Tju103和CTLA4-Ig诱导T淋巴细胞免疫耐受的比较

目的观察比较Tju103和CTLA4-Ig分别调节、诱导T淋巴细胞免疫耐受的作用.方法用经丝裂霉素处理的异基因正常BALB/c(H-2d)小鼠巨噬细胞做耐受诱导原,分别用Tju103和CTLA4-Ig体外训导、调节C57BL/6(H-2d)小鼠T淋巴细胞,通过增殖效应(单向混合淋巴细胞反应,MLR)和杀伤效应(MTT法),考察经训导的C57BL/6小鼠T淋巴细胞分别对BALB/c小鼠和CBA(H-2k)小鼠脾细胞以及EL9611(H-2d)红白血病细胞的增殖效应和杀伤效应的改变.结果经Yju103调节后,C57BL/6 小鼠T淋巴细胞对BALB/c和CBA小鼠脾细胞以及EL9611红白病细胞的增殖反应和杀伤效应抑制作用明显,未能诱导对已接触抗原(BALB/c小鼠)产生特异性免疫耐受.经CTLA4-Ig孵育后,C57BL/6 小鼠T淋巴细胞对这三种细胞的增殖反应和杀伤效应抑制作用明显;对已接触抗原呈现免疫耐受,而对第三种抗原(CBA小鼠)和EL9611白血病细胞保持反应性.结论在特异抗原存在下,Tju103和CTLA4-Ig均能明显抑制异基因小鼠T淋巴细胞增殖反应性和杀伤效应,但Tju103诱导非特异免疫抑制,而CTLA4-Ig诱导特异免疫耐受,更适合于诱导移植免耐受.     

小鼠同种异基因骨髓移植aGVHD动物模型的建立

目的：建立一个德定可靠的异基因骨髓移植（allogeneic bone marrow transplantation,Allo-BMT）急性移植物抗宿主病（acute graft-vetsus-host disease,aGVHD）动物模型,为Allo-BMT中aGVHD研究提供理想的实验平台。方法：以雄性C57BL／6（H-2b）为供鼠,雌性BALB／C（H-2d）为受鼠,受鼠致死性全身照射（total body irradiation,TBI）（8.0Gy）预处理后,在移植物中混合不同比例的供鼠骨髓细胞和脾细胞以引起不同程度的aGVHD。根据存活期、外周血白细胞计数、临床表现及病理检查等判断aGVHD程度。结果：单纯异基因骨髓组只有部分小鼠出现aGVHD,75％的小鼠长期存活（〉30d）。混合不同比例的供鼠骨髓细胞和脾细胞的Allo-BMT小鼠出现aGVHD的时间和程度均有差异,其中骨髓与脾细胞1.5：1或1：1混合组小鼠,均可在相对集中的时间内（8-15d）观察到典型的aGVHD临床和病理改变。结论：建立Allo-BMT aGVHD模型需要加入一定比例的脾细胞,供鼠骨髓与脾细胞1.5：1或1：1混合进行的Allo-BMT,可作为理想的aGVHD动物模型。     

TJU103对小鼠同种异基因造血干细胞移植模型GVHD的预防作用

目的探讨TJU103对小鼠同种异基因造血干细胞移植模型移植物抗宿主病(GVHD)是否起预防作用。方法(1)用单向混合淋巴细胞培养方法检测TJU103对T淋巴细胞增殖的影响;(2)以BALB/c(H-2d)、CB6F1(H-2d/b)分别作为完全异基因移植和半相合基因移植的受鼠,给予9.0Gy全身照射后4h,单纯照射组经尾静脉注射0.3mlD-Hank's液,不进行细胞移植;对照组经尾静脉注射C57BL/6小鼠混合细胞悬液(4.5×106骨髓细胞 3.0×107脾细胞),但不进行其他治疗处理;实验组经尾静脉注射C57BL/6小鼠混合细胞悬液(4.5×106骨髓细胞 3.0×107脾细胞) TJU103(终浓度25μg/ml),此后腹腔注射TJU10350μg/d,共7d,然后观察其造血恢复、植入及GVHD的情况。结果(1)TJU103可以显著抑制T细胞活化增殖,其抑制作用呈剂量依赖性,在25μg/ml浓度时,可以抑制83%细胞增殖。(2)由于没有给予GVHD防治措施,对照组受鼠GVHD的发生率和死亡率分别为10/10和10/10;而完全异基因移植实验组小鼠GVHD发生率仅为2/10,30d生存率增加至8/10(P<0.01),中位生存期延长至30d以上(P<0.01);半相合基因移植实验组小鼠GVHD发生率为1/10,30d生存率增加至9/10(P<0.01),中位生存期延长至30d以上(P<0.01)。结论TJU103不仅可以显著抑制体外T淋巴细胞增殖效应,而且可显著降低小鼠同种异基因造血干细胞移植模型GVHD的发生率。     

抗T细胞受体αβ单抗和抗CD80单抗联合供体骨髓移植诱导小鼠皮肤移植耐受

目的:探讨抗T细胞受体(T cell receptor, TCR)αβ单抗和抗CD80单抗联合供体骨髓细胞输注方法在同种异基因小鼠皮肤移植耐受诱导中的作用.方法:第0天,向C57BL/6小鼠(受体)尾静脉输注2×10⁸个BALB/c小鼠(供体)来源的骨髓细胞,同时腹腔注射抗TCRαβ单抗500 μg;第2天,向C57BL/6小鼠腹腔注射抗CD80单抗500 μg.第6天进行皮肤移植.在不同的时间对C57BL/6小鼠进行迟发型超敏反应测定和混合淋巴细胞反应(mixed lymphocyte reaction, MLR)分析,并进行MLR的白细胞介素-2(interleukin-2, IL-2)逆转实验、过继转移实验和嵌合体检查,探讨耐受形成的机制.结果:接受了抗TCRαβ单抗和抗CD80单抗联合供体骨髓移植的C57BL/6小鼠,供体皮肤移植物平均存活70 d;在迟发型超敏反应和混合淋巴细胞反应中均表现出显著的低反应性.IL-2逆转实验结果表明克隆不应答可能参与了移植耐受的形成.体内、体外过继转移实验均显示耐受C57BL/6小鼠脾细胞中存在抑制细胞的活性.嵌合体检查结果表明在耐受C57BL/6小鼠的胸腺和脾中均形成了混合嵌合体,嵌合体水平随时间下降.结论:抗TCRαβ单抗和抗CD80单抗联合大剂量供体骨髓细胞输注可以在组织相容性抗原完全不相同的成年小鼠间成功诱导出皮肤移植物的长期耐受.     

间充质干细胞联合NK细胞输注抑制DC减轻aGVHD

【目的】 观察骨髓MSC联合NK细胞输注对小鼠异基因骨髓移植后aGVHD的影响,初步探讨其作用机制?【方法】 分离C57BL/6小鼠骨髓细胞,体外培养MSC;取C57BL/6小鼠脾脏细胞,磁珠分选NK细胞;建立C57BL/6→BALB/c小鼠异基因骨髓移植模型,接受移植小鼠随机分为4组,每组10只小鼠,MSC和NK细胞移植组,MSC移植组,NK细胞移植组和培养基对照组?根据aGVHD分值,肝?脾?小肠aGVHD病理损害和平均存活时间,评价aGVHD的程度?ELISA法检测小鼠血清中IFNγ和IL-12的含量?流式细胞术检测受鼠脾脏细胞CD11c和CD86的表达情况?【结果】 根据GVHD分值,肝?脾?小肠aGVHD病理损害和平均存活时间综合评价,与培养基对照组比较,MSC联合NK细胞移植组,MSC移植组和NK细胞移植组,发生aGVHD的程度均减轻,以MSC联合NK细胞移植组减轻最为明显?IFNγ的含量,NK细胞组明显高于MSC联合NK细胞移植组,MSC移植组和培养基对照组?IL-12的含量,MSC联合NK细胞移植组低于NK细胞移植组和培养基对照组,MSC移植组低于培养基对照组?脾脏DC细胞的含量,MSC联合NK细胞移植组未成熟DC(CD11c+CD86-)和成熟DC(CD11c+CD86+)均低于MSC移植组,NK细胞组和培养基对照组?【结论】 MSC联合NK细胞输注降低DC细胞数量,减轻aGVHD,延长小鼠生存时间?     

非清髓性预处理联合髓腔内骨髓细胞输注诱导免疫耐受的实验研究

目的本研究通过非清髓性预处理方案联合髓腔内骨髓移植建立异基因小鼠免疫耐受模型,并探讨其诱导耐受的机理.方法受鼠C57BL/6(B6)于第0天接受60Coγ射线全身照射(TBI,550-cGy),4 h内输注雄性BALB/C(H2d)小鼠来源的骨髓细胞,2 d后腹腔注射环磷酰胺(CTX,200 mg·kg-1).通过皮肤移植、混合淋巴细胞反应(mixed lymphocyte reaction, MLR)检测耐受状态,并应用体外过继转移实验、IL 2逆转实验等探讨免疫耐受机制.结果经骨髓移植的B6小鼠对BALB/C小鼠的皮肤移植物平均存活时间超过300d,较空白组明显延长(P＜0.001);MLR结果证明B6小鼠获得供体特异性耐受,该耐受可以被IL 2逆转且可被过继转移;所有受鼠均未出现GVHD表现.结论非清髓预处理联合髓腔内骨髓移植可以有效诱导异基因小鼠的免疫耐受.     

Induction of specific immunosuppression of cardiac allograft rejection with monoclonal antibodies to CD44, LFA-1 and ICAM-1

In the p~ess of allograft rejechon, the interactionbetween multiple adhesitin molecules Promotes the development Of the inflallnnatory ~on that leads to aliceddestruchon. Ih both aamal and hUInan models of aliced~splantation, the expression of adheSion molecules, including letlkocyte funchon-associated antigen-l (on-l ),intel'Cellular adhesion molecule-l (ICal-l ), vascularcell adhesion molecule~l (VCAM~l) and CD44 over thesurfaCe of the endothelial cells has been shown tO increasedwi al…     

白细胞介素11联合粒细胞集落刺激因子对小鼠异基因骨髓移植后移植物抗宿主病影响的研究

目的探讨重组白细胞介素11(rhIL-11)联合重组粒细胞集落刺激因子(rhG-CSF)用于供者对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)的作用及其机制。方法建立同种异基因骨髓移植急性GVHD模型,以C57BL/6H-2b为供鼠,rhIL-11、rhG-CSF或二者联合应用作为动员剂,BALB/CH-2d受鼠经致死剂量(8·0Gy)照射后随机分为5组,A组为单纯照射组(10只),B组为GVHD对照组(18只),C组为G-CSF移植组(18只),D组为IL-11移植组(18只),E组为联合移植组(18只),在照射后4h移植供鼠骨髓细胞和脾细胞。观察移植后小鼠的生存期和病死率,应用流式细胞仪测定嵌合体形成情况,用四甲基偶氮唑盐(MTT)法检测混合淋巴细胞反应,酶联免疫吸附实验(ELISA)检测白细胞介素4(IL-4)、γ干扰素(IFN-γ)等细胞因子的分泌情况,并进行组间比较。结果E组小鼠生存时间明显长于其他各组(B、C、D),差异有统计学意义(P<0·01),30d存活率B、C、D、E组分别是:0、27·3%(3/11)、36·4%(4/11)、63·6%(7/11)。B组小鼠均出现GVHD病理改变,其程度与发生GVHD的时间密切相关,GVHD发生越早,病理改变越重;E组有3只小鼠在移植后20d内发生GVHD,病理程度改变较其他各组轻。与B、C、D组相比,E组移植后14d脾细胞培养上清中IFN-γ水平较低(92pg/ml±14pg/mlvs172pg/ml±31pg/ml、132pg/ml±23pg/ml、117pg/ml±28pg/ml,均P<0·01),IL-4的水平较高(36pg/ml±7pg/mlvs16pg/ml±4pg/ml、26pg/ml±4pg/ml、24pg/ml±4pg/ml,均P<0·05),D、E组肿瘤坏死因子α(TNF-α)水平相近,但明显低于B、C组(均P<0·01),并且E组对宿主抗原的淋巴细胞增殖均低于B、C、D组(0·22±0·08vs0·87±0·14、0·54±0·21、0·49±0·18,均P<0·01)。结论rhIL-11联合rhG-CSF用于供者能产生协同作用,减轻GVHD,其机制可能与受者体内T细胞向Th2细胞因子极化、炎症介质如TNF-α降低有关。     

人胎肝细胞对BALB／c小鼠免疫系统及其功能的影响

This study sought to better understand the effects of human fetal liver cells(FLC) on immunity. FLC were injected into BALB/c mice intraperitoneally. The design included four groups: negative control and three dose (125 x 10(5), 25 x 10(5), 5 x 10(5) FLC/day) groups. The results showed that FLC increased the thymus visceral coefficient, numbers of T-lymphocytes(TLC) and lymphocytes(LC) in blood, total number of spleen cells, the number of plaque forming cells(PFC) of spleen, the rate(%) of transformation and stimulus index of TLC of spleen, and the function of macrophages. FLC also effected the function of natural killer cells(NK cells) of BALB/c mice. The findings suggest that human FLC can strengthen the nonspecific immunity, humoral immunity and cellular immunity in mice. In this study, however, no effect of FLC on spleen visceral coefficient was noticed, and no sign of delayed type allergy was observed.     

异基因骨髓移植后小鼠虹膜睫状体内抗原递呈细胞的分布

目的:观察异基因骨髓移植后小鼠虹膜睫状体内抗原递呈细胞(APC)的分布情况。方法:绿色荧光蛋白(GFP)转基因C57BL/6J(H-2b)小鼠作为供体,经5.5Gy×2剂量照射24h的BALB/c(H-2d)小鼠作为宿主,于骨髓移植后21d和35d取出宿主小鼠眼球,分离得到虹膜睫状体,荧光显微镜下观察供体骨髓来源的细胞分布。同时采用免疫组织化学染色方法观察普通和移植后小鼠F4/80阳性细胞在虹膜中的分布规律。结果:异基因骨髓移植后21d和35d,宿主虹膜睫状体内可观察到大量呈树突状形态的绿色荧光细胞,呈“满天星”样散在分布于小鼠虹膜睫状体中,这些细胞F4/80抗体免疫组织化学荧光染色多为阳性。结论:虹膜睫状体中的APC细胞来源于骨髓,且大部分为F4/80阳性细胞;异基因骨髓移植后宿主虹膜睫状体中的APC细胞全部被供体来源的APC细胞取代。     

Graft-versus-leukemia effects from donor lymphocyte infusion after nonmyeloablative allogeneic bone marrow transplantation in mice

Background Nonmyeloablative allogeneic bone marrow transplantation has been used since the 1990s as a new hematological stem cell transplantation strategy for treating hematological diseases. The purpose of this study was to explore the graft-versus-leukemia (GVL) effects of donor lymphocyte infusions (DLIs) after nonmyeloablative allogeneic bone marrow transplantations, while assessing the declines in treatment-associated morbidity, mortality, and graft-versus-host disease (GVHD).Methods A total of 615 (H-2k) mice were injected with L615 tumor cells and received 500 cGy (60Coγ-ray) irradiation three days later, followed by an allogeneic bone marrow transplantation (allo-BMT). The allo-grafts consisted of 3×10（7） bone marrow cells and 1×10（7） spleen cells from BALB/C (H-2d) donor mice. Two days after the allo-BMT, the recipient mice were given 200 mg/kg of cyclophosphamide. Subsequently, recipient mice were infused with either donor spleen cells ［2×10（7）］ on day 14 or 21, or donor spleen cells ［5×10（7）］ pretreated with hydrocortisone and cyclosporin A (CsA) in vitro on day 14 post-BMT.Results The median survival time of mice that received DLI on day 21 and pretreated DLI on day 14 post-BMT was longer than that of controls and the day 14 DLI group (P&lt;0.01). No evidence of severe GVHD was observed in the day 21 DLI group nor in the day 14 treated DLI group. Mixed chimerism was confirmed in the day 14 DLI group, the day 14 treated DLI group, and the day 21 DLI group on the thirteenth day post-transplantation; full donor chimerism was observed two weeks after DLI.Conclusion Donor lymphocyte infusion after nonmyeloablative bone marrow transplantation may reduce transplantation-associated morbidity and mortality while strengthening graft-versus-leukemia effects.     

联合氟达拉滨和环磷酰胺的预处理减轻急性移植物抗宿主病的实验研究

 目的 探讨联合氟达拉滨与环磷酰胺的预处理对过继性淋巴细胞植活和急性移植物抗宿主病程度的影响。方法 受鼠为8~10周雌性BALB/C小鼠,供鼠为8~10周雄BALB/C×C57BL/6→F1小鼠。根据预处理方式的不同将受鼠分为5组（n=12）：联合应用氟达拉滨和环磷酰胺组、单用氟达拉滨组、单用环磷酰胺组、全身照射组以及空白对照组即应用生理盐水组。预处理结束后第2天过继性植入5×10⁷个供鼠脾细胞,检测供鼠脾淋巴细胞在受鼠外周血中的嵌合率,观察生存时间、急性移植物抗宿主病临床表现。结果 联合应用氟达拉滨和环磷酰胺组小鼠的脾淋巴细胞嵌合率与全身照射组相似,急性移植物抗宿主病的临床表现明显减轻（P<0.05）。结论 联合应用氟达拉滨和环磷酰胺能够促进过继性同种异基因淋巴细胞植活,可减轻急性移植物抗宿主病。     

全不相合与全相合骨髓细胞在致敏模型的归巢与植入

【目的】 探讨全不相合与全相合供者骨髓细胞在致敏模型的归巢与植入情况。【方法】 以异基因脾细胞输注致敏的BALB/c小鼠作为移植受者,致敏模型经8 Gy 60Co照射后分别经尾静脉移植1 × 107 C57BL/6或BALB/c供者的骨髓细胞。予CFSE标记供者骨髓细胞,并分别在移植后不同时间点(2、12及48 h),通过病理切片及组织细胞悬液动态示踪供者细胞在致敏受者各组织的分布。移植后记录各组的生存情况,每周监测造血重建与骨髓恢复情况。【结果】 归巢实验表明C57BL/6骨髓细胞在致敏受者股骨的分布随时间推移先增加而后减少,在脾脏的分布随时间推移呈进行性减少。BALB/c骨髓细胞在致敏模型股骨及脾脏的分布均随时间推移而增加。接受C57BL/6骨髓细胞的致敏模型于移植第12 ~ 15天内全部死亡,血象及骨髓象呈进行性下降;而接受BALB/c骨髓细胞的能长期存活,血象及骨髓象能迅速恢复。【结论】 全不相合的供者骨髓细胞在致敏模型中完全被排斥,而全相合供者的骨髓细胞能在致敏模型中能进行有效的归巢与植入。     

黄芪在同种移植排斥中对CD4+ CD25+ T细胞及Foxp3的影响

探讨黄芪(AST)在同种移植排斥中对CD4+CD25+T细胞动态变化及Foxp3表达的影响。方法：C57BL/6和BALB/c小鼠分别为供体和受体,建立皮肤移植模型。对照组：术后注射生理盐水;AST组：术后给予黄芪注射液;AST+脾细胞(SP)组：术前输注供体鼠脾细胞,术后给予黄芪注射液,CsA+SP组：术前输注供体鼠脾细胞,术后给予环孢素A（CsA）。术后逐日观察移植皮肤存活情况及小鼠一般状况;免疫荧光染色流式细胞术检测脾组织中CD4+CD25+T细胞动态变化;RT PCR检测Foxp3mRNA表达情况。结果：与对照组相比,AST组、AST+SP组、CsA+SP组移植皮片存活时间明显延长（P＜0.05）,移植后14?d,CD4+CD25+T细胞数量及Foxp3的相对表达量显著增多（P＜0.05）。结论：黄芪体内用药可延长同种移植物的存活时间,上调CD4+CD25+T细胞及Foxp3的活性。     

丙泊酚对电离辐射引起小鼠造血系统损伤的防护作用

目的：观察丙泊酚对电离辐射引起造血系统损伤防护作用。方法实验采用3种照射剂量：生存率实验照射剂量为7.5 Gy、脾结节形成实验照射剂量为6 Gy、外周血细胞计数和骨髓单侧股骨细胞计数实验照射剂量为2 Gy;生存率实验分为4组：7.5Gy组、7.5Gy+5 mg/kg丙泊酚组、7.5Gy+10 mg/kg丙泊酚组、7.5Gy+20 mg/kg丙泊酚组。其余实验分为4组：对照组、丙泊酚20 mg/kg组、照射组（2Gy或6Gy）、照射+丙泊酚20 mg/kg组。给药方式为照射前1 d、照射当天提前30 min各给药一次和照射后7d连续腹腔注射,每日1次,照射后第9天活杀小鼠,检测脾结节形成数,各类外周血细胞数及单侧股骨骨髓细胞数。结果丙泊酚20 mg/kg能提高受照射小鼠30 d生存率,增加受照射小鼠脾结节形成数、增加受照射小鼠外周血白细胞数、红细胞数、血红蛋白含量及小鼠单侧股骨骨髓有核细胞数,与照射对照组比较,结果有统计学意义（P＜0.05）。结论丙泊酚对电离辐射引起的造血系统损伤均有保护作用,其作用机制有待进一步研究。     

Anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways act as potent immunoregulatory cells in vitro and vivo

Background This study was to evaluate whether anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro and prolong cardiac allograft survival after adoptive transfer.Methods Anergic cells were induced in vitro by the addition of anti-CD154 and anti-CD80 monoclonal antibodies (mAbs) to primary MLR (mixed lymphocyte reaction) consisting of BALB/c as responder and C3H as stimulator. Anergic cells were added to a newly formed MLR in assessing the regulatory capacity and antigen specificity of anergic cells. The ability of anergic cells to respond to antigen and/or exogenous recombinant mouse interleukin-2 (rmIL-2) was tested. For in vivo studies, anergic cells were intravenously injected into 3.0-Gy γ-irradiated BALB/c mice immediately after heterotopic abdominal cardiac transplantation. To prolong allograft survival, recipient mice injected with anergic cells received rapamycin therapy ［1 mg·day（-1）·kg（-1）］.Results Anergic cells strongly suppressed the proliferation of naǐve BALB/c splenocytes against the original (C3H) stimulator in a dose-dependent manner, but they failed to suppress the proliferation of naǐve BALB/c splenocytes against the third-party （C57BL/6J） stimulator. The anergic state was reversed by both original (C3H) stimulator and additional exogenous IL-2. In in vivo studies, untreated irradiated BALB/c mice rejected C3H cardiac allografts with a mean survival time of (8.6±1.1) days, whereas those injected with the anergic cells rejected the allografts with a mean survival time of (11.8±1.9) days, which was slightly longer than that of the untreated mice. The protocol based on anergic cells injection plus rapamycin therapy could prolong allograft survival significantly ［(29.6±4.4) days］. Conclusions Anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro, and prolong cardiac allograft survival after adoptive transfer in the presence of rapamycin therapy. This procedure might be clinically useful for prolonging allograft survival if optimal protocols are developed.     

激活芳香烃受体抑制同种异体小鼠心脏移植物急性排斥反应

目的:验证在小鼠心脏移植中,2,3,7,8-四氯二苯二氧芑(TCDD)激活芳香烃受体(AHR)是否可以诱导调节性T细胞(Treg)扩增以及减轻急性排斥反应。方法:建立小鼠心脏移植模型,给予TCDD,观察对排斥反应及移植物生存期的影响。体外实验评估TCDD对Treg细胞比例的影响。检测受者体内Treg细胞比例及白细胞介素(白介素)-10表达水平。结果:TCDD激活AHR明显减轻心脏移植物内急性排斥反应,延长移植物存活时间[MST=(23.5±7.7)d]。体外实验中TCDD明显提升CD4+CD25+Foxp3+调节性T细胞比例[TCDD组(15.3±2.6)%;PBS组(4.7±2.4)%,P<0.01)],而受者体内脾脏和移植物内Treg细胞比例相比对照组也明显升高(P<0.05)。同时,TCDD明显提升了受者体内白介素-10的表达水平。结论:术前单次给予TCDD激活AHR可以明显抑制小鼠同种心脏移植物急性排斥反应,其机制可能与扩增Treg亚群有关。     

ICOS-Ig combined with CsA induces long term survival of cardiac allografts in mouse

Objective： To study the synergistic effect of ICOS-Ig combined with cyclosporine （CsA） on mouse heart transplantation and explore its therapeutic potential. Methods： ICOS-Ig fusion protein was generated by fusing the extraeellular portion of human ICOS and Fc portion of human IgG. To investigate the effect of ICOS-Ig on T-cell proliferation in vitro, ICOS-Ig or IgG was added to the primary MLR cultures （BALB/c spleen T cells as responder cells and irradiated C57BL/6 spleen cells as stimulator cells）. The cells responsiveness rates were detected by 3H-TdR methods. Then the T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 （donor） or C3H （third party） spleen cells as stimulator cells. To study the effect of ICOS-Ig on T-cell proliferation in vivo, CFSE-labeled C57BL/6 spleen cells were transferred to irradiated BALB/c mice. Mice were then treated with IgG, ICOS-Ig or CsA. Seventy two hours after transfer, the spleen cells of the mice were harvested for the detection of CD4^＋CFSE^＋ and CD8^＋CFSE^＋ by FACS. C57BL/6 mouse underwent transplantation of the hearts of BALB/c mouse and were then randomly divided into five equal groups： no treatment group, control lgG treated group （250 gg i.p. d2, 4, 6）, ICOS-Ig treated group （250 μg i.p. d2, 4, 6）, CsA treated group （10 mg/kg i.p. d0-6）, ICOS-Ig combined with CsA group. The cardiac allograft survival was monitored by daily palpation. Results： In primary MLR, ICOS-Ig inhibited T-cell proliferation, （inhibition ratio 58i8.2% in 50 μg/ml）. In secondary MLR, ICOS-Ig specifically inhibited donor spleen cells, which suggested ICOS-Ig could induce donor-specific hyporesponsiveness. In the CFSE dye assay, CD4^＋CFSE^＋ and CD8^＋CFSE^＋ in ICOS-Ig and CsA group was stronger than those in control group, which showed ICOS-Ig and CsA could inhibit the proliferation of allo-reactive T cells in vivo. In mouse heart transplantation model, survival was significantly prolonged in animals treated with ICOS-Ig or CsA as compared with controls. Moreover, ICOS-Ig combined with CsA group had even longer engraftment （〉100 d） than ICOS-Ig or CsA used alone. In histological examination, it was found that there were congestions and edemas in no treatment and IgG treated recipients, together with a lot of inflammatory cells infiltrated. Allogeneic hearts from ICOS-Ig and/or CsA immunized recipients revealed milder histological changes. It was revealed in mechanical analysis that splenic T cells from recipients also exhibited depressed mixed leukocyte reactions （MLR） and cytotoxic lymphocyte reactions （CTL）. Conclusion： These data suggest that ICOS-Ig combined with CsA induces a long-term survival of mouse cardiac allografts, whereas monotherapy is less effective in this regard. Thus, ICOS-Ig combined with CsA treatment may be a novel regimen to combat allograft rejection.     

父方否决细胞对小鼠半相合干细胞移植模型移植物抗宿主病的预防作用

目的 探讨父方否决细胞对小鼠半相合干细胞移植模型移植物抗宿主病(GVHD)是否起预防作用.方法 以B6CF1(H-2 b/d)作为半相合干细胞移植的受鼠,给予9.0 Gy全身照射后4 h,单纯照射组经尾静脉注射0.3 ml D-Hank's 液,不进行细胞移植;对照组经尾静脉注射C57BL/6小鼠混合细胞悬液(4.5×106骨髓细胞 3.0×107脾细胞),但不进行其他治疗处理;实验组于移植4 d后经尾静脉注射BALB/c小鼠脾细胞悬液(数量分别为5.0×106、1.0×107),然后观察其造血恢复、植入及移植物抗宿主病(GVHD)的情况.结果 由于没有给予GVHD防治措施,对照组受鼠GVHD的发生率和死亡率分别为10/10和10/10;而1/6量否决细胞组小鼠GVHD的发生率为5/10,30 d生存率增加至5/10(P＜0.01),5只因GVHD死亡的受鼠中位生存期为20 d,较对照组(中位生存期14 d)有显著性差异(P＜0.05);1/3量否决细胞组小鼠GVHD的发生率为2/10,30 d生存率增加至8/10,中位生存期延长至30 d以上(P＜0.05).结论 半相合干细胞移植后输注父方否决细胞,可以诱导特异性免疫耐受,显著降低半相合干细胞移植GVHD的发生率.     

B7反义肽预处理脾细胞诱导异基因嵌合体形成及延长移植物存活

目的　尝试以B7 CD2 8/CTLA4为靶点 ,用B7反义肽 (B7AP)预处理供者的脾细胞 ,免疫受者后结合输注供者骨髓细胞 ,诱导受体形成异基因嵌合体。方法　每只用 5× 10 6/ 10 0 μlB7AP预处理的供者小鼠 (C5 7BL/ 6 )脾细胞免疫受者小鼠 (BALB/c) ,诱导特异性同种低免疫应答反应 ;在此基础上每只受者小鼠输注供者骨髓细胞 2× 10 7/ 10 0 μl,诱导受者形成异基因嵌合体 ,以流式细胞术 (FACS)连续动态监测嵌合状态的维持情况并用小鼠心肌移植模型研究移植物存活的影响因素。结果　在受者体内成功诱导出了特异性同种低免疫应答反应 ,应答水平只有正常未免疫组的 4 3%(n =6 ,P <0 0 0 1) ;嵌合状态可以维持到 10 0d以上 (n =6 ) ,15 0d时嵌合率仍有 3.4 5 % ;嵌合小鼠进行同种异体小鼠心肌移植存活超过 10 0d(n =6 ) ,而对照组阿霉素组只能延长至 16d(n =6 )。结论　B7AP预处理小鼠脾细胞可诱导形成异基因嵌合体 ,并显著延长同种小鼠心肌移植存活的时间 ,为移植免疫的研究提供了一个简单易行的嵌合体平台技术 ,也为用B7AP在移植免疫中的应用进行了有益的尝试。