【作者】 郝磊；

【导师】 陈幸华；

【作者基本信息】 第三军医大学 ， 内科学， 2010， 博士

【摘要】 异基因造血干细胞移植(Allogeneic Hematopoietic Stem Cell Transplantation,Allo-HSCT)是目前治疗恶性血液病的有效方法。HLA(Human Leucocyte Antigen)相合的亲缘相关或无关供者是Allo-HSCT最合适的供者。然而仅有25%～30%的患者能找到HLA相合的亲缘供者;在无亲缘关系人群中找到HLA相合供者的概率是1/5万~1/10万,甚至更低。90%以上的患者能够找到HLA半相合的亲属(父母、子女、兄弟姐妹或堂表亲)供者,若能顺利跨越HLA限制,可为更多需接受移植治疗而无相合供者的患者获得治愈的机会。然而HLA半相合移植患者发生移植物抗宿主病(Graft Versus Host Disease,GVHD)的几率大约为80%左右(1),GVHD是Allo-HSCT的主要并发症之一,是导致移植失败的重要原因。寻求安全有效的防治Allo-HSCT后GVHD的新措施成为临床关注的焦点。骨髓来源多能间充质基质细胞(multipotent mesenchymal stromal cells, MSC)主要生物学功能包括支持造血细胞的增殖、分化和成熟,同时也具有对免疫细胞的调节作用。大量动物模型和临床研究证实MSC对GVHD有一定的防治作用。但由于骨髓收集方法相对困难、MSC的多向分化潜能等均限制了其在临床的应用。本室采用CD34+细胞分选,结合Dexter培养分离出脐带血中另外一群非造血细胞:人脐血源基质细胞(hUCB-derived stromal cells,hUCBDSC),且已经证实其具备体外支持造血的能力(3)。推测hUCBDSC可能具有和MSC相似的对免疫细胞调节作用的特性。本研究在构建MHC(Major Histocompability Complex)半相合移植小鼠GVHD模型基础上,着眼于启动GVHD异源反应的两个重要因素T细胞和DC细胞,从体外和体内探讨hUCBDSC的免疫调节作用以及可能机制。一、主要方法(一)体外实验1.采用6%明胶和1.077 g/L percoll分离液分离脐带血中的单个核细胞,磁珠分选CD34+细胞,含12.5%HS、12.5%FBS、10-6mol/L氢化可的松、10ng/mlSCF、10ng/ml bFGF的DMEM培养液培养hUCBDSC,37℃、5%CO2、饱和湿度恒温箱孵育。48h后全量换液,以后每周半量换液;待细胞融合度达到80%左右时,进行传代培养;2.采用流式细胞仪检测原代分离培养和冻存复苏后hUCBDSC细胞表面免疫分子HLA-Ⅰ、Ⅱ类分子,CD80,CD86,CD40和CD40L的表达率;3.采用70%percoll分离液和R&D systems公司的Mouse T cell Enrichment Columns进行小鼠脾脏T细胞的分选;4.分离小鼠骨髓单个核细胞后,rmGM-CSF和LPS诱导BM-DC的分化和成熟;5.将PHA或DC刺激的小鼠脾脏T细胞和不同浓度的hUCBDSC共培养, CCK-8检测细胞增殖情况;6.PHA刺激条件下小鼠T细胞和hUCBDSC共培养后,流式细胞仪检测T细胞的周期、凋亡、CD4+CD25+Foxp3+调节性T细胞(CD4+Treg)以及Th1/Th2亚群的变化。7.小鼠BM-DC和hUCBDSC共培养后,扫描电镜和透射电镜观察BM-DC形态和结构,流式细胞仪检测表面免疫分子MHC-Ⅱ、CD80、CD86的表达,CCK-8检测试剂盒检测其刺激异源T细胞增殖反应的能力。(二)体内实验1.受鼠C57BL/6×BALB/c F1接受750 cGy 60Coγ射线照射后4小时内经尾静脉注射供鼠C57BL/6来源的骨髓细胞BMC(1×107/只)、脾脏细胞SP(1×107/只),建立MHC半相合造血干细胞移植小鼠急性GVHD模型;2.受鼠移植BMC和SP的同时或1w后输注hUCBDSC(1×107/只),观察小鼠生存时间、GVHD临床表现和靶器官组织病理变化;3.细胞移植后1w、2w、3w和4w,流式细胞仪检测受鼠脾脏T细胞CD4+Treg、Th1/Th2亚群的比例和DC表面免疫分子MHC-II、CD80和CD86的表达。二、主要结果(一)体外实验1.hUCBDSC组成性高表达HLA-Ⅰ(84.1±2.9%),几乎不表达HLA-Ⅱ(1.6±0.3%)类分子,共刺激分子CD80(0.8±0.1%),CD86(0.8±0.1%),CD40(0.6±0.1%)和CD40L(0.5±0.1%)。细胞冻存复苏不影响上述分子的表达;2.hUCBDSC并不会引起小鼠T细胞的增殖,相反,以剂量依赖性方式抑制PHA或DC刺激的小鼠T细胞增殖;3.在PHA刺激72h后,处于G1期的T细胞占62.1±3.7%,S期占35.6±2.7%。而当与hUCBDSC共培养时, 84.3±3.6%T细胞处于G1期,12.3±1.5%T细胞处于S期。两者比较有显著统计学意义(p<0.01);4.PHA刺激单独培养的T细胞和与hUCBDSC共培养的T细胞中早期凋亡细胞的比例无明显差别(p>0.05);5.和单独培养的T细胞比较,与hUCBDSC共培养的T细胞中CD4+Treg亚群所占的比例显著增高(1.2±0.3% vs 12.1±1.4%,p<0.01);6.在PHA刺激时,T细胞单独培养或与hUCBDSC共培养5天后, Th1/Th2的比例分别为1.4±0.1%和0.7±0.1%,两者之间比较具有显著统计学意义(p<0.01);7.当BM-DC和hUCBDSC共培养时,电镜观察其表面突起显著减少,细胞器也不丰富。表面免疫分子MHC-II、CD80和CD86的表达显著降低,且其刺激异源T细胞增殖的能力也显著降低。(二)体内实验1.受鼠C57BL/6×BALB/c F1接受750 cGy TBI后输注供鼠C57BL/6来源的BMC和SP后,通过对生存期、临床表现和组织病理的观察,证实成功构建了MHC半相合HSCT小鼠GVHD模型;2.细胞移植后4h内或1w后输注hUCBDSC,小鼠长期生存率从20%分别提高到60%和40%,显著降低了小鼠GVHD临床评分和组织病理评分;3.细胞移植后各时相点流式细胞仪分析受鼠脾脏T细胞亚群和DC的变化,输注hUCBDSC的受鼠脾脏CD4+Treg比例增高、Th1/Th2亚群比值降低、DC表面免疫分子MHC-II、CD80和CD86的表达降低。三、结论1.hUCBDSC体外具有低免疫源性的特点,可以抑制PHA或DC刺激的小鼠T淋巴细胞的增殖。可能的作用机制包括阻止T细胞从G1期进入S期,诱导CD4+Treg的产生,降低Th1/Th2亚群的比例和对BM-DC成熟的影响。2.hUCBDSC可以延长MHC半相合造血干细胞移植后小鼠的生存期,减轻GVHD临床表现和靶器官组织病理变化,这可能与体内CD4+Treg的增多、Th1/Th2极化,以及不成熟DC的产生有关。更多还原

【Abstract】 IntroductionAllogeneic hematopoietic stem cell transplantation(Allo-HSCT)– using blood or marrow as the source– provides curative therapy for a substantial number of patients with hematological malignancies .HLA matched related or unrelated donors are the best selections. However, the odds are that only 25~30% of patients will have a matched sibling to act as donor, and the chance of finding a matched unrelated donor is even lower(one in 50~100 thousand). Another potential alternative donor is a genetically haploidentical family member. Such a donor is readily available in 90% of patients, which will increase the therapeutical opportunity for patients who have no HLA matched donors if the HLA barrier could be overcome suitably.80% of patients, who have received HLA haploidentical transplants, would suffer from graft verus host disease (GVHD).GVHD, the main complication after Allo-HSCT, accounts for morbidity and mortality post transplantation. To explore safe and efficient method to prevent incidence and severity of GVHD become the focus of scientists and clinicians. The critical function of multipotent mesenchymal stromal cells(MSC) include regulation of hematopoietic cell proliferation, differentiation and maturation. The interaction between MSC and immune cell has also been studied. It was demonstrated that MSC possess the ability to regulate immunity, evidenced by its effect on the GVHD prevention in large amount of animal model and clinical research. However, the application of MSC is limited in clinic due to relative difficult collection and multipotential differentiation. Our laboratory has previously isolated a novel population of adherent fibroblast-like cells from human umbilical cord blood (hUCB) CD34+cells, called hUCB-derived stromal cells (hUCBDSCs) and confirmed their supportive effect on hematopoisis in vitro. Previous studies have usually investigated human MSC-allogeneic immune cell reaction in vitro. To test the feasibility of replacing human immune cells with xenogeneic counterparts in vitro is of significance in establishing an animal model for further in vivo study of immunological properties of human MSC, such as their inhibitory effect on GVHD. In the present study, we focused on immunological properties of hUCBDSCs and their effect on xenogeneic immune cells in vitro and GVHD in mouse subjected to MHC haploidentical transplantation. MethodsPart A Experiment in vitro1. CD34+cells were isolated from human umbilical cord blood using 6% gelatin, percoll separating medium(density,1.077g/L) and magnetic bead selection and cultured in DMEM medium containing 12.5%HS, 12.5%FBS, 10-6mol/L hydrocortisone,10ng/mlSCF and 10ng/ml bFGF. Culture was replaced by fresh medium after 48 h, and then, it was demi-depopulated weekly and fresh medium was added to it. After the density of cells had risen above 80% confluence, hUCB-derived stromal cells were subcultured with 1:2ratio under the same culture medium and condition;2. The expression of HLA-I,HLA-II and costimulatory molecules such as CD80, CD86, CD40 and CD154 on freshly isolated and cryopreserved and resuscitated hUCBDSC was tested using flow cytometry;3. CD3+ T cells was isolated and enriched from spleen in mouse using 70% percoll combined with Mouse T cell Enrichment Columns;4. BM-DC was cultured and induced in medium containing rmGM-CSF and LPS after isolation of bone marrow mononuclear cells;5. hUCBDSC was cocultured with T cells stimulated by PHA or DC. The proliferation of the T cells was tested by CCK-8;6. hUCBDSC was cocultured with T cells stimulated by PHA. Cell cycle, apoptosis, the subset of CD4+Treg and Th1/Th2 in T cells were analyzed by flow cytometry;7. hUCBDSC was cocultured with BM-DC. Morphology was observed using electron microscope. The expression of MHC-Ⅱ、CD80、CD86 on BM-DC was tested by flow cytometry. T cells was stimulated by BM-DC after coculturation with hUCBDSC to analyze the function of BM-DC;Part B Experiment in vivo1. Recipient (B6×BALB/c)F1 mice were irradiated with a single dose of 750 cGy using a 60Coγ-ray source at a dose rate of approximately 31 cGy/min. BMC (1×107) and SP (1×10~7) were injected intravenously into recipient donor C57BL/6 mice within 4 hours of conditioning2. hUCBDSC(1×107) was infused simultaneously or on one week after transplantation of BMC and SP. Survival time, GVHD clinical appearance and pathohistological manifestation were observed;3. The subset of CD4+Treg and Th1/Th2 in splenic T cells and expression of MHC-Ⅱ、CD80、CD86 on splenic DC were analyzed in recipient mice using flow cytometry on 1w,2w,3w and 4w post transplantation.ResultsPart A Experiment in vitro1. 84.1±2.9% of hUCBDSC expressed HLA-I, but there was nearly no expression of HLA-II (1.6±0.3%). The levels of costimulator expression were as follows: CD80 (0.8±0.1%), CD86 (0.8±0.1%),CD40 (0.6±0.1%), and CD40L (0.5±0.1%), and cell cryopreservation did not influence the expression of aboved molecules;2. The addition of hUCBDSCs in in vitro culture did not evoke xenogeneic T-cell proliferation; on the contrary, it suppressed the proliferation induced by PHA or allogeneic dendritic cells;3. The proportion of T cell in G1 and S phase was 62.1±3.7% and 35.6±2.7% after T cells were stimulated by PHA for 72 hours. The percentage of T cells in G1 phase was increased to 84.3±3.6%, and in S phase was decreased to 12.3±1.5% when PHA stimulated T cells were cocultured with hUCBDSC;4. There are no significant difference statistically on the percentage of apopotic cell between T cells alone and T cells cocultured with hUCBDSC;5. There was a significant increase in the proportion of CD4+Treg (12.1±1.4% vs 1.2±0.3%, p < 0.01) when T cells were stimulated by PHA in the presence of hUCBDSCs compared with control;6. The ratio of Th1/Th2 subset in T cells alone was 1.4±0.1%, significantly higher than in T cells cocultured with hUCBDSC (0.7±0.1%, p<0.01);7. The surface projection and organelle in BM-DC was significantly decreased evidenced by observation using electron microscope. The expression of MHC-Ⅱ、CD80、CD86 on BM-DC was down-regulated. The stimulatory ability of BM-DC was significantly attenuated. Part B Experiment in vivo1. The MHC haploidentical HSCT GVHD model in mouse has been successfully constructed ecidenced by typical GVHD clinical appearance and pathohistological changes;2. The long-time survival ratio of recipient mice was increased from 20% to 60% and 40% after infusion of hUBDSC on day0 or on week. In addition, the GVHD clinical appearance has been attenuated significantly, and the injury degree of targeted tissue was significantly decreased.3. The subset of CD4+Treg in splenic T cells in recipient mice subjected to hUCBDSC infusion was increased significantly, and Th1/Th2 subset was decreased. Expression of MHC-Ⅱ、CD80、CD86 on splenic DC were down-regulated significantly.Conclusion1. HUCBDSC are immunoprivileged evidenced by lower or negligible expression of MHC-II, costimulatory molecules including CD80,CD86,CD40 and CD154 on cellular surface and non stimulatory effect on xenogeneic T cells. Interruption of cell cycle, induction of CD4+Treg, polarization of Th1/Th2 subset and interference with BM-DC maturation may be involved in its possible mechanisms rather than apoptosis.2. HUCBDSC could prolong survival time of mice subjected to MHC haploidentical HSCT, and attenuate GVHD clinical appearance and pathohistological change of target tissue. In accordance with results in vitro, possible mechanisms include induction of CD4+Treg, polarization of Th1/Th2 subset and interference with BM-DC maturation.更多还原