同种异体反应性淋巴细胞克隆疫苗回输诱导受体特异性免疫耐受研究

目的 探讨同种异体移植抗原特异性耐受诱导的新途径。方法 以同种异体反应性淋巴细胞克隆在体外培养条件下大量增殖后作为自身独特性疫苗,体内注射免疫家兔,以混合淋巴细胞反应来观察免疫前和免疫后对供体淋巴细胞刺激指数（SI）的变化。结果 同种异体反应性自身独特淋巴细胞疫苗具有显著诱导特异性免疫应答抑制现象,SI值显著降低（P＜0．005）,而对卡介苗诱导的特异性免疫应答并未因独特型淋巴细胞疫苗的应用下降。结论 同种异体反应性自身独特型淋巴细胞疫苗可能成为诱导抗原特异性免疫耐受的一种手段,作为心肺移植受体的术前准备。     

T细胞疫苗诱导大鼠同种异体肢体移植免疫耐受的实验研究

目的 探讨T细胞疫苗（TCV）诱导大鼠同种异体肢体移植特异性免疫耐受的作用。方法 制备受者Lewis大鼠针对供者DA大鼠的TCV，应用TCV，免疫正常Lewis大鼠共3次，每周1次。设TCV组和TCV未接种组，在接种前及接种后5d进行混合淋巴细胞培养（MLR）；设TCV组、CsA组和空白对照组，于接种后7d进行DA大鼠针对Lewis大鼠的同种异体肢体移植，在术后7d进行淋巴细胞毒检测，术后21d进行嵌合分析。结果 MLR显示，Lewis大鼠的脾细胞反应程度接种组显著低于未接种组（P〈0．01）；微量细胞毒测定显示，死亡细胞百分率在TCV组、CsA组、空白对照组3组比较性差异有统计学意义（P〈0．01）；嵌合分析显示经处理的Lewis大鼠脾脏，胸腺中检测出了DA大鼠源性的骨髓嵌合体。结论 T细胞疫苗可以抑制受者对供者的免疫应答；作为骨髓移植前预处理手段，T细胞疫苗接种后行吻合血管的骨髓移植成功地诱导出同种异体肢体移植嵌合耐受。     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受.方法将114只日本白色家兔和Dutch家兔分为对照组和实验组,日本白色家兔作为供体,Dutch家兔作为受体.对照组,在不使用免疫抑制剂的情况下,将12只日本白色家兔与12只Dutch家兔行相同面积的背部全厚皮肤互换移植,观察其成活时间.实验组,将45只日本白色家兔和45只Dutch家兔行全厚皮肤移植的同时行骨髓移植,然后将作为受体的Dutch家兔分为A,B,C,D四组,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植,观察移植皮肤的成活时间.结果对照组,供体与受体移植皮肤的平均成活时间分别为(12.0±1.7)天和(10.3±1.3)天.实验组,A,B,C,D四组移植皮肤的平均成活时间分别为(61.0± 7.2)、(80.7± 10.4)、(78.8± 12.7)、(88.0± 6.0)天.结论通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验,旨在为临床应用提供了理论基础及可靠依据,为同种组织重建提供一个新方法.     

骨髓输注和抗淋巴细胞球蛋白诱导对同种异体移植肾特异性免疫无反应性的临床研究

国外使用抗淋巴细胞球蛋白（ＡＬＧ）和输入供体特有骨髓（ＢＭ）诱导同种移植物的耐受力，并建立了动物模型。我们将这研究用于诱导特异性免疫无反应性病例２０例（骨髓组），并随机抽样２２例尸体肾移植患者作为对照组。骨髓组接受常规三联免疫抑制治疗，在１４天从ＡＬＧ诱导期后，停用１周，于第７天输入冷藏骨髓，随访５～４１个月。结果：１～６个月发生排斥反应２例（２／２０），均用大剂量激素逆转；另１例合并败血性肺炎死亡。对照组采用三联常规免疫抑制治疗剂量较骨髓组大，但不输入骨髓，结果同期发生排斥反应６例（６／２２），后期排斥反应２例，其中４例因排斥反应未能逆转而移植肾失功，有１例因合并心衰死亡。从两组结果分析，人的一年成活率两组无明显差别。移植肾一年成活率则骨髓组高于对照组（Ｐ＜０．０１），排斥反应发生率骨髓组明显低于对照组（Ｐ＜０．０５），且骨髓组常规免疫抑制剂用量较对照组小。     

肝特异性抗原的同种异体免疫原性

目的：研究肝特异性抗原的同种异体免疫原性及其意义。方法：用近交系F344大鼠的肝特异性抗原作为免疫原，通过小剂量长期足垫免疫、门静脉及胸腺大剂量免疫等不同的方法免疫近交系Lew大鼠，以环磷酰胺150mg/kg腹腔注射作为诱导淋巴细胞凋亡的阳性对照，用混合淋巴细胞（MLC）培养、混合淋巴细胞肝细胞（MLHC）培养，Western印迹、DNA凝胶电泳分析及TUNEL等检测手段，对外周及中枢淋巴器官的功能进行分析，以确定免疫后机体的免疫状态。结果：在取材时间点，各实验组均出现不同程度的特异性低免疫应答：MLC变化不明显，但MLHC却均较对照组明显降低，而各实验组Caspase-3的表达均明显增高，脾细胞的DNA凝胶电泳表现出抗原特异性的淋巴细胞凋亡，胸腺TUNEL结果，各实验组均出现不同程度的凋亡细胞。结论：肝特异性抗原具有同种异体免疫原性，可能是一种重要的移植抗原，为诱导同种移植免疫耐受开拓新的途径；对移植肝或细胞免疫原性的调变，不但要针对同种异体主要组织相容复合物（MHC）抗原，而且也应针对非MHC抗原。     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

目的 观察大鼠胸腺内注射异基因抗原在同种异体异基因股静脉移植免疫耐受中的作用.方法 将48只SD大鼠随机分为4组:自体股静脉移植组(A组)、异体股静脉移植组(B组)、异体股静脉移植免疫抑制剂组(C组)、胸腺内注射供体组织相容性(MHC)抗原后移植组(D组).于2周后进行影像学、组织学、免疫学检测.结果 组织学检测结果显示:D组、C组急性排斥反应损伤较轻,B组血管壁的各层结构破坏最重,可见大量炎性细胞浸润.B组受体大鼠血清干扰素(IFN)-γ浓度为(86.707±10.928)ng/L,显著高于A、C、D组[(29.328±4.170)、(69.076±8.059)、(63.355±4.895)ng/L,P<0.05];B组受体大鼠血清白细胞介素(IL)-4浓度为(23.656±3.369)ng/L,显著低于C、D组[(29.425±4.174)、(31.000±4.659)ng/L,P<0.05].结论 胸腺内注射异基因MHC抗原可诱导大鼠对同种异体血管移植的特异性免疫耐受.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

注射供者的肝匀浆提取液延长大鼠异位心脏移植存活时间

目的探讨注射供者的肝匀浆提取液对大鼠淋巴细胞功能及大鼠异位移植心的影响。方法以Wistar大鼠为供者,SD大鼠为受者。制作Wistar大鼠的肝匀浆提取液；建立大鼠同种异体异位心脏移植模型。(1)经受者阴茎静脉注射肝匀浆提取液0．3 ml,14d后取供、受者的血液,用四甲基偶氮唑盐(MTT)法分别测定受者对同一供者和无关供者的单向混合淋巴细胞反应(MLR)。(2)心脏移植术前2h经受者阴茎静脉注射肝匀浆提取液0．3 ml。心脏移植术后分别观察受者注射同一供者和无关供者的肝匀浆提取液后移植心脏的存活时间；心脏停跳后取移植心做病理检查及免疫组织化学检测。结果受者对同一供者和无关供者的单向MLR比较,前者明显减轻,吸光度A值分别为：0．434±0．034和0．522±0．015,两组比较,差异有统计学意义(P＜0．01)。心脏移植术前,受者接受同一供者和无关供者的肝匀浆提取液后,前者移植心脏存活时间延长,分别为(38．05±17．07)d和(9．86±2．67)d,两组比较,差异有统计学意义(P＜0．01)；且前者心肌出血、坏死程度更轻,心肌组织内IgM和IgG沉积更少。结论注射同一供者的肝匀浆提取液能特异性抑制相应个体抗原引起的淋巴细胞增殖反应,减轻大鼠移植心脏的排斥反应,明显延长其存活时间。     

复发性流产患者主动免疫治疗后淋巴细胞免疫表型的变化

目的探讨原因不明复发性流产(URSA)患者行淋巴细胞主动免疫治疗(LIT)后淋巴细胞免疫表型的变化对治疗效果的评估价值。方法采用流式细胞术分析URSA患者LIT前后外周血T淋巴细胞、B淋巴细胞、NK细胞和调节性T细胞免疫表型的变化(P0.05)。结果 25例URSA患者经LIT后成功妊娠16例,治疗后所有URSA患者(n=25)和妊娠成功组(n=16)外周血CD3+T细胞、CD4+HLA-DR+T细胞比例较治疗前均明显增加,CD4+T和CD3-CD56+NK细胞比例明显降低(P0.05);而治疗前后B细胞和Treg细胞、CD56bright CD16-NK、CD56dimCD16+NK、CD3+CD56+NKT及CD69+NK细胞比例则无明显变化(P0.05)。结论 LIT后外周血T细胞、NK细胞的比例发生了明显变化,CD4+T细胞、CD3-CD56+NK细胞比例降低和CD3+T细胞、活化CD4+T细胞增加也许有利于维持妊娠,T细胞和NK细胞的免疫表型有望作为LIT疗效评估的一个重要指标。     

耐受性树突状细胞延长大鼠移植脾存活时间

目的 观察供者来源的耐受性树突状细胞（DC）在脾移植中的作用，并探讨其作用机理。方法 以Wista大鼠为供者，SD大鼠为受者，建立同种颈部异位脾脏移植模型。（1）分离供者的骨髓细胞，分别采用白细胞介素4（IL-4）和白细胞介素10（IL-10）诱导培养出成熟的DC和耐受性DC，并在光镜下观察两者的细胞形态学差别。采用流式细胞术检测两者对共刺激分子CD86表达的差异，采用混合淋巴细胞反应比较其在体外刺激同种异体T淋巴细胞增殖的反应能力。（2）将受者随机分成4组，每组10只。单纯移植组：受者不经任何预处理仅进行脾移植。IL-10DC组：在移植前7d经受者尾静脉注射2×10^6／ml的经IL-10诱导的DC 1 ml。IL-4 DC组：在移植前7d经受者尾静脉注射2×10^6／ml的经IL-4诱导的DC 1 ml。空白对照组：在移植前7d经受者尾静脉注射无细胞的培养液1ml。观察各组移植术后发生急性排斥反应的时间。结果 （1）经IL-10诱导的骨髓细胞表现为未成熟树突状细胞的形态和特性，细胞体积大，但少见树突状突起，细胞表面低表达CD86分子，不能有效刺激T淋巴细胞的增殖。而经IL-4诱导的骨髓细胞为典型的成熟树突状细胞，细胞胞体大，并有树突状突起，细胞表面高表达共刺激分子CD86，可显著刺激T细胞的增殖。（2）IL-10 DC组发生急性排斥反应的时间较其他3组明显延迟（P〈0．01）；IL-4 DC组发生急性排斥反应的时间较单纯移植组和空白对照组明显提前（P〈0．05）；而单纯移植组与空白对照组间则无明显差异（P〉0．05）。结论 应用供者来源的耐受性树突状细胞能够延缓大鼠移植脾急性排斥反应的发生时间。     

未成熟树突状细胞联合雷帕霉素诱导小鼠肤移植免疫耐受

状细胞组、联合组的皮肤移植物存活时间分别为(6.9±1.9)、(10.3±3.0)、(17.0±3.4)、(20.8±3.6)d.方差分析提示组间差异有统计学意义(P＜0.05);S-N-K检验提示各组之间的差异均有统计学意义(P＜0.05).结论 供者骨髓来源未成熟树突状细胞可诱导小鼠皮肤移植免疫耐受;联合使用雷帕霉素可延长免疫耐受的持续时间.     

肾移植后供者细胞的迁移和嵌合

采用聚合酶链反应（ＰＣＲ）技术对１９８７年７月～１９９６年６月接受男性供肾的５７例女性受者的嵌合状态进行研究。发现嵌合总发生率为５４．４％（３１／５７）；生存期３年以上的受者嵌合发生率为６６．７％（１８／２７），而三年以内嵌合发生率为４３．３％（１３／３０），两者差异有显著性（Ｐ＜０．０５）；肾移植后２个月内，嵌合的形成尚不稳定。结果表明：随着肾移植受者生存期的延长，嵌合的发生率相应增高；嵌合反映了供者器官与受者免疫系统之间双向作用的关系，并与免疫耐受存在某种联系。     

Fragile maintenance of allograft tolerance induced by lymphocyte sequestration and co-stimulation blockade

The induction of long-term graft survival has been a goal for the last decade. Nevertheless, the issues of stable maintenance of allograft have not yet been evaluated thoroughly. Here, we studied new approaches for induction of tolerance by lymphocyte sequestration (FTY720) and co-stimulatory blockade (MR1) in skin graft model (DBA/2 to BALB/c), thus evaluating the mechanisms incorporated into the maintenance of allograft in proper function. FTY720 + MR1 treatment significantly prolonged graft survival than single agent treatment did, and induced long-term graft survival in 60% of recipients expressing the up-regulation of IL-4 and FoxP3. To assess the stability of graft maintenance, we performed the second transplantation on recipients that had shown long-term graft survival. While recipients accepted the second graft from the same strain of first donor, the recipients not only rejected the third-party skin (C57BL/6) promptly but also rejected the first graft soon after the third-party skin was transplanted. The expression patterns of IL-4 and FoxP3 were changed according to the strains of second graft in lymph nodes and in the first graft. T_reg cells from tolerant recipients effectively suppressed allo-antigen driven T cell proliferation, but T_reg cells from recipients primed with third-party antigen had significantly hampered suppressive capacity against previously tolerant antigens. Our data indicate that the combination treatment provides effective tool for the induction of long-term graft survival, and the maintenance of allograft in proper function is an actively regulated process.     

胸腺注射转染供者主要组织相容性复合物基因的T淋巴细胞诱导免疫耐受的实验

目的 探讨受者胸腺内注射可表达供者主要组织相容性复合物（MHC）基因的受者T淋巴细胞诱导的免疫耐受的效果。方法 将携带供鼠（C57BL／6，H－2^b）k位点基因cDNA的逆转录病毒载体质粒PXN（N2－B19－H－2K^b）经包装细胞PA317细胞包装为重组病毒后，感染体外培养的受鼠（Balb/c,H-2^d）T淋巴细胞，再将此可表达供者MHC抗原的受者T淋巴细胞（H－2K^db）回输受鼠胸腺内，然后将供鼠（H－2^b）的皮肤移植给受鼠（H－2^d），观察免疫耐受的诱导情况。结果 外源性MHC基因（H－2K^b）整合到靶细胞（H－2^d）染色体DNA，并有效地转录，在细胞膜上有H－2K^b分子表达；对照组移植皮肤平均存活时间（MST）为9d；注射单克隆抗体对照组MST为11d；第三供者组MST为11.5d；实验组（胸腺回输H－2K^db嵌合体T淋巴细胞）MST为35d；实验组脾细胞对刀豆素A（ConA）的增殖反应在正常范围，单向混合淋巴细胞反应对第三供者的脾细胞反应正常，但对特异供者的脾细胞无反应。结论 自身T淋巴细胞在胸腺内表达供者MHC抗原可在成年动物诱导出对供者移植的特异性免疫耐受，无非特异性免疫抑制。     

大鼠心脏移植与心肌细胞坏死

目的 探讨心肌细胞坏死在心脏移植中的发生和发展情况，为心脏移植的抗排斥反应治疗和预防提供依据。方法 用供体脾细胞（ＳＰＣ）和环磷酰胺（ＣＰ）预处理移植受体，ＨＥ染色对移植心脏的炎细胞浸润和心肌细胞坏死进行分析。结果 经ＳＰＣ和ＣＰ预处理后，移植心脏的存活时间明显延长，炎细胞浸润和心肌细胞变性坏死明显减轻。结论（１）急性排斥反应时，移植心脏内大量的炎细胞浸润和心肌细胞坏死。（２）大鼠心脏移植的急性排