白藜芦醇通过抑制内质网应激减轻大鼠脑缺血/再灌注损伤

目的:探讨白藜芦醇(Resveratrol,Res)对大鼠脑缺血/再灌注诱导的内质网应激反应的调控作用。方法:将72只健康雄性SD大鼠,采用随机数字表法分为3组(n=24):假手术组(S组)、脑缺血/再灌注模型组(I/R组)、Res组(R组)。采用阻断左侧大脑中动脉并阻断双侧颈总动脉30 min恢复灌注的方法制备大鼠脑缺血/再灌注模型。R组于缺血前7d给予腹腔注射Res(50 mg/kg),1次/日。对大鼠进行神经功能缺损评分后留取脑组织,采用TTC法检测脑梗死体积,干湿重法测定脑组织含水量,免疫组织化学及Western blot法检测大脑皮层内质网应激相关蛋白GRP78、p-PERK、CHOP的表达变化。结果:与S组比较,I/R组大鼠神经功能评分及脑含水量升高(P0.05),脑梗死体积增大(P0.05),皮层GRP78、p-PERK和CHOP蛋白表达均显著上调(P0.05)。与I/R组比较,P组大鼠神经功能评分及脑含水量降低(P0.05),脑梗死体积减小(P0.05),GRP78表达上调(P0.05),p-PERK和CHOP蛋白表达下调(P0.05)。结论:白藜芦醇通过抑制内质网应激反应,对大鼠脑缺血/再灌注损伤发挥保护作用。     

11,12-EET和缺血预处置对在体大鼠正常及再灌注心肌磷酸化ERK和p38MAPK表达的影响

目的:观察11,12-环氧二十碳三烯酸(11,12-EET)和缺血预处置对大鼠再灌注心肌组织磷酸化ERK1/ERK2和p38 MAPK表达的影响,了解大鼠心肌磷酸化ERK1/ERK2和p38 MAPK表达与预处置有否关系。方法: 使用雄性Wistar大鼠,通过结扎(60 min)和松开(30 min)冠状动脉左前降支,复制缺血/再灌注模型;采用缺血5 min,再灌注5 min两次造成缺血预处置。大鼠经手术并静脉给予6.24×10^-8mol/L 11,12-EET,稳定20 min,结扎冠脉复制缺血/再灌注模型。实验分5组:①正常组(norm);②假手术组(sham);③缺血再灌注组(I/R);④短阵缺血预处置组(SI+I/R);⑤11,12-EET预处置缺血/再灌注组(EET+I/R)。采用Western blot法测定心肌细胞外调节的蛋白激酶(ERK1/2)和p38 MAPK的表达程度,并观察再灌注过程中心功能的变化。结果: 再灌注30 min时,I/R组+dp/dt_max%、-dp/dt_max%和LVDP均显著低于sham组、SI+I/R组和EET+I/R组(P＜0.05);而I/R组大鼠心肌ERK1/2磷酸化表达明显高于sham组(P＜0.05),明显低于SI+I/R组和EET+I/R组(P＜0.05);I/R组大鼠心肌p38 MAPK磷酸化表达I/R组显著高于sham组、norm组、SI+I/R及EET+I/R组(P＜0.05)。结论:6.24×10^-8 11,12-EET mol/L具有保护心功能的作用,这种保护作用可能与大量激活磷酸化ERK1/2和抑制p38 MAPK有关。     

11，12-EET的延迟性心脏保护作用与磷酸化ERK1/2的关系

目的:观察11，12-EET延迟性保护作用对缺血再灌注大鼠心肌ERK活性及磷酸化ERK表达的影响，探讨其在11，12-EET延迟性保护中的作用。 方法:复制大鼠心肌缺血/再灌注模型；观察缺血/再灌注期间心脏收缩期左心室内压上升的最大变化速率（+dp/dtmax）及舒张期左心室内压下降的最大变化速率（-dp/dtmax）；采用免疫共沉淀法测定大鼠心肌组织中细胞外调节激酶（ERK）的活性，采用Western blotting法测定大鼠心肌组织中磷酸化ERK的表达。 结果:I/R组缺血60 min及再灌注30 min两个时段±dp/dtmax均低于sham组（P<0.05），24 hEET+I/R组缺血60 min及再灌注30 min两个时段±dp/dtmax明显高于I/R组（P<0.05），而24hEET+PD+I/R组缺血60 min及再灌注30 min两个时段±dp/dtmax明显低于24hEET+I/R组（P<0.05）。ERK的活性24hEET+I/R高于normal组，24hEET+I/R组低于24hEET+PD+I/R组。磷酸化ERK的表达I/R组高于normal组和sham组，24hEET+I/R高于I/R组，24 hEET+I/R组低于24hEET+PD+I/R组。 结论:外源性11，12-EET具有延迟性心脏保护作用，大量激活磷酸化的ERK参与这种保护作用。     

肢体缺血后处理通过激活MAPK途径保护大鼠海马区缺血性神经元

目的探讨肢体缺血后处理对缺血性神经元损伤的作用,观察大鼠海马CA1区ERK1/2的磷酸化及其蛋白表达的变化情况。方法健康成年雄性SD大鼠,随机分为假手术组(sham)、全脑缺血再灌组(四动脉闭塞全脑缺血模型ischemia/reperfusion,I/R)、肢体缺血后处理组(limb ischemia postconditioning,Lpost)、肢体缺血后处理+ERK1/2抑制剂组(Lpost+PD98059)。光镜下焦油紫染色法观察各组大鼠海马CA1区神经元存活情况;Western blot法检测海马CA1区ERK1、2磷酸化及蛋白表达情况。结果 Western blot结果显示脑缺血再灌注各时间点Lpost组p-ERK1/2水平均较I/R组高,且于再灌注后10min、1d出现两个高峰,给予ERK1/2上游激酶抑制剂PD98059后能够抑制再灌注后1dp-ERK1/2水平的升高;而各时间点各组ERK1/2水平之间无明显差异。焦油紫染色结果显示再灌注1d后,I/R组与sham组相比,海马CA1区存活神经元数量明显减少;Lpost组较I/R组海马CA1区存活神经元数量明显增加;而给予ERK1/2上游激酶抑制剂PD98059后(Lpost+PD98059)神经元生存数量较Lpost组大幅下降。结论肢体缺血后处理具有抗大鼠海马CA1区缺血性神经元损伤的作用,激活ERK1/2通路可能是其发挥保护作用机制之一。     

异丙酚对脑缺血再灌注损伤大鼠内质网应激的影响及作用机制

目的 探讨异丙酚对脑缺血再灌注(I/R)损伤大鼠的神经保护作用及机制。方法 30只SD雄性大鼠随机分为假手术组(Sham)、缺血再灌注组(I/R)及异丙酚组，每组10只。采用Zea Longa评分评估神经功能损伤；HE染色观察大鼠大脑缺血区形态结构；流式细胞仪与TUNEL检测海马神经元凋亡率；免疫荧光检测葡萄糖调控蛋白78 (GRP78)、C/EBP同源蛋白(CHOP)表达；Western blot检测脑组织内质网应激相应蛋白p-PERK、p-EIF-2a、ATF4和CHOP的表达。结果 与Sham组相比，I/R组大鼠神经功能评分明显升高、海马神经元凋亡率显著升高、p-PERK、p-eIF2α、ATF4和CHOP蛋白表达明显上调；给予异丙酚治疗后，神经功能缺损评分、海马神经元凋亡率均显著降低，p-PERK和p-eIF2α、ATF4和CHOP蛋白的表达降低。结论 异丙酚通过抑制p-PERK-p-elF2α-ATF4-CHOP通路和内质网应激保护脑缺血再灌注损伤大鼠的脑神经。     

rAAV9介导CaMEK基因转导减轻缺血再灌注损伤诱导的心肌细胞凋亡的研究

目的 观察重组9型腺相关病毒(recombinant adeno-associated virus serotype 9,rAAV9)携带CaMEK基因转导心肌细胞是否能减轻缺血再灌注损伤诱导的心肌细胞凋亡.方法 分离培养SD大鼠心肌细胞,建立I/R模型,rAAV9-CBA-CaMEK转染心肌细胞.实验分组:(1)正常对照组;(2) I/R组;(3) I/R+rAAV9-CBA-CaMEK组;(4) I/R+rAAV9-CBA-CaMEK+PD98059组.Westem blot测定心肌细胞ERK1/2磷酸化水平及Caspase-3、Bax蛋白的表达.结果 分离培养的原代心肌细胞经鉴定结果阳性,Western blot分析显示rAAV9介导CaMEK基因转染心肌细胞P-ERK1/2的表达高峰时间在第5天,并可减轻缺血再灌注诱导的心肌细胞凋亡,而给予PD98059处理后,这种心肌细胞保护作用消失.结论 rAAV9介导的CaMEK基因转染可显著减轻缺血再灌注诱导的心肌细胞凋亡.     

西洋参茎叶总皂苷通过抑制过度内质网应激减轻大鼠心肌缺血/再灌注损伤

 目的：观察西洋参茎叶总皂苷（PQS）对大鼠心肌缺血/再灌注（ischemia/reperfusion, I/R）损伤的保护作用,并从内质网应激(ERS)的角度探讨其可能的分子机制。方法：采用SD大鼠心肌I/R模型,随机分为正常对照组、模型组与药物预处理组,检测血流动力学及血清心肌肌钙蛋白T（cTnT）含量,以氯化三苯基四氮唑（TTC）和伊文思蓝双染法检测心梗面积,采用HE染色和TUNEL法分别评估心肌病理损伤和心肌细胞凋亡程度,Western blotting法检测心肌组织凋亡调节因子以及内质网应激相关蛋白的表达。结果：与I/R组相比,PQS+I/R组平均动脉压降低32.0%（P<0.05）,左室±dp/dtmax分别升高64.0%和35.0%（P<0.05）,血清cTnT含量降低53.3%（P<0.05）,坏死面积/缺血面积(AN/AAR)百分比降低65.5%（P<0.05）,心肌细胞凋亡率降低54.9%（P<0.05）;心肌组织病理损伤程度减轻;抗凋亡蛋白Bcl-2表达升高110.0%（P<0.05）,促凋亡蛋白Bax表达降低47.8%（P<0.05）,钙网蛋白(CRT)表达降低43.4%（P<0.05）,C/EBP同源蛋白（CHOP）和剪切后的caspase-12蛋白表达分别降低38.6%和23.7%（P<0.05）。结论：PQS可显著减轻大鼠心肌I/R损伤,其机制与降低I/R诱导的CRT过表达,抑制CHOP、caspase-12等内质网凋亡通路激活,从而抑制过度ERS介导的细胞凋亡有关。     

缺血后处理抑制缺血再灌注大鼠心肌内质网应激相关凋亡

目的:研究缺血后处理(I-postC)对缺血/再灌注(I/R)大鼠心肌组织Caspase-12和CHOP(CEBP Homologous Protein)的影响,从内质网应激角度探讨I-postC抑制I/R心肌细胞凋亡的机制。方法:采用Wistar大鼠在体心脏I/R模型,以TUNEL法检测细胞凋亡,并检测血浆乳酸脱氢酶(LDH)和肌酸激酶(CK-MB)含量及心肌组织超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,免疫印迹法检测心肌组织Caspase-12、CHOP、Bcl-2和Bax表达。结果:I-postC显著抑制I/R所致的心肌细胞凋亡(较I/R组低74.3%,P<0.01),上调Bcl-2表达(较I/R组高53.2%,P<0.05)并抑制Bax表达上调(较I/R组低46.1%,P<0.05);与I/R组比较,I-postC组心肌细胞LDH和CK-MB漏出减少(分别较I/R组低37.6%和34.3%,P<0.01),心肌组织MDA含量降低(较I/R组低38.4%,P<0.05),SOD活性上调(较I/R组高18.3%,P<0.05);I-postC抑制I/R所诱导的Caspase-12活化和CHOP表达上调(分别较I/R组低41.7%和33.3%,P<0.01)。结论:I-postC通过抑制Caspase-12活化和CHOP过表达,减轻内质网应激相关凋亡,保护大鼠I/R心肌。     

Akt与ERK1/2在吡格列酮保护乳鼠心肌缺血再灌注损伤中的关系

目的:研究Akt与ERK1/2在吡格列酮保护乳鼠心肌缺血再灌注损伤作用中的关系。方法:体外培养1~3日龄的Wistar乳鼠心室肌细胞,通过缺氧3 h,复氧3 h,建立心肌细胞缺血再灌注损伤模型。实验分为空白对照组(C组)、缺血再灌注组(I/R组)、缺血再灌注+吡格列酮组(I/R+P组)、缺血再灌注+吡格列酮+Akt抑制剂mk2206组(I/R+P+MK组)、缺血再灌注+吡格列酮+ERK1/2抑制剂PD98059组(I/R+P+PD组)。应用免疫印迹和免疫荧光检测各组细胞p-Akt和p-ERK1/2的表达变化。结果:与I/R组比较,I/R+P组p-Akt和p-ERK1/2均增加;I/R+P+MK组的p-Akt显著减少,而p-ERK1/2却明显增加;I/R+P+PD组的p-ERK1/2显著减少,而p-Akt却明显增加。各组间总Akt和ERK1/2的表达无明显变化。结论:吡格列酮可激活Akt和ERK1/2对缺血再灌注的心肌有保护作用,该过程中Akt和ERK1/2相互之间有影响。     

抗衰老Klotho蛋白减轻大鼠乳鼠心肌细胞缺氧/复氧损伤

目的:观察抗衰老Klotho蛋白对大鼠乳鼠原代心肌细胞缺氧/复氧(H/R)损伤的作用并探讨其作用机制。方法:建立大鼠乳鼠心肌细胞H/R模型,并将心肌细胞分为正常对照组、H/R模型组和不同浓度(0.1μmol/L、1μmol/L和10μmol/L)Klotho作用H/R组。观察各组心肌细胞H/R前后搏动频率变化,利用MTT方法检测细胞存活率;测定各组心肌细胞H/R后LDH、CK、AST的漏出量及MDA含量、SOD活性;流式细胞术检测各组心肌细胞的凋亡率;real-time PCR检测各组心肌细胞中内质网应激标记及凋亡相关分子GRP78、CRT和CHOP和caspase-12 mRNA的表达情况;Western blot法检测心肌细胞内内质网应激凋亡蛋白CHOP和caspase-12蛋白表达及Akt磷酸化水平。结果:与正常对照组相比,H/R模型组中心肌细胞搏动频率和细胞存活率显著下降,细胞凋亡率显著升高(P0.05);LDH、CK、AST和MDA含量升高而SOD活性显著降低(P0.05);GRP78、CRT、CHOP和caspase-12 mRNA表达显著增高(P0.05);CHOP和caspase-12蛋白表达随之增高而Akt的磷酸化水平显著降低(P0.05)。与H/R模型组相比,抗衰老Klotho蛋白作用H/R心肌细胞后,心肌细胞搏动频率和细胞存活率显著升高,细胞凋亡率逐渐降低(P0.05),LDH、CK、AST和MDA含量下降而SOD活性显著增高(P0.05),GRP78、CRT、CHOP和caspase-12 mRNA的表达逐渐降低(P0.05),CHOP和caspase-12蛋白表达也随之降低,而Akt磷酸化水平显著增加(P0.05)。结论:抗衰老Klotho蛋白能够提升H/R损伤后心肌细胞的存活率,抑制细胞凋亡,通过抵抗氧化应激和过度内质网应激反应发挥作用,并与激活Akt磷酸化有关。     

喉癌组织中FEZ1／LZTS1基因的表达及其临床意义

目的：探讨FEZ1／LZTS1基因在喉鳞状细胞癌中的表达缺失及其与临床病理的关系。方法：采用逆转录聚合酶链反应（RT—PCR）法,分析50例原发性喉癌组织和癌旁组织中FEZ1／LZTS1基因的表达情况。结果：FEZ1／LZTS1基因在癌旁组织中表达率明显高于喉癌组织（P〈0．01）;在喉癌组织中FEZ1／LZTS1基因阳性表达率随其病理分级、临床分期升高而降低（P〈0．01）,而与患者年龄、性别和原发灶部位无关。结论：FEZ1／LZTS1基因的表达缺失可能对LSCC的发生发展起重要作用,是LSCC发展中的重要分子事件。     

Morphological aspects of LFA-1/ICAM-1 and VLA4/VCAM-1 adhesion pathways in human lymph nodes

Monoclonal antibodies specific for the adhesion molecules participating in lymphocyte homing, lymphocyte function associated antigen-1 (LFA-1) and very late antigen 4 (VLA4), and their respective ligands, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), were used to characterize their expression pattern in human lymph nodes by immunohistochemical and immunoelectron microscopic techniques. The location of LFA-1-positive lymphocytes and selective expression of ICAM-1 on the luminal plasma membrane of high endothelial venule endothelium suggested that the LFA-1/ICAM-1 adhesion pathway participates only in the initial step of the lymphocyte migration process. Lymphocytes passing through endothelium appear not to be influenced by this pathway. VCAM-1 was detected occasionally on the endothelium of high endothelial venules in the hyperplastic lymph nodes in the mesentery, but not in peripheral lymph nodes. VLA4-positive lymphocytes tended to be more frequently observed within high endothelial venules in mesenteric lymph nodes than in peripheral ones. Strong expression of both ligands, ICAM-1 and VCAM-1, was noted on the plasma membrane of follicular dendritic cells, and was especially prominent on their labyrinthine folding, and on the interdigitating cells in the paracortex. Furthermore, both LFA-1-and VLA4-positive lymphocytes localized around these cells. This suggests that LFA-1/ICAM-1 and VLA4/VCAM-1 adhesion pathways play an important role in the lymphocyte recognition of antigen-presenting cells.     

PD-1/PD-L1抑制性通路在病毒性感染中的研究进展

杀伤性T细胞(CTL)是病毒感染过程中主要的效应细胞.在急性病毒性感染中,CTL细胞通过杀伤病毒感染的细胞,分泌抗病毒细胞因子从而有效清除病毒.而在慢性病毒感染过程中,病毒特异性CTL发生克隆耗竭,细胞数量少,细胞功能受损,分泌细胞因子能力下降.目前证明,CD28家族成员Programmed Death-1(PD-1)是一种重要的细胞表面分子,能够抑制CTL细胞功能,参与外周免疫耐受,维持T细胞克隆耗竭,保护机体不受免疫病理损伤.在慢性病毒性感染中,T细胞表面的PD-1与其受体PD-L1/2结合后,传递抑制性信号,下调外周T细胞功能.阻断PD-1通路,能够重建CD8+T细胞功能,抑制病毒复制,促进病毒清除.因此,通过干扰PD-1/PD-L1抑制途径进行免疫调节,可能是处理慢性病毒感染性疾病的一个新方法.     

PD-1/PD-L1 blockade in renal cell cancer

Introduction: Immunotherapy using checkpoint inhibitors is providing significant benefit to patients with renal cell carcinoma (RCC), both in overall survival and tolerability of treatment. Given the recent approval of the first checkpoint inhibitor in RCC, this review discusses the background and clinical data for checkpoint inhibition in RCC.

Areas covered: This review introduces and discusses the basic biologic mechanisms of checkpoint inhibitor function and focuses on the current evidence in clinical trials for the use of immunotherapy in RCC.

Expert commentary: Immunotherapy has been a mainstay of therapy in RCC, but the recent approval of nivolumab with ORR of 25% and durable responses has provided a transformative new therapeutic option.     

Expanding the Mutational Spectrum of CRLF1 in Crisponi/CISS1 Syndrome

Crisponi syndrome (CS) and cold‐induced sweating syndrome type 1 (CISS1) share clinical characteristics, such as dysmorphic features, muscle contractions, scoliosis, and cold‐induced sweating, with CS patients showing a severe clinical course in infancy involving hyperthermia associated with death in most cases in the first years of life. To date, 24 distinct CRLF1 mutations have been found either in homozygosity or in compound heterozygosity in CS/CISS1 patients, with the highest prevalence in Sardinia, Turkey, and Spain. By reporting 11 novel CRLF1 mutations, here we expand the mutational spectrum of CRLF1 in the CS/CISS1 syndrome to a total of 35 variants and present an overview of the different molecular and clinical features of all of them. To catalog all the 35 mutations, we created a CRLF1 mutations database, based on the Leiden Open (source) Variation Database (LOVD) system ( https://grenada.lumc.nl/LOVD2/mendelian_genes/variants ). Overall, the available functional and clinical data support the fact that both syndromes actually represent manifestations of the same autosomal‐recessive disorder caused by mutations in the CRLF1 gene. Therefore, we propose to rename the two overlapping entities with the broader term of Crisponi/CISS1 syndrome.     

ApoE^-/-小鼠动脉粥样硬化斑块中NHE1与Netrin-1表达上调

目的探讨ApoE^-/-小鼠动脉粥样硬化斑块组织及小鼠巨噬细胞系(RAW 264.7)是否同时表达钠氢交换体1(NHE1)及神经轴突导向因子-1(netrin-1),以及两者之间共定位、相互影响情况。方法制作小鼠动脉粥样硬化斑块模型,HE染色检测主动脉粥样斑块形成;免疫荧光及显微共聚焦检测动脉粥样斑块中NHE1、netrin-1表达及共定位;用氧化型低密度脂蛋白(Ox-LDL)干预RAW264.7细胞,Western blot检测不同浓度及时间干预下, NHE1和netrin-1蛋白表达;设计NHE1基因小干扰RNA(siRNA),通过脂质体转染导入RAW264.7细胞,RT-qPCR及Western blot分别检测靶向作用于NHE1基因的小干扰RNA(siRNA)的抑制效率。结果小鼠动脉硬化斑块中NHE1表达上调(P<0.05),且NHE1与netrin-1存在部分共定位情况;Ox-LDL可上调RAW264.7细胞NHE1及netrin-1蛋白表达,且呈一定时间-浓度依赖性(P<0.05);构建的NHE1 siRNA能有效抑制RAW264.7细胞NHE1表达(P<0.05);有效敲降RAW264.7细胞NHE1表达能使Ox-LDL干预下升高的NHE1、netrin-1蛋白表达下调(P<0.05)。结论小鼠动脉粥样硬化斑块及Ox-LDL干预的小鼠巨噬细胞系(RAW264.7)中NHE1、netrin-1表达上调且存在共定位规律,netrin-1表达受NHE1表达的影响。     

Role of LFA-1/ICAM-1 in interleukin-2-stimulated lymphocyte proliferation

Major adhesion routes between lymphoid cells involve the receptor/ligand pairs LFA-l/ICAM-1 and CD2/LFA-3, in addition to VLA or CD44 molecules. In this study we evaluated the role of these adhesion receptors in the proliferative response of lymphoid cells to interleukin-2 (IL-2). Blocking studies were performed with a panel of monoclonal antibodies (mAb) directed against these adhesion molecules. Selective inhibition of recombinant (r)IL-2-induced cell proliferation was observed with mAb directed against the a or /3 subunit of LFA-1 or to its ligand ICAM-1. Interestingly, rIL-2-induced proliferation was also inhibited by NKI-L16, an anti-la antibody known to enhance cell-cell interaction. Resting lymphocytes were preferentially susceptible to the inhibition, particularly in an early phase of culture and when stimulated with a relatively low dose of rIL-2. By using mAb that specifically could block distinct rIL-2 activation pathways, LFA-l/ICAM-1 interaction was found to be required for p55 IL-2 receptor (IL-2R)-mediated interaction of rIL-2 with its high-affinity receptor, but not for p75 IL-2R-mediated responses. Furthermore, it was shown that the rIL-2 response of T lymphocytes, but not of natural killer cells, was dependent on LFA-l/ICAM-1 interaction. This suggests that LFA-l/ICAM-1 interaction is required for an optimal rIL-2 response of cells capable of IL-2 secretion. Our data provide evidence for the hypothesis that adhesion receptor-directed release of IL-2 may result in a locally high concentration of IL-2 that triggers high-affinity IL-2R signaling and up-regulates p55 IL-2R to enhance cytokine responsiveness.