的增长,抑制作用却并不显著(P>0.05); BMSCs同样可显著抑制B细胞产生抗CII抗体(P<0.05);BMSCs及其分化的软骨细胞发挥抑制作用时的同样并不会引起B细胞凋亡;TGF-β1在BMSCs及其分化的软骨细胞抑制B细胞的作用中也发挥重要的作用。
5.异体BMSCs治疗大鼠CIA的效果静脉给予CIA大鼠异体BMSCs并不会导致大鼠死亡,反而能够显著缓解CIA的病程发展(即关节肿胀变化过程)(P<0.05)以及显著降低CIA大鼠血清中抗CII抗体的水平(P<0.05);关节组织病理学检查证实,BMSCs能显著缓解CIA大鼠关节滑膜增殖和关节软骨的破坏程度(P<0.05),治疗越早,疗效越明显。
结论:
在成功建立BMSCs培养、鉴定以及诱导向软骨分化的研究体系的基础上,证明异体BMSCs及其诱导的软骨细胞在体外具有抑制RA CII特异性T、B细胞的作用,静脉移植BMSCs能够显著缓解大鼠CIA并减轻关节软骨的破坏。
Objective:
Rheumatoid arthritis (RA) is a chronic and systemic inflammatory auto-immune disease characterized by pain, swelling, and inflammation of joints resulting in progressive destruction of cartilage and bone. Though the mechanisms underlying remain to be clarified, cumulative evidence suggests that T cells, B cells and multiple pro-inflamatory cytokines are all involved in the pathogenesis of RA. Thus, that inhibiting antigen-reactive T-cell/B-cell activities and antagonizing pro-inflammatory cytokines are used as critical methods for RA threatment, which also have been proved effective by the successful using biological agents targeting at T cells, B cells and multiple pro-inflamatory cytokines in RA patients. However, these biological agents have not been got satisfying results in the treatment of some patients with refractory RA, and then autologous bone marrow transplantation (Auto-BMT) has been used as an important substitution. Though arthritis in patients with refractory RA has been notably allevated by auto-BMT, the prostecdtive efficacy of auto-BMT is still unknown. Moreover, RA patients-derived stem cells are recently considered to have some functional defects,which raised some suspection on the role of auto-BMT in RA threatment. On the orther hand, cartilage-repairing in the inflammation-infiltrated RA joints is another aspect for RA therapy, which still has not been well-studied so far.
The multilineage potential of bone mesenchymal stem cells (BMSCs) and their immunosuppressive nature suggest that allogenic BMSCs may be promising candidates for cartilage repairing and immunotherapy in the treatment of RA. BMSCs-based tissue engineering has obtained satisfactory results in repairing the articular cartilage defects in osteoarthritis (OA) models and patients. In addition, allogenic BMSCs also have been successfully used to treat graft-versus-host diseases (GVHD) in patients who received bone marrow transplantation (BMT) and alleviated a T-cell-mediated auto-immune diseas-experimental autoimmune encephalomyelitis (EAE) in an animal model. However, cell therapy for RA using allogenic BMSCs has not been well-studied.
In the present study, we firstly isolated BMSCs from healty adult bone marrow and induced theses cells to differentiate into chondrocyte/cartilage both in vitro and in vivo after expansion in vitro. Then, we explored the therapeutic potentials of BMSCs in RA treatment: the effects of BMSCs on the responses of CII-reactive T cells/ B cells from peripheral blood (PB)/synovial fluid (SF) of RA patients in vitro and on the collagean induced arthritis model by i.v administration were evaluated; we also investigated whether MSCs-differentiated chondrocytes (BMSCs-chondrocytes) possess the same immunological properties as MSCs to explore their cartilage-repairing potentiality in the inflammation-infiltrated RA joints for preventing cartilage re-destruction.
Methods (five parts):
1. Isolation and identification of BMSCs Bone marrow samples (3~5ml) were obtained from healthy adult human donors, and BMSCs were separated by centrifugation in percoll solution followed by adherence to the plastics cultured in vitro; then, the cell surface antigen were detected by flow cytometry (FCM) and their multilineage potential was confirmed through inducing BMSCs to differentiate into osteoblast and adipocytes; in addition, the morphology, cell growth curve, cell cycle and senescence staining were explored in cells from different passages; furthermore, the microstructure of BMSCs were also observed under transmission electron microscope(TEM).
2. Chondrogenic differentiation of BMSCs in vitro and in vivo The micro-cell aggregate was get by centrifugate and was induced by TGF-β1, dexamethasone(Dex) and Vitamin C(Vit C); 21 days later, the micro-cell aggregate were embedded in paraffin, sectioned and stained with HE staining, toluidine blue staining and immunohistochemical staining with anti-type II collagen (CII); the expression of CII in cell plasm were detected by Western blot and The expression of pro CoII mRNA were detected by RT-PCR; in addition, BMSCs-CII scaffod compounds were constructed in vitro followed by cultured in chondrogenesis-inducing medium, and the chondrogenic differentiation of these compounds were observed 8 weeks later after implanting subcutaneously in the athymic mice.
3. Influence of BMSCs and BMSCs-chondrocytes on CII-reactive T cells in vitro Paired peripheral blood mononuclear cells and synovial fluids mononuclear cell were isolated from patients with RA; then the effects of both MSCs and MSCs-chondrocytes on proliferation, activation-antigen expression (CD69 and CD25), and cytokines production (IFN-γ, TNF-α, TGF-β1, IL-17A, IL-10 and IL-4) of CII-reactive T cells and T-cell subsets in RA patients were investigated with the stimulation of CII or not by 3H-thymidine-incorporate detection, flow cytometry and ELISA respectively; CD3/Annexin V staining was used to evaluate T-cell apoptosis in the inhibition by FCM; the role of TGF-β1 underlying the inhibition was also investigated.
4. Influence of BMSCs and BMSCs-chondrocytes on CII-reactive B cells in vitro Paired peripheral blood mononuclear cells and synovial fluids mononuclear cell were isolated from patients with RA; then the effects of both MSCs and MSCs-chondrocytes on proliferation, anti-CII antibody production,B-cell subsets in RA patients were investigated with the stimulation of CII or not by BrdU-incorporate detection, flow cytometry and ELISA respectively; CD19/Annexin V staining was used to evaluate B-cell apoptosis in the inhibition by FCM; the role of TGF-β1 underlying the inhibition was also investigated.
5. Influence of BMSCs by I.V on CIA Rats were isolated and expanded firstly; Collagen-induced arthritis (CIA) model was induced by intradermal injection with chicken CII in complete Freund’s adjuvant (CFA) into SD rats,and a second injection of CII in CFA was administered on 8 day after primary immunization; CIA rats received BMSCs by i.v administration were divided into 2 groups according to the time point they received BMSCs after primary immunization: on 7 day and 21 day, respectively; CIA rats who did not receive BMSCs served as positive control; the effects of allogenic BMSCs on the development of arthritis were investigated by a arthritis scoring system and the titre of anti-CII antibody in serum at different time point, together with a histopathological scoring system in ankle joints after rats being sacrificed.
Results:
1. Isolation, identification of BMSCs and its biological properties The population we isolated showed a fibroblast-like morphology, and expressed multiple cell surface antigens while not expressed cell antigen of hemopoietic stem cell; these cells also retained the capacity to differentiate into osteocytes and adipocytes, which confirmed that the cells we isolated were BMSCs; both morphology and the results of FCM showed that the cells were 90 % and 98 % homogeneous respectively in passage 2 and passage 3; however, BMSCs were found senenscence during sequential passages; in addition, BMSCs were found to differentiate into osteoblast precursors during long-term culture; under TEM,we also found BMSCs were composed of 2 subpopulatons:nave cells and mature cells.
2. BMSCs could be successfully induced to differentiate into chondrocytes in vitro and in vivo 21 days later, the HE result showed that the post-inductive cells exhibited a chondrocyte-like morphology. The cells stained with toluidine blue and Col II were positive in the extracelluar matrix. The result of Western blot and RT-PCR showed that the Col II and its pro Col II mRNA were only expressed in post-inductive cells; 8 weeks later after implantation into athymic mice, BMSCs-CII scaffods compounds showed a cartilage-like morphology.
3. BMSCs and BMSCs-chondrocytes suppressed CII-reactive T cells in vitro Allogenic BMSCs failed to elicit positive responses of CII-reactive T cells, whereas significantly suppressed CII-stimulated T-cell proliferation and activation-antigen expression in a dose-dependent fashion without inducing T-cell apoptosis; the inhibition was still observed even BMSCs were added as late as 3 days after the initiation of stimulation; moreover, BMSCs inhibited T cells in producing IL-17A, IFN-γand TNF-α(P<0.05), while up-regulated the levels of IL-10 and TGF-β1 (P<0.05) and prevented IL-4 from decreasing(P<0.05); furthermore, Th1 and Th17 were found to be significantly suppressed by BMSCs(P<0.05) while Treg were not significantly upregulated (P>0.05); TGF-β1 was confirmed to play a critical role in inhibition. throughout our study, MSCs-chondrocytes shared the similar properties with MSCs.
4. BMSCs and BMSCs-chondrocytes suppressed CII-reactive B cells in vitro Allogenic BMSCs also failed to elicit positive responses of CII-reactive B cells, whereas significantly suppressed CII-stimulated B-cell proliferation and anti-CII antibody prduction without inducing B-cell apoptosis; in addition, BMSCs were also found significantly inhibited the up-regulation of CD27+IgG+non-swiched memory B cells with the stimulation of CII and down-regulation of CD27-IgG+ na?ve B cells(P<0.05); as for CD27+IgD- memory B cells, its elevation in the presence of CII was still suppressed by BMSCs, but insignificantly (P>0.05); TGF-β1 also played a critical role in the inhibition; throughout our study, MSCs-chondrocytes shared the similar properties with MSCs.
5. BMSCs alleviated arthritis in CIA BMSCs administration by i.v. fialed to result in the death of CIA rats for graft-versus-host diseases (GVHD), whereas siginificantly allevated arthritis and prevent the development of joint inflammation in the CIA rats(P<0.05); the titre of anti-CII antibody in CIA serum was also significantly suppressed by BMSCs(P<0.05); furthermore, BMSCs were found to significantly prevented the proliferation of synovium and destruction of cartilage in CIA rat(sP<0.05); in addition, the ealier using BMSCs for therapy ,the better it wil be;
Conclusion:
1. We successfully established the methods in our labaratory for BMSCs isolatation、culture and identification in vitro, and even successffuly induced BMSCs to differentiate into chondrocyte/cartilage both in vitro and in vivo;
2. We found allogenic BMSCs and they differentiated chondrocytes could significantly suppressed the responses of CII-reactive T cells and B cells in vitro;
3. We found allogenic BMSCs significantly alleviated the development of arthritis and destruction of cartilage in CIA rats;
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