【作者】 陈峰；

【导师】 吴德沛；

【作者基本信息】 苏州大学 ， 内科学， 2007， 博士

【摘要】 作为一种具有多向分化潜能（向成骨细胞、成脂细胞、成软骨细胞及成肌细胞等多种组织分化）的成体干细胞,间充质干细胞（Mesenchymal stem cell,MSC）最初在组织工程中以“种子细胞”的角色参与组织修复和再生而倍受重视。近期研究发现MSC还具有特殊的造血调控及免疫调节作用,并在造血重建、移植免疫和基因治疗等多方面展现出良好的应用前景。在少数的几个Ⅰ期临床试验中已观察到了令人鼓舞的结果,如HSC/MSC共移植能明显促进受体的造血恢复,减少移植相关并发症,尤其是可降低GVHD的发生。但目前MSC联合移植防治GVHD的研究尚处于起步阶段,还有许多问题亟待解决。如MSC移植后在受体内发挥免疫调节效应的机制迄今仍不明确,部分体外研究、动物实验及Ⅰ期临床试验的结论也还存在着不一致,已报道的少数临床资料多不是随机或对照研究,近期还有研究显示MSC共移植并不能证实减少了GVHD的发生和减轻了严重程度,这些都提示MSC对GVHD的防治作用可能有限或需配合其它措施,基于HSC/MSC共移植对部分高GVHD风险（如单倍型或非血缘供体移植）和高植入失败风险（如脐血及非清髓移植）的病例可能是一种极有临床应用前景的移植策略,是否可以通过基因转染修饰的MSC移植来增强其体内免疫调节效应?近年MSC已成为基因治疗中理想的靶细胞之一,可稳定表达多种外源性目的基因,且回输后植入反应弱,因此通过基因转染MSC进行基因治疗理论上是可行的。既往的研究已明确,GVHD的中心环节是免疫活性T细胞的激活,而发病的重要因素及介质是细胞因子分泌异常,细胞因子失调通过级联放大效应诱导“细胞因子灾难”,是各种因素加重GVHD的最终共同通路,利用Ⅱ类细胞因子负调控Th1细胞及单核/巨噬细胞功能,诱使Th1反应向Th2方向“漂变”,减少GVHD致病因子（如IL-1,IL-2,IFN-γ,TNF-a,NO等）生成,是控制GVHD而保留GVL效应的有效策略之一。白细胞介素10（IL-10）作为一种理想的抗炎因子,可抑制T淋巴细胞和单核/巨噬细胞等炎症细胞的激活及多种炎症因子的合成,而从起始到效应阶段都可抑制Th1型细胞免疫反应,更提示IL-10在干预GVHD的发生和进程中具有潜在的重要作用。近期研究认为基于IL-10的抗炎特性及IL-10与树突状细胞（DC）、调节性T细胞（Treg）的联系,对于诱导外周免疫耐受及植入存活都是有益的,已有研究者发现IL-10分泌的低水平与GVHD的高发生率有关。由此,我们设想如以IL-10基因转染修饰的骨髓MSC共移植,能否与IL-10协同发挥免疫抑制效应,联合细胞治疗与基因治疗的优势,阻止或减轻GVHD?但由于伦理学和方法学的限制,使我们无法在人体内进行相关的MSC移植免疫学研究。基于上述设想,本研究拟解决以下主要问题:1.分离、培养小鼠骨髓间充质干/祖细胞（mMSC/MPC）,确保其良好的增殖能力和多向分化潜能,研究其生物学特征,并对相关生物学特性进行初步研究,为其后进行基因转染修饰及应用于动物模型做准备。2.构建并制备载有mIL-10基因的复制缺陷型腺病毒载体,并证实该病毒载体可高效转染mMSC/MPC并获得高效稳定的体外表达,以符合体内实验要求。3.建立适宜的H-2单倍型骨髓移植aGVHD模型,在此基础上分别进行MSC/MPC及mIL-10基因转染修饰MSC/MPC的共移植,观察相应的免疫生物学效应。通过如上研究探讨MSC/MPC移植能否在受体内诱发免疫耐受及其可能的相关机制,观察IL-10基因转染修饰的MSC/MPC能否协同发挥免疫调节活性,增强免疫耐受效应,并对其在移植中的免疫生物学行为进行初步研究,为潜在的GVHD防治新策略提供理论及实验的依据。第一部分:小鼠骨髓间充质干/祖细胞体外培养体系的优化及相关生物学研究方法:无菌剥离SPF级雌性C57BL╱6小鼠股骨与胫骨,以培养基反复强力冲洗并刮擦髓腔内膜后收集冲洗液,加入碎骨片振荡冲洗,过200目滤网制备为单细胞悬液。分别以3种完全培养基（低糖DMEM完全培养基,低糖DMEM/MCDB201完全培养基,Mesencult完全培养基）重悬,以1×10⁶/cm²的密度接种于25cm²滤膜培养瓶,转细胞培养箱,37℃,5%C02,100%饱和湿度条件下培养。24小时后全量换液,去除未贴壁细胞,此后每3日换液一次。镜下贴壁细胞约70%-80%汇合时消化,以2.5～5×10³/cm²密度接种传代。对不同代数的培养细胞进行如下检测:倒置显微镜下形态学特点观察;不同培养体系小鼠成纤维细胞样集落形成单位（CFU-F）的检测;计数绘制细胞生长曲线及倍增时间;流式细胞仪细胞周期检测;流式细胞仪检测细胞表面标记;体外定向诱导向成骨、成脂肪分化,鉴定多向分化潜能;RT-PCR法检测细胞mIL-10mRNA;双抗体夹心ELISA法检测细胞培养上清中mIL-10。采用SPSS 11.5 FOR WINDOWS统计处理软件分析,检验水准α=0.05。结果:收集经骨片冲洗的小鼠骨髓冲出液接种培养,经传代培养3代以上后细胞呈现为均质性较好的短梭形细胞,漩涡或放射状生长;优化选择Mesencult完全培养基为MSC/MPC培养体系;细胞可传代生长10代以上保持生物学特性稳定,选择P3代培养细胞为实验用细胞;对数生长期的群体倍增时间（Td）为31.1±0.6小时;细胞汇合80%-90%时G0/G1期细胞百分率80.3%,G2期细胞12.4%,S期细胞7.3%;P3代细胞表面抗原高表达CD29、CD44、CD105、Sca-1,极低表达CD45,低到中度表达CD31;P3代细胞经体外定向诱导培养,可分化为成骨及脂肪细胞,证实所培养细胞具备多向分化潜能;RT-PCR法检测培养细胞mIL-10mRNA,双抗体夹心ELISA法检测细胞培养上清中mIL-10,均未能证实mMSC/MPC体外表达分泌mIL-10。第二部分:mIL-10重组复制缺陷型腺病毒载体的构建制备及转染mMSC/MPC表达方法:以pORF5-mIL-10质粒为模板,PCR扩增回收mIL-10 cDNA,经一系列酶切和连接反应,将其定向插入腺病毒载体pSGCMV中,构建pSGCMV-mIL10质粒;此质粒与5型腺病毒质粒pBGHE3通过脂质体Lipofectamine2000共转染HEK293细胞,经胞内同源重组产生重组复制缺陷型腺病毒Ad5-mIL-10;经纯化后的Ad5-mIL-10在HEK293细胞中大量扩增,并通过氯化铯密度梯度离心法纯化,制备病毒液,50%组织培养感染剂量法（TCID50）测定病毒滴度;以Ad5-EGFP腺病毒确定转染mMSC/MPC的最宜感染复数（MOI）,Ad5-mIL10腺病毒体外转染mMSC/MPC;RT-PCR法检测Ad5-mIL10转染mMSC/MPC后mIL10的mRNA表达,ELISA法检测Ad5-mIL10转染mMSC/MPC培养上清中mIL-10。结果:扩增产物经酶切鉴定、PCR扩增、DNA测序均证实为小鼠白介素10;构建制备的重组腺病毒Ad5-mIL-10经PCR鉴定正确,滴度达2.5×10¹⁰pfu/ml;Ad5-mIL-10以最宜MOI值200转染mMSC/MPC时能获得高效稳定的体外表达,符合体内实验要求。第三部分:mIL-10基因转染修饰骨髓间充质干/祖细胞在H-2单倍型骨髓移植aGVHD模型中的生物免疫学效应方法:以雌性C57BL/6小鼠为供鼠,雄性F1（CB6F1）小鼠为受鼠,建立H-2单倍型骨髓移植aGVHD模型:对受鼠进行直线加速器6mvX射线一次性全身照射,总剂量12Gy,剂量率100cGy/min。制备供鼠1:0,1:1,1:2,1:3四种比例的骨髓单个核细胞（MNC）/脾MNC混合悬液（骨髓MNC取1×10⁷/只）,每比例为一实验组（n=15）。受鼠于照射后4小时内经尾静脉回输相应混合悬液,移植后进行一般情况观察,定期体重监测、外周血象及嵌合体测定,GVHD临床综合评分,组织病理学半定量评分,以综合评判各组aGVHD效应,最终确定引出理想aGVHD状态的相关参数,建立H-2单倍型骨髓移植aGVHD模型;选择1×10⁵/只和5×10⁵/只两个剂量梯度作为MSC/MPC移植剂量,在H-2单倍型骨髓移植aGVHD模型中,分别以MSC/MPC及mIL-10基因转染修饰的MSC/MPC进行共移植实验,对相应的4个共移植实验组及aGVHD对照组进行移植后一般情况观察,定期体重监测,外周血象及嵌合体测定,GVHD临床综合评分,组织病理学半定量评分,对各组GVHD状况进行综合评价,并选择移植后14天、28天、35天、60天及濒死前等不同的时间点,采集制备各组实验鼠血清,以ELISA法进行mIL-10、mIL-4、mINF-γ及mTNF-a定量检测。采用SPSS 11.5 FOR WINDOWS统计软件分析数据,检验水准α=0.05。结果:雄性F1（CB6F1）小鼠为受鼠,经直线加速器6mvX射线一次性全身照射（总剂量12Gy,剂量率100cGy/min）,接受雌性C57BL/6供鼠1×10⁷骨髓MNC/3×10⁷脾MNC（每只）联合输注时,可在相对集中的时间内（+24d～+28d）稳定地引出典型aGVHD,临床及病理程度较一致,死亡时间相对集中（+27d～+33d）,以此为参数建立H-2单倍型骨髓移植aGVHD模型;体内共移植实验结果显示:以1×10⁵/只MSC/MPC共移植时可降低aGVHD的发生及严重程度（P＜0.05）,提高细胞剂量（5×10⁵/只）并不能增强实验效应（P＞0.05）;同细胞剂量的mIL-10基因转染修饰MSC/MPC共移植能更为显著地减低aGVHD的发生及严重程度（P＜0.01）,但并不能降低甚至可能会加重cGVHD的发生及严重程度,提高细胞剂量其效应无显著差别（P＞0.05）。细胞因子检测显示:MSC/MPC共移植在+14d明显上调受鼠体内IL-4水平（P＜0.01）,对IL-10影响不显著（P＞0.05）,同时明显减低INF-γ和TNF-a水平（P均＜0.05）;mIL-10基因转染修饰的MSC/MPC共移植较MSC/MPC更高地上调了受鼠体内IL-10水平（P＜0.01）,同时协同上调IL-4水平（P＜0.05）,更显著地减少了INF-γ生成（P＜0.01）,但IL-10水平的上升可能也增加了受鼠发生及加重cGVHD的风险。结论:1.成功分离、培养了正常C57BL/6小鼠的骨髓间充质干/祖细胞,生物学特性稳定,具有良好的增殖能力和向成骨、脂肪细胞的分化潜能,可用于进行基因转染修饰及应用于动物模型。2.成功构建了重组AD5-mIL-10复制缺陷型腺病毒载体系统,并证实对mMSC/MPC具有较高的转染效率并获得高效稳定的体外表达.。3.成功建立了雌性C57BL/6小鼠→F1（CB6F1）雄性小鼠方向的H-2单倍型骨髓移植aGVHD模型。1×10⁵/R MSC/MPC共移植可降低aGVHD的发生及严重程度,提高细胞剂量（5×10⁵/只）并不能增强效应。不同程度地上调受鼠IL-4水平（但对IL-10影响不显著）,同时明显减低INF-γ和TNF-a水平,这可能是MSC/MPC移植减轻aGVHD的机制之一。1×10⁵/只mIL-10基因转染修饰MSC/MPC共移植能更显著地减低aGVHD的发生及严重程度,但并不能降低甚至可能会加重cGVHD的发生及严重程度,提高细胞剂量其效应无显著差别;mIL-10基因转染修饰MSC/MPC较未修饰者更高地上调了受鼠IL-10水平,同时协同上调体内IL-4水平,更为显著地减少INF-γ生成,可能是最终显著降低aGVHD发生及严重程度的原因,但IL-10水平上调也增加了受鼠发生及加重cGVHD的风险。更多还原

【Abstract】 As an adult stem cell with the ability of multiple lineage differentiations（including osteoblasts,adipocytes,chondrocytes,and myoblasts）,mesenchymal stem cells have received considerable attention and been a perfect "seed cell" in regenerative medicine.Recent research has showed that mesenchymal stem cells have some special efficacy on hematopoietic reconstitution and immunomodulation,which making them favourable perspective in clinical application.The encouraging results have been observed in limited clinical trials,co-transplantation with MSC and HSC enhanced hematopoietic engraftment,attenuated transplant-related complications,especially the incidence and severity of GVHD.However,the mechanism of immunomodulation has not been completely resolved,some latest studies suggested that the efficacy of MSC on prophylaxis and treatment for severe acute GVHD was limited,even not confirmable.Interleukin-10 is an ideal anti-inflammatory cytokine,inhibits the activation of T lymphocytes,monocytes/macrophages,and the synthesis of various inflammatory cytokines.IL-10 represents a substancial suppressor of the cellular immune response of Th1 type,which implys its potential effects on preventing the occurrence and development of GVHD.Because of its anti-inflammatory properties and its association with dendritic cells and regulatory T cells,IL-10 should have substantial benefit on the induction of immune tolerance and graft survival.Holler’s research showed that decreased IL-10 production correlated with a subsequent high incidence of GVHD.We assumed that with the combination of cell therapy and gene therapy,the co-transplantation with marrow-derived mesenchymal stem/progenitor cells modified with mIL-10 gene will prevent or attenuate GVHD more effectively in coordination with IL-10,but it hard to carry out in human because of the limitation of ethics and technology.In our research,firstly we purified and cultured murine marrow-derived mesenchymal stem/progenitor cells（MSCs/MPCs） with active proliferation activity and multidifferentiation potential,and studied its biological characteristic and corresponding properties.Then,a mlL-10 recombinant adenoviral vector was constructed Successfully and proved to be high efficient in transfecting mMSC/MPCs.The mMSCs/MPCs modified with mIL-10 gene expressed mIL-10 protein efficiently in vitro.Thirdly,we established an eligible model of murine acute GVHD after H-2 haploidentical bone marrow transplantation.On the basis of the model,MSCs/MPCs and MSCs/MPCs modified with mIL-10 gene were transplanted respectively to observe the immunobiologic effects.Section 1:The optimization of culture system ex vivo of mMSC/MPC,the corresponding characterization of mMSC/MPC.Methods:Bone marrow cells were collected by flushing the femurs and tibias from female C57BL/6 mice with medium repeatedly and thoroughly,while scraping the endosteum Of the bone,then the collection were vibrated with the bone fragments.The mononuclear cells were made and plated in the 25cm² culture flasks,at a concentration of 1×10⁶/cm²,with three different complete medium respectively:DMEM-LG medium supplemented with 15%FBS,DMEM -LG/MCDB201 medium supplemented with 15% FBS,and Mesencult basal medium supplemented with 20%MSC Stimulatory Supplement.Flasks were maintained in incubator with 5%CO2 at 37℃.After 24h,nonadherent cells were eliminated by medium changing,then the medium were changed every 3 days.The cells were trypsinized when tending to 70%-80% confluences,and replated at a concentration of 2.5～5×10³/ cm².The characters of different cell passages,such as morphology,the CFU-F with different medium,cell growth curve,cell cycle,phenotype were demonstrated.The ability of multidifferentiation along adipocytic,osteoblastic pathways were also assayed.The expression of IL-10mRNA in the cells were detected by RT-PCR and IL-10 protein in the culture supernatant by ELISA.The calculations and statistical comparisons were performed using the software SPSS 11.5 and the reported P was 2 sided（a =0.05）.Results:The cell population consisted of spindle,shaped cells in confluent cultures after three consecutive passaging,and seemed homogeneous by light microscopy.The Mesencult complete medium was the optimal culture system.The biological stabilities were maintained till 10 passages above,and the P3 cells were choosed to be the suitable object for the experiment.Cell-doubling time was 31.1±0.6 hours in log phase.Cell cycle analyses revealed that 80.3%of the P3 cells at G0/G1 phase,12.4%, 7.3%at G2 and S phase respectively,when they tended to 80%-90% confluences.Flow-cytometric analysis showed that the P3 cells were high positive for CD29、CD44、CD105、Sca-1,moderate positive for CD31,and nearly negative for CD45. The cells can be differentiated into osteoblast,adipocyte in vitro.We failed to identify IL-10 mRNA of the cells by RT-PCR and protein of IL-10 in the culture supernatant by ELISASection 2:The construction of mIL-10 recombinant adenoviral vector,the transfection and mIL-10 expression of mMSC/MPCs ex vivoMethods:The mIL-10 cDNA was removed and amplificated from pORF5-mIL-10 plasmid through PCR,then inserted into adenoviral vector pSGCMV to construct the recombinant plasmid,pSGCMV-mIL10.The recombinant plasmid were mixed with human serum type v adenoviral plasmid（pBGHE3） and co-transfected into HEK 293 cell line through Lipofectamine 2000.The replication-incompetent adenovirus containing mIL-10 was harvested from the supernatant of HEK 293 cell line.The adenovirus was produced sufficiently in HEK 293 cell line and purificated by CsCl gradient centrifugation.The titer of the recombinant adenovirus was assayed through the method of TCID50.The optimal MOI for mMSC/MPC was identified by the adenovirus Ad5-EGFP.The expression of IL-10mRNA in the mMSC/MPC transfected was detected by RT-PCR and IL-10 protein in the culture supernatant by ELISA. Results:The amplification production was verified by restrictive endonucleases analysis,PCR,DNA sequencing.The recombinant adenovirus containing mIL-10 was verified by PCR,and the titer of adenovirus was 2.5×¹⁰pfu/ml.the mMSCs/MPCs transfected with mIL-10 recombinant adenoviral vector expressed mIL-10 protein efficiently ex vivo at the MOI of 200.Section 3:The establishment of a model of murine acute GVHD after H-2 haploidentical bone marrow transplantation,the immunobiologic effects of mMSCs/MPCs modified with mIL-10 gene in the model when co-transplanted.Methods:The female C57BL/6 mice as donor,male CB6F1 mice were used as recipients to establish aGVHD model of murine after H-2 haploidentical BMT.The recipients received 12Gy（100cGy/min） total body irradiation（6mv,X-ray） as the lethal conditioning.Bone marrow MNC（1×10⁷/a recipient mouse） and splenic MNC（0,1×10⁷,2×107,3 10⁷/a recipient mouse,respectively） were transplanted by tail vein infusion into irradiated recipients in 4 hours.Four groups were divided（n=15） according to the different MNC mixture.The general state,survival time,loss of weight,WBC counting in peripheral blood,chimerism,clinical signs were observed after BMT regularly.The severity of aGVHDwas assessed with a clinical GVHD scoring system and a semiquantitative system for histologic examination,and the corresponding parameters were determined for an eligible acute GVHD model.On the basis of the model,MSCs/MPCs and mMSC/MPCs modified with mIL-10 gene were transplanted respectively to observe the immunobiologic effects. Four groups were divided according to the different dose of cells（1×10⁵ and 5×10⁵/a recipient mouse）.The general state,survival time,loss of weight,WBC counting in peripheral blood,chimerism,clinical signs,were observed regularly,and the severity of aGVHD was assessed similarly.The serum of recipient mice was collected on the day 14,28,35 after transplantation and in extremis,then be used to detect cytokines of mIL-10, mIL-4,mINF-γand mTNF-a by ELISA.Results:Under the condition of TBI（X-ray,6mv,total dose:12Gy,dose rate:100 cGy/min）, typical aGVHD clinical signs were induced stably in limited days（+24d～+28d） when recipient mice were infused with 1×10⁷ bone marrow MNCs plus 3×10⁷ splenic MNCs from the donor mice.The clinical and histologic degree was relatively coincident,and the incidence of aGVHD in 35 days were 100%,all the recipient mice died within +27d～+33d.Accordingly,the C57BL/6→CB6F1 model of munne aGVHD after H-2 haploidentical BMT was set up successfully.The co-transplant of 1×10⁵ MSCs/MPCs seemd to decrease the incidence and severity of aGVHD（P＜0.05）,while increased cell dose（5×10⁵） failed to enhance the effect significantly（P＞0.05）.The co-transplant of 1×10⁵ MSCs/MPCs modified with mIL-10 gene showed the more significant decreasing incidence and severity of aGVHD than MSCs/MPCs（P＜0.01）.While it failed to decrease,even augment the incidence and severity of cGVHD,similarly,increased cell dose（5×10⁵） failed to enhance the effect significantly（P＞0.05）,The detection of cytokines showed that the co-transplant of MSCs/MPCs up-regulate the level of IL-4 significantly in 14 days after transplant（P＜0.05）,decrease the level of INF-γand TNF-a significantly（P＜0.05）,while no significant effect on IL-10（P＞0.05）.The co-transplant MSCs/MPCs modified with mIL-10 gene showed the significant increase of IL-10（P＜0.05）,the corresponding increase of IL-4（P＜0.05）,and more dramatic decrease of INF-γ（P＜0.01）.Conclusions:1.mMSCs/MPCs from C57BL/6 mice have been isolated,cultured and purified ex vivo successfully.With the stable biologic characterization,active proliferation and multiple differentiated abilities,the mMSCs/MPCs could be used in gene-transfection and animal model experiment.2,mlL-10 recombinant replication-incompetent adenoviral vector system was constructed successfully,and mMSCs/MPCs could be gene-transfected with high efficiency by the adenoviral vector.The mMSCs/MPCs transfected with mIL-10 recombinant adenoviral vector expressed mIL-10 protein efficiently and stably ex vivo.3.The C57BL/6→CB6F1 model of murine aGVHD after H-2 haploidentical BMT was established successfully.The co-transplant of 1×10⁵ MSCs/MPCs could decrease the incidence and severity of aGVHD,while increased cell dose（5×10⁵） failed to enhance the effects significantly.MSCs/MPCs up-regulated the level of IL-4 significantly in 14 days after transplant,decreased the level of INF-γand TNF-a significantly（no significant effect on IL-10）,it maybe the one of the mechanisms of MSCs/MPCs alleviating aGVHD.The co-transplant of 1×10⁵ MSCs/MPCs modified with mIL-10 gene showed the more significant effects on decreasing incidence and severity of aGVHD than MSCs/MPCs,while it failed to decrease,even augment the incidence and severity of cGVHD.Similarly,increased cell dose（5×10⁵） failed to enhance the effects significantly.The significant increase of IL-10,the corresponding increase of IL-4 and more dramatic decrease of INF-γmay accounted for the possible reason,while the upregulation of IL-10 may increase the risk of cGVHD.更多还原