【作者】 眭建；

【导师】 许化溪；

【作者基本信息】 江苏大学 ， 农产品加工与贮藏工程， 2007， 博士

【摘要】 背景支气管哮喘是一种慢性气道炎症性疾病,属于超敏反应性质,多种炎症细胞、炎症介质和细胞因子与其发生发展有关,其中辅助T细胞亚群（Th1/Th2）的失衡、Th2细胞数量增加、功能活化起着关键作用。树突状细胞是目前已知的功能最强的抗原递呈细胞（APC）,系已知唯一能活化初始T细胞的APC,是识别内外源性抗原、调节天然免疫和获得性免疫的关键因素。已知分布于气道的DC是呼吸道免疫防御的前哨细胞,在通过胞饮、吞噬或受体介导的内吞三种方式摄取吸入性抗原后逐渐成熟,趋化因子、共刺激分子和MHCI工类分子表达增加,而FcγRII、FcγRI和FcεRI等表达减少,并于24小时内从气道迁移至纵隔淋巴结产生初级免疫应答,促使初始Th细胞（Th0）向Th1或Th2分化。Tho的活化需DC参与的抗原识别信号和协同刺激信号,其分化方向受控于抗原刺激后DC分泌的细胞因子,IL-12和IFN—γ是诱导Th0向Th1分化的重要细胞因子。许多试验显示DC的免疫调节作用与其细胞膜表面TLR的表达有关,一般认为相应P/NP通过TLR4激活DC主要引起IL-12p70、IFN-γ介导蛋白（IP-10）等细胞因子大量增加。鉴于树突状细胞TLR4对于T细胞分化的调节作用,若将TLR4基因通过腺病毒载体导入抗原（如LPS）激发的DC,可促使DC分泌较多的IFN-γ等,上调Th0表达的IL-12Rβ2和TLR4,从而利于Th0向Th1分化。目的将转染了TLR4基因和LPS致敏的DC输入OVA诱导的过敏性哮喘模型小鼠体内,以促进Th1的分化,逆转过敏性哮喘的Th亚群失衡,从而通过TLR4基因转移DC干预的免疫调节方式探讨哮喘基因治疗的新方法。方法1.利用RT-PCR方法,从Balb/c小鼠脾组织细胞mRNA中扩增TLR4基因全长cDNA,并将其连接到pcDNA3.1（+）载体上,进行测序和核酸分析。2.采用AdMax包装系统,将克隆了外源基因的腺病毒穿梭质粒与携带了腺病毒大部分基因组的包装质粒共转染293细胞,利用Cre/loxP系统的作用实现重组,产生重组腺病毒。RT-PCR方法检测AdV-TLR4mRNA在传代DC中的表达、Western Blot方法检测AdV-TLR4蛋白的表达。3.RT-PCR方法检测AdV-TLR4转染的DCIL-12mRNA、IL-4mRNA和IFN-γmRNA表达的变化;ELISA方法检测AdV—TLR4转染的DC培养上清IL-12、IL-4和IFN-Y的表达水平;流式细胞仪分析AdV—TLR4转染的DC表面免疫特性标志蛋白Ia、CD40、CD80和CD 86的改变。4.0.1%OVAAl（0H）₃溶液皮下注射、1%OVA的生理盐水溶液雾化吸入的方法制备小鼠哮喘模型;福尔马林组织固定、包埋、切片,HE染色和AB/PAS复染进行小鼠肺组织学形态观察,Gimsa-Wright染色观察BALF中Eos;骨髓细胞分离,红细胞裂解液处理,GM-CSF和IL-4诱导、RMPI1640完全培养基培养的方式获取DC,行哮喘小鼠DCTLR4mRNA和TLR4蛋白表达检测;ELISA方法检测小鼠脾细胞IL-4、IL-5、IFN-Y和血浆IgE含量、人血浆IL-4和IgE含量、BALF中IL-4和IgE含量;流式细胞仪方法检测脾细胞CD4+CD25+细胞百分比。结果1.扩增得到的小鼠TLR4基因cDNA全长2508bp,编码536个氨基酸,blast结果分析表明,该cDNA与Genbank中发表的序列（NM021297）具有100%的同源性。2.重组质粒pDC316—mTLR4的PCR及酶切鉴定表明,TLR4基因被定向插入;与pBHGF35骨架质粒共转染293细胞并包装成病毒,经病毒DNA的PCR检测,证实已完成对重组病毒的包装;重组病毒转染小鼠DC,其TLR4mRNA和TLR4蛋白均明显表达。3.诱导Th1细胞分化的IL-12、IFN-γ在Ad-TLR4转染DC后48小时表达量明显增高,而诱导Th2细胞分化的IL-4表达量明显降低;转染细胞的PCR检测显示IL-12 mRNA、IL-4 mRNA和IFN-Y mRNA的变化与相应蛋白表达量的变化具有一致性;LPS实验进一步证实显示,LPS对转染细胞表达细胞因子的影响与LPS有关,LPS（+）转染DC组上述细胞因子改变的幅度高于LPS（-）组,LPS具有促进和加强Ad-TLR4-DC的某些生物学特性的改变。流式细胞检测表明Ad5F35-mTLR4组DC细胞CD 86表达较对照组和Ad5F35一mCMV-EGFP明显增高;进一步的实验显示,Ad5F35-mTLR4转染DC LPS（+）组Ia、CD40和CD80表达较相应LPS（-）明显增高;LPS（+）对照组CD80和CD 86表达也较相应LPS（-）组明显增高。LPS具有促进和加强Ad-TLR4-DC细胞表面某些免疫特性标志蛋白的改变。4.TLR4 DC悬液给予过敏性哮喘模型小鼠,显示表现为杯状细胞增生减少、气道内粘液减少,WBC数、Eso比率和IL-4水平均降低,而免疫调节性T细胞CD4+CD25+细胞百分率增高,显示TLR4 DC具有明显的抗支气管哮喘作用。TLR4 DC抗支气管哮喘的机制,推测与TLR4 DC激发使具有调节T细胞分化的细胞因子改变有关,如IFN-γ增加可促使Th1分化;TLR4DC进入机体还促使了免疫调节性T细胞CD4+CD25+增加,免疫调节性T细CD4+CD25+可增加机体对过敏物质的耐受性,进而可减轻其炎症反应。结论1.成功构建了小鼠TLR4基因的全长编码区cDNA。2.重组病毒感染滴度为2.3×10⁸ IU/ml,可用于体内外动物实验。在本研究中作为TLR4基因干预对机体Th1/Th2平衡影响的研究工具。3.LPS促进Ad-TLR4-DC细胞表面Ia、CD40、CD80和CD 86免疫特性标志蛋白改变,促进DCIL-12、IL-4和IFN-γ表达改变,推测这些改变与DCTLR4信号传导有关。4.TLR4 DC对OVA诱导的支气管炎症反应有一定抑制作用,LPS与TLR4有一定的协调作用,其机制与DC的免疫调节作用有关。更多还原

【Abstract】 BackgroundBronchial asthma(BA)refers to a chronic inflammatory disorder of the airways,resulting from mostly hypersensitivity.Its pathogenesis and development are considered related to diverse inflammatory cells, mediators and cell factors,and the imbalance of T cell subgroup (Th1/Th2),increase in Th2 cell number and its activated function may play a key role.Dendritic cell(DC)is up to now the most potent antigen presenting cell(APC)and is also the unique one to activate initial T cell, especially it is a critical factor to recognize exterior and interior antigen and regulate innate and adaptive immunity.DCs distributed in airways serve as the guard cells of respiratory system and turn mature after taking in inhaled antigen by pinocytosis,pahgocytosis and receptor-mediated endocytosis,manifested with increasing expression of chemotatic factors,costimulatory molecules and MHCII molecules, and decreasing expression of Fcγ～RII、FcγRI and Fcε1,which would move to mediastinal lymph node from airways within 24 hours to produce primary immune response,promoting initial Th cells(Th0)to transform into Th1 or Th2.The antigen recognition signal and costimulatory signal related to DC are necessary to Th0 activation because it is controlled by the cytokines produced by DCs stimulated by antigen.For example,IL-12 and IFN-γare key factors to induce Th0 into Th1.A great many studies indicated that DC’s immune regulation was related to the expression of TLR on the surface of cell membrane. It is generally known that PAMP may activate DCs by TLR4,resulting in considerable secretion of cytokines such as IL-12p70 and IFN-γmediated protein(IP-10).In use of this mechanism,that is,TLR4 of DCs may promote the differentiation of T cells,we made a trial to induce gene TLR4 via adenovirus vector into DCs stimulated by antigen such as LPS,so that DCs could produce much more cytokines like IFN-γto up-regulate the expression of IL-12Rβ2 and TLR4 from Th0,which may induce Th0 into Th1.ObjectiveWe infused DCs(transfected by gene TLR4 and stimulated by LPS)into the mice with allergic asthma induced by OVA in order to promote the differentiation of Th1,and reverse the imbalance of T cell subgroup caused by allergic asthma.From this study,we expect to find a new gene therapy for asthma through intervention of DC transfected with TLR4.Method1.The full length cDNA of TLR4 gene was obtained by using RT-PCR method from mRNA of Balb/c mouse spleen cells and then was connected with pcDNA3.1(+)carrier for sequencing and nucleotide analysis.2.With the aid of AdMax package system,293 cells were transfected with both adenovirus shuttle plasmid(with exogenous gene cloned)and packaging plasmid(with most adenovirus genomes),and then they were recombined via Cre/loxP system into recombinant adenovirus.AdV-TLR4 mRNA expression level in passage DCs was detected by RT-PCR assay and AdV-TLR4 protein level was detected by Western Blot method.3.The expression of AdV-TLR4-transfected DCIL-12mRNA、IL-4mRNA and IFN-γmRNA were detected by RT-PCR;the expression of IL-12、IL-4 and IFN-γin culture supernatant of AdV-TLR4-transfected DCs were detected by ELISA assay;specific immunity marker proteins on the surface of AdV-TLR4-transfected DCs including Ia,CD40,CD80 and CD 86 were detected by flow cytometry.4.Asthma mouse model was prepared with hypodermical injection of 0.1 %OVA Al(OH)3 solution and atomization of 1%OVA normal saline solution; tissue fixation with formalin,embedment,slice,HE stain and AB/PAS re-stain for mouse lung tissue morphologic observation.Eos in BALF was observed after Gimsa—Wright stain;the DCs were obtained through bone marrow cells separation,red blood cells treatement with lysate,induction of GM-CSF and IL-4,and RMPI 1640 culture,the expression level of both DCTLR4 mRNA and TLR4 protein of asthma mice were detected;IL-4、IL-5、IFN-γ,serum IgE level of mice spleen cells,human serum IL-4 and IgE,and IL-4 and IgE in BALF were detected via ELISA;CD4+ and CD25+ spleen cells percentage were detected with flow cytometry.Result1.Mouse TLR4 gene cDNA obtained from PCR has a full length of 2508bp and encodes 536 amino acids.The result of blast indicated this cDNA was 100%homologous with the one(sequence NO.NM021297)reported in Genbank.2.PCR and enzyme digestion identification of recombinant plasmid showed:gene TLR4 was set correctly;pBHGF35 backbone plasmid was transfected into 293 cells and was packaged into virus.PCR assay proved the package of recombinant virus was finishend;the recombinant virus was transfected into mouse DCs and the TLR4 mRNA and TLR4 proteins were highly expressed.3.1.L-12 and IFN-γ(inducing Th1 differentiation)were highly expressed 48 hours after DCs were transfected by Ad-TLR4,while,IL-4(inducing Th2 differentiation)showed a decreasing expression;PCR assay of transfected DC showed that the changes in IL-12 mRNA,IL-4 mRNA and IFN-γmRNA were consistent with the changes in corresponding proteins expression;LPS test indicated that LPS could effect the cytokines produced by transfected cells which were changed more greatly in LPS(+)group than that in LPS(-). LPS could also enhance some bionomics of Ad-TLR4-DC.2.Cytometry assay showed that CD86 was expressed more highly in Ad5F35-mTLR4 group than that in both control and Ad5F35-mCMV-EGFP group. Simultaneously,Ia,CD40 and CD80 of DC transfected by Ad5F35-mTLR4 were expressed much more highly in LPS(+)group than that in LPS(-)group; CD80 and CD86 in LPS(+)were more highly expressed than that in LPS(-) group.LPS may promote and strengthen the change of specific immunity marker proteins on the surface of Ad-TLR4-DCs.4.When TLR4 DC suspension was given to the mice with allergic asthma,the result showed a decrease in beaker cell proliferation,mucosal fluid in airways,WBC,Eso ratio and IL-4 level.However the percentage of CD4+CD25+ cells increased,which indicating TLR4DC could resist bronchial asthma.The mechanism is possibly related to the cytokines induced by TLR4DCs and the former can promote T cells differentiation,for instance, IFN-γmay induce T11 differentiation;TLRC4DC may also increase immune-regulating T cells CD4+CD25+ that enhance the toleration of allergen and minimize the inflammation.Conclusion1.Full-length coding region cDNA of mouse TLR4 gene was successfully established.2.Recombinant viral infection titer is 2.3×108 IU/ml and it can be utilized in animal experiment in vivo and in vitro.3.LPS can lead to the change of specific immunity marker proteins on the surface of AdV-TLR4- DC including Ia,CD40,CD80 and CD 86,and it also result in the change of expression of IL-12,IL-4 and IFN-γsecreted by DC.These changes are presumed to be related to signal conduction of DCTLR4.4.TLR4DC may inhibit the bronchial inflammation induced by OVA. Plus,LPS and TLR4 show a coordination which is possibly related to immune regulation of DC.更多还原