骨髓间充质干细胞的IDO对小鼠异基因骨髓移植中GVHD效应影响的实验研究
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摘要
近30年来,异基因骨髓移植(allogeneic bone marrow transplantation ,Allo-BMT)已经成功应用于治疗恶性血液病、严重免疫缺陷病和骨髓造血功能衰竭等疾病。至今为止,急性移植物抗宿主病(acute graft-versus-host disease ,aGVHD)仍是移植相关死亡的主要原因,是移植的主要障碍。特别是在接受HLA配型不全相合的无关供者移植的患者中,常发生严重的急性GVHD。虽然应用免疫抑制药物和T细胞去除的方法能有效的控制GVHD,但治疗的同时会引起严重的并发症,比如感染和增加疾病复发风险。因此许多研究者一直致力于寻找一些防止GVHD发生并能避免免疫抑制引起的并发症的方法。
骨髓间充质干细胞(mesenchymal stem cells , MSCs)是骨髓中能分化为成骨细胞、脂肪细胞和成肌细胞,具有多向分化潜能的细胞。研究已经证实MSCs能产生许多促进造血细胞归巢、增殖和分化的细胞因子,因此在促进造血恢复中发挥重要的作用。而且,MSCs不表达MHC II类分子或共刺激分子,具有较弱的抗原性。研究也表明MSCs具有免疫调节的特性,可以减轻GVHD。但对于MSCs发挥免疫负调节作用、减轻GVHD的机制仍不清楚。
吲哚胺2,3-过氧化酶(indoleamine 2,3-dioxygenase IDO)是色氨酸沿犬尿氨酸代谢途径降解的限速酶.可以降解T细胞周围的色氨酸。而T细胞的细胞周期中有一个对周围色氨酸水平极为敏感的调节点,色氨酸的缺乏使其停滞在mid-G1期,从而抑制活化的T细胞的增殖。在IFN-γ的作用下,机体内的多种细胞如成纤维细胞、上皮细胞、巨噬细胞均可产生IDO。近来,人们还发现母胎界面滋养层细胞中IDO在避免胎儿受母体排斥中具有重要作用。因此,抗原提呈细胞上的IDO被公认为抑制机体T细胞对异体抗原发生免疫应答的主要机制之一。基于此,我们试图体外分离培养及扩增小鼠的MSCs,检测IFN-γ作用下的MSCs上IDO mRNA和IDO蛋白的表达,以及IDO在MSCs介导同种混合淋巴细胞增殖反应的作用;通过建立小鼠异基因骨髓移植模型,观察具有IDO表达的MSCs在小鼠异基因骨髓移植中抑制同种异体免疫反应、降低GVHD发生的体内效果,探讨MSCs的IDO活性参与MSCs发挥免疫负调节作用的机理,为临床MSCs防治GVHD提供可靠的实验依据。
第一部分小鼠骨髓间充质干细胞的分离培养及其生物学特性的研究
目的:探讨小鼠骨髓来源的间充质干细胞(MSCs)的体外分离、纯化、扩增和多向分化条件。
方法:获取小鼠骨髓细胞,以IMDM作为培养基进行培养和纯化细胞,瑞氏染色和电镜观察形态;FACS检测其免疫表型和细胞周期;体外诱导成骨细胞、脂肪细胞、神经样细胞分化,Von Kossa染色、油红O染色、免疫组织化学法检测细胞向成骨、成脂肪细胞、神经样细胞分化情况。
结果:小鼠骨髓来源的细胞呈纤维样贴壁生长,瑞氏染色和电镜观察具有MSCs特征;FACS检测结果显示,表达MSCs相关的抗原CD29、CD44、CD105,而CD31、CD13、CD34、CD45、I-ab为阴性;体外诱导成骨细胞、脂肪细胞、神经样细胞分化成功。
结论:体外成功地分离及培养了小鼠MSCs。
第二部分IDO在小鼠骨髓间充质干细胞的表达及对T细胞增殖的影响
目的:探讨γ-干扰素(IFN-γ)作用下小鼠MSCs上吲哚胺2,3-双加氧酶(IDO)表达情况及其活性对异基因T淋巴细胞增殖的影响。
方法:以不同浓度的IFN-γ(0、20、50、100、200 U/ml)作用小鼠MSCs,以RT-PCR和Western blot分别检测IDO mRNA和IDO蛋白表达,以反相高效液相色谱法(HPLC)检测IDO活性;分别以丝裂霉素C灭活的C57BL/ 6鼠脾细胞为刺激细胞,BALB/C鼠脾细胞为反应细胞建立混合淋巴细胞培养体系(MLRs)。有或无1-甲基色氨酸(1-MT)存在下, MSCs/ MLRs共培养体系中C57BL/ 6鼠MSCs为1×105、5×104、1×104、5×103(MSCs:反应T细胞数量比为1:5,1:10,1:50,1:100)。以MTT法检测T淋巴细胞增殖率,HPLC法检测各MLRs体系上清中IDO活性。
结果: IFN-γ作用后, MSCs出现IDO mRNA、IDO蛋白表达以及功能性的IDO活性,且IDO mRNA、IDO蛋白表达量和IDO活性与IFN-γ浓度呈剂量依赖性;当MSCs为1×10~5、5×10~4、1×10~4(即MSCs:反应T细胞为1:5,1:10,1:50)时,与对照组比较,T淋巴细胞的增殖率显著降低(P<0.05),IDO活性显著升高(P<0.05);加入1-甲基色氨酸(1-MT)后,T淋巴细胞的增殖率及IDO活性均恢复。当MSCs为5×103(即MSCs:反应T细胞为1:100)时,与对照组比较,T淋巴细胞的增殖率轻度升高(P>0.05),IDO活性无明显变化(P>0.05)。
结论: MSCs上的IDO表达与T淋巴细胞的增殖密切相关,MSCs在体外可能通过IDO活性发挥免疫抑制作用。
第三部分小鼠异基因骨髓移植急性移植物抗宿主病模型的建立
目的:建立小鼠异基因骨髓移植急性移植物抗宿主病(aGVHD)模型,为下一步异基因骨髓移植中aGVHD研究提供理想的实验参照。
方法:采用近亲系的雄性C57BL/6(H-2b)小鼠为供鼠、60Co致死量照射的雌性BALB/C(H-2d)小鼠为受鼠建立异基因骨髓移植模型,以混合移植2:1的异基因骨髓细胞与外周脾细胞作为aGVHD组,同时设立空白对照组(无骨髓细胞及外周脾细胞植入)和单纯Allo-BMT组(无外周脾细胞植入)。根据临床表现、存活期及病理改变等判断GVHD程度。
结果:单纯Allo-BMT组无典型的急性GVHD表现;aGVHD组受鼠GVHD的临床评分在10分左右,存活期6-24天,临床和病理均可在相对集中的时间内观察到典型的急性GVHD改变。
结论:成功地建立了小鼠异基因骨髓移植急性移植物抗宿主病(aGVHD)模型。
第四部分MSCs的IDO对小鼠异基因骨髓移植中aGVHD效应的影响
目的:观察具有IDO表达的MSCs在小鼠异基因骨髓移植中降低aGVHD发生的体内效果,探讨MSCs上的IDO活性参与其发挥免疫抑制作用的机制。
方法:采用近亲系的雄性C57BL/6(H-2b)小鼠为供鼠、60Co致死量照射的雌性BALB/C(H-2d)小鼠为受鼠建立不同的移植组:aGVHD组(A组)、大剂量MSCs组(B组)、小剂量MSCs组(C组)和1-MT预处理组(D组)。根据临床表现和病理改变判断GVHD程度,并比较不同移植组的存活率、GVHD发生情况和Th1类细胞因子(IL-2、IFN-γ)和Th2类(IL-4、IL-10)细胞因子分泌情况。
结果: B组受鼠,GVHD的临床评分在4分左右,无组织病理改变,全部长期存活,与A组比较差异显著(P<0.05)。与A组比较,B组受鼠血清中Th1类细胞因子显著降低,Th2类细胞因子显著升高(P<0.05)。A、C、D三组之间的GVHD临床评分、病理变化、生存率及细胞因子水平无统计学差异(P>0.05)。
结论:在小鼠Allo-BMT中,同种异基因MSCs的IDO活性可促进Thl向Th2的偏离,进而产生抑制GVHD效应,推测MSCs在体内同样通过IDO活性发挥免疫抑制作用。
Over the last 30 years allogeneic bone marrow transplantation (Allo-BMT) has become an accepted curative therapy for a variety of malignant hematopoietic diseases, severe immunologic deficiency diseases and hematopoietic failure of bone marrow. To date, the chief obstacle to the deployment of this treatment in other than life-threatening conditions is graft-versus-host disease (GVHD), and in particular acute GVHD, which is most severe in patients receiving matched unrelated donor transplants. The immunosuppressive drugs and T-cell depletion is effective at controlling GVHD, but these regimens can lead to serious complications from infection and an increased risk of leukemia relapse after Allo-BMT. Many researchers have therefore sought to develop techniques to prevent GVHD that avoid the complications incurred by immunosuppression.
Mesenchymal stem cells (MSCs) are multipotent precursors present in bone marrow, capable of differentiating into osteoblasts, adipocytes, and myoblasts. Some studies have confirmed MSCs could constitutively secrete a variety of cytokines that promote the homing or proliferation and differentiation of hematopoietic cells, and accordingly play important roles in promoting hematopoietic recovery. Moreover, MSCs are poor antigen-presenting cells and do not express MHC class II or co-stimulatory molecules, studies have also demonstrated MSCs could exhibit immunoregulatory properties in vitro or in vivo. Due to the distinct immunophenotype profile MSCs is concerned as a prophylaxis for GVHD, but the molecular mechanisms responsible for the immunosuppressive effects of MSCs have not been unequivocally identified.
Indoleamine 2,3-dioxygenase(IDO) is the initial and rate-limiting enzyme of the kynurenine pathway of degradation of L-tryptophan. IDO functionally catabolizes conversion from tryptophan .Tryptophan is necessary to the survival and proliferation of T cells. In the absence of tryptophan, the T cells cycle is arrested at a mid-G1 point. Following stimulation with IFN-γ, it is induced in various types of cell lines and cell types such as fibroblasts, epithelial cells and macrophages. Recently, it has been implicated IDO synthesized by the macrophages of placenta plays an important role in the prevention of allogeneic fetal rejection. So in professional antigen-presenting cells (APCs) expression of IDO has recently been identified as a major immunosuppressive mechanisms that inhibit T-cell activation and proliferation to autoantigens and alloantigens.
Basing on these, we tried to investigate the IDO mRNA and IDO protein expression in murine bone marrow-derived MSCs induced by IFN-γ, and the capacity of IDO activity in MSCs to induce allogeneic T cell proliferation; The in vivo effects of MSCs expressing IDO on acute graft-versus-host disease (aGVHD) in murine allogenic bone marrow transplantation (Allo-BMT) model and its underlying mechanisms were also investigated in this paper.
PartⅠStudy on isolation and biological characteristics of mesenchymal stem cells from murine bone marrow
Objective: To investigate the isolation, purification, expansion and biological characteristics of mesenchymal stem cells (MSCs) derived from murine bone marrow in vitro.
Methods: MSCs were isolated from murine bone marrow and cultured in IMDM medium. The morphology of the cells was observed by Wright’s staining and electron microscope. Cell cycle and immunophenotype were investigated by flow cytometry. Assays of adipogenic and osteogenic differentiation were performed in vitro, then, Von Kossa stain, Oil-redO staining and immunohistochemistr were used to indentified to the induced-cells.
Results: The cells from murine bone marrow displayed a fibroblast-like morphology adhering to the culture plate. FACS showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34, and I-ab were negative. Adipocyte, osteocyte and nerve-like cells differentiation were induced successfully.
Conclusion: MSCs derived from murine bone marrow were successfully isolated and cultured in vitro. PartⅡStudy on the effect of IDO in MSCs on allogeneic T-lymphocyte proliferation Objective: To study the effect ofγ-interferon (IFN-γ) on the IDO mRNA and IDO protein expression of MSCs and the effect of IDO activity in MSCs on allogeneic T-lymphocyte proliferation. Methods: IDO mRNA and IDO protein expression of MSCs induced by IFN-γat various concentration (0、20、50、100、200 U/ml) were analyzed by RT-PCR method and Western blot method respectively ,and IDO activity was determined by high-performance liquid chromatography (HPLC); MLRs cultures were set up with mitomycin C–treated C57BL/ 6-derived splenocytes as stimulators and BALB/c-derived splenocytes as responder cells. In MSCs/MLRs coculture experiments, MLRs were performed on a layer of 1×105,5×104,1×104 or 5×103 C57BL/ 6-derived MSCs (MSCs :responder T cells =1:5,1:10,1:50,1:100 ) with or without 1-methyl-D-trytophan(1-MT). T-lymphocytes proliferation was determined by MTT assays and IDO activity in coculture supernatant was determined by HPLC. Results: IDO mRNA expression, IDO protein expression and IDO functional activity were found in MSCs induced by IFN-γ, and the amount of expression and IDO activity increased gradually with the concentration of IFN-γ; When cocultured with 1×10~5、5×10~4、1×10~4 MSCs (MSCs :responder T cells =1:5,1:10,1:50),T- lymphocytes proliferation decreased apparently and IDO activity increased obviously compared with control group(p<0.05);In parallel experiments, 1-MT was added. T-lymphocytes proliferation and IDO activity restored partially. While cocultured with 5×10~3 MSCs (MSCs :responder T cells =1:100)T- lymphocytes proliferation raised lightly and IDO activity had no change compared with control group(p>0.05) .
Conclusion: IDO activity in MSCs is relation to T-lymphocyte proliferation, and MSCs may perform their immunosuppressive function by IDO activity in vitro.
PartⅢEstablishment of acute graft-versus-host disease model in murine allogeneic bone marrow transplantation
Objective: To establish murine acute graft-versus-host disease (aGVHD) model for the study of aGVHD in allogeneic bone marrow transplantation (Allo-BMT).
Methods: Lethally irradiated BALB/C (female,H-2d) murine transplanted with 1×107 C57BL/6 (male,H-2b ) bone marrow cells and 5×106 peripheral splenocytes (bone marrow cells and peripheral splenocytes ratio was 2:1) acted as GVHD group. The following two groups were simultaneously used: blank control group, no bone marrow cells and peripheral splenocytes transplanted; Allo-BMT group, no peripheral splenocytes transplanted. Evaluated the degree of GVHD according to clinical and pathological data.
Results: Typical aGVHD could not be induced in GVHD group. GVHD clinical scores of recipient in GVHD group was about 10, and all murine survived 6 to 24 days after transplantation. Clinical signs and pathological changes of aGVHD were seen in a relative localized period.
Conclusion: Murine lethal aGVHD model was successfully established.
PartⅣEffect of IDO in MSCs on aGVHD in murine allogeneic bone marrow Transplantation
Objective: Study the in vivo effects of MSCs expressing IDO on acute graft-versus-host disease (aGVHD) in murine allogenic bone marrow transplantation (Allo-BMT) model and explore the mechanism responsible for the immunosuppressive effects of MSCs.
Methods: Various Allo-BMT groups were established with lethally irradiated BALB/C (female, H-2d) murine and C57BL/6(male, H-2b) : GVHD group(Group A); Cotransplanted with large dose MSCs(Group B); Cotransplanted with low dose MSCs(Group C); Cotransplanted with large dose MSCs pre-deal by 1-MT(Group D). The degree of GVHD ,survival rates, histopathological changes and helper T-cell type l [Th1(IL-2、IFN-γ)] and type 2 [Th2(IL-4、IL-10)] cytokines level were observed in all the groups after BMT.
Results: It showed that GVHD clinical scores of recipient in group B was about 4, and no histopathological changes were seen, and the survival rate was 100%. The degree of GVHD was evidently lessened and survival rates was significantly higher compare with group A (P<0.05); In group B, Th1 cytokines level decreased and Th2 cytokines level increased significantly compare with group A (P<0.05). But GVHD clinical scores , histopathological changes , survival rate and cytokines level were not obviously different among Group A, C and D (P>0.05).
Conclusion: IDO activity in alloreactive MSCs shift the Thl/Th2 balance toward a Th2-production of type-2 cytokines dominant phenotype, then reduced the incidence of GVHD. We supposed that MSCs may perform their immunosuppressive function by IDO activity in vivo.
骨髓间充质干细胞(mesenchymal stem cells , MSCs)是骨髓中能分化为成骨细胞、脂肪细胞和成肌细胞,具有多向分化潜能的细胞。研究已经证实MSCs能产生许多促进造血细胞归巢、增殖和分化的细胞因子,因此在促进造血恢复中发挥重要的作用。而且,MSCs不表达MHC II类分子或共刺激分子,具有较弱的抗原性。研究也表明MSCs具有免疫调节的特性,可以减轻GVHD。但对于MSCs发挥免疫负调节作用、减轻GVHD的机制仍不清楚。
吲哚胺2,3-过氧化酶(indoleamine 2,3-dioxygenase IDO)是色氨酸沿犬尿氨酸代谢途径降解的限速酶.可以降解T细胞周围的色氨酸。而T细胞的细胞周期中有一个对周围色氨酸水平极为敏感的调节点,色氨酸的缺乏使其停滞在mid-G1期,从而抑制活化的T细胞的增殖。在IFN-γ的作用下,机体内的多种细胞如成纤维细胞、上皮细胞、巨噬细胞均可产生IDO。近来,人们还发现母胎界面滋养层细胞中IDO在避免胎儿受母体排斥中具有重要作用。因此,抗原提呈细胞上的IDO被公认为抑制机体T细胞对异体抗原发生免疫应答的主要机制之一。基于此,我们试图体外分离培养及扩增小鼠的MSCs,检测IFN-γ作用下的MSCs上IDO mRNA和IDO蛋白的表达,以及IDO在MSCs介导同种混合淋巴细胞增殖反应的作用;通过建立小鼠异基因骨髓移植模型,观察具有IDO表达的MSCs在小鼠异基因骨髓移植中抑制同种异体免疫反应、降低GVHD发生的体内效果,探讨MSCs的IDO活性参与MSCs发挥免疫负调节作用的机理,为临床MSCs防治GVHD提供可靠的实验依据。
第一部分小鼠骨髓间充质干细胞的分离培养及其生物学特性的研究
目的:探讨小鼠骨髓来源的间充质干细胞(MSCs)的体外分离、纯化、扩增和多向分化条件。
方法:获取小鼠骨髓细胞,以IMDM作为培养基进行培养和纯化细胞,瑞氏染色和电镜观察形态;FACS检测其免疫表型和细胞周期;体外诱导成骨细胞、脂肪细胞、神经样细胞分化,Von Kossa染色、油红O染色、免疫组织化学法检测细胞向成骨、成脂肪细胞、神经样细胞分化情况。
结果:小鼠骨髓来源的细胞呈纤维样贴壁生长,瑞氏染色和电镜观察具有MSCs特征;FACS检测结果显示,表达MSCs相关的抗原CD29、CD44、CD105,而CD31、CD13、CD34、CD45、I-ab为阴性;体外诱导成骨细胞、脂肪细胞、神经样细胞分化成功。
结论:体外成功地分离及培养了小鼠MSCs。
第二部分IDO在小鼠骨髓间充质干细胞的表达及对T细胞增殖的影响
目的:探讨γ-干扰素(IFN-γ)作用下小鼠MSCs上吲哚胺2,3-双加氧酶(IDO)表达情况及其活性对异基因T淋巴细胞增殖的影响。
方法:以不同浓度的IFN-γ(0、20、50、100、200 U/ml)作用小鼠MSCs,以RT-PCR和Western blot分别检测IDO mRNA和IDO蛋白表达,以反相高效液相色谱法(HPLC)检测IDO活性;分别以丝裂霉素C灭活的C57BL/ 6鼠脾细胞为刺激细胞,BALB/C鼠脾细胞为反应细胞建立混合淋巴细胞培养体系(MLRs)。有或无1-甲基色氨酸(1-MT)存在下, MSCs/ MLRs共培养体系中C57BL/ 6鼠MSCs为1×105、5×104、1×104、5×103(MSCs:反应T细胞数量比为1:5,1:10,1:50,1:100)。以MTT法检测T淋巴细胞增殖率,HPLC法检测各MLRs体系上清中IDO活性。
结果: IFN-γ作用后, MSCs出现IDO mRNA、IDO蛋白表达以及功能性的IDO活性,且IDO mRNA、IDO蛋白表达量和IDO活性与IFN-γ浓度呈剂量依赖性;当MSCs为1×10~5、5×10~4、1×10~4(即MSCs:反应T细胞为1:5,1:10,1:50)时,与对照组比较,T淋巴细胞的增殖率显著降低(P<0.05),IDO活性显著升高(P<0.05);加入1-甲基色氨酸(1-MT)后,T淋巴细胞的增殖率及IDO活性均恢复。当MSCs为5×103(即MSCs:反应T细胞为1:100)时,与对照组比较,T淋巴细胞的增殖率轻度升高(P>0.05),IDO活性无明显变化(P>0.05)。
结论: MSCs上的IDO表达与T淋巴细胞的增殖密切相关,MSCs在体外可能通过IDO活性发挥免疫抑制作用。
第三部分小鼠异基因骨髓移植急性移植物抗宿主病模型的建立
目的:建立小鼠异基因骨髓移植急性移植物抗宿主病(aGVHD)模型,为下一步异基因骨髓移植中aGVHD研究提供理想的实验参照。
方法:采用近亲系的雄性C57BL/6(H-2b)小鼠为供鼠、60Co致死量照射的雌性BALB/C(H-2d)小鼠为受鼠建立异基因骨髓移植模型,以混合移植2:1的异基因骨髓细胞与外周脾细胞作为aGVHD组,同时设立空白对照组(无骨髓细胞及外周脾细胞植入)和单纯Allo-BMT组(无外周脾细胞植入)。根据临床表现、存活期及病理改变等判断GVHD程度。
结果:单纯Allo-BMT组无典型的急性GVHD表现;aGVHD组受鼠GVHD的临床评分在10分左右,存活期6-24天,临床和病理均可在相对集中的时间内观察到典型的急性GVHD改变。
结论:成功地建立了小鼠异基因骨髓移植急性移植物抗宿主病(aGVHD)模型。
第四部分MSCs的IDO对小鼠异基因骨髓移植中aGVHD效应的影响
目的:观察具有IDO表达的MSCs在小鼠异基因骨髓移植中降低aGVHD发生的体内效果,探讨MSCs上的IDO活性参与其发挥免疫抑制作用的机制。
方法:采用近亲系的雄性C57BL/6(H-2b)小鼠为供鼠、60Co致死量照射的雌性BALB/C(H-2d)小鼠为受鼠建立不同的移植组:aGVHD组(A组)、大剂量MSCs组(B组)、小剂量MSCs组(C组)和1-MT预处理组(D组)。根据临床表现和病理改变判断GVHD程度,并比较不同移植组的存活率、GVHD发生情况和Th1类细胞因子(IL-2、IFN-γ)和Th2类(IL-4、IL-10)细胞因子分泌情况。
结果: B组受鼠,GVHD的临床评分在4分左右,无组织病理改变,全部长期存活,与A组比较差异显著(P<0.05)。与A组比较,B组受鼠血清中Th1类细胞因子显著降低,Th2类细胞因子显著升高(P<0.05)。A、C、D三组之间的GVHD临床评分、病理变化、生存率及细胞因子水平无统计学差异(P>0.05)。
结论:在小鼠Allo-BMT中,同种异基因MSCs的IDO活性可促进Thl向Th2的偏离,进而产生抑制GVHD效应,推测MSCs在体内同样通过IDO活性发挥免疫抑制作用。
Over the last 30 years allogeneic bone marrow transplantation (Allo-BMT) has become an accepted curative therapy for a variety of malignant hematopoietic diseases, severe immunologic deficiency diseases and hematopoietic failure of bone marrow. To date, the chief obstacle to the deployment of this treatment in other than life-threatening conditions is graft-versus-host disease (GVHD), and in particular acute GVHD, which is most severe in patients receiving matched unrelated donor transplants. The immunosuppressive drugs and T-cell depletion is effective at controlling GVHD, but these regimens can lead to serious complications from infection and an increased risk of leukemia relapse after Allo-BMT. Many researchers have therefore sought to develop techniques to prevent GVHD that avoid the complications incurred by immunosuppression.
Mesenchymal stem cells (MSCs) are multipotent precursors present in bone marrow, capable of differentiating into osteoblasts, adipocytes, and myoblasts. Some studies have confirmed MSCs could constitutively secrete a variety of cytokines that promote the homing or proliferation and differentiation of hematopoietic cells, and accordingly play important roles in promoting hematopoietic recovery. Moreover, MSCs are poor antigen-presenting cells and do not express MHC class II or co-stimulatory molecules, studies have also demonstrated MSCs could exhibit immunoregulatory properties in vitro or in vivo. Due to the distinct immunophenotype profile MSCs is concerned as a prophylaxis for GVHD, but the molecular mechanisms responsible for the immunosuppressive effects of MSCs have not been unequivocally identified.
Indoleamine 2,3-dioxygenase(IDO) is the initial and rate-limiting enzyme of the kynurenine pathway of degradation of L-tryptophan. IDO functionally catabolizes conversion from tryptophan .Tryptophan is necessary to the survival and proliferation of T cells. In the absence of tryptophan, the T cells cycle is arrested at a mid-G1 point. Following stimulation with IFN-γ, it is induced in various types of cell lines and cell types such as fibroblasts, epithelial cells and macrophages. Recently, it has been implicated IDO synthesized by the macrophages of placenta plays an important role in the prevention of allogeneic fetal rejection. So in professional antigen-presenting cells (APCs) expression of IDO has recently been identified as a major immunosuppressive mechanisms that inhibit T-cell activation and proliferation to autoantigens and alloantigens.
Basing on these, we tried to investigate the IDO mRNA and IDO protein expression in murine bone marrow-derived MSCs induced by IFN-γ, and the capacity of IDO activity in MSCs to induce allogeneic T cell proliferation; The in vivo effects of MSCs expressing IDO on acute graft-versus-host disease (aGVHD) in murine allogenic bone marrow transplantation (Allo-BMT) model and its underlying mechanisms were also investigated in this paper.
PartⅠStudy on isolation and biological characteristics of mesenchymal stem cells from murine bone marrow
Objective: To investigate the isolation, purification, expansion and biological characteristics of mesenchymal stem cells (MSCs) derived from murine bone marrow in vitro.
Methods: MSCs were isolated from murine bone marrow and cultured in IMDM medium. The morphology of the cells was observed by Wright’s staining and electron microscope. Cell cycle and immunophenotype were investigated by flow cytometry. Assays of adipogenic and osteogenic differentiation were performed in vitro, then, Von Kossa stain, Oil-redO staining and immunohistochemistr were used to indentified to the induced-cells.
Results: The cells from murine bone marrow displayed a fibroblast-like morphology adhering to the culture plate. FACS showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34, and I-ab were negative. Adipocyte, osteocyte and nerve-like cells differentiation were induced successfully.
Conclusion: MSCs derived from murine bone marrow were successfully isolated and cultured in vitro. PartⅡStudy on the effect of IDO in MSCs on allogeneic T-lymphocyte proliferation Objective: To study the effect ofγ-interferon (IFN-γ) on the IDO mRNA and IDO protein expression of MSCs and the effect of IDO activity in MSCs on allogeneic T-lymphocyte proliferation. Methods: IDO mRNA and IDO protein expression of MSCs induced by IFN-γat various concentration (0、20、50、100、200 U/ml) were analyzed by RT-PCR method and Western blot method respectively ,and IDO activity was determined by high-performance liquid chromatography (HPLC); MLRs cultures were set up with mitomycin C–treated C57BL/ 6-derived splenocytes as stimulators and BALB/c-derived splenocytes as responder cells. In MSCs/MLRs coculture experiments, MLRs were performed on a layer of 1×105,5×104,1×104 or 5×103 C57BL/ 6-derived MSCs (MSCs :responder T cells =1:5,1:10,1:50,1:100 ) with or without 1-methyl-D-trytophan(1-MT). T-lymphocytes proliferation was determined by MTT assays and IDO activity in coculture supernatant was determined by HPLC. Results: IDO mRNA expression, IDO protein expression and IDO functional activity were found in MSCs induced by IFN-γ, and the amount of expression and IDO activity increased gradually with the concentration of IFN-γ; When cocultured with 1×10~5、5×10~4、1×10~4 MSCs (MSCs :responder T cells =1:5,1:10,1:50),T- lymphocytes proliferation decreased apparently and IDO activity increased obviously compared with control group(p<0.05);In parallel experiments, 1-MT was added. T-lymphocytes proliferation and IDO activity restored partially. While cocultured with 5×10~3 MSCs (MSCs :responder T cells =1:100)T- lymphocytes proliferation raised lightly and IDO activity had no change compared with control group(p>0.05) .
Conclusion: IDO activity in MSCs is relation to T-lymphocyte proliferation, and MSCs may perform their immunosuppressive function by IDO activity in vitro.
PartⅢEstablishment of acute graft-versus-host disease model in murine allogeneic bone marrow transplantation
Objective: To establish murine acute graft-versus-host disease (aGVHD) model for the study of aGVHD in allogeneic bone marrow transplantation (Allo-BMT).
Methods: Lethally irradiated BALB/C (female,H-2d) murine transplanted with 1×107 C57BL/6 (male,H-2b ) bone marrow cells and 5×106 peripheral splenocytes (bone marrow cells and peripheral splenocytes ratio was 2:1) acted as GVHD group. The following two groups were simultaneously used: blank control group, no bone marrow cells and peripheral splenocytes transplanted; Allo-BMT group, no peripheral splenocytes transplanted. Evaluated the degree of GVHD according to clinical and pathological data.
Results: Typical aGVHD could not be induced in GVHD group. GVHD clinical scores of recipient in GVHD group was about 10, and all murine survived 6 to 24 days after transplantation. Clinical signs and pathological changes of aGVHD were seen in a relative localized period.
Conclusion: Murine lethal aGVHD model was successfully established.
PartⅣEffect of IDO in MSCs on aGVHD in murine allogeneic bone marrow Transplantation
Objective: Study the in vivo effects of MSCs expressing IDO on acute graft-versus-host disease (aGVHD) in murine allogenic bone marrow transplantation (Allo-BMT) model and explore the mechanism responsible for the immunosuppressive effects of MSCs.
Methods: Various Allo-BMT groups were established with lethally irradiated BALB/C (female, H-2d) murine and C57BL/6(male, H-2b) : GVHD group(Group A); Cotransplanted with large dose MSCs(Group B); Cotransplanted with low dose MSCs(Group C); Cotransplanted with large dose MSCs pre-deal by 1-MT(Group D). The degree of GVHD ,survival rates, histopathological changes and helper T-cell type l [Th1(IL-2、IFN-γ)] and type 2 [Th2(IL-4、IL-10)] cytokines level were observed in all the groups after BMT.
Results: It showed that GVHD clinical scores of recipient in group B was about 4, and no histopathological changes were seen, and the survival rate was 100%. The degree of GVHD was evidently lessened and survival rates was significantly higher compare with group A (P<0.05); In group B, Th1 cytokines level decreased and Th2 cytokines level increased significantly compare with group A (P<0.05). But GVHD clinical scores , histopathological changes , survival rate and cytokines level were not obviously different among Group A, C and D (P>0.05).
Conclusion: IDO activity in alloreactive MSCs shift the Thl/Th2 balance toward a Th2-production of type-2 cytokines dominant phenotype, then reduced the incidence of GVHD. We supposed that MSCs may perform their immunosuppressive function by IDO activity in vivo.
引文
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