Rab9与NPC1在小胶质细胞内的定位研究

目的:观察Rab9与NPC1蛋白在小胶质细胞内的定位及其与溶酶体、高尔基体和晚期内体的关系。方法:以小鼠小胶质细胞系BV-2为对象,采用特异性识别Rab9、NPC1的抗体和溶酶体、高尔基体、晚期内体的特异性表面标记进行荧光免疫细胞化学染色,在激光共聚焦显微镜下观察。结果:Rab9和NPC1共定位于小胶质细胞胞浆内,Rab9表达于溶酶体、高尔基体和晚期内体。结论:Rab9对NPC1蛋白的正常功能可能有重要影响。     

Rab9与NPCI在小胶质细胞内的定位研究

目的:观察Rab9与NPC1蛋白在小胶质细胞内的定位及其与溶酶体、高尔基体和晚期内体的关系.方法:以小鼠小胶质细胞系BV-2为对象,采用特异性识别Rab9、NPC1的抗体和溶酶体、高尔基体、晚期内体的特异性表面标记进行荧光免疫细胞化学染色,在激光共聚焦显微镜下观察.结果:Rab9和NPC1共定位于小胶质细胞胞浆内,Rab9表达于溶酶体、高尔基体和晚期内体.结论:Rab9对NPC1蛋白的正常功能可能有重要影响.     

IL-4对小鼠小胶质细胞ICAM-1表达的影响

目的探讨白细胞介素-4(IL-4)对小鼠小胶质细胞炎症因子细胞间粘附分子-1(ICAM-1)表达的影响。方法用IL-4与小鼠小胶质细胞共同孵育,观察不同处理组细胞形态的变化。ELISA法检测IL-4孵育小胶质细胞培养上清中ICAM-1的浓度,同时采用RT-PCR技术检测细胞ICAM-1mRNA水平。结果 IL-4与小胶质细胞共同孵育后细胞形态随孵育时间发生变化,贴壁细胞逐渐减少,IL-4减少小胶质细胞分泌ICAM-1并下调mRNA的表达水平。结论 IL-4可能通过抑制ICAM-1的产生及分泌而参与小胶质的细胞迁移过程,从而在中枢神经系统中发挥神经保护作用。     

PD-1/PD-L1在非小细胞肺癌中的研究进展及展望

PD-1(programmed cell death-1,程序性死亡受体1)与其配体PD-L1(programmed cell death-ligand 1,程序性死亡配体1)属于CD28/B7家族,是一对共刺激分子,具有负性调控作用。PD-1通过与其配体PD-L1结合调节肿瘤的微环境,使肿瘤细胞免于机体免疫系统的监视和清除。目前已有较多研究显示PD-1/PD-L1在非小细胞肺癌组织中的表达水平与患者的临床病理因素及预后存在显著的相关性。在非小细胞肺癌的治疗领域,以PD-1/PD-L1为代表的免疫治疗成为继手术治疗、化疗、放疗、分子靶向治疗之后的新焦点。PD-1/PD-L1抑制剂在一系列非小细胞肺癌临床试验中也显示出了巨大的临床潜力。本文就PD-1/PD-L1的生物学结构及其在非小细胞肺癌中的作用机制、研究进展及展望作一综述。     

三阴乳腺癌组织中TUFT1和Rab5-GTP的表达水平及临床意义

目的 研究三阴乳腺癌(TNBC)组织中TUFT1和Rab5-GTP的表达水平,分析两种蛋白表达的相关性及TUFT1与患者临床病理特征的关系。方法 选取2021年3月至2021年12月住院手术的TNBC患者80例,收集其癌组织。免疫组化法检测TUFT1和Rab5-GTP在癌组织中的表达情况,分析两种蛋白表达的相关性;评估TUFT1与TNBC患者临床病理特征之间的关系。结果 在TNBC组织中,观察到TUFT1主要在癌细胞胞膜上和胞质内均有染色,以胞质内染色最强,Rab5-GTP在胞质内染色,两种蛋白的表达呈正相关(P<0.05);TUFT1表达与患者年龄无关(P>0.05),而与肿瘤大小、组织学分级、腋窝淋巴结转移均呈正相关(均P<0.05)。结论 TUFT1和Rab5-GTP的表达水平与三阴乳腺癌病程的进展关系密切。     

细胞因子对白血病细胞B7-1基因表达及诱导外周血T细胞活化的影响

目的：观察γ干扰素（ＩＦＮγ）、白细胞介素（ＩＬ）１０、ＩＬ１２对转染Ｂ７１白血病细胞Ｂ７１基因表达及其诱导健康人外周血单个核细胞（ＰＢＭＣ）产生ＩＦＮγ、ＩＬ２的影响。方法：用逆转录聚合酶链反应（ＲＴＰＣＲ）和酶联免疫吸附反应（ＥＬＩＳＡ）方法检测ＩＦＮγ、ＩＬ２基因表达和蛋白质合成。结果：发现ＩＦＮγ、ＩＬ１２增强ＰＢＭＣ产生ＩＬ２、ＩＦＮγ，ＩＬ１０抑制其产生，同时发现ＩＦＮγ、ＩＬ１２增强转染Ｂ７１白血病细胞Ｂ７１基因表达，ＩＬ１０抑制其表达。结论：细胞因子ＩＦＮγ、ＩＬ１０、ＩＬ１２影响转染Ｂ７１白血病细胞诱导ＰＢＭＣ产生ＩＦＮγ、ＩＬ２，其可能是通过影响Ｂ７１基因表达实现的     

B7-H1基因在白血病细胞中的表达及其临床意义

本研究通过检测B7-H1基因在多种白血病细胞中的表达水平,探讨其临床意义。采用SYBR GreenⅠ实时定量PCR法检测9株白血病细胞系、4株IFN-γ体外诱导后的白血病细胞系,59例原代急性白血病细胞、10例原代白血病细胞诱导生成的树突状细胞(DC)以及2株实体肿瘤细胞系,10例正常人骨髓单个核细胞中B7-H1mRNA的表达水平,并对59例急性白血病患者的治疗反应与其白血病细胞中B7-H1基因表达水平之间的相关性进行了分析。结果显示:B7-H1 mRNA在白血病细胞系、原代急性白血病细胞中的表达水平较低,在IFN-γ体外诱导后的白血病细胞系、原代白血病细胞诱导生成的DC中的表达水平明显上调,在化疗后未获完全缓解(CR)的患者白血病细胞中B7-H1 mRNA表达水平明显高于化疗后获得CR的患者。结论:B7-H1基因在白血病细胞中的表达水平较低,但在某些因素作用下其表达水平明显上调。白血病细胞中B7-H1基因表达水平与患者的治疗反应有显著相关性。     

非小细胞肺癌组织MIP-1表达及其与树突状细胞浸润的关系

目的探讨非小细胞肺癌组织MIP-1表达及其与树突状细胞浸润的关系。方法选择74例非小细胞肺癌组织标本,采用荧光原位杂交法检测MIP-1的表达,免疫组织化学检测趋化因子受体CCR1、CCR7的表达。结果肺癌细胞胞质及胞膜表达MIP-1,同时检测到肺癌组织中有CCR1和CCR7阳性树突状细胞的浸润。MIP-1、CCR1和CCR7阳性树突状细胞的表达和肺癌分期相关。肺癌组织中MIP-1阳性细胞表达率与CCR1阳性树突状细胞数量呈正相关,而与CCR7阳性树突状细胞数量呈负相关。结论非小细胞肺癌组织MIP-1表达与肿瘤浸润树突状细胞表面分子CCR1、CCR7的表达关系密切。     

基质细胞衍生因子1及受体CXCR7在肿瘤中的作用

基质细胞衍生因子(SDF)-1是一种由骨髓基质细胞合成释放的趋化因子,属于CXC亚家族成员.多年来一致认为趋化因子受体4(chemokine receptor 4,CXCR4)是SDF-1的唯一受体,然而研究发现SDF-1同样能与另一受体CXCR7相结合,并且在肿瘤的发生发展中具有重要作用.本文就SDF-1与CXCR7...     

丙型肝炎病毒E1基因在COS-7中的表达

目的：了解丙型肝炎病毒（HCV）E1基因在真核细胞中的表达情况。方法：采用基因重组技术,构建含HCVE1基因的重组表达质粒pEE1/HisC-E1,经酶切和PCR鉴定后,用脂质体转染法将重组质粒导入COS-7进行表达,经蛋白印迹实验鉴定表达产物。结果：重组表达载体pEF1/HisC-E1在COS-7中获得表达,表达的E1糖蛋白约29kD,具有抗原性,结论：HCVE1基因在真核细胞高效表达,为进一步研究HCVE1糖蛋白的生物学特性奠定基础。     

Differential Expression of Rab5 and Rab7 Small GTPase Proteins in Placental Tissues From Pregnancies Affected by Maternal Coronavirus Disease 2019

PurposeThe majority of pregnancies affected by maternal coronavirus disease 2019 (COVID-19) do not result in fetal transmission. However, several studies have identified parenchymal changes in their placental tissues, suggesting a placental response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the maternal–fetal interface. Although many COVID-19 placental studies have focused on the expression of the canonical SARS-CoV-2 entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2, further characterization of subcellular molecules involved in viral trafficking have not yet been investigated in these tissues. Of interest are Rab proteins, a family of small GTPase proteins that direct intracellular transport between different endocytic organelles. Rab5 and Rab7 in particular have previously been implicated in HIV and cytomegalovirus invasion of placental trophoblast cells in vitro; the localization of these molecules has not been fully characterized within the human maternal–fetal interface, however, or within placental tissues from SARS-CoV-2–infected pregnancies.MethodsUsing fluorescent immunohistochemistry, Rab5 and Rab7 placental localization and comparative fluorescence intensity were explored in a cohort of placental tissues from pregnancies affected by maternal COVID-19 disease (COVID, n = 15) compared with contemporary control subjects (Control, n = 10). Fluorescence intensity was quantified by using corrected total cell fluorescence values.FindingsWithin placental villi, Rab5 was consistently localized in syncytiotrophoblast and cytotrophoblast cells. Rab5 had significantly higher mean (SEM) fluorescence intensity in the COVID cohort (Control, 1.96 [0.16]; COVID, 2.62 [0.09]; P = 0.0014). In contrast, although Rab7 was also localized within placental villous syncytiotrophoblast and cytotrophoblast cells, mean (SEM) Rab7 fluorescence intensity was significantly downregulated in COVID vs Control placentas (Control, 35.9 [4.1]; COVID, 20.1 [0.52]; P = 0.0001).ImplicationsThis differential expression of Rab5 and Rab7 suggests that placental endocytic pathways may be altered at the maternal–fetal interface in pregnancies affected by maternal SARS-CoV-2 infection. As key molecules governing intracellular vesicle transport, including viral trafficking, Rab GTPase proteins may be of interest for ongoing studies examining placental responses to COVID-19 in pregnancy.     

Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund’s adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats

The elusiveness of the mechanism underlying pain is a major impediment in developing effective clinical treatments. We examined whether the phosphorylation of spinal serum- and glucocorticoid-inducible kinase 1 (SGK1) and downstream glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1)/Rab4-dependent GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling play a role in inflammatory pain. After intraplantar injection of complete Freund’s adjuvant (CFA), we assessed thermal hyperalgesia using the Hargreaves test and analyzed dorsal horn samples (L4-5) using Western blotting, coprecipitation, and immunofluorescence. CFA administration provoked behavioral hyperalgesia along with SGK1 phosphorylation, GluR1 trafficking from the cytosol to the membrane, and phosphorylated SGK1 (pSGK1)-GRASP-1, GRASP-1-Rab4, and Rab4-GluR1 coprecipitation in the ipsilateral dorsal horn. In the dorsal horns of hyperalgesic rats, CFA-enhanced pSGK1 was demonstrated to be colocalized with NeuN, GRASP-1, Rab4, and GluR1 by immunofluorescence. GSK-650394 (an SGK1 activation antagonist, 1, 10, and 30 μM, 10 μL/rat, intrathecally) dose-dependently prevented CFA-induced pain behavior and the associated SGK1 phosphorylation, GluR1 trafficking, and protein-protein interactions at 1 day after CFA administration. Intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, an AMPAR antagonist, 1, 3, and 10 μM, 10 μL/rat) attenuated the hyperalgesia and GluR1 trafficking caused by CFA; however, it had no effect on SGK1 phosphorylation. Small interfering RNA targeting Rab4 hindered the CFA-induced hyperalgesia and the associated GluR1 trafficking and Rab4-GluR1 coprecipitation. Our results suggest that spinal SGK1 phosphorylation, which subsequently triggers the GRASP-1/Rab4 cascade, plays a pivotal role in CFA-induced inflammatory pain by regulating GluR1-containing AMPAR recycling in the dorsal horn.     

钙调蛋白在C型Niemann-Pick病小鼠Purkinje细胞的表达

目的:探讨钙调蛋白在C型Niemann-Pick病(NPC)小鼠Purkinje细胞的表达。方法:采用免疫组织化学和Westen Blot技术检测3种钙调蛋白Calbindin D28k、IP3R1、SERCA2b在4、6、8周龄NPC小鼠小脑部的表达,以同周龄WT(野生型)小鼠作为对照组。结果:CalbindinD28k和IP3R1在6、8周龄时表达较WT小鼠显著下降,在4周龄时无明显减少,SERCA2b在8周龄时表达较WT小鼠显著下降。结论:3种钙调蛋白表达的下降可能导致钙失衡,参与神经元变性。     

B7-H1在人骨髓间充质干细胞上的表达及其对T淋巴细胞增殖的影响

目前关于人骨髓间充质干细胞（hBMMSC）的免疫调节机制尚不完全清楚。为探讨B7-H1在hBMSC上的表达水平及hBMMSC的免疫调节机制是否与B7-H1介导的信号通路（B7-H1／PD-1）有关,首先分离、培养、鉴定hBMMSC,采用流式细胞术、RT—PCR、Western—blot检测B7-H1在hBMMSC上的表达水平。进一步采用混合淋巴细胞培养法（MLC）观察hBMMSC对T淋巴细胞增殖的抑制作用,然后使用功能级的抗B7-H1单克隆抗体阻断B7-H1．用CCK-8试剂盒检测阻断前后T淋巴细胞增殖活性。结果表明：hBMMSC高表达B7-H1分子;表达B7-H1的hBMMSC能有效地抑制T淋巴细胞的增殖,其抑制作用与hBMMSC数量呈剂量依赖性;使用抗B7-H1单克隆抗体阻断B7-H1后hBMMSC对T淋巴细胞增殖的抑制作用显著降低,抑制率由64．1％降低至38．75％。结论：B7-H1在hBMMSC上高表达,B7-H1介导的信号通路（B7-H1／PD-1）参与了hBMMSC的免疫调节作用。     

重组人血小板生成素基因的COS—7细胞及在小鼠体内的转移与表达

为了观察重组血小板生成素(thrombopoietin,TPO)基因在COS-7细胞及在小鼠体内的表达,应用DNA重组技术构建了含TPO基因的真核表达质粒pcd2/TPO,用脂质体法将其转染COS-7细胞,用裸质粒DNA直接注射加电脉冲的方法将pcd2/TPO质粒转移到小鼠骨骼肌中.用RT-PCR及ELISA法检测到TPO基因在COS-7细胞的瞬时表达,MTT法显示其有刺激TPO依赖细胞系增殖的活性.RT-PCR及免疫组织化学染色可检测到TPO基因在转基因小鼠骨骼肌的表达,ELISA法定量检测转基因组小鼠血清TPO浓度为(1 185±264)ng/L,明显高于正常小鼠的TPO浓度(250±76)ng/L.本实验实现了TPO基因在小鼠体内和COS-7细胞的转移,为今后TPO基因治疗的研究奠定了实验基础.     

鼻咽癌癌组织中Midkine基因表达的研究

目的研究Midkine(MK)基因在鼻咽癌(NPC)组织中的表达。方法用Trizol法提取鼻咽癌组织和正常鼻咽组织的RNA,经RT-PCR获得扩增的MK DNA,溴化乙锭琼脂糖凝胶电泳检测PCR产物。结果45例鼻咽癌组织中有37例在447 bp处出现1条MK的特征条带。7例鼻咽部正常组织均表达阴性。24例颈淋巴结转移患者中20例鼻咽癌组织MK mRNA表达阳性(表达率为83.33%),21例无颈淋巴结转移中17例鼻咽癌组织MK mRNA表达阳性(表达率为80.95%)。结论MK mRNA在鼻咽癌组织特异表达,MK有望作为鼻咽癌筛查的指标之一。