骨髓来源半成熟树突状细胞诱导大鼠小肠移植免疫耐受

Objective To study MyD88 silent semi-mature DC (simDC) inducing immune tolerance in rat small bowel transplantation model. Methods All rats were randomly divided into three groups (n=6). Seven days before transplantation of intestine from F344 donors, Wistar recipient rats were injected with simDC (group A) , imDC (group B) and saline ( group C) through the penile dorsal vein respectively.Small bowel transplantation was performed and the survival time of recipients was observed. Serum IL-2 and IFN-γ levels were assayed. Results The survival time of recipients in group A was ( 13.7±1.2) days, which was significantly longer than that in groups B [(8.0±1.0) days,P<0. 05] and C [(6. 0±0. 8) days P<0. 05]. The serum levels of IL-2 and IFN-γ in group A were lower than in groups B and C (P<0. 05). Conclusion Depending on the unique phenotypic and functional features of sem-DC,they can induce the transplantation tolerance and prolong the survival of intestinal allografts after transplantatin.     

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.     

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.     

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.     

骨髓来源半成熟树突状细胞诱导大鼠小肠移植免疫耐受

目的 观察髓样分化因子88(MyD88)沉默的半成熟树突状细胞(DC)诱导大鼠小肠移植模型免疫耐受.方法 供体乃44大鼠,受体Wistar大鼠.随机分为3组(n=6).A组(半成熟DC组)移植前7d经阴茎背静脉注射半成熟MyD88沉默DC(2×106细胞数),B组(未成熟DC组)移植前7 d经阴茎背静脉注射未成熟DC(2×106细胞数)和C组(阴性对照组)移植前7 d经阴茎背静脉注射1 ml生理盐水.3组均于注射7 d后行大鼠异位节段性小肠移植术,观察统计各组受体存活情况,检测受体血清中的促炎症因子白细胞介素(IL)-2、干扰素(IFN)-γ等的变化.结果 半成熟MyD88沉默DC组[(13.7±1.2)d,n=6]较未成熟DC组[(8.0±1.0)d,n=6,P<0.05]和阴性对照组[(6.0±0.8)d,n=6,P<0.05)存活时间显著延长,同时受体血清中IL-2和IFN-γ等促炎症因子的表达明显降低(P<0.05),病理改变亦较轻微.结论 MyD88沉默的半成熟DC具有独特的表形和功能,能够诱导受体产生特异性免疫耐受,显著延长受体术后的存活时间.     

用MyD88 siRNA制备半成熟树突状细胞致免疫耐受的实验研究

目的 探讨利用Toll样受体关键蛋白转录水平抑制剂髓细胞样分化标记物（myeloid differentiation marker 88，MyD88）siRNA，制备半成熟树突状细胞（dendritic cell，DC）的表型和致耐受功能。方法 取BALB／c小鼠骨髓接种、培养，加入浓度为10ng／ml的重组小鼠粒细胞巨噬细胞集落刺激因子（rmGM—CSF）诱导，培养至第8d将DC分为3组，空白对照组：于培养过程中不加其它任何物质；LPS组：加入终浓度为1μg／ml的LPS；实验组：加入MyD88 siRNA4h后，再加入1μg／ml的LPS。采用流式细胞术检测3组DC的表型，用酶联免疫吸附测定（ELISA）检测培养上清的白细胞介素10（IL-10）、白细胞介素12（IL-12）的含量，初次和再次混合淋巴细胞实验检测DC刺激T细胞增殖能力，并观察术后鼠移植心存活时间。结果 空白对照组DC表型为CD11c^＋、CD25、CD40^low、CD80^low、CD86^low和MHC-Ⅱ^low未成熟表型DC，再经LPS刺激后成为成熟表型DC；而实验组DC表型为CD11c^＋、CD25^-、CD40^mid、CD80^low、CD86^low和MHC-Ⅱ^mid。半成熟表型，其IL-10／IL-12比率明显增高；可诱导同种异型T细胞无反应性，并能延长移植心存活时间。结论 MyD88siRNA转染结合LPS刺激可使未成熟DC进一步分化为一种半成熟DC，较未成熟DC拥有更稳定有效的致耐受特性。     

输注转染MyD88siRNA基因的供者树突状细胞延长小鼠移植心存活时间

目的 探讨输注供者来源的转染了髓样分化因子88(MyD88)siRNA基因的树突状细胞(DC)在延长同种小鼠移植心存活时间中的作用及机制.方法 以脂质体为载体,将化学合成的MyD88siRNA导入BALB/c小鼠(供者)骨髓来源的DC中,制备转染MyD88siRNA基因的DC(MyD88siRNA-DC).随机将27只C57BL/6小鼠(受者)平均分为磷酸盐缓冲液(PBS)对照组、培养8 d的DC(Day8-DC)组及MyD88siRNA-DC组,分别将PBS、Day8-DC及MyD88siRNA-DC输注至受者体内.于输注后第7、14和21天时应用免疫双荧光染色法观察供者DC在受者脾脏内的存活情况;混合淋巴细胞反应(MLR)测定受者脾脏内T淋巴细胞对供者同种抗原的反应性.另取27对供、受者(BALB/c小鼠和C57BL/6小鼠),通过袖套管技术建立颈部异位心脏移植模型,随机平均分为PBS对照移植组、Day8-DC移植组及MyD88siRNA-DC移植组,各组于移植前7 d分别经受者门静脉注射0.5 ml PBS、2.0× 106个Day8-DC及2.0× 106个MyD88siRNA-DC.于输注后第7天,观察各组移植心的存活时间;病理检查观察排斥反应程度;酶联免疫吸附试验测定受者血清巾Th1及Th2型细胞因子[γ干扰素(INF-γ)、白细胞介素(IL)-12、IL-4和IL-10]水平的变化.结果 MyD88siRNA-DC在受者脾脏内的存活时间明显延长,MyD88siRNA-DC组受者脾脏内T淋巴细胞对供者抗原的反应性最低(P＜0.01).PBS对照移植组、Day8-DC移植组及MyD88siRNA-DC移植组移植心的存活时间分别为:(6.67±1.37)d、(13.67±2.25)d和(24.50±4.42)d,与PBS对照移植组相比,Day8-DC移植组移植心存活时间延长(P＜0.01),而MyD88siRNA-DC移植组移植心存活时间较Dby8-DC移植组进一步延长(P＜0.01);MyD88siRNA-DC移植组移植心排斥反应病理分级最低,受者血清中INF-γ和IL-12水平显著降低(P＜0.01),而IL-4和IL-10水平明显升高(P＜0.01).结论 输注转染MyD88siRNA基因的供者DC能够延长同种小鼠移植心的存活时间;其机制可能与诱导受者Th1/Th2免疫偏移及形成供、受者微嵌合状态有关.     

TLR4/MyD88信号通路在膝骨关节炎滑膜炎中的作用

Toll样受体4是重要的固有免疫模式识别受体,通过识别损伤相关分子模式及病原相关分子模式,启动髓样分化因子 88依赖途径后激活NF-κB信号通路,引发炎性介质及细胞因子的分泌,触发固有免疫应答,在膝骨关节炎滑膜炎的发生中发挥作用。研究TLR4/MyD88信号通路在膝骨关节炎滑膜炎中的作用,以期更深入的阐明膝骨关节炎滑膜炎发病机制,为膝骨关节炎滑膜炎的诊疗提供理论依据和参考。     

树突状细胞MyD88介导热休克蛋白60的信号转导

目的观察热休克蛋白印（HSP60）刺激对树突状细胞（DC）活性的影响，探讨髓性分化因子88（MyD88）在介导HSP60信号转导中的作用。方法培养小鼠骨髓源性DC，分为对照组、HSP60组及RNA干扰组，对照组在正常条件下继续培养2d，HSP60组加入终质量浓度为10mg／L的HSP60，RNA干扰组于加入MyD88 siRNA4h后给予10mg／L HSP60刺激，均继续培养2d。流式细胞术检测DC细胞表面CD11c、CD80、CD86及MHC-Ⅱ分子的变化，ELISA法检测DC分泌肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）和白细胞介素-12（IL-12）的浓度，免疫细胞化学检测DC MyD88、核因子-κB（NF-κB）的表达，Western blot检测DC MyD88的变化，混合淋巴细胞培养检测T细胞增殖能力。结果3组CD11c阳性率差异无统计学意义（P〉0．05），分别为78．65％、82．40％及85．72％，HSP60组CD80、CD86及MHC-Ⅱ分子的阳性率分别为70．28％、76．49％及38．24％，较对照组（阳性率分别为28．42％、34．56％及16．28％）及RNA干扰组（阳性率分别为32．16％、40．47％及20．76％）显著增高（P〈0．05）；HSP60组TNF-α、IFN-γ及IL-12浓度显著高于对照组及RNA干扰组（P〈0．05）；HSP60组可见MyD88高表达及NF-κB核移位；HSP60组SI显著高于对照组及RNA干扰组（P〈0．01）。结论HSP60通过MyD88依赖的途径活化DC，MyD88是HSP60信号转导中的重要调节分子。     

TLR4介导大鼠肾脏缺氧复氧炎症反应依赖于MyD88

目的：探讨MyD88是否参与了TLR介导大鼠肾脏缺氧复氧（hypoxia/reoxygenation,H/R）引起的炎症反应.方法：体外分离获得Wistar大鼠原代肾小管上皮细胞（proximal tubule epithelial cells,PTECs）,建立H/R模型.采用TLR4siRNA干扰PTECs,检测白细胞介素8（interleukin-8,IL-8）和肿瘤坏死因子α（tumor necrosis factor α,TNF-α）的表达.之后,加入髓样分化因子88（myeloid differentiation factor 88,MyD88）的抑制剂ST2825后,检测IL-8和TNF-α的表达.结果：TLR4表达沉默后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）;加入ST2825后,IL-8、TNF-α和MyD88的表达均显著下降（P〈0.05）,且抑制MyD88活性后显著降低细胞的凋亡水平（P〈0.05）.结论：MyD88参与了TLR4介导的大鼠肾脏缺氧复氧引起的炎症反应.     

MyD88依赖途径在膝骨关节炎进程中的基因表达相关性研究

目的 :探讨MyD88依赖途径的相关基因TLR4、NF-κB与MyD88在膝骨关节炎(osteoarthritis,OA)不同病程下大鼠滑膜组织的表达特点及相关性。方法:将60只Wistar大鼠随机分为6组:空白组(N)、假手术组(F)和模型组[2周组(2W)、4周组(4W)、8周组(8W)、12周组(12W)],每组各10只。各模型组以Hulth法建立大鼠膝OA动物模型,空白组不予手术,假手术组仅打开关节腔。按上述设计时间收集样本,经总RNA的提取及反转录后,用Realtime PCR法对各组滑膜组织中TLR4、NF-κB及MyD88的相对表达量进行检测,分析其相关性。结果 :假手术组各基因的mRNA表达与空白组相比差异均无统计学意义((P0.05);而各模型组较空白组各基因表达增强(P0.05)。相关性分析显示:MyD88与TLR4及NF-κB的mRNA相关性系数分别为0.91和0.86,差异有统计学意义(P0.05)。结论:MyD88与TLR4、NF-κB的表达均呈显著的正相关性,在通路上起重要的枢纽作用,可通过其表达预测通路上其他基因的表达情况,进一步明确膝OA病理机制。     

Ift88 regulates Hedgehog signaling,Sfrp5 expression,and β‐catenin activity in post‐natal growth plate

Primary cilia are present on most cell types including chondrocytes. Dysfunction of primary cilia results in pleiotropic symptoms including skeletal dysplasia. Previously, we showed that deletion of Ift88 and subsequent depletion of primary cilia from chondrocytes resulted in disorganized columnar structure and early loss of growth plate. To understand underlying mechanisms whereby Ift88 regulates growth plate function, we compared gene expression profiles in normal and Ift88 deleted growth plates. Pathway analysis indicated that Hedgehog (Hh) signaling was the most affected pathway in mutant growth plate. Expression of the Wnt antagonist, Sfrp5, was also down‐regulated. In addition, Sfrp5 was up‐regulated by Shh in rib chondrocytes and regulation of Sfrp5 by Shh was attenuated in mutant cells. This result suggests Sfrp5 is a downstream target of Hh and that Ift88 regulates its expression. Sfrp5 is an extracellular antagonist of Wnt signaling. We observed an increase in Wnt/β‐catenin signaling specifically in flat columnar cells of the growth plate in Ift88 mutant mice as measured by increased expression of Axin2 and Lef1 as well as increased nuclear localization of β‐catenin. We propose that Ift88 and primary cilia regulate expression of Sfrp5 and Wnt signaling pathways in growth plate via regulation of Ihh signaling. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 350–356, 2013