【作者】 苏纪权；

【导师】 张兴义；

【作者基本信息】 吉林大学 ， 外科学， 2007， 博士

【摘要】 目的:探讨RNAi (RNA interference)技术沉默树突状细胞CD40基因,建立DC(Dentritic cells)细胞培养及转染方法,对体外脾淋巴细胞的影响,对IL-2、IFN-γ、IL-4及IL-10分泌的影响,为寻求治疗急性移植排斥反应提供基础研究。方法:1.DC细胞培养:培养参照Giannoukakis等报道的方法[Molecular Therapy 2000;1(5):430-437]。从Dark Agouti (DA)供体大鼠股骨中获取的骨髓细胞在含2ml RPMI-1640的24孔培养板(2×106/孔)中培养,其中含抗生素10%(v/v)小牛血清FCS。加入基因重组大鼠GM-CSF(20ng/mL)以扩增未成熟DC。除GM-CSF外,加入基因重组大鼠IL-4 (20 ng/mL)以获得成熟DC。在选定的培养孔,于培养开始时分别加入pSilencer1:U6-CD40 siRNA和pSilencer空白质粒转染细胞,以鉴定siRNA-CD40抑制由IL-4诱导的DC成熟的能力和其抑制DC中CD40基因表达的能力。每2天更换富含细胞因子的培养液;轻微旋动培养板后,吸除一半旧培养液,再加入等量且含细胞因子的新鲜培养液。因此,非贴壁的粒细胞将被清除又可确保那些已生长出的松散地附在弹性帖壁巨嗜细胞单层上的DC细胞簇不被同时移除。10天后收集那些自动从DC细胞簇离开的未帖壁DC。2.大鼠Silencer2.1-U6- siRNA-CD40重组质粒的构建与鉴定(1)siRNA靶位点的选择及编码siRNA DNA模板的体外合成根据GeneBank(AF241231)大鼠CD40基因mRNA的已知序列,寻找符合特征的靶序列,合成分别针对其三个编码区合成两条DNA寡核苷酸链(分别命名为CD40 -1,-2,-3)并用RNA structure 4.11软件对其二级结构预测,每条模板链的组成包括19个碱基正义链和与其互补的19个碱基的反义链,正反链之间有9个碱基(TTCAAGAGA)间隔,反义链之后有5个T作为转录终止信号,在模板链的两端加入Hind III及BamH I酶切位点。合成两条链在退火缓冲液(100mM K-acetate, 30mM HEPES-KOH pH 7.4, 2 mMMg-acetate)作用下退火(90℃,3min,37℃,1hr),形成双链结构。待连接。(2)pSilencer2.1-U6载体线性化及回收pSilencer?2.1-U6 neo质粒:全长4521bp,含有U6启动子,氨苄抗性,含NEO基因,可由新霉素筛选稳定转染。pEGFP质粒:带有增强绿色荧光蛋白的原核克隆和表达载体,全长3.4kb,含有LacZ启动子,氨苄抗性;同时与Silencer2.1-U6-siRNA重组共转染,估计细胞体系的转染效率。将pSilencer2.1-U6用BamH I及Hind III分别消化,对消化后产物经1%琼脂糖凝胶电泳鉴定,将含有线性化质粒的凝胶在紫外灯下切下,放入1.5ml离心管中,加入3倍体积的融胶液,45℃-55℃水浴5-10min,使胶完全融化,加入10μl玻璃奶(通常1μgDNA用1μl玻璃奶),混匀,45℃-55℃冰水浴5-10min,其间每隔2-3min混匀一次,5,000转/min离心30-60s,弃上清,加入400μl洗液,轻弹管底,5,000转/min离心30s,弃上清,重复上述步骤一次,吸净洗液,室温干燥,加入10-30μl无菌双蒸水,将玻璃奶悬起,45℃-55℃水浴5-10min,10,000转/min离心1-2min,回收上清用λDNA mark做标准品定量,待连接。(3)载体与siRNA模板链连接连接体系中模板与载体的摩尔数比约为3-5:1,充分混匀,加入T4连接酶16℃反应过夜。连接产物可立即转化感受态细胞或于-20℃保存。同时进行空载体自身环化作为阴性对照。(4)感受态细胞的制备与连接产物的转化取-70℃保存的GM109大肠杆菌接种于平板,次日挑取单菌落接种于LB固体培养基中,370C培养过夜。用无菌签挑取单菌落,转到3mlLB液体培养基中,37℃300rpm振荡2h,取1ml菌液接种到100ml LB培养基中,37℃继续培养待OD600大约为0.4时取出,冰浴10min。将菌液转到灭菌的预冷50ml离心管中,4℃4000g离心10min回收细菌,重悬于10mlCaCl2(0.1mol/L)中,冰浴30min , 4℃4000离心10min ,弃上清,加4 ml预冷的CaCl2(0.1mol/L),置于4℃,16h后用于转化试验,或加入甘油至终浓度10%,-70℃保存备用。取100μl的感受态细菌,加入5μl pSilencer2.1-U6与siRNA连接产物,用空载体作对照,轻轻旋转混匀,冰浴30min,42℃热休克90s,快速放置冰浴中2min,加入800μl LB培养基(不含Amp),37℃振荡培养60min,吸100μl培养液滴于含Amp的LB平板涂匀,待表面液体吸收后,倒置平皿37℃培养16-20h。(5)阳性重组克隆的筛选(碱裂解法)15,000rpm离心10min,弃上清,加入冰冷的75%乙醇洗一次,离心室温干燥,用含RNase(50μg/ml)的双蒸水10μl充分溶解沉淀。用M13F(-40): 5’-GTTTTCCCAGTCACGAC-3’ Rev: 5’-GAGTTAGCTCACTCATTAGGC-3’ primer测序。(6)重组质粒的转染DC细胞用RPMI1640培养基加10%小牛血清37℃,5% CO2培养。用100 ml培养瓶,当贴壁细胞达到80%-90%融合,用无血清培养基洗三遍,将脂质体15μl加无菌水85μl室温放置10分钟,重组质粒18μg用无菌水加至总体积100μl室温放置10分钟,两者混合室温放置10分钟后加入细胞无血清培养液中,用pEGFP质粒与psilencer1.0-U6-CD40-siRNA按1:20的比例以慢病毒为载体共转染,以标记阳性转染的细胞;空质粒对照用psilencer1.0-U6质粒,另外一组只用脂质体加入培养细胞内;于转染后24-72小时,加G418(新霉素)进行稳定转染阳性克隆筛选,筛选21天后收集细胞,做Western blot分析。3.Western blot测定RNAi干涉后CD40基因在DC中的表达情况裂解RNAi干涉后的DC,用常规Western blot方法检测CD40表达情况。4.单向混合淋巴细胞反应(MLR)采用3HTdR掺入法。刺激物为RNAi干涉后的DC;反应物为Lewis大鼠的脾细胞。最后用自动液闪计数仪测定每分钟脉冲数值。5.通过ELISA的方法检测大鼠淋巴细胞分泌细胞因子的情况。6.统计学分析所得数据以均数±标准差表示,用SPSS软件包(10.0版)进行统计学处理,多组资料均数比较采用F检验,若差异有显著性,则进一步进行q检验。结果:1.成功从大鼠骨髓细胞中体外诱导培养出树突状细胞。2.通过感染含有RNAi序列的慢病毒可以抑制树突状细胞中CD40的表达。3.RNAi沉默后树突状细胞体外活化脾细胞的能力减弱。4.RNAi干涉后的DC活化的淋巴细胞分泌IL-2和IFN-γ的量显著减少结论:应用RNAi技术沉默DC上的CD40,切断第二信号传递使T细胞沉默,不被激活,达到抑制急性免疫排斥反应。更多还原

【Abstract】 As a kind of powerful antigen presenting cell(APC),dendritic cells(DCs) are investigated more and more with aidding T and B lymphocyte’s immunological function and found that they have important means in the happening and develpping process of transplantation reject reaction, rheumatoid disease,tumor, allergy and autoimmune disease.In the local tissue of these diseases the DCs have change with quantity and or or surface marks,then that leads to produce of autoantibody,form of rheumatoid factor or defect of local immune function.In present,the more using of DCs is immune of anti-tumor and anti- transplantation reject reaction,but the research with utilizing RNA interference(RNAi) silences DCs to inhibit actue immunol- ogical reject reaction of transplantation is fewer.RNA interference(RNAi) is technolagy of gene silence of posttrans- cription,small or short inference RNA(siRNA) can target some kinds of monitoring progress of posttranscription, identify mRNA which has the homology sequence,then specifily cut the mRNA and obstruct its interpretation function.The investigation uses the RNAi silence CD40 of DCs which gets from the bone marrow of the rats,which effects the expression of the DCs.Through the test in vitro to investigate the inhibitional contribution of lymphocyte of spleen through interferencing the expression of CD40. Object To investigate RNAi silenceing CD40 gene treats actue immunological reject reaction of transplantation,and commit some contributions for treatment of the actue immunological reject reaction of transplantation.Method1. the cultivation of DCsDCs were cultivated as regular method. Photos of DCs’growth were taken every day. The activation and maturation markers of dendritic cell were detected by flow cytometer at 12th day.2. preparation of recombinant virus vector expressing CD40 siRNAUsing CD40 gene sequences from GenBank, selected suitable target site, synthesized loignucleotides as DNA template encoding CD40 siRNA, annealed and ligated into lentivirus expressing vector to construct recombinant virus vector expressing CD40 siRNA.3. Construction of T293 cell which expressed CD40 gene and RNAi this cell with recombinant lentivirus vector expressing CD40 siRNAT293 transfection cell expressing CD40 gene were constructed as a toll cell to explor the method of RNAi. The T293 cell which expressed CD40 gene were transfected with recombinant lentivirus vector. To determine the expression of the CD40 after transfection, Western blot analyses with samples extracted from transfected and control cells were performed.4. Expression of CD40 gene in DCs which are interferenced by RNAi is determined by Western blot.The CD cells were transfected with recombinant lentivirus vector. To determine the expression of the CD40 after transfection, Western blot analyses with samples extracted from transfected and control cells were performed.5. one-way mix lymphocyte reactionsplenocytes cells of the rat were isolated, and were mix-cultured with DC interferenced by RNAi. Proliferation of splenocytes were assayed by 3H-TdR incorporate method. ELISA for IFN-γ、IL-2、IL-4、IL-10 were performed to determine the secretion of related cytokine. Statistical analysisData were wxpressed as mean±SE. Comparison between groups was performed with student’s t testχ2 analysis using a sigmastat statistical software package(SPSS,Chicatgo,IL). P<0.05 was taken as showing siginificance.Results①DCs are successfully induced and cultivated from the bone marrow cells of the rat in vitro. The condition of DCs’activation and maturation was well and these DCs could be used in the following experiment.②The recombinant lentivirus vector expressing CD40 siRNA was constructed, and confirmed by restriction enzyme digest and sequence analysis.③T293 transfection cell expressing CD40 gene were constructed. Transfection with recombinant lentivirus vector inhibited the CD40’s expression of T293 transfection cell. The results from Western blot for the samples from T293 cells after transfection demonstrated that the recombinant lentivirus vector expressing CD40 siRNA could specifically reduce CD40 expression. ④Transfection with recombinant lentivirus vector inhibited the CD40’s expression of DCs. The results from Western blot for the samples from DCs after transfection demonstrated that the recombinant lentivirus vector expressing CD40 siRNA could specifically reduce CD40 expression.⑤The results of 3H-TdR incorporate method showed that Proliferation of splenocytes in CD40 siRNA groups was significantly lower than that of control groups. ELISA exhibited that the cytokine IFN-γ、IL-2、IL-4、IL-10 in siRNA groups were significantly lower than those in control groups.Conclusion :1. The method of inducing DCs from bone marrow of rat in vitro is safe and convenient.2. The technology of RNAi can interferences the expression of CD40 in the second signal pathway of DCs,and absence of the stimulus signal will lead to inability or apoptosis of T lymphocyte.3. Through the technology of interference CD40 of DCs,two RNAi sequences with interference well are successfully got, and the result consistents with what is expected. The test in vitro demonstrate that the ability of activating immune cell of DCs which are interferenced by RNAi obviously decreases.更多还原