外源性IL-6促进IL-6基因敲除小鼠移植肝再生的研究

目的 观察外源性重组白细胞介素6(rIL-6)对白细胞介素6(IL-6)基因敲除小鼠原位肝移植后存活时间和移植肝再生过程的影响.方法 动物为C57BL/6野生型小鼠和IL-6基因敲除小鼠,分为4组进行实验:基因敲除对照组,肝移植供、受者均为IL-6基因敲除小鼠；野生型对照组,以IL-6基因敲除小鼠为供者,野生型C57BL/6小鼠为受者；基因敲除rIL-6组,供、受者均为IL-6基因敲除小鼠,肝移植前1h于受者皮下注射rIL-6,1 mg/kg;野生型rIL-6组,以IL-6基因敲除小鼠为供者,野生型C57BL/6小鼠为受者,受者应用rIL-6,用法和用量同前组.观察受鼠的存活情况.获取移植肝,进行组织学和免疫组织化学检查.结果 供肝冷缺血时间约为50 min.基因敲除对照组受鼠平均存活2.2d,野生型对照组受鼠平均存活1.9 d,基因敲除rIL-6组受鼠平均存活＞17.6 d,野生型rIL-6组受鼠平均存活＞20.4 d.各组间存活时间的差异均有统计学意义(P＜0.01).基因敲除对照组和野生型对照组受鼠移植肝有片状坏死和肝细胞气球样变,有极少数溴脱氧尿核苷染色阳性的细胞存在.基因敲除rIL-6组和野生型rIL-6组受鼠的移植肝无细胞坏死等改变,有散在的肝细胞溴脱氧尿核苷染色阳性.结论 外源性的IL-6能够延长IL-6基因敲除小鼠肝移植后的存活时间,促进其移植肝细胞再生.     

小鼠50%体积肝脏原位移植实验模型的建立

目的 介绍一种小鼠50%体积的原位肝脏移植实验模型.方法 选择同系雄性C57BL/6小鼠,供、受体各28只.实验分为50%体积移植组和全肝移植对照组.50%肝脏移植物通过切除左侧肝叶获得.供肝经门静脉灌注4℃威斯康星大学保存液.血管重建时,肝上腔静脉端端吻合,门静脉和肝下腔静脉采用袖套法吻合.不作动脉重建.术后观察移植物的存活时间.组织病理学检查肝脏移植物的组织结构和细胞形态,免疫组化检测肝细胞复制情况.结果 小鼠肝脏移植手术成功率为100%.50%体积的小鼠肝脏移植受体存活率为90%(＞15 d).全肝移植受体存活率为100%(＞15 d).所有受体手术无肝期保持在23 min内.术后组织学检查移植肝组织结构良好,全肝移植后肝脏再生反应不明显.50%体积的肝脏移植后激发明显的再生反应.结论 小鼠50%体积的肝脏移植实验模型存活率高,术后激发明显的再生反应.该模型可应用于缺血再灌注损伤和肝脏再生等基础研究.     

NAD+介导的SIRT1去乙酰化活性在小鼠内毒素性急性肺损伤中的作用

目的评价烟酰胺腺嘌呤二核苷酸(NAD+)介导的沉默信息调节因子1(SIRT1)去乙酰化活性在小鼠内毒素性急性肺损伤(ALI)中的作用。方法 SPF级健康雄性C57BL/6小鼠25只, 6～8周龄, 体重20～25 g, 野生(WT)型10只, NAD+合成途关键酶烟酰胺单核苷酸腺苷酸转移酶1(NMNAT1)敲除(KO)型15只, 采用随机数字表法, 将WT型小鼠分为2组(n=5):对照组(WT+C组)和ALI组(WT+ALI组);将KO型小鼠分为3组(n=5):对照组(KO+C组)、ALI组(KO+ALI组)和ALI+ NAD+前体物质烟酰胺单核苷酸(NMN)组(KO+ALI+NMN组)。静脉注射LPS 15 mg/kg制备内毒素性ALI模型, KO+ALI+NMN组注射静脉注射LPS前1 h腹腔注射NMN 500 mg/kg。各对照组给予等容量生理盐水。注射LPS或生理盐水后12 h时, 取腹主动脉血标本行血气分析, 后处死小鼠留取肺组织, 测定肺湿重/干重(W/D)比值, 光镜下观察肺组织病理学改变, 并行肺损伤评分;采用ELISA法检测肺组织IL-6、IL-1β、TNF-α含量,...     

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.     

保存不同时间的大鼠部分肝脏移植后的肝细胞再生

目的 探讨保存不同时间的大鼠部分肝脏移植后的肝细胞再生及其可能机制.方法 采用近交系雄性Lewis大鼠为供、受者,按照实验设计分别将供肝于4℃UW液中保存1 h(冷缺血1 h组)、8 h(冷缺血8 h组)和16 h(冷缺血16 h组).然后进行原位肝移植.移植肝恢复血流前,用3-0丝线结扎供肝左侧中央叶、左外叶及尾状叶,保留右侧的肝叶.即可制成大鼠50%体积肝脏(以下简称"半肝")原位移植模型.术后观察各组移植肝的存活情况和肝细胞再生情况;采用逆转录聚合酶链反应测定肝组织中自细胞介素-6(IL-6)和肿瘤坏死因子α(TNF_n)的表达情况;采用Western印迹法检测肝组织中信号传导与转录因子-3(STAT-3)表达情况;采用免疫组织化学染色检测移植肝组织中细胞周期素DI(Cyelin D1)的表达和肝细胞摄取溴脱氧尿核苷(BrdU)情况.结果 各组手术成功率均为100%.与冷缺血1 h组相比,冷缺血8 h组和冷缺血16 h组移植肝组织中TNF-a(F=67.45,P<0.05)和IL-6(F=287.73,P<0.05)的表达明显增加.STAT-3的表达也明显增强.肝移植后24 h.冷缺血8 h组在胞浆和细胞核内均有Cyclin D1的表达.而冷缺血16 h组移植肝组织中未见明显的Cyclin D1表达.移植后24 h.冷缺血16 h组的BrdU染色阳性的肝细胞数无明显增多,而在冷缺血8 h组可见BrdU染色阳性的肝细胞明显增多(t=19.40,P<0.05).结论冷保存一定时限的大鼠部分肝脏在移植后可获得肝细胞再生,此过程可能通过TNF-α/IL,16/sTAT-3/Cyclin D1/DNA合成的途径进行调节;当冷保存时间达16 h后,肝细胞不能对肝脏再生早期信号起反应.     

供者肝脏非实质细胞输注诱导免疫耐受模型的建立

目的探讨供者肝脏非实质细胞输注诱导受者特异性免疫耐受的效果.方法将2×107个C3H/HE小鼠的肝脏非实质细胞通过尾静脉输入C57BL/6小鼠的体内,48*!h后给其腹腔注射环磷酰胺200*!mg/kg,18*!d后接受C3H/HE小鼠的皮片移植,观察皮片的存活情况.于肝脏非实质细胞输注前及输注后18*!d、30*!d、60*!d进行供、受者混合淋巴细胞培养.结果15只输注C3H/HE小鼠肝脏非实质细胞的C57BL/6小鼠,其移植皮片的存活时间明显延长(74.6*!d±9.7*!d,对照组仅存活10*!d±0.4*!d);混合淋巴细胞培养的结果证实受者的T淋巴细胞对供者小鼠淋巴细胞的反应程度明显降低.结论肝脏非实质细胞可以有效诱导免疫耐受.     

β-arrestin-2通过上调PI3K/Akt信号抑制自噬以减轻小鼠肝脏缺血-再灌注损伤

目的  验证β-arrestin-2是否通过上调PI3K/Akt信号抑制自噬对小鼠肝脏缺血-再灌注损伤（IRI）发挥保护作用。方法  将C57BL/6背景的β-arrestin-2基因敲除（KO）及野生型（WT）小鼠各12只,随机分为KO小鼠假手术组（KO+sham组）,KO小鼠IRI组（KO+IRI组）,WT小鼠假手术组（WT+sham组）和WT小鼠IRI组（WT+IRI组）,每组各6只。分别建立70%肝脏IRI模型或进行假手术处理,于肝脏再灌注或手术后6 h进行相关研究。采用免疫组织化学（免疫组化）染色检测凋亡信号蛋白裂解半胱氨酸天冬氨酸蛋白酶3（cleaved Caspase-3）、增殖信号蛋白Ki-67及PI3K/Akt通路信号蛋白p-Akt的表达。结果  免疫组化染色结果显示,与相应的sham组比较,KO+IRI组和WT+IRI组小鼠肝组织中的cleaved Caspase-3、Ki-67和p-Akt阳性细胞计数均增加（均为P < 0.01）;与WT+IRI组比较,KO+IRI组小鼠肝组织中cleaved Caspase-3阳性细胞计数增加,Ki-67和p-Akt阳性细胞计数均减少（均为P < 0.05）。结论  β-arrestin-2可减轻小鼠IRI后肝细胞凋亡、促进其损伤修复,其通过上调PI3K/Akt信号抑制自噬来减轻小鼠肝脏IRI。     

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.     

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.     

Donor IL-6 deficiency evidently reduces memory T cell responses in sensitized transplant recipients

BackgroundResistance of tolerance induction in sensitized transplantation is mainly caused by generation of memory T cells. It is unknown whether alteration of graft niche such as level of pro-inflammatory cytokines can affect generation of memory T cells.MethodsIL-6 deficient or wild-type (WT) C57BL/6 heart grafts were transplanted into pre-sensitized wild-type BALB/c recipients. Frequencies of memory T cells in the peripheral blood, grafts, and spleen were evaluated.ResultsWe revealed that deficiency of donor IL-6 could significant prolong sensitized allograft survival. Compared with counterpart of WT group, frequency of effector memory CD4 + T cells (CD4 + CD44 + CD62L-) in the peripheral blood was significantly lower in the IL-6 KO group (p = .026) at day 3 post-transplantation. Frequency of effector memory CD8 + T cells (CD8 + CD44 + CD62L-) in the peripheral blood was significantly lower in the IL-6 KO group (p < .0001) at day 3 post-transplant in comparison to that of WT group. No significant difference of central memory T cells was found between these groups. Histology demonstrated that deficiency of donor pro-inflammatory cytokine IL-6 (IL-6 KO group) preserved cardiac architecture with a mild infiltration of lymphocytes, whereas wild-type donor (control group) caused an evident lymphocytic infiltration within myocardial fibers of grafts and destruction of cardiac structure.ConclusionDeficiency of pro-inflammatory IL-6 of donor graft could effectively prolong sensitized allograft survival, which was caused by a remarkable decrease of peripheral memory T cells rather than central memory T cells. This unveiled mechanism of targeting IL-6 signaling pathway might provide a novel insight into preventing allograft rejection for sensitized transplant recipients.     

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.     

鼠纤维介素在小鼠心脏移植急性排斥反应中的表达及其意义

目的　探讨小鼠同种心脏移植急性排斥反应期间鼠纤维介素 (mfgl2 )在移植心脏组织中的表达及其与组织病理学改变的关系。方法　采用BALB/c小鼠到C5 7BL/ 6小鼠的颈部异位心脏移植作为同种排斥反应模型 ,以抗mgfl2多克隆抗体干预 ,并设同系小鼠心脏移植对照组。采集移植心组织标本作病理学检查 ,以免疫组织化学方法测定mfgl2在移植心脏组织细胞上的表达 ,并对mgfl2的表达进行半定量分析。结果　同系移植对照组移植心脏组织结构正常 ,未见mfgl2表达 ;同种移植组移植心脏组织出现进行性坏死 ,大量单个核细胞浸润 ,并伴有mfgl2的表达 ,且血管内皮细胞表达mfgl2 ;抗mfgl2抗体干预组移植心脏组织损伤较轻 ,移植物的存活时间延长 (P <0 .0 1) ,巨噬细胞、淋巴细胞的浸润和mfgl2表达量也显著减少。结论　mfgl2表达水平与排斥反应所致移植心脏病理损害程度相关 ;抗mfgl2抗体干预能显著减少移植心脏巨噬细胞、淋巴细胞的浸润 ,明显延长移植心脏存活时间。     

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.     

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.     

抗RANTES单抗联合环孢素诱导小鼠二次移植心脏长期存活

目的 探讨抗RANTES单克隆抗体(单抗)联合环孢素(CsA)在抗小鼠心脏二次心脏移植排斥反应中的作用。 方法 以BALB/c小鼠为供体,C57BL/6小鼠为受体构建小鼠心脏二次移植模型。按随机数字表法分为4组：对照组(生理盐水组)、实验A组(抗RANTES单抗治疗组)、实验B组(CsA治疗组)及实验C组(抗RANTES单抗+CsA联合治疗组),每组6只。观察各组小鼠二次移植心脏存活时间;应用组织病理学观察各组移植心脏急性排斥反应的程度;采用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测移植心脏组织中RANTES、白细胞介素(IL)-2、IL-10、干扰素(IFN)-γ和转录生长因子(TGF)-β基因的信使核糖核酸(mRNA)的相对表达量;采用酶链免疫吸附试验(ELISA)检测RANTES、IFN-γ、IL-2、IL-10和TGF-β在血清中的含量。 结果 所有实验小鼠二次心脏移植全部成功复跳。与对照组比较,实验A、B、C组的移植心脏存活时间明显延长(均为P＜0.01)。移植心脏病理学染色可见,实验C组炎症细胞的浸润较对照组、实验A组、实验B组明显减少。与其它3组比较,实验C组的移植心脏组织RANTES、IFN-γ、IL-2的mRNA表达量均明显减少,而IL-10、TGF-β的mRNA表达量均明显增加(均为P＜0.05)。与其它3组比较,实验C组的血清中RANTES、IL-2、IFN-γ含量均明显降低,而血清中IL-10、TGF-β含量均明显升高(均为P＜0.05)。 结论 联合应用抗RANTES单抗和CsA可更有效诱导小鼠对二次心脏移植的免疫耐受,延长移植物的存活时间。      

Effect of renal fibrosis after macrophage depletion in C3-deficient unilateral ureteral obstruction mice

Objective To investigate the effect and mechanism of renal fibrosis after macrophage depletion in C3-deficient unilateral ureteral obstruction mice. Methods Renal interstitial fibrosis model was established by unilateral ureteral obstruction (UUO) in male C3-deficient mice and age-matched C57BL/6 WT mice (8-12 weeks of age). Mice were randomly divided into 4 groups, including sham operation in wild type group（WT/sham）(n=18), UUO operation in wild type group（WT/UUO）(n=18), sham operation in C3-deficient group（C3KO/sham）(n=18), and UUO operation in C3-deficient group（C3KO/UUO）(n=18). The expression of complement C3 was detected by immunohistochemical staining and renal interstitial macrophages were assessed by immunofluorescence staining. Tubulointerstitial fibrosis was observed by both HE staining and Masson staining after 14 days of UUO. Collagen accumulation and score of tubulointerstitial injury were obtained. Wild type and C3-deficient UUO mice were treated by liposome clodronate in early or late stage respectively and then interstitially infiltrated macrophages and renal fibrosis were analysed. Mice were sacrificed randomly at 3,7,14 days after UUO and obstructed kidneys were collected. Macrophage phenotype was detected by double-labeling immunofluorescence with F4/80 and iNOS for the M1, F4/80 and CD206 for the M2 macrophage subpopulation. iNOS, Arg-1 and CD206 were also detected by western blot. Results C3 deficient mice exhibited attenuated renal fibrosis, reduced collagen accumulation and tubulointerstitial injury score compared with WT mice (P＜0.01). Meanwhile, macrophage depletion in early or late stage of UUO reduced renal fibrosis in WT mice, but had no effect on C3-deficient UUO mice. Decreased accumulation of M1 macrophages and expression of iNOS, increased accumulation of M2 macrophages and expression of Arg-1, CD206 were found in C3 deficient mice compared with WT mice in early stage of UUO (P＜0.01). Conclusion Renal fibrosis is not reduced after depletion of macrophages in C3 deficient UUO mice due to the altered macrophage polarization.     

C-Jun N-Terminal Kinase 2 Promotes Graft Injury via the Mitochondrial Permeability Transition After Mouse Liver Transplantation

The c-Jun N-terminal kinase (JNK) pathway enhances graft injury after liver transplantation (LT). We hypothesized that the JNK2 isoform promotes graft injury via the mitochondrial permeability transition (MPT). Livers of C57BL/6J (wild-type, WT) and JNK2 knockout (KO) mice were transplanted into WT recipients after 30 h of cold storage in UW solution. Injury after implantation was assessed by serum ALT, histological necrosis, TUNEL, Caspase 3 activity, 30-day survival, and cytochrome c and 4-hydroxynonenal immunostaining. Multiphoton microscopy after LT monitored mitochondrial membrane potential in vivo . After LT, ALT increased three times more in WT compared to KO (p < 0.05). Necrosis and TUNEL were more than two times greater in WT than KO (p < 0.05). Immunostaining showed a >80% decrease of mitochondrial cytochrome c release in KO compared to WT (p < 0.01). Lipid peroxidation was similarly decreased. Every KO graft but one survived longer than all WT grafts (p < 0.05, Kaplan-Meier). After LT, depolarization of mitochondria occurred in 73% of WT hepatocytes, which decreased to 28% in KO (p < 0.05). In conclusion, donor JNK2 promotes injury after mouse LT via the MPT. MPT inhibition using specific JNK2 inhibitors may be useful in protecting grafts against adverse outcomes from ischemia/reperfusion injury.