异基因骨髓间充质干细胞移植对EAE小鼠的治疗作用

目的 探讨异基因骨髓间充质干细胞(BMSC)对实验性自身免疫性脑脊髓膜炎(EAE)的治疗作用及相关免疫调节机制.方法 用髓鞘少突胶质细胞糖蛋白(MOG)多肽片段诱导建立C57BL/6J小鼠EAE模型;分离纯化培养BALB/c小鼠BM-SC,对EAE模型小鼠进行移植,移植前后对EAE小鼠进行神经功能评分;流式细胞术检测小鼠淋巴器官CD4+ CD25+ Foxp3+T细胞(Treg)的数量;荧光定量RT-PCR检测小鼠脾脏IL-2、IL-4、IL-17和IL-23 mRNA水平的表达.结果 异基因BMSC移植后小鼠神经功能评分明显改善;BMSC移植组小鼠胸腺、脾脏、淋巴结Treg数量显著增加;脾脏中IL-2、IL-17表达显著下降,同时IL-4、IL-23表达显著增加.结论 异基因BMSC移植通过调节免疫系统的Treg数量和CD4+T细胞分泌细胞因子的水平对EAE起治疗作用.     

骨髓异常增生综合征患者骨髓来源的间充质干细胞免疫功能异常

目的 研究骨髓异常增生综合征(myelodysplastic syndromes,MDS)难治性贫血(refractory anemia,RA)型患者(MDS-RA)和健康成年人骨髓来源的间充质干细胞(bone marrow derived mesenchymal stem cell,BMSC)的免疫学特征,比较它们是否存在免疫功能的异常.方法 分离MDS-RA和健康成年人来源的间充质干细胞,分别检测它们对T细胞周期、活化、抑制和增殖的作用情况.结果 MDS-RA的BMSC抑制T细胞增殖和活化能力均减弱,但是抑制活化的T细胞凋亡的作用反而增强.结论 MDS-RA患者骨髓来源的MSC存在明显的免疫调节功能缺陷,MDS-RA患者骨髓微环境可能存在免疫异常,如果使用MDS患者自体的MSC移植治疗可能不是一种很好的选择.对于MDS患者最好是选用异基因的MSC移植.     

异基因骨髓源间充质干细胞对BXSB小鼠T、B细胞的影响

目的：探讨异基因骨髓源间充质干细胞（Bonemarrow derived mesenchymal stem cells，BMSCs）对BXSB小鼠T、B细胞增殖、活化及功能成熟等方面的影响。方法：采用。H．TdR掺入法、FACS法、EIJSA等方法检测BMSCs对BXSB小鼠T、B细胞的影响。结果：BALB／c小鼠的BMSCs在不影响BXSB小鼠T细胞诱导活化的基础上抑制其诱导增殖；可降低由ConA诱导的CD4^＋IL^＋细胞的数量，而提高由ConA诱导的CD4^＋IFN-γ^＋细胞数量。对于B细胞，BALB／c小鼠的BMSCs可以抑制其增殖、活化及IgG的分泌。另外，BALB／c小鼠的BMSCs可以抑制BXSB小鼠B细胞上CIMOL的异位表达。结论：异基因BMSCs对自身免疫病小鼠的T、B细胞有一定的调节作用。     

小鼠骨髓间充质干细胞的分离与培养

探索在体外分离培养小鼠骨髓间充质干细胞 (Bone m arrow m esenchym al stem cells,BMSCs)的最适条件。以密度梯度离心技术及贴壁筛选法相结合 ,在体外分离 BAL B/C小鼠的 BMSCs,通过 MTT法测定了不同离心力、不同换液时间、不同血清浓度、不同种植密度等因素对 BMSCs分离和培养的影响。在 5 0 0 g× 30 min的离心力下分离细胞 ,加入 10 %的胎牛血清 ,原代按 (12～ 2 0 )× 10 5/m l的细胞密度接种 ,接种 2 4 h后换液 ,继代种植密度为 6 .4～ 2 5 .6× 10 4 /ml时最适宜细胞生长 ;在此条件下 ,细胞快速增殖 ,传代后 8h贴壁达 90 %以上 ,2 4 h便进入对数生长期 ,直至第 5 d,细胞约增殖 3倍。本研究建立了在体外分离培养骨髓间充质干细胞的细胞生物学方法和技术参数。     

骨髓间充质干细胞研究进展

骨髓间充质干细胞是干细胞领域的研究热点之一.虽然近几年来有关间充质干细胞的研究已取得了很大进展,但仍有很多问题有待进一步解决.主要对间充质干细胞的异质性、分化与融合的问题、修复组织的机制、免疫性及研究方法等问题进行了综述.     

骨髓和脂肪来源的间充质干细胞免疫调节作用的比较

目的比较脂肪源间充质干细胞(AMSCs)和骨髓来源间充质干细胞(BMSCs)的免疫调节能力的差异。方法用流式细胞仪分析AMSCs和BMSCs的表型。通过MSCs和淋巴细胞共培养体系,检测MSCs对T细胞增殖、周期、活化、凋亡以及Th细胞分化的影响。结果表型分析结果显示AMSCs和BMSCs的表型基本一致。AMSCs和BMSCs都能抑制T细胞的增殖和早期活化,并在调节辅助性T细胞亚群的分化上作用类似。但在MSC对活化后T细胞的凋亡影响方面,BMSCs能够抑制活化T细胞的凋亡,而AMSC没有这种作用。结论 AMSCs和BMSCs对T淋巴细胞的影响基本类似,其对活化T细胞凋亡影响的差异还有待进一步的机制研究。     

骨髓和脂肪来源间充质干细胞的免疫调节作用

背景：脂肪来源的间充质干细胞是否具有和骨髓来源间充质干细胞类似的免疫调节作用? 目的：观察骨髓来源和脂肪来源间充质干细胞的免疫学特征。 方法：分离骨髓和脂肪来源的间充质干细胞,分别检测它们对T细胞周期、活化、抑制和增殖的作用情况。 结果与结论：骨髓来源和脂肪来源的间充质干细胞同样具有抑制T细胞增殖的能力,在有丝分裂原刺激和混合淋巴细胞反应的T细胞增殖中这种作用都是具有剂量依赖性的,在1︰2时有极强的抑制作用,但是在1︰100时这种作用基本消失,在共培养时骨髓来源和脂肪来源的间充质干细胞都可以使更多的T细胞被抑制在G0/G1期,同时也可以抑制T细胞的早期活化,但是上述作用脂肪来源的间充质干细胞均较骨髓来源间充质干细胞弱,且脂肪来源的间充质干细胞并不具有抑制T细胞凋亡的作用。     

骨髓间充质干细胞免疫学特性的研究进展

随着免疫学和移植学的发展,人类对自身免疫性疾病认识的不断提高和对器官移植的日益接受,免疫抑制剂在临床医学中的应用愈来愈广泛。它们能提高患者的生存质量,延长患者的寿命,但是其昂贵的费用、易引起感染和肿瘤等的副作用明显地限制了它的临床应用。为此,免疫学专家和移植学专家一直在探索理想的诱导特异性耐受的方法和途径,以便减少免疫抑制剂的应用剂量和副作用,乃至部分或甚至完全替代免疫抑制剂。近20多年来,成体干细胞的可塑性现象引起人们的极大兴趣。其中骨髓间充质干细胞(bone marrow mesenchymal stemcell,MMSC)的功能作用和…     

间充质干细胞在T细胞免疫反应中的调节作用

间充质干细胞(MSCs)独特的免疫调节作用以T细胞为主,在混合淋巴细胞反应中,通过周期阻滞抑制T细胞增殖,但不引起T细胞凋亡增加、活化受抑。同时MSCs还能降低反应体系中的CD8+T细胞和Th1细胞,升高Th2细胞以抑制炎症反应,从而在T细胞介导的自身免疫性疾病中发挥治疗作用。     

骨髓间充质干细胞调节心脏移植大鼠的免疫功能

BACKGROUND:Heart transplantation is an effective method for treatment of end-stage heart failure, but immune rejection that seriously impact therapeutic effacicy is easy to occur after transplantation. OBJECTIVE:To investigate the regulatory effect of bone marrow mesenchymal stem cells on the immune function of rats undergoiong heart transplantation. METHODS:Twenty Lewis rats were enrolled as donors, and 20 Wistar rats as recipients. Heart transplantation models were established in the Wistar rats. These 20 model rats were randomized into cell transplantation and control group with 10 rats in each group. Forty-eight hours after heart transplantation, rats in the cell transplantation group were given bone marrow mesenchymal stem cell suspension (1 mL, 2×10⁸ cells/L) via the tail vein, while rats in the control group were given normal saline in the same dose. Then, the expression levels of serum interleukin-2, interleukin-10 and percentage of CD4⁺, CD8⁺, CD4⁺/CD8⁺, CD4⁺CD25^high, CD4⁺CD25^high Foxp3⁺ T cells in the venous blood were detected in the two groups at 7 days after cell transplantation. Additionally, rat myocardial tissues were taken and observed pathologically. RESULTS AND CONCLUSION:The survival time of the cell transplantation group was significantly longer than that of the control group (P < 0.05). The expression level of interleukin-2 showed no significant difference between the two groups (P > 0.05), but the level of interleukin-10 in the cell transplantation group was significantly higher than that in the control group (P < 0.05). Compared with the control group, the percentage of CD4⁺/CD8⁺, CD4⁺CD25^high, CD4⁺CD25^high Foxp3⁺ and CD4⁺ T cells was significantly higher, and the percentage of CD8⁺ T cells was significantly lower in the cell transplantation group (P < 0.05). Histopathological findings showed that there were a small amount of infiltrated lymphocytes in the cell transplantation group with the presence of slight bleeding and edema, and these inflammatory reactions were milder than those in the control group. These findings indicate that bone marrow mesenchymal stem cell transplantation can effectively reduce the rejection in rats undergoing heart transplantation.     

骨髓间充质干细胞对异体外周血B淋巴细胞的免疫调节作用

目的 探讨骨髓间充质干细胞(mesenchymal stem cells, MSC)在体外对异体外周血B淋巴细胞的免疫调节作用.方法 用密度梯度离心法从骨髓中分离、培养MSC,从外周血中分离单个核细胞,L-亮氨酸甲酯去除单核细胞,以2-氨乙基硫脲溴化物(AET)处理绵羊红细胞(SRBC)的花环形成法,去除T淋巴细胞获得纯化的B淋巴细胞.用羊抗人IgM单克隆抗体(anti-IgM)刺激与或未与MSC或其培养上清共培养3d的B淋巴细胞,应用MTT法测8淋巴细胞的增殖,ELISA法测培养上清中免疫球蛋白IgG、lgM的产生,应用流式细胞术分别检测与MSC共培养24、48h后B淋巴细胞的凋亡.结果 MSC及其培养上清抑制由anti-IgM诱导的B淋巴细胞的增殖和分泌IgG、IgM,且随着MSC细胞数量及其培养上清浓度的增加,这种抑制越明显.流式细胞术结果显示,与MSC共培养不同时间的B淋巴细胞的凋亡变化无统计学意义,MSC抑制B淋巴细胞的增殖存在暂时性和可逆性.结论 MSC对异体外周血8淋巴细胞存在免疫调节作用,并且这种调控机制是复杂的,不仅与MSC细胞数量有关,还与细胞间的相互作用和MSC分泌的细胞因子有关.     

The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate along different mesenchymal lineages including those forming bone, cartilage, tendon, fat, muscle and marrow stroma that supports hematopoiesis. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries and for hematopoietic disorders by both local and systemic application. In the present study, rat marrow-derived MSCs were ex vivo culture-expanded, labeled with (111)In-oxine, and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in excised organs, including liver, lungs, kidneys, spleen and long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected primarily in the lungs and then secondarily in the liver and other organs. When sodium nitroprusside was used, more labeled MSCs cleared the lungs resulting in a larger proportion detected in the liver. Most importantly, the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator.     

骨髓间充质干细胞移植对脑梗塞大鼠神经功能恢复及突触素表达的影响

目的:探讨骨髓间充质干细胞(BMSC)移植对脑缺血大鼠神经功能恢复和脑组织中突触素(synaptophysin)mRNA表达的影响.方法:72只清洁级成年雄性SD大鼠,随机分为假手术组(Sham组)、局灶性脑缺血组(MCAO组)、溶剂对照组(vehicle组)和BMSC治疗组(BMSC组),每组18只,采用改良Zea-Longa线栓法制作大脑中动脉栓塞(MCAO)模型,BMSC移植前用DAPI标记,BMSC组于造模成功1 d后经侧脑室注射入BMSC(1×106),vehicle组则注射同等剂量的PBS,术后4 d、7 d和14 d通过平衡木行走、转棒上行走及网屏抓握进行神经功能评估,观察其恢复状况,并断头取脑,免疫组织化学及RT-PCR法检测突触素表达情况.结果:BMSC组缺血周边区脑组织中观察到DAPI染色的阳性细胞.BMSC组术后7 d和14 d神经功能评估明显优于MCAO组和vehicle组(P＜0.05 or P＜0.01);BMSC组术后4 d、7 d和14 d脑组织中突触素表达较MCAO组和vehicle组明显增高(P＜0.01).结论:BMSC移植可以促进脑缺血大鼠神经功能的恢复,促进缺血脑组织中突触素生成,BMSC移植促进脑缺血大鼠神经功能恢复部分是通过促进脑组织中生成突触素发挥作用.     

多发性骨髓瘤患者骨髓间充质干细胞免疫调节功能异常及其在骨髓瘤骨病中的作用

目的 探讨多发性骨髓瘤(MM)患者骨髓间充质干细胞(MSCs)免疫调节功能及其对骨髓瘤骨病的影响.方法 分离MM患者和正常对照MSCs,流式细胞技术比较二者的免疫表型.Real-time PCR检测实验组及对照组MSCs的TGF-β1、TGF-β2、TGF-β3、IL-6、IL-3、TNF-α、FasL和NF-κB配体的受体激活物(RANKL)表达量.共培养系统与流式细胞术检测MSCs对T细胞增殖、凋亡和早期活化标志CD25和CD69表达的影响.Von kossa染色、real-time PCR和Western blot等技术检测T细胞对正常对照MSCs向成骨细胞分化的影响.结果 MM来源和正常对照MSCs具有相似的形态学和免疫表型.MM来源的MSCs表达TGF-β1、IL-6、IL-3、TNF-α和RANKL较正常对照MSCs增加,而TGF-β2、TGF-β3和FasL的表达下降.MM来源的MSCs对T细胞增殖的抑制作用明显减弱.正常对照MSCs较MM来源MSCs使更多T细胞静止于G0/G1期.正常对照MSCs明显促进T细胞凋亡,而MM来源的MSCs对T细胞的促凋亡作用减弱.正常对照MSCs明显抑制T细胞活化分子表达,而MM患者的MSCs此抑制作用明显降低.与MM患者的MSCs共培养后的T细胞以及直接来源于MM患者的T细胞均可以明显抑制正常对照MSCs向成骨细胞分化.结论 MM患者的MSCs免疫调节功能较正常对照MSCs降低,主要表现为抑制T细胞活化的功能减弱,而活化的T细胞可抑制MSCs向成骨细胞分化,此可能为骨髓瘤骨病发病机制之一.     

骨髓间质干细胞研究进展

全能干细胞和多能干细胞具有高度自我更新能力和多向分化潜能 ,是组织工程及细胞和基因治疗中重要的靶细胞。胚胎干细胞是从早期胚胎中分离的 ,具有向机体各种组织细胞分化的潜能 ,但其自身的免疫原性以及取材困难 ,限制了它在临床上的应用。近年来研究发现 ,除了胚胎干细胞外 ,机体内还存在一些多能干细胞 ,它们具有一定的自我更新和分化能力 ,如来源于脑室管膜的神经干细胞可分化为神经元和神经胶质细胞[1] ;骨髓中的造血干细胞可分化为红细胞、粒细胞、巨噬细胞等各种血细胞。而骨髓间质干细胞作为非造血组织干细胞[2 ] ,除了参与构成支…     

骨髓问充质干细胞联合胰岛移植对受体鼠树突状细胞成熟和功能的影响

目的:研究骨髓问充质干细胞(MSCs)对骨髓细胞(BMC)分化为成熟树突状细胞(DCs),以及MSCs联合胰岛移植对受体鼠BMC来源DCs成熟和功能的影响,以探讨MSCs如何通过DCs发挥免疫抑制作用的机制.方法:将BALB/c小鼠分离纯化骨髓MSCs按比例加入C57BL/6小鼠BMC,在含重组鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和重组鼠白介素4(rmIL-4)的培养条件下制备DCs,加脂多糖(LPS)促DCs成熟,检测各组DCs免疫表型的变化,抗原摄取及其分泌白细胞介素-12的能力.观察MSCs联合胰岛移植的同种异基因糖尿病模型小鼠的血糖和组织学变化,并取受体鼠BMC体外诱导为DCs,检测成熟DCs免疫表型、抗原摄取和分泌IL-12能力.结果:体内外实验证明MSCs可使BMC来源的DCs表型CD11c,成熟表面标志CD83、协同刺激分子CD86和I-A<'b>表达明显降低(P<0.05),抗原摄取和分泌IL-12能力显著下降(P<0.01).MSCs联合胰岛移植,可抑制同种异基因受体鼠免疫排斥反应的发生.结论:MSCs可通过抑制BMC来源的DCs的成熟和功能,降低DCs抗原摄取和分泌IL-12能力,诱导免疫耐受,减轻对移植胰岛的排斥反应的发生.     

Xenotransplantation of long-term-cultured swine bone marrow-derived mesenchymal stem cells

Swine-derived MSCs were efficiently isolated and extensively expanded using a low fetal serum content growth medium to which selected growth factors were added. After > or =96 cell population doublings (PDs), MSCs were devoid of cytogenetic abnormalities. In vitro chondrogenic and osteogenic differentiation capacity was preserved after 80 PDs. To test therapeutic efficacy, 1 x 10(6) 80-PD MSCs were injected directly into the peri-infarct zone of hearts of immunodeficient (non-obese diabetic/severe combined immunodeficient) mice at the time of acute myocardial infarction. Engrafted MSCs survived in the infarcted hearts for at least 4 weeks. Echocardiography at 2 and 4 weeks postinfarction revealed a significant preservation of the left ventricular ejection fractions of infarct hearts receiving MSCs compared with infarct hearts receiving saline. Peri-infarct zone capillarity was better preserved in MSC-treated hearts than other infarct groups of hearts, but infarct size was comparable in all groups. Only rare engrafted MSCs expressed cardiac-specific or endothelial cell-specific markers. Hence, 80-PD MSCs retained the capacity to promote functional improvement in the infarcted heart despite minimal differentiation of MSCs into cardiomyocytes or endothelial cells. These data suggest that the beneficial effects of MSC transplantation most likely result from the trophic effects of MSC-released substances on native cardiac and vascular cells. The capacity to massively expand MSC lines without loss of therapeutic efficacy may prove to be useful in the clinical setting where "off the shelf" MSCs may be required for interventions in patients with acute coronary syndromes.