CTLA4Ig与CD40Ig双基因局部共转染对异种大鼠移植肾Bcl-2/Bax的影响

目的:探讨CTLA4Ig与CD40Ig双基因局部转染对异种移植肾Bcl-2/Bax的影响,探讨其诱导免疫耐受的机制。方法:以PcDNA3.1(+)-CTLA4Ig和PcDNA3.1(+)-CD40Ig为载体,通过脂质体lipo2000将CTLA4Ig与CD40Ig双基因转入豚鼠肾脏,以豚鼠为供者,SD大鼠为受者,行异种肾移植手术。将豚鼠到SD大鼠的异种肾移植模型分为pcDNA3.1空载体组(第1组)、CD40Ig基因局部转染组(第2组)、CTLA4Ig基因局部转染组(第3组)和CD40Ig、CTLA4Ig双基因局部转染组(第4组)。Western blot检测移植肾HA-CTLA4Ig、HACD40Ig蛋白表达和移植肾Bcl-2、Bax的表达情况,观察移植肾病理改变。结果:术后第5天,第4组移植肾淋巴细胞浸润明显少于第2组和第3组,第4组移植肾Bcl-2蛋白表达明显升高(P0.05),而Bax蛋白表达则显著降低(P0.05)。结论:CTLA4Ig与CD40Ig双基因局部转染供肾可降低Bax表达,上调Bcl-2表达,诱导移植肾免疫耐受。     

共刺激信号阻断剂基因局部转染对大鼠移植肾存活的影响

目的　观察CTLA 4Ig基因局部转染延长移植肾存活的效能。 方法　以CTLA 4Ig基因重组腺病毒为载体 ,将CTLA 4Ig基因转入BN大鼠肾脏。以BN大鼠为供者 ,Lewis大鼠为受者 ,行同种肾移植术。用经CTLA 4Ig基因转染的供肾移植给受者为转染组 ;用未转染CTLA 4Ig基因的供肾移植给受者为对照组。观察移植肾存活时间和术后肾功能变化。结果　转染组移植肾存活 (32± 8.0 )d ,对照组移植肾存活 (8.5± 1.4 )d ,转染组存活时间明显延长 ;转染组术后血清肌酐较同期对照组明显为低。结论　CTLA 4Ig基因局部转染供肾可明显延长移植肾的存活时间。     

CTLA4Ig与CD40Ig局部共转染诱导异种大鼠移植肾免疫耐受的影响

CD28-B7和CD40-CD40L共刺激分子系统是T细胞依赖的免疫反应启动和维持所必不可少的基本信号系统.缺乏或阻断共刺激信号,将会引起T细胞的无反应性或细胞凋亡.我们用CTLA4Ig和CD40Ig双基因局部转染大鼠肾脏后进行异种移植,观察双基因局部共转染能否诱导移植肾免疫耐受.     

负载Ad-CTLA4Ig的未成熟树突状细胞延长大鼠移植肾存活时间的探讨

目的 探讨负载细胞毒性淋巴细胞抗原4免疫球蛋白基因重组腺病毒(Ad-CTLA4Ig)的受者未成熟树突状细胞(DC)对大鼠移植肾存活时间的影响.方法 选择雄性SD大鼠为供者,雄性Wistar大鼠为受者,建立大鼠肾移植模型.将受者随机分为4组,每组12只.制备Ad-CTLA4Ig及受者未成熟DC悬液,37℃混合孵育6 h,于移植前7 d,实验组经腹腔内注射负载Ad-CTLA4Ig的DC悬液;Ad-CTLA4Ig对照组、重组腺病毒空载体(Ad-VG)对照组和生理盐水(NS)对照组分别经腹腔内注射1 ml Ad-CTLA4Ig、Ad-VG和NS.观察各组移植肾的存活时间、组织形态学改变、受者血液中腺病毒中和抗体滴度、血清CTLA4Ig水平及混合淋巴细胞反应(MLR)的变化.结果 实验组移植肾存活时间为(94.6±9.0)d,较各对照组显著延长:[Ad-CTLA4Ig对照组为(39.6±10.6)d,Ad-VG对照组为(8.6±2.8)d,NS对照组为(8.4±2.6)d],差异均有统计学意义(P＜0.01),实验组移植肾组织损伤程度较轻,血液中腺病毒中和抗体滴度及混合淋巴细胞反应指数均较各对照组显著减少;实验组血清CTLA4Ig水平较Ad-CTFLA4Ig对照组显著升高.结论 负载Ad-CTLA4Ig的受者未成熟DC可减少腺病毒中和抗体,维持CTLA4Ig的稳定表达,从而延长大鼠移植肾的存活时间.     

移植物局部转染CTLA4Ig基因对大鼠小肠移植排斥反应的预防作用

目的探讨CTLA4Ig基因在移植小肠局部转染表达及表达产物对排斥反应的预防作用。方法大鼠小肠移植前,经肠系膜上动脉给供肠灌注脂质体包裹的CTLA4IgcDNA重组质粒,术后应用组织免疫学及逆转录聚合酶链反应(RTPCR)检测移植小肠中CTLA4Ig转基因产物的表达,并行组织病理学检查,同时设立无CTLA4Ig基因转染的对照组。结果对照组(n=8)的移植小肠在术后7～14d全部坏死;实验组(n=18)7只受鼠的移植小肠于术后56～90d坏死,11只受鼠的移植小肠于术后90d时仍存活良好。对照组移植物组织切片见单核细胞广泛浸润,肠壁肌层及黏膜出血坏死,绒毛及隐窝结构消失;实验组存活超过90d者组织切片仅见少量单核细胞浸润,黏膜轻度增厚,但无明显的肠绒毛缩短及隐窝细胞减少等改变。组织免疫学及RTPCR结果显示,在移植后28d内移植小肠中有CTLA4Ig转基因产物的表达,但第56d及90d则检测不到其表达。结论经肠系膜上动脉体外灌注脂质体包裹的CTLA4IgcDNA重组质粒可有效转染移植小肠,CTLA4Ig基因在移植小肠局部转染对术后的排斥反应具有预防作用。     

重组腺相关病毒载体介导CTLA4Ig转染延长同种大鼠心脏移植存活时间

目的　研究重组腺相关病毒载体介导 CTLA4Ig(rAAV CTLA4Ig)全身转染对大鼠同种心脏移植的影响。方法　以BN大鼠为供者,Lewis大鼠为受者,建立心脏移植模型。实验分为两组。对照组:供、受者不给予任何处理;转染组:受者于心脏移植前30 d,通过尾静脉全身注射 1×109斑点形成单位(PFU)的 rAAV CTLA4Ig。观察移植心存活时间;ELISA法检测血清 CTLA4Ig、干扰素 γ(IFN γ)和白细胞介素 4(IL 4)水平;免疫组织化学法(SABC法)检测移植心组织中 CD4 和CD8 T细胞的浸润。对移植心存活时间明显延长的受者,用其脾细胞与供者脾细胞进行混合淋巴细胞培养,观察受者对供者的免疫反应状态。结果　转染组移植心存活时间较对照组明显延长(P<0.05);转染组血清CTLA4Ig蛋白一直维持在26.67~35.47 mg/L,移植后转染组血清 IFN γ水平下调,血清 IL 4水平上调(P<0.05);对照组出现典型的急性排斥反应表现,转染组心肌组织基本正常,间质内无炎性细胞浸润或血管外周及心肌间质内有局灶性炎性细胞浸润,未见坏死;对照组移植心组织中浸润的CD4 和CD8 T细胞数量明显高于转染组(P<0.01);转染组受者对供者的脾细胞增殖反应明显低于对照组(P<0.05)。结论　重组腺相关病毒载体可以介导 CTLA4Ig基因的持续表达,通过全身途径转染受者可以明显延长     

环孢素对共刺激阻断剂延长移植肾存活效应的影响

目的:探讨环孢素(CsA)对共刺激阻断剂CTLA4Ig延长移植肾存活效应的影响。方法:肾移植大鼠分为对照组(第1组)、CTLA4Ig组(第2组)、CsA CTLA4Ig组(第3组)、CTLA4Ig IL-2组(第4组)和CsA CTLA4Ig IL-2组(第5组),观察术后血肌酐(Scr)、移植肾病理改变、移植肾存活时间。结果:与第1组、第4组相比,第2组、第3组、第5组移植肾存活时间显著延长(P<0.01),其中,第3组移植肾存活时间最长(66.1±10.6)d;术后15天,第2组Scr显著低于第3组、第5组(P<0.05);术后30天,第3组、第5组Scr显著低于第2组(P<0.01);术后30天,第3组、第5组移植肾淋巴细胞浸润明显少于第2组。结论:CsA可增强CTLA4Ig延长移植肾存活的效应,对外源性IL-2逆转CTLA4Ig的效应具有抵抗作用。     

基因转移CTLA4-Ig和抗CD154抗体在异种胰岛移植排斥反应中的作用

目的 探讨基因转移细胞毒性T细胞相关抗原4免疫球蛋白(CTLA4-Ig)和抗T细胞分化群154(CD154)抗体在异种胰岛移植排斥反应中的作用及机理.方法 建立人-大鼠异种胰岛移植模型,用携带CTLA4-Ig基因的重组腺病毒感染移植胰岛细胞,并用抗CD154抗体进行治疗,观察糖尿病大鼠胰岛移植后血糖变化、生存情况及移植物病理形态学改变,检测移植物CTLA4-Ig、胰岛素的表达和移植大鼠白细胞介素2(IL-2)、肿瘤坏死因子(TNF)-α的水平变化.结果 (1)糖尿病大鼠移植后2 d血糖降至正常,对照组血糖平均在移植后8 d升高,抗体治疗组、转染组和联合治疗组血糖分别在18、25和36 d升高.(2)对照组、抗体治疗组、转染组和联合治疗组的移植物存活时间分别为(10.0±2.1)d、(22.0±8.2)d、(28.0±6.5)d和(37.0±9.3)d,各组间比较差异有统计学意义(P＜0.05);移植大鼠生存时间分别为(21.0±5.7)d、(35.0±6.5)d、(48.0±8.5)d和(65.0 ±12.5)d,各组间比较差异有统计学意义(P＜0.05).(3)对照组在移植后1周内,IL-2、TNF-α的水平均急剧上升,较移植前显著升高(P＜0.01).(4)各治疗组移植物见成片的胰岛细胞团,未见淋巴细胞浸润,转染组和联合治疗组移植物可见CTLA4-Ig和胰岛素的表达.结论 基因转移CTLA4-Ig和抗CD154抗体均可抑制异种胰岛移植排斥反应,二者联合效果优于单独使用.     

CTLA4Ig基因对大鼠胰岛移植后排斥反应的治疗作用

目的　研究CTLA4Ig基因在糖尿病大鼠体内表达及其产物对胰岛移植物存活的作用。方法　利用Lipofectin载体包裹CTLA4IgcDNA质粒后转染鼠胰岛和肌肉细胞 ,检测移植后CT LA4Ig表达和T淋巴细胞转化率。结果　胰岛移植术后 7dT淋巴细胞转化试验 ,实验组 (A组 )和对照组 (B组 )每分钟脉冲数 (cpm)分别为175 .7± 98.2 ,2 5 4.4± 116 .3 ,两组比较差异显著 (P <0 .0 5 )。A组胰岛移植第 7d ,2只大鼠血清CTLA4Ig呈阳性 (阳性率 2 0 % )。A、B两组胰岛移植后血糖维持正常时间分别为 (14.8± 12 .3)d和 (3 .6± 5 .1)d ,两组比较差异显著 (P <0 .0 5 )。A、B两组大鼠平均存活时间分别为 (2 4.0± 10 .8)d和 (10 .8± 4.8)d ,两组比较 ,差异有极显著性 (P <0 .0 1)。结论　脂质体包裹的CTLA4IgcDNA转染肌细胞和胰岛细胞 ,可以在受体大鼠胰岛细胞或肌肉组织中表达 ,其表达产物可使胰岛移植物和受体鼠存活时间明显延长 ,抑制细胞免疫活性 ,发挥其治疗排斥反应的作用     

供心体外转染腺病毒介导的CD40Ig融合基因延长小鼠移植心脏存活时间

目的探讨体外转染CD40Ig融合基因对小鼠移植心脏存活时间的影响。方法构建携带小鼠CD40胞外段和人IgGFc融合基因的重组腺病毒载体(AdCD40Ig),以BALB/c小鼠为供者,C57BL/6小鼠为受者,建立小鼠腹部异位心脏移植模型,实验组供心移植前在体外以AdCD40Ig灌注,转染CD40Ig基因,另设空载体转染对照组、非转染对照组和近交系对照组(供、受者均为近交系C57BL/6小鼠)。术后观察移植心的存活及移植物中炎症细胞浸润情况,采用酶联免疫吸附试验(ELISA)检测受者体内CD40Ig融合蛋白表达情况,流式细胞仪检测受者体内产生γ干扰素(IFN-γ)的脾细胞。结果实验组移植心的存活时间达(15.8±0.7)d,明显长于空载体转染对照组和非转染对照组(P<0.01)。术后第2d,实验组受者体内CD40Ig融合蛋白表达最高,1周后明显降低。术后第7d,实验组移植心组织中浸润的炎症细胞明显比未处理对照组和空载体对照组少。实验组产生IFN-γ的CD4+和CD8+T淋巴细胞分别为(2.18±0.16)%和(10.82±0.74)%,与近交系对照组接近,明显低于未处理对照组和空载体对照组(P<0.01)。结论供心体外转染CD40Ig融合基因可有效抑制移植后受者体内同种T淋巴细胞的增殖,并延长移植心的存活时间。     

Prolongation of renal allograft survival in rats by replication-defective recombinant adenovirus-mediated coexpression of CD40L and CTLA4Ig

BACKGROUND: Blockade of costimulatory signals has been shown to prolong allograft survival. The aim of the present study was to investigate the effect of simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways by replication-defective adenovirus-mediated expression of secretable extracellular domain of human CD40L (shCD40L) and CTLA4Ig to prolong rats renal allograft survival. METHODS: We constructed Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig. Coexpression of shCD40L and CTLA4Ig was evaluated by confocal laser scanning microscopy. The function of these two molecules was examined in human mixed lymphocyte reactions (MLRs) in vitro and in experimental BN-to-LEWIS rat renal transplantation in vivo. RESULTS: Successful construction of Adv-shCD40L-IRES2-CTLA4Ig was confirmed by polymerase chain reaction. Coexpression of shCD40L and CTLA4Ig on human kidney cell line HK-2 cells after transfection was detected by direct immunofluorescence staining. Human MLR was inhibited to 52.2%+/-0.6% and 42.1%+/-0.2% of the vehicle control by Adv-shCD40L and Adv-CTLA4Ig, respectively. Adv-shCD40L-IRES2-CTLA4Ig resulted in further inhibition of MLR to 22.0%+/-0.2% of vehicle control. Transfection with Adv-shCD40L or Adv-CTLA4Ig alone prolonged renal graft survival to 24.8+/-2.5 days and 27.3+/-3.6 days, respectively, as compared to vehicle-treated controls (7.8+/-0.3 days). Cotransfection of both genes extended graft survival to 41.8+/-3.7 days. CONCLUSIONS: Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig, achieved simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways, Adv-shCD40L-IRES2-CTLA4 by Ig synergistically inhibited human T-cell proliferation in MLR, and prolonged rats renal allograft survival.     

Prolongation of islet allograft survival by coexpression of CTLA4Ig and CD40LIg in mice

BACKGROUND: B7/CD28 and CD40/CD40L have been well established as important costimulatory pathways. Cytotoxic T lymphocyte-associated antigen-4 (CTLA4) delivers negative signals to antigen-presenting cells to down-regulate proinflammatory responses and competitively inhibits the binding of B7 and CD28. Signals from the CD40/CD40L costimulatory pathway also play an important role in acute rejection of organ grafts. METHODS: Recombinant adenoviruses Ad-sCD40LIg-IRES2-CTLA4Ig, Ad-CTLA4Ig, and Ad-sCD40LIg were constructed to express sCD40LIg and CTLA4Ig simultaneously or separately as described previously. Streptozocin-induced diabetic BALB/c mice were injected with recombinant adenovirus, receiving approximately 500 donor islets isolated from C57BL/6 mice under the left kidney capsule. Five groups were assigned according to the treatment: nontreated group, Ad-Shuttle-CMV-treated group, Ad-CTLA4Ig-treated group, Ad-sCD40LIg-treated group, and Ad-sCD40LIg-IRES2-CTLA4Ig-treated group. The islet graft mean survival time (MST) was evaluated in the present study. RESULTS: Compared to the islet graft MST of the nontreated group (7.3+/-0.82 days) or Ad-Shuttle-CMV-treated group (7.2+/-1.47 days), the Ad-CTLA4Ig-treated and Ad-CD40LIg-treated islet graft survivals in recipients were 56.3+/-13.71 days (P<.01) and 47.3+/-15.64 days (P<.05), respectively. The islet graft MST was dramatically prolonged to 116.3+/-20.32 days in the Ad-sCD40LIg-IRES2-CTLA4Ig-treated group (P<.01). CONCLUSION: Simultaneous blockade of the CD40/CD40L and B7/CD28 costimulatory pathways via coexpression of sCD40LIg and CTLA4Ig mediated by replication-defective adenovirus may be an acceptable method to induce immune tolerance.     

Long-term acceptance of rat cardiac allografts on the basis of adenovirus mediated CD40Ig plus CTLA4Ig gene therapies

BACKGROUND: We have previously demonstrated that blockade of either CD80/86-CD28 or CD40-CD154 costimulatory pathways by using adenovirus vector coding CTLA4Ig (AdCTLA4Ig) or CD40Ig (AdCD40Ig) genes induced donor-specific tolerance in rat liver transplantation. In this study, we asked whether these gene-therapy-based costimulation blockade would induce tolerance in cardiac transplantation. METHODS: Heterotopic heart transplantation was performed in a full major histocompatibility complex (MHC) barrier combination of ACI (RT1avl) to Lewis (LEW, RT1l) rats. Vector (1 x 10(9) plaque forming unit [PFU]), AdLacZ, AdCTLA4Ig, or AdCD40Ig, was administered intravenously to recipient animals immediately after grafting, and graft survival, serum CTLA4Ig/CD40Ig levels, and graft histology were assessed. Tolerance was determined by secondary skin-graft challenging. RESULTS: Allografts of both untreated and AdLacZ controls were promptly rejected within 7 days, whereas a single treatment with AdCTLA4Ig or AdCD40Ig significantly prolonged median graft survival to 55.5 and 28.5 days, respectively. In contrast, the combined AdCTLA4Ig and AdCD40Ig gene therapy maintained high CTLA4Ig and CD40Ig levels through the posttransplant period and allowed long-term cardiac allograft survival for more than 270 days. However, both donor and third-party skin grafts were rejected in the animals who harbored cardiac grafts over 150 days. Also, typical features of chronic rejection were evident in the long-term surviving grafts. CONCLUSION: Simultaneous blockade of CD28 and CD154 pathways by AdCTLA4Ig plus AdCD40Ig induces a strong immunosuppression that allows long-term acceptance of full MHC mismatched cardiac graft in rats. This strategy, however, was not enough to induce tolerance to skin grafts and to avoid chronic rejection, as shown in the liver-transplantation model.     

Combined gene therapy with adenovirus vectors containing CTLA4Ig and CD40Ig prolongs survival of composite tissue allografts in rat model

BACKGROUND: The blockade of costimulatory signal pathway by anti-CD40 ligand antibody or cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) prolongs allograft survival in various vascularized organ transplantations. Because of the short half life of these agents, repeated administration of proteins is required to achieve significant graft survival. Furthermore, there is limited information regarding the effect of cosimulatory blockade on the survival of composite tissue allografts. Therefore, we examined the effect of adenovirus-mediated gene transfer of CTLA4Ig or CD40Ig gene or both in composite tissue allotransplantation. METHODS: The hind limbs removed from male ACI rats (RT1 ) were transplanted into female Lewis rats (RT1 ) heterotopically. The recombinant adenovirus carrying CTLA4Ig (AxCTLA4Ig) or CD40Ig (AxCD40Ig) was intravenously administered after limb transplantation. RESULTS: Limb allograft survival was significantly prolonged by either AxCTLA4Ig or AxCD40Ig treatment at 1 x 10 plaque forming unit (mean survival time [MST] of 39.4+/-6.0 and 13.0+/-2.9, respectively) compared with the adenovirus vector containing beta-galactosidase-treated group (MST of 4.8+/-0.8). Combination of AxCTLA4Ig and AxCD40Ig led to significant prolongation of graft survival (MST of 49.2+/-6.6). Serum levels of CD40Ig were higher in rats treated with combination therapy than those treated with AxCD40Ig alone, whereas the serum levels of CTLA4Ig in rats treated with AxCTLA4Ig alone and AxCTLA4Ig and AxCD40Ig combined were very similar. CONCLUSION: This study indicates that an adenovirus-mediated gene therapy of CTLA4Ig or CD40Ig has a therapeutic potential for preventing rejection in composite tissue transplantation. Furthermore, a combination therapy of AxCTLA4Ig and AxCD40Ig was even more effective in preventing acute rejection and prolonging the survival of allografted limbs without apparent complication.     

CTLA4Ig combined with anti-LFA-1 prolongs cardiac allograft survival indefinitely

CTLA4Ig and anti-LFA-1 are members of a new generation of immunomodulatory drugs which inhibit important signaling pathways in T cell activation. Both substances target molecules which have pivitol functions in the activation of CD4+ and CD8+ T cells and have been theorized to have an interdependent relationship. These drugs have been used independently in various treatment regimens and have shown great promise in prolonging the survival of allografts. In order to test whether these substances have synergistic or potentiating effects when combined, we performed mixed lymphocyte reactions, skin transplantation and vascularised heterotopic heart transplantation in the Balb/c (H-2(d)) to C3H/HeJ (H-2(k)) strain combination. When anti-LFA-1 and CTLA4Ig were combined at low doses, there was a substantial inhibition of lymphocyte proliferation. When each drug was used as a mono-therapy in skin graft recipients, there was no significant effect on median graft survival (anti-LFA-1, 15 days; CTLA4Ig, 16 days) when compared to untreated controls (13 days), whereas a combination of anti-LFA-1 and CTLA4Ig extended graft survival significantly to 32 days. Untreated vascularised heart grafts rejected at a median of 8 days, CTLA4Ig-treated mice rejected at a median time of 79 days and anti-LFA-1-treated mice rejected at 43 days (n = 9). When CTLA4Ig and anti-LFA-1 were combined, all animals had functioning heart grafts at 100 days after transplantation. Histological analysis of combined-therapy hearts showed no signs or only minor changes associated with chronic rejection. In conclusion, these results indicate a synergistic effect of combining anti-LFA-1 with CTLA4Ig in inhibiting lymphocyte proliferation and prolonging the survival of fully MHC-mismatched allografts.     

Prevention of Acute Lung Allograft Rejection in Rat by CTLA4Ig

CTLA4 immunoglobulin (CTLA4Ig), which binds with a high affinity to B7-1 and B7-2, interrupts T-cell activation by inhibiting costimulatory signal. CTLA4Ig has been used in hopes of achieving antigen-specific tolerance induction in several solid organ transplants. In lung allograft rejection, however, its use has been controversial in terms of its effect on prevention of rejection. In the present study, the effect of murine CTLA4Ig on rat-lung allograft rejection was investigated. Rat left-lung transplantation was performed in an RT1 incompatible donor (Brown Norway; BN)-recipient (F344) combination. All allografts (n = 12) without any treatment were rejected within 7 days after transplantation. A single injection of murine form CTLA41g at a dose of 100 microg intraperitoneally (ip) or intravenously (iv) on day 1 post-transplantation achieved long-term graft survival (>90days) in 2/5 (40%) and 3/8 (38%), respectively. Moreover, 6/7 (86%) allografts in rats that received iv injection of 500 microg CTLA4Ig survived more than 90days. Allograft survival in the CTLA4Ig 500 microg iv recipient group was significantly longer than that in the no-treatment control or control immunoglobulin group (p <0.01). Four out of seven recipients bearing functional allografts for more than 90 days with the CTLA4Ig treatment accepted donor-specific skin grafts, whereas all third-party skin grafts (n=3) were rejected. Prevention of rat-lung allograft rejection could be achieved by intravenous administration of CTLA4Ig, resulting in long-term allograft survival with acceptance of donor-specific skin grafts.     

CTLA4Ig和西罗莫司短期联合使用延长异种胰岛移植物的存活

目的 观察细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig)与西罗莫司(SRL)联用阻断共刺激通路对异种胰岛移植物存活的影响.方法 取C57BL/6小鼠,腹腔注射链佐星,制成糖尿病模型.采用随机单位组设计分组法将糖尿病小鼠分为7组,各组均于小鼠左肾包膜下移植SD大鼠胰岛300胰岛当量.CTLA4Ig组分别于移植当天及移植后第2、4、6天腹腔注射CTLA4Ig 0.5 mg/d;SRL组分别于移植当天及移植后第1、2天给予SRL灌胃,0.2 mg·kg-1·d-1,其后隔天用药1次,共用2周;MRI组分别于移植当天及移植后第2、4天腹腔注射仓鼠抗小鼠CD154单克隆抗体(MR1)0.5 mg/d;CTLA4Ig和SRL联用组(SRL联用组)、CTLA4Ig和MRl联用组(MR1联用组)以及CTLA4Ig、MR1和SRL联用组(三药联用组)各药物的剂量与用法同上述各组;对照组仅行胰岛移植,不予以药物.观察至移植后200 d,通过监测受者血糖水平来判断排斥反应的发生情况.记录各组移植物的存活(即无排斥反应)时间.发生排斥反应者,或未发生排斥反应、移植物存活时间＞200 d者,取移植胰岛,行HE染色及免疫荧光染色,进行组织学观察.结果 对照组移植物存活时间中位数为17 d,该组最终均发生排斥反应.SRL组、MRl组和CTLA4Ig组移植物存活时间中位数分别为34 d、98 d和77 d.均明显长于对照组(P＜0.05),三组中分别有90%(9/10)、62.5%(5/8)和83.3%(5/6)的小鼠发生排斥反应.SRL联用组移植物存活时间中位数为130 d,明显长于上述4组(P＜0.01),有50%(3/6)的小鼠发生排斥反应.MR1联用组以及三药联用组移植物存活时间中位数均＞200 d,分别有42.9%(3/7)和25%(2/8)的小鼠发生排斥反应.组织学检查结果显示,对照组发生排斥反应时,其移植胰岛破坏严重,可见大量CD4+和CD8+淋巴细胞及巨噬细胞浸润,并可见IgG、IgM和补体C3沉积.其它组发生排斥反应者的组织学改变与对照组相似.SRL联用组存活200 d的小鼠,其移植胰岛组织中未见或仅有少量炎症细胞浸润,胰岛素和胰高血糖素染色阳性,未见IgG、IgM和补体C3沉积.结论 短期联合使用CTLA4Ig和SRL能显著延长小鼠体内大鼠来源的胰岛的存活时间.