靶向OX40基因的siRNA抑制大鼠肝脏移植排斥反应的研究

目的 探讨RNA干扰阻断OX40-OX40L共刺激通路对大鼠移植肝存活时间的影响.方法 制作原位肝脏移植物大鼠模型,供体DA大鼠,受体Lewis大鼠.移植术毕时将5 ml含5×109pfu的pLVTHM-OX40-siRNA重组慢病毒经阴茎背静脉在10秒内注射受鼠体内,术后观察受鼠存活时间,移植肝脏进行组织病理学检查,采用ELISA法检测大鼠外周血IL-2、IFN-γ的水平,并进行受者大鼠脾细胞对供者大鼠脾细胞的混和淋巴细胞反应(MLR). 结果转染组大鼠肝脏移植物的存活时间为(74.0±9.3)d,明显长于对照组(7.3±0.5)d和未转染组(7.5±0.5)d;转染组移植肝组织炎细胞浸润、间质水肿、肝组织坏死程度较对照组和未转染组减轻;耐受组大鼠脾脏中T淋巴细胞刺激T淋巴细胞增殖的能力较对照组降低(t=25.1,P＜0.01);移植术后第7 d,转染组血清IL-2浓度为(46±8.4)pg/ml,IFN-γ浓度为(202.7±14.6)pg/ml,均显著低于对照组和未转染组(分别t=176.4,45.5,P＜0.01).结论 转染靶向OX40的siRNA,可通过阻断OX40-OX40L共刺激通路,抑制大鼠肝脏移植后的排斥反应,延长大鼠移植肝的存活时间.     

供体肝脏转染CD40抑制大鼠肝移植急性排斥反应的研究

在器官移植排斥反应中 ,细胞免疫起重要的作用。T细胞激活必须接受抗原呈递细胞 (antigenpresentingcell,APC)的双重信号激活 ,其中第二信号为T细胞抗原特异性激活所必需 ,若缺乏共同刺激信号 ,可导致T细胞的免疫无应答甚至凋亡(apoptosis) [1] 。本实验研究利用供体肝脏转染pCDNACD4 0Ig ,供体肝脏表达CD4 0Ig ,阻断共同刺激通路 ,观察CD4 0Ig对同种异体肝移植排斥反应的抑制作用。材料和方法1 .实验材料 :TUNEL试剂盒购于德国宝灵曼公司 ,鼠抗人CD4 0单克隆抗体购于Zymed公司 ,PcDNA 3.1CD4 0Ig为人CD4 0胞外区与人IgGFC…     

输注转染IL-2基因同源短发夹型RNA的淋巴细胞抑制大鼠肝移植急性排斥反应

目的 探讨输注转染白细胞介素2(IL-2)基因同源短发夹型RNA(shRNA)的受者淋巴细胞对大鼠肝移植后急性排斥反应的影响.方法 构建针对大鼠活化T淋巴细胞IL-2基因编码区的shRNA表达质粒,体外转染受者(BN大鼠)淋巴细胞.以Lewis大鼠为供者、BN大鼠为受者,进行肝移植,干扰组的受者于移植术中经门静脉回输转染IL-2-shRNA的BN大鼠淋巴细胞;环孢素A组(CsA组)的受者移植术中经门静脉输入生理盐水,肝移植后应用CsA肌肉注射;生理盐水对照组的受者移植术中经门静脉输入生理盐水;空载体对照组的受者移植术中经门静脉输入转染空载体的BN大鼠淋巴细胞.术后第7天,处死BN大鼠,取移植肝组织,观察组织和细胞超微结构改变情况以及肝细胞凋亡情况,同时测定肝功能指标、肝组织中IL-2基因的表达以及血清IL-2、肿瘤坏死因子a(TNF-α)和γ干扰素(IFN-γ)水平.计算各组受者1周存活率.结果 移植后第7天,生理盐水对照组和空载体对照组的移植肝组织中可见中、重度急性排斥反应改变,而干扰组和CsA组病理切片中无或仅有轻度急性排斥反应征象.干扰组凋亡肝细胞数为(23±3.2)/mm2,CsA组为(26±4.0)/mm2,均明显低于生理盐水对照组和空载体对照组(P＜0.05).干扰组和CsA组的IL-2mRNA表达明显低于生理盐水对照组和空载体对照组(P＜0.05);干扰组和CsA组的血清IL-2、TNF-α和IFN-γ水平较空载体对照组和生理盐水对照组分别有不同程度降低(P＜0.05).干扰组和CsA组的丙氨酸转氨酶和总胆红素均明显低于空载体对照组和生理盐水对照组(P＜0.05).而白蛋白则高于空载体对照组和生理盐水对照组(P＜0.05).干扰组受者1周存活率为87.5%(7/8),明显高于生理盐水对照组的37.5%(3/8)和空载体对照组的37.5%(3/8,P＜0.05).结论 输注转染IL-2-shRNA的受者淋巴细胞可抑制大鼠肝移植后的急性排斥反应,但这种作用是短期的.     

RNA编辑酶ADAR1在大鼠肝移植排斥反应中的变化

目的观察RNA编辑酶ADAR1在大鼠肝移植排斥反应中的表达变化。方法实验分为4组①同基因移植组(n=15),取Wistar大鼠的肝脏原位移植给Wistar大鼠;②异基因移植组(n=15),取SD大鼠的肝脏移植给Wistar大鼠;③异基因移植 FK506治疗组(n=15),取SD大鼠的肝脏移植给Wistar大鼠,术后肌注FK506,2mg/(kg·d);④对照组(n=15),对Wistar大鼠不行肝移植,仅行开、关腹手术。建立大鼠原位肝移植模型,分别于术后第3、5及7d各处死5只大鼠,取脾脏组织,用RT-PCR方法检测ADAR1 mRNA的表达变化。结果移植后各组大鼠肝脏、脾脏病理变化随时间发展而呈进行性变化,异基因移植组病理变化最明显。ADAR1 mRNA表达在异基因移植组的各个时相点明显高于同基因移植组和异基因移植 FK506治疗组(P<0.001),于第5d时最明显。结论在大鼠原位肝移植发生急性排斥反应时,ADAR1增高程度与排斥反应的强度变化趋势一致。FK506可以抑制ADAR1的表达,明显减轻移植肝组织的急性排斥反应。     

小干扰RNA在肝癌治疗中的应用

小干扰RNA(small interfering RNA,siRNA)是21～23 nt的短双链RNA,将哺乳动物细胞内特异基因的mRNA特异性降解而使基因失活,引起转录后沉默该基因的作用.siRNA技术在人类恶性肿瘤的基因治疗有特异而强烈的作用,具有非常广阔的情景,但其稳定性是有待解决的新问题.本文针对siRNA在肝癌治疗中的研究现状进行综述.     

小干扰RNA技术与骨质疏松症治疗

以骨量减少、骨微结构破坏为主要特征的骨折疏松症及其所致骨折严重影响人均寿命及生活质量,目前的治疗方法因多种原因并不能很好地解决相关问题。RNA干扰是一种序列特异的转录后基因沉默技术,通过小干扰RNA技术可以特异性调控蛋白表达,达到靶向治疗目的。该文就小干扰RNA技术在骨质疏松症治疗方面的研究作一综述。     

靶向CD40的shRNA干扰抗大鼠异体肢体移植急性排斥反应的实验研究

目的 探讨靶向CD40的RNA干扰对大鼠异体肢体移植急性排斥反应的影响. 方法以纯系SD大鼠为供体,纯系Wistar大鼠为受体,行同种异体右后肢移植.27只大鼠肢体移植后随机分为三组,A组:注射入梭华.Sofast.siCD40-2/pSilencer载体复合物600 μL;B组:注射Sofast-pSilencer4.1-CMV neo空载体复合物600 μL;C组:注射生理盐水600μL,以上均通过阴茎背静脉注射.观察移植物排斥反应征象及存活情况,并于第7天对产生免疫耐受大鼠进行混合淋巴细胞反应,同时进行组织学检查. 结果与B、C组相比,A组移植物发生排斥反应的时间及存活时间均显著延长,差异有统计学意义(P＜0.01)(＞13 d),未见排斥反应征象;B、C组均于术后近期发牛排斥反应.A组大鼠对供体的淋巴细胞呈现低反应性,移植的供体同系大鼠的肢体得以存活. 结论术后不应用免疫抑制剂的情况下,靶向CD40的shRNA干扰可以抗大鼠异体肢体移植急性排斥反应.     

小干扰RNA对小鼠淋巴细胞cbl-b基因表达的抑制作用

目的 设计合成及筛选自身免疫关键基因--cbl-b (Casitas B-cell lineage lymphoma-b,cbl-b)基因小分子干扰RNA(siRNA).方法 应用RNA设计软件,模拟cbl-b小鼠cbl-b mRNA二级结构,设计并合成针对cbl-b mRNA的4对21核苷酸(nt)siRNA,96孔板转染小鼠淋巴细胞,以空白及转染非特异siRNA(与cbl-b mRNA无同源性的21nt siRNA)作为对照,应用蛋白免疫印迹(Western blot)方法 检测小鼠淋巴细胞cbl-b蛋白表达.结果 cbl-b基因siRNA工作浓度为100nmol/L时转染小鼠淋巴细胞转染率最高,可达(87.48±1.94)%,平均荧光强度最强,可达33.09±1.77.与对照相比,转染cbl-b siRNA的小鼠淋巴细胞蛋白表达明显下调,以siRNA-4最显著,抑制率达85%,转染非特异性siRNA的小鼠淋巴细胞cbl-b蛋白表达水平无明显变化.结论 得到cbl-b siRNA转染小鼠淋巴细胞最佳转染条件,成功筛选能高效抑制cbl-b蛋白表达的siRNA-4,其有效抑制时间约为48h,有望通过沉默cbl-b基因直接活化淋巴细胞,增强机体主动免疫杀伤肿瘤细胞.

Abstract：

Objective To design, synthesize screen small interfering RNA (siRNA) targeting to Casitas B-cell lineage lymphoma-b (cbl-b).Methods Four pairs of 21 nucleotide siRNAs directed to Cbl-b mRNA were designed and synthesized by utilizing RNA design software to simulate secondary structure of cbl-b mRNA in mice. These siRNAs were respectively transfected into lymphocytes in 96 shadows mask by oligofectamine package, and untreated and unspecific siRNA-transfected lymphocytes served as controls. The expression of cbl-b protein was detected by Western blotting.Results When the work concentration of siRNA was 100 nmol/L, transfection efficiency of lymphocytes was highest, up to (87.48±1.94)% and the mean fluorescence intensity was strongest, up to 33.09±1.77. Compared with bland controls, the expression of cbl-b protein level was markedly down-regulated in siRNA-transfected lymphocytes. The inhibitory rate of the siRNA of the target-4 was highest, up to 85%. The expression of cbl-b protein in unspecific siRNA-transfected lymphocytes had no significant changes.Conclusion siRNA-4, which can highly effectively inhibit protein expression of cbl-b gene, was screened successfully, and its inhibition effect can maintain near 48 h. It is hopeful that the cbl-b siRNA will activate lymphocytes directly by cbl-b gene silencing, and kill tumor by activate immunization.     

小干扰RNA与口腔癌研究进展综述

口腔癌是口腔外科常见恶性肿瘤,近年来的研究显示其发生、发展、侵袭转移和预后与体内小干扰RNA表达水平密切相关。因此本文通过对小干扰RNA产生机制、与肿瘤的发生、与口腔癌前病变、与口腔癌及与口腔癌的治疗等方面综述如下。     

小干扰RNA对大鼠T淋巴细胞CD28基因表达的抑制作用

目的探讨小干扰RNA(siRNA)对大鼠T淋巴细胞共刺激分子CD28基因表达及干扰后的T细胞的功能的影响。方法设计针对目标基因的siRNA,转染大鼠T淋巴细胞,逆转录-聚合酶链反应((RT_7PCR)方法检测CD28 mRNA水平,MLR检测转染后的T淋巴细胞的增殖能力,ELISA法检测细胞因子水平。结果siRNA转染大鼠T淋巴细胞后,抑制CD28分子的表达,半定量RT-PCR检测显示T淋巴细胞CD28的mRNA均有明显的下降,siRNA转染48 h后,siRNA-1、siRNA-2、siRNA-3、siRNA-4组CD28基因表达均受到抑制,其抑制率最高为(88.56±4.70)%,T细胞增殖能力、IL-2,IFN-γ水平明显低于对照组。结论siRNA可以特异性抑制大鼠T淋巴细胞共刺激分子CD28基因的转录和表达,降低大鼠T细胞的增殖能力,抑制了IL-2,IFN-γ细胞因子的分泌水平。     

阻断OX40和CD40协同共刺激信号延长小鼠胰岛移植物存活时间

目的 研究阻断OX40/OX40L和CD40/CD154L协同共刺激通路对小鼠胰岛移植物存活的影响及其机制.方法 以DBA/2小鼠为供者,C57BL/6小鼠为受者,制作胰岛移植模型.受鼠分为4组.(1)对照组,注射IgG; (2)抗OX40组,注射抗OX40L单克隆抗体;(3)抗CD154组,注射抗CD154单克隆抗体;(4)联合治疗组,注射抗OX40L单克隆抗体和抗CD1 54单克隆抗体.记录各组胰岛移植物平均存活时间(MST).将CD154敲除小鼠处死,取其脾脏T淋巴细胞,体外检测活化T淋巴细胞表面OX40的表达;在活化T淋巴细胞中加入不同浓度的抗OX40L单克隆抗体,体外检测T淋巴细胞增殖情况.结果 对照组胰岛移植物MST为19 d,抗CD154组胰岛移植物MST为48 d(P＜0.05);抗OX40组胰岛移植物MST为22 d,与前两组相比较,差异无统计学意义(P＞0.05);联合治疗组胰岛移植物MST＞ 150 d,高于另外3组(P＜0.05).66％的胞表达OX40,较初始T淋巴细胞的表达率高(2％,P＜0.05);加入抗OX40L单克隆抗体后,T淋巴细胞增殖受抑制且呈剂量依赖性.结论 阻断OX40/OX40L和CD40/CD154L双通路可诱导小鼠胰岛移植物长期存活, 其发挥作用的关键机制是抑制了T淋巴细胞的增殖.     

siRNA干扰OX40/OX40L通路对小鼠移植心脏的免疫保护作用

目的 观察真核表达载体介导siRNA对小鼠移植心脏的免疫保护作用.方法 在C57/BL6J→BALB/C组合[主要组织相容性复合体(MHC)不合]、DBA/2→BALB/C组合[次要组织相容性复合体(mHC)不合]中分别于小鼠心脏移植术后第1、4天胸腺注射生理盐水,pRNAT-CTL阴性对照载体和pRNAT-OX40 siRNA,观察移植心脏存活情况.术后第7天取移植心脏行病理学检查,取血清检测心肌酶谱,取脾脏行逆转录.聚合酶链反应(RT-PCR)和Western blot检测OX40mRNA表达和OX40膜蛋白表达.结果 在C57/BL6J→BALB/C组合中(MHC不合),pRNAT-OX40siRNA组移植心脏生存时间较生理盐水组和阴性对照组无明显延长,移植心脏病理学检查无明显改善.而在DBA/2→BALB/C组合中(mHC不合),pRNAT-OX40 siRNA组小鼠移植心脏生存时间较生理盐水组和阴性对照组显著延长,病理改变显著减轻,乳酸脱氢酶(LDH)、谷草转氨酶(AST)、肌钙蛋白(Tn)以及脾脏OX40 mRNA表达和OX40膜蛋白表达与空白对照组和阴性对照组比较明显下降.结论 在DBA/2→BALB/C组合中(mHC不合),单独阻断OXdO/OX40L通路对小鼠移植心脏有保护作用.在C57/BL6J→BALB/C的组合中(MHC不合),单独阻断OX40/OX40L通路未发现对移植心脏的保护作用.     

OX40 (CD134) blockade inhibits the co-stimulatory cascade and promotes heart allograft survival

BACKGROUND: CD134 (OX40) is a member of the tumor necrosis factor receptor superfamily that is expressed as a late event in T-cell activation and contributes to the generation of immunologic memory. CD134 blockade effectively ameliorates inflammation in models of autoimmune disease, but its role in transplantation has been less well studied. METHODS: The authors used an OX40-immunoglobulin (Ig) fusion protein to examine the contribution of this co-stimulatory molecule to the in vitro alloimmune response and studied the ability of CD134 blockade to prevent cardiac allograft rejection in mouse models of heart transplantation using strains representing both major histocompatibility complex (MHC) (BALB/c to CBA/Ca) and minor histocompatibility complex (mHC) (B10.BR to CBA/Ca) antigen mismatches. RESULTS: CD134 upregulation on in vitro alloactivated T cells was delayed compared with CD69 and CD25, and inhibition of T-cell proliferation was critically dependent on the timing of OX40-Ig administration. Heart allograft survival in a fully allogeneic, MHC-mismatched strain combination was not influenced by CD134 blockade alone, but OX40-Ig treatment in the mHC-mismatched model resulted in long-term graft survival (median survival time extended from 14 days to >100 days). Early mononuclear cell infiltration of the graft was similar in both rejecting and long-surviving heart grafts, but OX40-Ig treatment appeared to delay cellular infiltration. CONCLUSIONS: These results show that CD134-CD134L interaction plays an important role in the co-stimulatory cascade and that blockade of this molecular interaction may be of therapeutic value in helping to prevent allograft rejection.     

抑制记忆性T淋巴细胞共刺激分子OX40延长小鼠胰岛移植物的存活时间

目的 观察抑制记忆性T淋巴细胞共刺激分子OX40延长胰岛移植小鼠存活时间的作用,探讨其作用机制.方法 采用免疫磁珠法制备初始性、类记忆性及记忆性CD8+T淋巴细胞,并采用逆转录聚合酶链反应法检测3种T淋巴细胞上OX40的表达量.将C57BL/6小鼠脾脏T淋巴细胞经尾静脉注射给Rag-/-小鼠.将Rag-/-小鼠分为3组,对照组:给予同型IgG;治疗组:给予抗OX40L单克隆抗体;基因敲除组:输注的T淋巴细胞由OX40基因敲除的C57BL/6小鼠供给.T淋巴细胞输注6周后,使用链脲霉素诱导建立糖尿病模型,然后以DBA/2小鼠为供者,进行胰岛移植,移植后观察和比较3组间胰岛移植物的存活时间.结果 OX40在初始性、类记忆性和记忆性T淋巴细胞上的相对表达量分别为2.87、111.24和146.15,类记忆性和记忆性T淋巴细胞间OX40表达量的差异无统计学意义(P＞0.05),但均显著高于初始性T淋巴细胞(P＜0.01).对照组、治疗组和基因敲除组胰岛移植物的存活时间分别为21、130和125 d,治疗组和基因敲除组间胰岛移植物存活时间的差异无统计学意义(P＞0.05),二者均显著长于对照组(P＜0.05).结论 OX40在记忆性T淋巴细胞表面高表达,且阻断OX40共刺激分子通道可明显延长胰岛移植物的存活时间,抑制OX40/OX40L共刺激通道可能是诱导胰岛移植免疫耐受的关键点之一.

Abstract：

Objective To investigate the role of OX40 in the mechanisms of memory T cells in islet transplant tolerance.Methods The expression of OX40 on native, like memory and memory CD8+T cells was detected by RT-PCR. Splenic T cells from B6 mice were injected into Rag-/- mice via the tail vein, and the Rag-/- mice were divided into three groups (n=8 each): control group, given IgG; treatment group, given anti-OX40L; and OX40 knock-out group, given T cells from OX40 knock-out B6 mice spleen. All recipients were induced into diabetes mellitus model after adoptive transfer. Islet transplantation was performed on all Rag-/- mice as recipients. The mean survival time of islet was observed.Results The expression of OX40 in native T cells, like memory T cells and memory T cells was 2.87, 111.24 and 146.15 respectively. The expression of OX40 in like memory and memory T cells was higher than in native T cells (P<0.05). Comparison with control group , The mean survival time of the DBA/2 islet allografts in treatment group (130 days) and OX40 knock-out group (125 days) was significantly longer than in control group (21 days, P<0.05).Conclusion The OX40 expression is high in memory T cells. The mean survival time of the islet allografts can be prolonged by blocking OX40/OX40L pathway. OX40/OX40L pathway may be the key point of transplant tolerance.     

OX40-IgG1融合基因重组表达载体的构建及鉴定

目的:构建含人OX40-IgG1融合基因的真核表达载体pDC315-0X40Ig,表达具有生物学活性的OX40Ig融合蛋白,为诱导异体复合组织移植免疫耐受的基因治疗作准备.方法:全基因合成目的基因OX40Ig,即人0X40胞外段序列及人IgG1 Fc段序列,将该片段插入到pUC57(+)载体后,亚克隆至真核表达载体pDC315(+),构建pDC315-0X40Ig重组质粒.构建的重组质粒经PCR、酶切及测序鉴定后,用脂质体法转染于NIH/3T3细胞.以SDS-PAGE与Western Blotting检测细胞培养上清中OX40Ig融合蛋白的表达情况;MTT法观察OX40Ig对混合淋巴细胞反应(MLR)的抑制作用.结果:合成的目的基因全长1320bp,构建的重组质粒经PCR,双酶切及测序鉴定正确.SDS-PAGE与Western Blotting证实OX40Ig融合蛋白在3T3细胞中实现表达,表达产物相对分子量为48000左右,与理论预期值相符.体外实验证实OX40Ig蛋白能够抑制异基因淋巴细胞的MLR.结论:成功构建了人OX40-IgG1融合基因真核表达载体pDC315-OX40Ig,体外实验证实表达的OX40Ig蛋白可抑制淋巴细胞增殖,为干预同种异体复合组织移植排斥反应奠定了基础.     

血清可溶性OX40配体与晚期胃癌生存期的关系研究

目的探讨晚期胃癌患者血清可溶性OX40配体（sOX40L）水平与生存期的关系,为胃癌免疫治疗提供思路。 方法选取武汉市红十字会医院和华中科技大学同济医学院附属协和医院2015年1月至2017年5月收治的115例晚期胃癌患者为观察组,同期健康体检者91名为对照组。ELISA法检测两组血清sOX40L水平,Kaplan-Meier曲线分析sOX40L水平与晚期胃癌患者生存时间的关系,再行危险因素的Cox回归分析。 结果观察组sOX40L水平高于对照组（P<0.05）,其中高水平组（sOX40L≥20.58 ng/L）58例,低水平组（sOX40L<20.58 ng/L）57例,sOX40L水平与患者TNM分期、淋巴结转移、肝转移有关（P<0.05）。高水平组的中位总生存时间为6.65个月（95% CI：5.89~7.42）,短于低表达组的9.47个月（95% CI：8.59~10.36）,差异有统计学意义（P< 0.001）。年龄≥60岁、sOX40L高表达、TNM Ⅳ期、淋巴结转移、肝转移均是晚期胃癌不良预后的独立危险因素（P<0.05）。 结论sOX40L在晚期胃癌患者血清中呈高表达,其水平与TNM分期、淋巴结转移、肝转移有关,是胃癌患者不良预后的独立危险因素。     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.