【作者】 辛海明；

【导师】 彭毅志；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 研究背景及研究目的临床上同种异体皮肤移植是目前大面积深度烧伤患者早期创面覆盖最直接、最有效的治疗方法,但是由于皮肤的强烈抗原特性,导致移植后3周左右外源皮肤就会发生不可逆的排斥反应,极大地限制了自体微粒皮混合大张异体皮移植效果。如何成功诱导出皮肤移植后的免疫耐受,临床上至今未发现非常有效的措施。树突状细胞(dendritic cells,DCs)是目前已知的功能最强大的专职抗原提呈细胞,在免疫反应中发挥了极其重要的作用。DCs的分化发育过程伴随着DCs由未成熟的前体细胞分化发育为成熟细胞,细胞的形态、表面标志以及生物学功能等都发生了相应变化。未成熟树突状细胞(immature dendritic cells,imDCs)因为其功能上最显著的特点就是可以诱导T淋巴细胞的特异性低应答而成为当前研究免疫耐受策略的热点,这也在我们前一个国家自然科学基金项目(No.30271341)中得到证实。利用imDCs或者基因修饰DCs进行的联合移植实验已经在肾、肝、心脏、胰腺和小肠等器官中取得了令人较为满意的效果,这为皮肤移植实验提供了非常有利的依据。DCs的发育成熟过程伴随着其表型的变化,包括摄取能力的减弱和MHC类分子、共刺激分子的表达上调,最终变成强大的抗原提呈细胞。研究发现imDCs在炎性介质作用下逐渐迁移成熟的过程中,其表面趋化因子受体-7(Chemokine receptor-7,CCR7)表达上调。通过CCR7与其配体MIP3β(即CCL19)和SLC(即CCL21)的相互作用,引导DCs趋化迁移至淋巴结,由此CCR7被认为是成熟树突状细胞(mature dendritic cells,mDCs)迁移归巢完成抗原递呈从而激活初始T细胞的主要受体之一。目前CCR7趋化迁移的能力在部分恶性肿瘤的转移中得到证实,研究表明CCR7严格调控了肿瘤细胞的转移。肿瘤细胞这种非随机的、有组织器官选择性的转移过程类似于免疫刺激过程中DCs的定向迁移。基于CCR7具有非常重要的趋化迁移功能,若DCs成熟度越高,CCR7表达更强,则免疫系统呈现更为强劲的免疫应答,反之则诱导免疫耐受。然而,由于imDCs缺乏CCR7的表达,向淋巴结的趋化迁移能力较弱,长时间处于非T细胞区易受各种炎症因子或者外来抗原的刺激而发育为mDCs,严重影响其诱导免疫耐受的效果。基于以上分析,本研究拟通过构建含有小鼠CCR7的重组腺病毒,尝试在imDCs上建立CCR7的表达,使imDCs获得原本mDCs才具有的高效迁移能力,避免imDCs在外周组织向成熟状态的分化,有效地确保其诱导免疫耐受功能的发挥,为临床上大面积深度烧伤患者早期创面覆盖引起的免疫排斥提供新的解决方法和思路。研究方法1、小鼠骨髓源树突状细胞的培养及鉴定小剂量rmGM-CSF和rmIL-4联合体外诱导培养BALB/c小鼠骨髓来源的imDCs,第5天收获得到一定数量的imDCs,加入rmTNF-α刺激细胞成熟而获得mDCs。通过光学显微镜及扫描电镜观察细胞的形态学变化,应用流式细胞仪检测细胞表面标志分子的表达水平。2、CCR7基因转染小鼠骨髓源未成熟树突状细胞及其功能鉴定采用梯度离心法将Ad空载体和Ad-ccr7腺病毒转染入imDCs,通过光学显微镜和扫描电镜来观察细胞形态学上的变化,应用流式细胞仪检测细胞表面的相应标志物,从而来观察Ad-ccr7腺病毒转染对小鼠骨髓源imDCs的成熟状态及形态的影响。采用Real-time PCR法、细胞免疫荧光法、Western Blot法检测Ad-ccr7腺病毒转染前后小鼠骨髓源树突状细胞CCR7mRNA及蛋白水平的表达变化。通过体外趋化试验和混合淋巴细胞反应比较观察Ad-ccr7腺病毒转染前后小鼠骨髓源DCs的体外迁移能力和体外刺激T淋巴细胞增殖能力。3、CCR7基因转染imDCs对小鼠异基因皮肤移植的影响在小鼠异基因皮肤移植实验中,我们将供者源imDCs、供者源mDCs、供者源imDCs+Ad、供者源imDCs+Ad-ccr7和供者源imDCs+Ad-ccr7+IL-10通过腹腔注射入受体小鼠体内,观察各组细胞对小鼠异基因移植皮片存活状况的影响,并通过组织学检查观察移植皮片的结构改变。实验结果1、小鼠骨髓源DCs的培养及鉴定结果体外培养的DCs经光学显微镜及扫描电镜观察发现:imDCs表面光滑,凹凸不平,毛刺状突起较少,而mDCs表面伸出许多较长的树枝样突起,突起长短不一,形态各异。流式细胞仪检测发现在imDCs中CD11c、CD80、CD83、MHCⅡ的阳性率分别为24.7%、9.4%、20.5%和20.5%,而在mDCs中CD11c、CD80、CD83、MHCⅡ的阳性率分别为86.5%、88.4%、76.9%和92.4%。这表明体外培养的DCs符合其典型形态学特征和表面标志水平。2、CCR7基因转染对小鼠骨髓源imDCs的影响2.1采用高速离心法将Ad-ccr7腺病毒转染入小鼠骨髓源imDCs内,其感染效率在MOI达到100时约有70%左右。在扫描电镜中可见imDCs转染腺病毒空载体和Ad-ccr7后,细胞形态不规则,表面粗糙,细胞表面的不规则膜性树枝状突起较未转染imDCs增多,而IL-10组细胞膜表面不规则突起明显减少,这说明IL-10可一定程度地抑制imDCs的成熟。小鼠骨髓源imDCs经腺病毒转染2天后,流式细胞术检测发现imDCs+Ad组细胞中CD11c、CD80、CD83、MHCⅡ的阳性率分别为40.0%、27.7%、28.5%和37.8%。imDCs+Ad-ccr7组细胞中CD11c、CD80、CD83、MHCⅡ的阳性率分别为37.8%、27.4%、21.3%和37.0%,而imDCs+Ad-ccr7+IL-10组细胞中CD11c、CD80、CD83、MHCⅡ的阳性率分别为20.5%、15.5%、21.6%和32.9%。这表明转染Ad空载体和Ad-ccr7腺病毒可促使imDCs表面标志略微升高,即向成熟方向发展,但加入细胞因子IL-10可略微下调这些表面标志的表达,在一定程度上抑制imDCs的成熟。2.2 Real-time PCR结果显示, imDCs中CCR7mRNA水平较低, mDCs中CCR7mRNA含量是imDCs的1.544倍。imDCs转染Ad空载体后,其CCR7mRNA含量是imDCs的1.115倍,imDCs转染Ad-ccr7腺病毒后,其CCR7mRNA含量是imDCs的2.941倍,而IL-10组中CCR7mRNA含量为imDCs的1.556倍,接近正常mDCs含量。这也说明Ad-ccr7腺病毒转染可以明显增加未成熟树突状细胞CCR7mRNA水平的表达,同时IL-10在一定程度上可以抑制转染过程中imDCs的成熟。2.3细胞免疫荧光结果显示imDCs和imDCs+Ad组细胞不表达CCR7,而mDCs细胞可表达CCR7。imDCs转染Ad-ccr7腺病毒后CCR7的表达增加,说明CCR7基因有效转入imDCs并表达蛋白。Western Blot结果通过Qulitity One软件分析显示:imDCs、imDCs+Ad、imDCs+Ad-ccr7、imDCs+Ad-ccr7+IL-10和mDCs中CCR7蛋白表达的相对含量分别为0.09、0.15、0.96、0.46和0.35。结果表明,imDCs和imDCs+Ad组中CCR7在蛋白水平几乎不表达,而转染Ad-ccr7腺病毒后imDCs的CCR7蛋白含量明显上升,其蛋白相对含量可以高于正常mDCs的含量。加入IL-10可以抑制imDCs朝成熟方向转化,但是其CCR7蛋白的表达含量仍可以达到mDCs的水平。2.4体外迁移实验结果表明imDCs细胞的体外趋化迁移率均低于5%,而mDCs、imDCs+Ad-ccr7、imDCs+Ad-ccr7+IL-10组细胞在CCL19作用下体外趋化迁移率均较imDCs组明显上升(aP<0.05)。而CCL19+anti-CCR7mAb组,在CCL19对抗剂anti-CCR7mAb作用下迁移率均有下降(dP <0.05),但仍高于各自的空白组。这表明能够表达CCR7蛋白的转染病毒后imDCs及mDCs可在其配体CCL19的作用下增强体外迁移能力,同时anti-CCR7mAb可以在体外部分的拮抗其配体CCL19的作用。2.5混合淋巴细胞反应结果表明,imDCs以1:5比率与同种异体未致敏T淋巴细胞混合后imDCs组刺激T淋巴细胞增殖能力较弱,刺激指数为1.6±0.1,而mDCs组刺激T淋巴细胞增殖能力明显增强,刺激指数为5.6±0.3(aP<0.05)。腺病毒转染后细胞刺激T细胞增殖的能力较imDCs组增强但是弱于mDCs组,刺激指数分别为3.4±0.2和3.1±0.2,但是加入IL10可以明显降低刺激能力,刺激指数为1.8±0.1(bP<0.05)。表明腺病毒转染可部分增强imDCs的刺激增殖能力,但还是弱于mDCs,IL-10可抑制其促增殖能力。3、CCR7基因转染imDCs对小鼠异基因移植皮片的影响3.1通过与对照组(NS)相比较,供者源imDCs、供者源imDCs+Ad-ccr7、供者源imDCs+Ad-ccr7+IL-10组皮肤移植物的MST均明显延长(aP<0.05),而供者源mDCs和imDCs+Ad组MST与对照组相比无显著性差异(P>0.05),说明单纯使用供者源mDCs对移植皮片的存活无明显影响,但供者源未成熟DC的应用可延长皮片的存活时间。同时,供者源imDCs+Ad-ccr7+IL-10组MST较供者源imDCs组显著延长(bP<0.05),而供者源imDCs+Ad-ccr7组MST较供者源imDCs组差异不明显(P>0.05),显示供者源imDCs在转染腺病毒Ad-ccr7后,其诱导免疫耐受的能力较转染前提高,加入IL-10更能够显著提高皮片存活的时间。3.2组织切片活检显示皮肤组织结构较清楚,纤维组织结构排列有序,成纤维细胞多,真皮层内血管腔丰富,结构完整,大小不一,局部有炎性细胞浸润。表明皮片生长情况良好,白细胞的趋化功能正常。结论1、小鼠骨髓来源细胞在应用低剂量rmGM-CSF和rmIL-4联合刺激诱导后获得具有典型形态特征的imDCs和mDCs。2、Ad-ccr7腺病毒成功转染入小鼠骨髓源imDCs内。转染后小鼠imDCs在形态学及细胞表面标志分子上可向成熟状态分化,但IL-10可一定程度地抑制imDCs的成熟。3、Real-time PCR结果表明Ad-ccr7腺病毒转染可以明显增加未成熟树突状细胞CCR7mRNA水平的表达,同时IL-10在一定程度上可以抑制转染过程中imDCs的成熟。4、细胞免疫荧光及Western Blot结果表明,转染Ad-ccr7腺病毒后imDCs的CCR7蛋白含量明显上升。加入IL-10可以抑制imDCs朝成熟方向分化,但其CCR7蛋白的表达含量仍可以达到mDCs的水平。5、体外迁移实验表明imDCs细胞的体外趋化迁移率均较低,而转染Ad-ccr7腺病毒后imDCs在CCL19作用下体外趋化迁移率均较imDCs组明显上升。同时anti-CCR7mAb可以在体外部分的拮抗其配体CCL19的作用。6、混合淋巴细胞反应实验显示imDCs组刺激T淋巴细胞增殖能力较弱,而mDCs组刺激T淋巴细胞增殖能力明显增强。Ad-ccr7腺病毒转染后其刺激T细胞增殖的能力较imDCs组增强但是弱于mDCs组,加入IL10可以明显降低刺激能力。7、小鼠异基因皮肤移植实验表明,单纯使用供者源mDCs对移植皮片的存活无明显影响,供者源imDCs在转染Ad-ccr7腺病毒后可明显延长皮片的存活时间,加入IL-10更能够显著提高皮片存活的时间。更多还原

【Abstract】 Background and objectiveAllogeneic skin transplantation is the most effective method in the early coverage of extensive burn wounds. However, the host versus graft reaction (HVGR) after transplantation leads to irreversible rejection about 3 weeks later, which limits the effection of skin transplantation. And there isn’t any effective treatment to induce immune tolerance after transplantation. Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APC) in vivo, which play critical roles in immune reponse. Dendritic cells differentiate from immature dendritic cells (imDCs) to mature dendritic cells (mDCs), in the process, morphological characteristics, surface markers and biological functions in DCs have changed accordingly. The most distinguished ability of imDCs is inducing T cell anergy and promote alloantigen-specific tolerance, which has been verified in our last National Natural Science Foundation of China (NSFC) subjest (No.30271341). Based on the advantages, combined transplantation using imDCs or genetically-modified imDCs have seen satisfied results in kidney、liver、heart、pancreatic islet and small intestine transplantation.The phenotypical changes that take place during maturation include the blunting of the endocytic ability and the up-regulation of MHCs and costimulatory molecules that eventually transform these DCs into potent APC. Maturation takes place concomitantly with the migration of the DCs from their niches in the peripheral tissues to the lymph nodes where they arrive through the lymphatics. In this regard, during maturation the expression of the chemokine receptor-7 (CCR7) is up regulated. Through interaction with ligand MIP3β(CCL19) and SLC (CCL21), CCR7 guides the migratory DCs to the nodes, presents antigens to naive T cells and activates immune response. Recent studies have also showed that CCR7 plays an important role in cancer metastasis. Such function of CCR7 is kind of like oriented migration in DCs. If DCs became more matured, they express more CCR7, the immune system will present stronger response. Otherwise, immune tolerance is induction. However, imDCs lack the expression of CCR7 and direct less migratory DCs to lymph nodes. Long time for residing in non-T cell area put imDCs into high risks sensing of“danger signals”(tissue damage、inflammatory cytokines or pathogens), which starts the differentiation of maturation and change the important tolerance inducing functions of imDCs.Base on the the analysis above, the present study was designed to build expression of CCR7 in imDCs through the construction of recombinant adenovirus carrying murine CCR7 gene, which will afford imDCs high chemotactic ability possessed by mDCs, inhibit the maturation of imDCs in peripheral tissues and guarantee the induction of immune tolerance of imDCs in the early coverage of extensive burn woundsMethods1、Cultivation and identification of dendritic cells derived from murine marrow ImDCs isolated from BALB/c murine marrow was induced and proliferated by rmGM-CSF and rmIL-4, and harvested amount of imDCs after 5 days, obtained mDCs through the stimulation of rmTNF-α. The change of cell morphology was observed through the light microscope and the scanning electron microscope, the expression level of cell surface markers were detected application flow cytometry.2、CCR7 gene transfection immature dendritic cell derived from murine marrow and identification of its functionTransfected the Ad-ccr7 adenovirus into immature dendritic cells through gradient centrifugation methods, observed the change of cell morphology through the light microscope and the scanning electron microscope and detected the expression level of cell surface markers application flow cytometry after Ad-ccr7 adenovirus transfection it, it will be useful to observed the influence of mature state and morphology after transfection. The expression change of CCR7 mRNA and protein in immature dendritic cells after Ad-ccr7 adenovirus tansfection were detected by Real-time PCR、cell immumofluorescence and Western Blot methods. The migration ability and the stimulate proliferation ability of T lymphocyte by immature dendritic cells after Ad-ccr7 adenovirus transfection was observed by migration experiment and mixed lymphocyte reaction in vitro.3、Influence of imDCs after CCR7 gene transfection to murine allogene skin transplantionIn murine allogene skin transplantion experiments, we injected the donor imDCs、donor mDCs、donor imDCs+Ad、donor imDCs+Ad-ccr7 and donor imDCs+Ad-ccr7+IL-10 into receptor murine, observed the influence of each group cells to murine allogene skin transplantion graft survive state and observed the construction change of skin grafts through histology detection.Results1、The results of cultivation and identification of dendritic cells derived from murine marrowThe dendritic cells cultivated in vitro were observed by light mircroscope and scanning electron microscope, we discovered: the surface of immature dendritic cell was slick、rugosity and less sentus ecphyma, but the surface of mature dendritic cell was stretched out many branch ecphymas, which was different longer and strange form. The flow cytometry founded that the positive percentage of CD11c、CD80、CD83、MHCⅡin immature dendritic cell was 24.7%、9.4%、20.5% and 20.5%, but the positive percentage in mature dendritic cell was 86.5%、88.4%、76.9% and 92.4%. These demonstrated the dendritic cells cultivation in vitro consistented with the typical morphology characteristic and the level of surface markers.2、Influence of CCR7 gene transfection to immature dendritic cells derived from murine marrow2.1 We transfected Ad-ccr7 adenovirus to immature dendritic cells derived from murine marrow by high speed centrifugation method, the transfection efficiency up to 70% as MOI in 100. The morphology of immature dendritic cell after transfection Ad void vector and Ad-ccr7 adenovirus by scanning electron microscope was rugosity, rough hard sphere, the irregularity dendritic processes more than imDCs un-transfection, but the irregularity dendritic processes decreased in IL-10 group, this illustration IL-10 can slightly inhibited the maturation of imDCs. The flow cytometry founded that the positive percentage of CD11c、CD80、CD83、MHCⅡin imDCs+Ad was 40.0%、27.7%、28.5% and 37.8%, the positive percentage of CD11c、CD80、CD83、MHCⅡin imDCs+Ad-ccr7 was 37.8%、27.4%、21.3% and 37.0%, but the positive percentage in imDCs+Ad-ccr7+IL-10 was 20.5%、15.5%、21.6% and 32.9%. These demonstrated Ad-ccr7 adenovirus transfection can spur the surface marker of imDCs slightly increased, development to maturity direction, but IL-10 can slightly down regulated the expression of surface markers and inhibited the maturation of imDCs.2.2 Results of Real-time PCR manifested, the expression level of CCR7 mRNA in imDC was low, and the expression level of CCR7 mRNA in mDCs was 1.544 fold than imDCs. The expression level of CCR7 mRNA in imDCs after Ad void vector transfection was 1.115 fold than imDCs, the expression level of CCR7 mRNA in imDCs after Ad-ccr7 adenovirus transfection was 2.941 fold than imDCs, but the expression level of CCR7 mRNA in IL-10 group was 1.566 fold than imDCs, approach the normal level in mDCs. These illustrated the expression level of CCR7 mRNA in imDCs after Ad-ccr7 adenovirus transfection can increased obviously, meanwhile IL-10 can slightly inhibited the maturation of imDCs during transfection.2.3 Results of cell immunofluorescence displayed there has no expression of CCR7 in imDCs and imDCs+Ad group, but it had expression of CCR7 in mDCs. The expression of CCR7 in imDCs after Ad-ccr7 adenovirus transfection was increased, it means CCR7 gene had transfected imDCs and expressed the protein of CCR7. The results of Western Blot analysed by software Qulitity One manifested: the expression relative amount of CCR7 protein in imDCs、imDCs+Ad、imDCs+Ad-ccr7、imDCs+Ad-ccr7+IL-10 and mDCs were 0.09、0.15、0.96、0.46 and 0.35 respectively. There almost has no expression of CCR7 protein in imDCs and imDCs+Ad group, but the expression of CCR7 in imDCs after Ad-ccr7 adenovirus transfection upgraded obviously, the expression relative amount of CCR7 protein more than the normal expression in mDCs. IL-10 can inhibitted the imDCs differentiation toward maturation, but the expression of CCR7 protein can achieve the level of mDCs.2.4 The results of migration experiment in vitro displayed the migration percentage of imDCs less than 5%, but the migration percentage in mDCs、imDCs+ Ad-ccr7 and imDCs+Ad-ccr7+IL-10 group which affected by CCL19 were increased obviously than imDCs(aP<0.05). But in CCL19+anti-CCR7 mAb group, the migration percentage decreased by CCL19 antagonizer anti-CCR7 mAb, it higher than its control group respectively (dP <0.05).These indicated that the migration ability in vitro of imDCs after Ad-ccr7 adenovirus transfection and mDCs which expressed CCR7 protein can enhanced by its ligand CCL19, meanwhile anti-CCR7 mAb can rivalied the effection of its ligand CCL19 in vitro.2.5 The results of mixed lymphocyte reaction demonstrated, imDCs mixed the T lymphocytes at rate 1:5, the proliferation ability of imDCs stimulate to T lymphocytes was lower, SI was 1.6±0.1, but the proliferation ability of mDCs stimulate to T lymphocytes increased significantly, SI was 5.6±0.3 (aP<0.05). The proliferation ability of imDCs after adenovirus transfection was higher than imDCs and lower than mDCs, SI was 3.4±0.2 and 3.1±0.2 respectively. IL-10 can decreased the proliferation ability, SI was 1.8±0.1 (bP<0.05). Adenovirus transfection can partly enhance the proliferation ability of imDCs, but it still lower than mDCs, IL-10 can inhibited the proliferation ability.3、Influence of imDCs after CCR7 gene transfection to murine allogene skin transplantion3.1 Compare to control group, the MST of skin grafts in donor imDCs、donor imDCs+Ad-ccr7、donor imDCs+Ad-ccr7+IL-10 group were prolonged obviously (aP<0.05), but the MST of donor mDCs and imDCs+Ad group had no difference(P>0.05). It means the donor mDCs had no obviously influence on the MST of skin grafts, but the donor imDCs can prolong the MST of skin grafts. Meanwhile the MST of donor imDCs+Ad-ccr7+IL-10 group was significant prolongation than the MST of donor imDCs, but the MST of donor imDCs+Ad-ccr7 group has no obviously difference than donor imDCs, it manifestation the ability of induction immune tolerance of imDCs after Ad-ccr7 adenovirus transfection was higher than before the transfection, IL-10 can significant extended the MST of skin grafts.3.2 The clear epithelial structure and infiltration of inflammatory cells were observed in specimens.Conclusion1. The cells we cultured displayed typical morphlogical features of imDCs and mDCs, which were induced with lower rmGM-CSF and rmIL-4 from murine marrow.2. We successfully transfected Ad-ccr7 adenovirus to immature dendritic cells derived from murine marrow. The morphology and surface marker of imDCs after Ad-ccr7 adenovirus transfection can differentiated towards maturity direction, but IL-10 can slightly inhibited the maturation of imDCs.3. Results of Real-time PCR manifested the expression level of CCR7 mRNA in imDCs after Ad-ccr7 adenovirus transfection can increased obviously, meanwhile IL-10 can slightly inhibited the maturation of imDCs during transfection.4. Results of cell immunofluorescence and Western Blot displayed the expression of CCR7 in imDCs after Ad-ccr7 adenovirus transfection upgraded obviously. IL-10 can inhibitted the imDCs differentiated towards maturation, but the expression of CCR7 protein can achieved the level of mDCs.5. The results of migration experiment in vitro displayed the migration percentage of imDCs was lower, but the migration percentage in imDCs after Ad-ccr7 adenovirus transfection which affected by CCL19 were increased obviously than imDCs. Meanwhile anti-CCR7 mAb can slightly rivalied the effection of its ligand CCL19 in vitro.6. The results of mixed lymphocyte reaction demonstrated the proliferation ability of imDCs stimulate to T lymphocytes was lower, but the proliferation ability of mDCs stimulate to T lymphocytes increased significantly. The proliferation ability of imDCs after adenovirus transfection was higher than imDCs and lower than mDCs, IL-10 can decreased the proliferation ability.7. Results of murine allogene skin transplantion experiment demonstrated the MST of skin grafts in donor mDCs had no significant difference, but the donor imDCs after Ad-ccr7 adenovirus transfection can prolonged the MST of skin grafts, IL-10 can significant extended the MST of skin grafts.更多还原