弓形虫可溶性抗原混合液延长小鼠移植心脏存活时间

目的 探讨弓形虫可溶性抗原混合液(STAgs)延长小鼠移植心脏存活时间的作用及其作用机制.方法 通过在冰浴中超声粉碎弓形虫速殖子制备弓形虫STAgs.实验分为3组,每组受者9只.STAgs组和急性排斥反应(AR)组:供者为Balb/c小鼠,受者为C57BL/6小鼠,移植前4 d两组受者分别皮下注射STAgs 5μg和磷酸盐缓冲液100μl,同系对照组供、受者均为C57BL/6小鼠,术前未进行任何处理.分组后建立小鼠颈部异位心脏移植模型.术后观察移植心脏存活时间,术后第7天每组处死3只受者,获取移植心脏行病理学检查观察排斥反应,采用免疫组织化学检测移植心中CD4+和CD8+T淋巴细胞.结果 同系移植组在观察终点100 d时均存活,AR组和STAgs组移植心脏存活时间分别为(6.7±0.5)和(70.8±3.5)d,3组间两两比较,差异均有统计学意义(P＜0.05).术后第7天,同系移植组、AR组和STAgs组移植心排斥反应分级分别为0级、Ⅲ～Ⅳ级和0～Ⅰ级;免疫组织化学检测显示STAgs组CD4+和CD8+T淋巴细胞比例明显少于AR组,差异有统计学意义(P＜0.05).结论 弓形虫STAgs能显著延长小鼠移植心脏的存活时间,减轻移植心脏的排斥反应,县体机制可能与弓形虫STAgs可影响TH1/TH2比例相关,也可能通过刺激机体产生脂氧素A4抑制树突状细胞活化发挥作用.

Abstract：

Objective To investigate the effects of T. gondii soluble tachyzoite antigen (STAgs) on the survival time of mouse heart allograft and the possible mechanism. Methods The STAgs were prepared by pulverizing T. gondii tachyzoite with ultrasound on ice. Cervical heterotopic heart transplantations were done by using Balb/c mice as donors, and C57BL/6 mice as recipients.The recipients were classified randomly into three groups: syngeneic group, acute rejection group and STAgs-treated group. The recipients in acute rejection group and STAgs-treated group were injected subcutaneously with 0. 1 ml PBS and 0. 1 ml (5 μg) STAgs at the 4th day before transplantation respectively, and those in syngeneic group were not subjected to any treatment. The grafts were observed daily by cervical palpation, and the total cessation of cardiac contraction was defined as the endpoint. The heart allografts were harvested at the 7th day after transplantation for pathological examination and immunohistochemical staining for CD4+ T, CD8+ T. Results The recipients in syngeneic group were all alive at the 100th day after transplantation. The average survival time in acute rejection group and STAgs-treated group was (6.7± 0.5) days and (70.8± 3.5) days,respectively (P＜0.05). HE staining showed that the rejection on the 7th day after transplantation in syngeneic group, acute rejection group and STAgs-treated group was fallen into 0 degree, Ⅲ-Ⅳ degree and 0- Ⅰ degree, respectively. Immunohistochemical staining revealed that the CD4+ T and CD8+T were markedly down-regulated in STAgs-treated group as compared with those in acute rejection group. Conclusion T. gondii STAgs can significantly prolong the survival time of mouse heart allograft and inhibit the rejection probably by changing the ratio of TH1/TH2, or inhibiting the effect of dendritic cells by inducing the lipoxin A4.     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.

Abstract：

Objective To investigate the expression of CXCR6 in allograft rejection and effect of CXCL16/CXCR6 interaction on allograft survival Methods Intra-abdominal heterotopic heart transplantation was performed using wild type (WT) Balb/c mice (H-2d) (allogeneic) as donors or WT C57BL/6 mice (B6, H-2b) (syngeneic) as donors, and using WT B6 mice as recipients. The intragraft expression of CXCR6 and expression of CXCR6 in CD8+ T cells of the spleens from syngeneic and allogeneic recipients were examined. The allogeneic recipients were further divided into the experimental group (n = 5) and control group (n = 6) randomly. The experiment group and control group were injected with anti-CXCL16 mAb or control mAb respectively until rejection occurred. The cardiac allograft survival in experimental group and control group was evaluated. Results Rejected allografts showed higher expression of CXCR6 than syngeneic cardiac grafts. More importantly,expression of CXCR6 in CD8+ T cells was also up-regulated by allograft rejection. However, injection of anti-CXCL16 mAb could not inhibit cytotoxic activity of CD8+ T cells. Moreover, experimental group could not prolong the cardiac graft survival time as compared with control group. Conclusion Expression of CXCR6 in CD8+ T cells is up-regulated in allograft rejection.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

热休克蛋白60诱导小鼠皮肤移植耐受的实验研究

目的探讨热休克蛋白60（heat shock protein60，HSP60）对小鼠移植皮片成活的影响及其机制。方法选用60只C57BL／6（H．2b）小鼠为受体，45只BALB／C（H．2d）小鼠、15只CBA／N（H-2^k）为供体，均为8～12周龄近交系雌性小鼠。受体小鼠剪下1cm&#215;1cm全层皮肤，干纱布清创，即为植床：随机分为4组，每组15只。A组：无菌取供体BALB／C（H．2d）小鼠背部1cm&#215;1cm全层皮片，刮去皮下组织后移植至受体小鼠背部；B组：受体小鼠背部皮下注射0．1mL不完全弗氏佐剂（imcompleted Freund’s adjuvant，IFA），2周后行BALB／C（H-2d）小鼠皮肤移植：C组：受体小鼠背部皮下注射经0．1mLIFA乳化后的50gtgHSP60，2周后行BALB／C（H-2^d）小鼠皮肤移植：D组：受体小鼠背部皮下注射经0．1mL IFA乳化后的50μg HSP60，2周后行CBA／N（H-2^k）小鼠皮肤移植。B、C、D组供体皮肤移植方法同A组。术后观察移植皮片成活时间；移植术后7、25d行单向混合淋巴细胞反应及混合淋巴细胞培养上清液细胞因子检测；术后7d行迟发型超敏反应测定。结果A、B、C、D组移植皮片成活时间分别为（12．4&#177;0．5）、（11．6&#177;0.8）、（29.3&#177;2．6）及（27．6&#177;2．1）d：A、B组与C、D组比较，差异有统计学意义（P〈0．05），A、B组间及C、D组间比较，差异无统计学意义（P〉0．05）。皮肤移植术后7d，A、B、C、D组混合淋巴细胞反应每分钟放射脉冲数（counts of perminute impulse，cmp）值分别为12836&#177;1357、11876&#177;1265、6581&#177;573及6843&#177;612；A、B组与C、D组比较，差异有统计学意义（JP〈0．05）：A、B组间及C、D组间比较差异无统计学意义（JP〉0．05）；术后25d，各组cpm值分别为13286&#177;1498、12960&#177;1376、11936&#177;1265及12374&#177;1269，各组间差异无统计学意义（P〉0．05）。皮肤移植术后7d，C、D组混合淋巴细胞培养上清液IL-10高于A、B组，IL-2、干扰素γ（interferonl，，IFN-γ）低于A、B组（JP〈0．05），A、B组间及C、D组间差异均无统计学意义（JP〉0．05）：术后25d，各组IL-2、IL-10及IFN-γ差异无统计学意义（P〉0．05）。皮肤移植术后7d，A、B、C、D组受体小鼠对供体小鼠脾细胞的迟发型超敏反应为（0．84&#177;0．09）、（0.81&#177;0．07）、（0．43&#177;0．05）及（0．46&#177;0．03）mm：A、B组与C、D组比较，差异有统计学意义（P〈0．05），A、B组间及C、D组间差异无统计学意义（P〉0．05）。结论HSP60对免疫耐受的诱导和维持有一定作用。     

第三方骨髓间充质干细胞对同种异体皮肤移植的影响

目的 探讨第三方骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)对同种异体皮肤移植的影响.方法 40只雌性C57BL/6和50只雄性BALB/C小鼠分别作为皮肤移植的供体和受体.将50只BALB/C小鼠随机分为5组,每组10只:空白对照组,直接进行皮肤移植;给予受体环磷酰胺组(Cyelophosphamide,CP组);给予受体SD大鼠BMSCs移植组(SD-BMSCs组);给予受体CP+SD-BMSCs移植组(CP+SD-BMSCs组);CM-DiI荧光标记组.CP+SD-BMSCs组给予大剂量CP腹腔注射,200 mg/kg,2 d,移植当天自受体小鼠尾静脉注射1×10~6个SD-BMSCs.检测项目包括:观察皮片存活时间、单向混合淋巴反应、组织HE染色、组织荧光镜检,流式细胞仪检测受体外周血单核细胞中SD-BMSCs的含量等.结果 CP+SD-BMSCs组皮肤移植物存活时间(14.5±1.9)d,空白对照组为(8.O±0.8)d,CP组为(11.1±1.4)d,SD-BMSCs组为(10.9±1.4)d,CP+SD-BMSCs组皮肤移植物存活时间明显比后3组延长(P<0.05).混合淋巴反应结果显示,BMSCs对淋巴细胞的增殖抑制作用存在量效关系.HE染色发现CP+SD-BMSCs组不但有血管生成,淋巴细胞浸润也较少.CM-DiI标记的SD-BMSCs能够在BALB/c小鼠体内存活30 d以上,SD-BMSCs组与CP+SD-BMSCs组外周血流式细胞仪均检测到SD-BMSCs.结论 BMSCs能够在异种受体体内存活较长时间;第三方BMSCs能够延长同种异体移植物的存活时间;BMSCs能够抑制异种T淋巴细胞的活化增殖.     

细胞毒性T淋巴细胞相关抗原4诱导新鲜异体骨移植免疫耐受的实验研究

目的　利用外源性细胞毒性T淋巴细胞相关抗原4 ( cytotoxic T lymphocyte- associated antigen 4immuno globlin,CTL A4 - Ig)阻断T细胞反应的第2信号,诱导新鲜同种异体骨移植免疫耐受。　方法　采用近交系小鼠BAL B/C 6 6只作为骨移植的受体,进行异位肌袋一次及第2次骨移植。一次移植分为3组,每组18只,一组移植C5 7BL/6小鼠骨骼,注射L6 (对照品) ,为AL组;一组移植C5 7BL/6小鼠骨骼,注射CTL A4 - Ig,为AC组;一组移植同系BAL B/C小鼠骨骼,注射PBS缓冲液,为AB组。在第2、4及6周,进行血淋巴细胞亚群分析,血清抗体测定,供体细胞及抗原二次刺激实验及组织学观察。另取12只BAL B/C小鼠,进行第2次移植实验,供体为C5 7BL/6小鼠的骨骼,注射CTL A4 - Ig后2周,分别移植C5 7BL/6 ( BC组)和C3H( BH组)小鼠的骨骼,检测产生免疫耐受的特异性。　结果　AL组与AB组比较,产生了强烈的免疫排斥反应,移植后2、4及6周,CD4 T细胞的增殖明显增加( P<0 .0 5 ) ,血清抗体明显增多( P<0 .0 5 ) ,供体细胞及骨抗原二次刺激的细胞增殖也明显增加( P<0 .0 5 ) ;AC组免疫排斥反应较轻,在CD4 T细胞的增殖、血清抗体及二次刺激的细胞增殖方面均与AB组相似( P>0 .0 5 ) ;组织学观察也显示,异体骨周围的淋巴细胞浸润消失,软骨诱导及新骨形     

自体骨髓间质干细胞对异基因小鼠皮肤移植免疫排斥的影响

目的 观察自体骨髓间质干细胞(bone marrow stromal cells,BMSCs)对异基因小鼠皮肤移植的影响.方法 建立C57BL/6至BALB/c小鼠皮肤移植急性排斥模型,密度梯度离心法分离与贴壁法富集BALB/c小鼠BMSCs,自尾静脉输注.实验分3组,每组10只小鼠.A组:正常BALB/c小鼠+自体BMSCs,B组:仅行皮肤移植,C组:皮肤移植后BALB/c小鼠+自体BMSCs.检测各时间段皮肤组织病理表现以及相关细胞因子浓度变化.结果 由密度梯度离心法与贴壁法分离的细胞具有BMSCs的一般特征,C组较B组移植后皮肤免疫排斥反应明显减轻,IL-2、IFN-γ等细胞因子浓度在术后第7天(F=248 954.6,P＜0.05;F=148 311.7,P＜0.05)、第14天(F=117 372.3,P＜0.05:F=126 743.3,P＜0.05)均有降低.结论 异基因小鼠皮肤移植术后输注自体BMSCs可以抑制免疫排斥反应,其机制可能与减少IL-2、IFN-γ等相关细胞因子分泌有关.     

Beta7 integrins contribute to skin graft rejection

BACKGROUND: Because integrins alpha4beta7 and alphaEbeta7 contribute to epidermotropism of T-cells during skin inflammation, we sought to study their role in skin allograft rejection. METHODS: Wild-type (WT) (beta7+/+) and beta7 gene knockout (beta7-/-) C57BL/6 (H-2(b)) mice and SJL/J (H-2(s)) mice served as donors and recipients of allogeneic skin grafts. An anti-integrin beta7 subunit mAb (FIB504.64) was used to treat WT beta7+/+ C57BL/6 recipients of skin grafts from SJL/J mice. RESULTS: WT C57BL/6 recipients acutely rejected skin from SJL/J mice in 13 days. In contrast, the survival of SJL/J skin on either beta7-/- gene knockout or WT C57BL/6 recipients treated with anti-beta7 subunit mAb, was prolonged by 6 to 7 additional days (P<0.01). The survival of skin allografts from either beta7-/- or beta7+/+ C57BL/6 mice received by SJL/J recipients was not prolonged (P >0.05). CONCLUSIONS: Beta7 integrins contribute to skin graft rejection, in accord with their role in mediating the epidermotropism of T-cells during skin inflammation.     

Targeted gene disruption of the heat shock protein 72 gene (hsp70.1) in the donor tissue is associated with a prolonged rejection-free survival in the murine skin allograft model

It is known that the expression level of the heat shock protein (HSP) is elevated in allograft tissues, but the specific role of the HSP in the acute rejection has not been elucidated. This study aims to determine how and when the HSP72 molecule works immunologically in the process of acute allograft rejection from a skin graft model in which HSP72 (hsp70.1 gene) knock out (KO) mice were adopted either as a donor or as a recipient. In experiment I, tail skin was grafted from either the HSP72 KO C57BL/6 mice or wild-type C57BL/6 mice--as the allograft control--onto the trunk of B10BR mice. The grafts were observed for any signs of rejection until the 14th day after the graft. The survival of the grafted skin from the two donor groups was analyzed by a log-rank method. The grafted skin was observed for the degree of rejection using light microscopy. In addition, the degree of apoptosis was assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). A mixed lymphocyte reaction (MLR) was observed with HSP72 KO C57BL/6 splenocytes as the stimulator and B10BR mice lymphocytes as the responder cells. The cytokine levels, such as interleukin-2 (IL-2) and interferon-gamma, were measured from an MLR supernatant using enzyme-linked immunosorbent assay (ELISA). In experiment II, the tail skin was grafted in the opposite direction from the B10BR mice to the HSP72 KO or wild-type C57BL/6 mice, and the grafts were observed for any signs of rejection, as defined in experiment I. The absence of HSP72 expression was observed in the HSP72 KO mice lymphocytes after heat stress (42 degrees C) using Western blot analysis. In experiment I, the survival of the skin grafts from the HSP72 KO C57BL/6 mice was 12+/-1.3 days (median+/-S.E.), which was significantly longer than that from the allograft control donor (9+/-0.6 days; p=0.03). This coincided with the microscopic finding of the graft tissues. The MLR response of the CD4+ lymphocytes stimulated by HSP72 KO C57BL/6 splenocytes was lower, and the interferon-gamma concentration from the MLR supernatant was also lower. On Day 9, 3.7+/-1.1(mean+/-S.D.) TUNEL-positive cells per unit high-power field (HPF) were observed from the HSP72 KO C57BL/6 skin, which was significantly lower than that from the control skin (8.7+/-2.1 cells/HPF; p=0.045). In contrast, in experiment II, there was no difference in the survival of the skin graft either from the B10BR to HSP72 KO C57BL/6 mice or from the B10BR to the wild-type C57BL/6 mice. In summary, the survival of the skin grafts from the HSP72 KO C57BL/6 mice was prolonged, and the degree of rejection was lower than that from the allograft control. This means that the presence of HSP72 in the donor tissue is related to the up-regulation of acute rejection from the recipient immune system. The HSP72 KO mice as the donor were shown to have decreased level of antigen presentation to the recipient CD4+ lymphocytes. Therefore, the HSP72 molecule from the donor cells is believed to be related to the induction of the recipient immune reaction via alloantigen presentation.     

胸腺内注射可溶性异基因主要组织相容性复合物抗原诱导皮肤移植特异性耐受

用３ｍｏｌ／ＬＫＣｌ从Ｃ５７ＢＬ／６小鼠脾细胞提取可溶性主要组织相容性复合物（ＭＨＣ）抗原，注射到ＢＡＬＢ／Ｃ受体鼠胸腺内，诱导了成年小鼠对该异基因小鼠皮肤移植物的耐受。除在胸腺注射当天及第３天给予抗Ｔ细胞单克隆抗体外，不使用免疫抑制剂。实验组移植皮肤平均存活时间（ＭＳＴ）为８３天，对照组ＭＳＴ为１１天。诱导耐受的小鼠对第３供体的移植皮肤仍正常排斥（ＭＳＴ为１２天）。单向混合淋巴细胞反应，耐受小鼠脾脏淋巴细胞对特异供体的脾细胞无反应，对第３供体的脾细胞反应正常，对丝裂原刺激的增殖反应正常。显示诱导的耐受是供体特异性的，无非特异性免疫抑制。     

肝细胞移植治疗急性肝功能衰竭的免疫排斥研究

目的研究肝细胞腹腔移植治疗急性肝功能衰竭的免疫排斥反应。方法用胶原酶灌注法分离幼猪及BALB/c和C57BL/6小鼠肝细胞;用腹腔注射CCl4的方法建立C57BL/6急性肝功能衰竭模型;将实验动物分为同基因移植组、同种异基因移植组和异种移植组。各组动物肝细胞移植入急性肝功能衰竭小鼠腹腔内,观察受体小鼠存活情况,比较各组动物T细胞亚群、免疫球蛋白水平和细胞因子的变化。结果①受体小鼠存活情况:同基因移植组为8/10,同种异基因移植组为6/10,异种移植组为3/10,同基因移植组和同种异基因移植组受体的生存情况明显好于异种移植组(P<0.05)。②T细胞亚群变化:移植后7 d内,同基因移植组外周血中CD4+和CD8+T细胞均无明显变化,同种异基因移植组CD4+T细胞于移植后3 d时达高峰(P<0.05),而CD8+T细胞7 d内无明显变化,异种移植组CD4+和CD8+T细胞移植后7 d内均明显升高,且于移植后3 d时达高峰(P<0.05)。③免疫球蛋白水平:同基因移植组血清免疫球蛋白IgM和IgG水平在移植后0.5、1和3 d时未见明显变化,同种异基因移植组和异种移植组的IgM在移植后1 d时达高峰(P<0.05),而IgG在移植后3 d时达高峰(P<0.05)。④血清细胞因子水平:移植后7 d,同种异基因移植组和异种移植组的IFN-γ、TNF-α及IL-2血清浓度明显高于同基因移植组(P<0.05);异种移植组的IL-6血清浓度明显高于同基因移植组和同种异基因移植组(P<0.05)。结论无论同种还是异种肝细胞移植,初期均发生了强烈的CD4+及CD8+T细胞参与的细胞免疫和较强体液免疫反应。     

记忆性CD4+T淋巴细胞对小鼠腹腔移植心脏排斥反应的影响

目的 探讨记忆性CD4+T淋巴细胞对来自同一抗原特异性移植心脏急性排斥反应的影响.方法 将C57BL/6小鼠的皮肤移植给Balb/c小鼠,使其致敏,3个月后,取受者的脾脏,应用小鼠记忆性CD4+T淋巴细胞纯化试剂盒纯化记忆性CD4+T淋巴细胞.同时取未进行皮肤移植的Balb/c小鼠,同法获取非致敏CD4+T淋巴细胞.以C57BL/6小鼠为供者,正常Balb/c小鼠为受者,行腹腔心脏移植,移植前3周,实验组受者经阴茎背静脉注射记忆性CD4+T淋巴细胞;非致敏对照组受者经阴茎背静脉注射非致敏CD4+T淋巴细胞;空白对照组不注射任何T淋巴细胞.各组均于移植前1 d、移植当天及移植后1 d给予环孢素A.术后记录移植心脏存活时间;取移植心脏,进行病理学观察.结果 实验组、非致敏对照组和空白对照组移植心脏存活时间分别为(8.5±1.5)d、(25.7±5.5)d和(21.2±9.2)d,实验组明显短于另两组(P<0.05).移植心脏急性排斥反应的病理学分级,实验组为3.43±0.68,非致敏对照组为1.29±0.46,空白对照组为1.31±0.49,实验组病理学分级明显高于非致敏对照组和空白对照组(P<(0.05).结论 当记忆性CIN+T淋巴细胞再次接触同一抗原特异性的移植心脏时,可促进急性排斥反应的发生,且对常规剂量的环孢素A不敏感.