狼疮肾炎患者外周血单个核细胞内白细胞介素-6 gp80与gp130基因表达的研究

目的　了解狼疮肾炎 (LN)患者外周血单个核细胞 (PBMCs)白细胞介素 6 (IL 6 )与IL 6受体 (gp80与gp130 )的mRNA表达情况 ,并分析它们之间及其与自身抗体的关系。 方法　采用逆转录聚合酶链反应 (RT PCR)方法检测 18例活动期LN患者、16例非活动期LN患者和 10名正常人PBMCs内IL 6、gp80与 gp130的mRNA表达水平。 结果　①活动期LN患者、非活动期LN患者及正常人PBMCsIL 6mRNA检出阳性率各为 72 2 %、37 5 %和 10 0 %,前者显著高于后两者 ,而后两者PBMCsIL 6mRNA的检出阳性率差异则无显著性 ;这三组人群PBMCs内gp80mRNA与 gp130mRNA的检出阳性率均无统计学差异。②活动期LN患者PBMCs中IL 6mRNA表达水平高于非活动期LN患者 ,后者又高于正常人 ;活动期LN患者PBMCs中有较高水平的gp80mRNA与 gp130mRNA表达 ,而非活动期LN患者PBMCsgp80mRNA与 gp130mRNA表达水平 ,与正常对照比较则无统计学差异。③活动期LN患者PBMCs内IL 6mRNA、gp80mRNA和gp130mRNA相对表达水平互呈正相关 ,但在非活动期LN患者中则未见这种正相关关系。活动期LN患者PBMCs内IL 6、gp80和gp130基因表达水平与血清ANA、抗ds DNA抗体和抗Sm抗体均无显著的相关性。 结论　活动期LN患者PBMCs内IL 6及其受体gp80和 gp130的mRNA表达均上调 ,且受体 g     

人工合成gp210多肽抗原在原发性胆汁性肝硬化中的应用

目的 尝试用gp210抗原的羧基末端氨基酸序列合成多肽做抗原,探索一种简单实用的抗gp210抗体的检测方法.方法 采用棋盘试验法建立抗gp210抗体的酶联免疫吸附试验(ELISA)检测方法,分别用ELISA方法与免疫印迹法检测原发性胆汁性肝硬化(PBC)组与对照组的抗gp210抗体.结果 gp210抗原的工作浓度为5 μg/ml.血清抗gp210抗体的吸光度(A)值>0.61(x+3s)为阳性.两种方法在检测抗gp210抗体之间差异无统计学意义(p=0.617),二者之间有很强的相关性(r=0.868,P=0.000).结论 人工合成gp210多肽抗原与天然纯化抗原敏感性和特异性基本一致,可以用于临床检测抗gp210抗体.     

依那普利对实验性心肌梗死大鼠左室gp13O基因表达的影响

目的:研究急性心肌梗死(AMI)后左室重塑(LVRM)的发生与gp130基因异常表达的关系及依那普利对gp130表达的影响.方法:AMI后24h存活的73只雄性Wistar大鼠随机分为AMI组及依那普利治疗组,AMI组又依照术后1、3、7、14、21、28 d分为6个时间点,另设假手术组及正常对照组.各8只.依那普利组于术后第2天起灌胃给药(10mg·kg-1·d-1),连续4周.行心脏标本病理分析,分别采用放射免疫法和氯氨T法测定左室非梗死区(LVNIZ)血管紧张素Ⅱ(AngⅡ)和LVNIZ羟脯氨酸含量(HC),用RT-PCR法检测LVNIZ gp130 mRNA的表达.最终获完整资料大鼠76只,于上述各组分别为9、8、8、9、9、8、9、8和8只.结果:①与正常及假手术组相比,AMI组LVRM参数:心脏重量(HW)、左室重量(LVW)、左室重量指数(LVWI)、心肌细胞横径(TDM)、HC均显著增加(P＜0.05～0.01);②AMI组LVNIZ AngⅡ明显升高,与LVRM相关性好(P＜0.01);③LVNIZ gp130 mRNA表达于AMI第1天即开始明显增加,第7天达高峰,以后有所下降,但第28天时仍明显高于对照组(P＜0.05～0.01);④相关分析显示LVNIZ gp130mRNA表达水平与LVNIZ AngⅡ、LVRM 参数间呈正相关(P＜0.05～0.01);⑤与AMI第28天组相比,依那普利组LVRM明显减轻,LVNIZAngⅡ显著下降,gp130 mRNA表达明显下调(P＜0.05～0.01).结论:大鼠AMI后LVNIZ gp130 mRNA的过度表达与AMI后LVRM的发生密切相关,依那普利改善LVRM的机制还可能与抑制gp130基因的过度表达有关.     

带绿荧光蛋白的人类免疫缺陷病毒被膜蛋白gp160的表达

人类免疫缺陷病毒1(HIV1)被膜蛋白Env是HIV感染和HIV免疫研究中的一个重要部分[1],通过表达前体蛋白gp160,经蛋白酶裂解形成Env蛋白的膜外部分gp120和跨膜部分gp41。随着分子克隆技术的应用,已有许多gp160和gp120表达结构被合成。但需要特殊的标记技术才能快速、敏感、特异和直观地反映Env成分的表达。本研究旨在结合增强的绿荧光蛋白(EGFP)的荧光表达[2],尝试合成适合于定性、定量和定位测定的gp160表达结构。材料与方法一、质粒与细胞pDM128含有位于HIV1分子克隆SF2的6606～8396bp的Env编码序列(gp160),SV40启动子、SF2的vpu…     

热休克蛋白gp96在慢性乙型肝炎组织中的检测及其意义

对慢性乙型肝炎进行gp96检测 ,探讨gp96表达状况与肝炎病变进展过程的相关性及其在抗乙型肝炎病毒免疫反应中的作用。采用免疫组织化学 (IHC)方法 ,对 77例慢性乙型肝炎病例的活检肝组织标本进行gp96检测 ,其中轻度慢性肝炎者 37例 ,中度 2 0例 ,重度 2 0例。以 16例非肝炎肝组织作为本实验对照。gp96在对照组正常肝组织和慢性乙型肝炎中的平均阳性细胞率分别为 0 5 3%和 15 94 % ,两组间具有非常显著性差异。在慢性肝炎轻、中、重三组 ,阳性细胞平均率分别为 6 14 %、2 1 39%及 2 9 18% ,统计学检验表明组与组之间有显著差异 ,gp96表达率随着炎症程度的加重而上升。提示gp96与机体抗乙肝病毒的免疫应答程度有一定的相关性     

热休克蛋白gp96在原发性肝癌组织中的检测及其意义

热休克蛋白gp96的发现为肿瘤和病毒性肝炎等传染性疾病的免疫治疗开辟了新的途径。用免疫组织化学方法对gp96存原发性肝癌组织中的表达进行检测，探讨gp96在抗肝癌免疫反应中的作用。     

抗HIV-1 gp41单克隆抗体的制备及意义

目的为HIV-1感染的检测和研究提供依据。方法以基因工程重组抗原HIV-1 gp41蛋白免疫BALB／c小鼠,利用常规杂交瘤技术和间接ELISA法制备抗gp41蛋白的单克隆抗体（mAb）,用ELISA法鉴定所得mAb的Ig亚类、效价及特异性。单抗经饱和硫酸铵（SAS）纯化和HRP标记后,利用双抗体夹心ELISA筛选检测gp41蛋白的最佳配伍单抗,分别包被ELISA板及作为酶标mAb,建立双mAb夹心ELISA法。结果经细胞融合、筛选、克隆化,获得了12株可分泌高效价mAb的杂交瘤细胞。纯化的腹水mAb经配对试验,选出2株mAb：7D4和9G2-HRP建立了双mAb夹心ELISA法,检测HIV-1 gp41抗原的灵敏度是1μg/L。结论获得12株效价高的抗HIV-1 gp41的mAb,建立了特异性强、灵敏度较高的检测HIV-1 gp41抗原的双抗体夹心ELISA法。     

N-糖基化修饰蛋白gp73与肝硬化患者Child-Pugh的关系

目的评价N-糖基化修饰蛋白gp73的血清水平与肝硬化患者Child-Pugh之间的关系及其可能的临床意义。方法所观察的患者均为2009年1月~2010年10月在北京地坛医院门诊及住院肝硬化患者,所有患者均为HBsAg阳性。观察患者共610例,其中男性患者448例,年龄17~82岁,平均48岁;女性患者162例,年龄18~76岁,平均55岁。入组肝硬化患者的Child-Pugh分级主要依据血清白蛋白、总胆红素、凝血酶原活动度、腹水及肝性脑病评价等5项指标。血清gp73浓度采用ELISA法检测。结果伴随肝功能衰退,血清gp73水平也相应升高。血清gp73水平Child-Pugh为C级的患者（255.78 ng/mL±100.89 ng/mL）显著高于B级（203.30 ng/mL±99.15 ng/mL）和A级的患者（125.28 ng/mL±67.05 ng/mL）。血清gp73与白蛋白之间呈显著负相关（r=-0.52,P〈0.0001）。结论 HBV感染肝硬化患者血清gp73水平随肝功能恶化而升高。     

一例HIV-1急性感染者奠基病毒的gp160基因序列特征及其假病毒构建

目的观察急性感染期艾滋病病毒I型（HIV-1）gp160的全长基因序列及感染特征。方法从一例处于Feibig I期HIV-1感染者血浆中提取RNA,扩增gp160全长基因并测序,分析其生物学信息;将gp160全长基因与pcDNA3.1His/V5真核表达载体连接,构建Env-pcDNA3.1真核表达质粒,与骨架质粒pNL4-3.Luc.R-E-共转染293细胞,包装出假病毒。用包装的假病毒感染ghost细胞,测定感染细胞的荧光素酶活性（RLU）,鉴定假病毒的感染活性。结果成功扩增出gp160全长基因,嗜性预测为CCR5,N-糖基化位点数与标准株HXB2相同,但gp120糖基化程度更高,氨基酸变异主要集中在V1-V5区。假病毒感染试验显示,RLU值达到7log。结论获得了处于急性感染期的HIV-1gp160基因序列和高感染活性的假病毒。     

抗gp210抗sp100抗体与原发性胆汁性肝硬化活动性的相关性

目的 评估原发性胆汁性肝硬化(PBC)患者血清中抗gp210和抗sp100抗体与PBC活动性的关系.方法 采集72例PBC患者的静脉血,以酶联免疫吸附试验(ELISA)检测抗gp210、抗sp100的水平,分析患者的临床资料.结果 抗gp210阳性患者白蛋白水平较阴性组降低,Mayo危险评分较后者升高,差异均有统计学意义(分别为P＜0.01和P＜0.05).随着疾病好转,抗gp210抗体多呈下降趋势.抗sp100阳性组与阴性组在血清生化指标、Mayo危险评分方面差异均无统计学意义.结论 抗gp210阳性患者病情较重,需加强随访,积极治疗.     

Expression and regulation of gp130 messenger ribonucleic acid in cultured immature rat Sertoli cells

The expression of glycoprotein 130 (gp130) was studied in rat primary Sertoli cells by Northern blot analysis. Gp130 mRNA of 9.0 and 7.5 kb were detected in a variety of rat tissues including the testis. Gp130 mRNAs were detected in isolated, immature Sertoli cells. The levels of gp130 were significantly stimulated by the addition of interleukin-1-beta (IL-1-beta) or interleukin-6 (IL-6) but not by the addition of follicle-stimulating hormone (FSH). IL-6 activates intracellular signaling by binding with a receptor consisting of an 80-kDa ligand-binding protein, IL-6 receptor (IL-6R), and a second 130-kDa protein, gp130. As previously reported, expression of IL-6R mRNA in rat Sertoli cells was stimulated not only by IL-1-beta and IL-6 but also by FSH. In contrast, gp130 mRNA expression was not stimulated by FSH in our analysis. These data suggest that gp130 expression may be regulated by more than 1 mechanism and that production of gp130 and IL-6R, the 2 components of the IL-6 receptor system, may be regulated, at least in part, by a different pathway.     

Functional interaction of the gp80 and gp130IL-6 receptors in human B cell malignancies

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin's malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recog-nizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the dis-appearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.     

Suppression of bone resorption by madindoline A,a novel nonpeptide antagonist to gp130

IL-6 is a multifunctional cytokine involved in regulation of differentiation, antibody production, and growth of certain types of tumor cells. Its excessive production plays a major role in pathogenesis of multiple myeloma and postmenopausal osteoporosis. In the course of a screening program aimed at IL-6 inhibitor from microbial products, we found madindoline A (MDL-A) and madindoline B, which have a fuloindoline structure with diketocyclopentene bound to the methyl group. MDL-A has no cytotoxic activities. It inhibited only activities of both IL-6 and IL-11 without affecting the IL-6-specific signal transduction cascade, JAK2/STAT3. In a dose-dependent manner [(3)H]MDL-A binds to gp130, which is a signal transducing 130-kDa glycoprotein, but formation of the trimeric complex IL-6/IL-6 receptor/gp130 was not inhibited, suggesting that MDL-A suppresses dimerization of trimeric complexes. Not only did MDL-A markedly inhibit IL-6- and IL-11-induced osteoclastogenesis in vitro, but it also inhibited IL-6-stimulated serum amyloid A production and bone resorption in an experimental model of postmenopausal osteoporosis in vivo by a different mechanism from that of 17beta-estradiol. Here we show that MDL-A has a highly selective inhibitory effect on IL-6 and IL-11 activities by inhibiting a gp130 activity while suppressing bone loss in ovariectomized mice. MDL-A is anticipated as a lead compound for treatment of hormone-dependent postmenopausal osteoporosis, which has no serious side effects, and as a new mechanism of action, gp130 blocking.     

Graves病患者甲状腺组织内IL-6及gp130表达增强

对8例Graves病(GD)甲状腺组织内白细胞介素6(IL-6)及其受体(gp80、gp130)表达进行研究,发现GD组甲状腺组织内IL-6mRNA、gp130mRNA表达增高;GD中甲状腺滤泡细胞和淋巴细胞胞浆IL-6表达水平显著升高.提示GD患者甲状腺内IL-6/gp130信号通路的激活可能参与GD的发病.     

Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production

Regulation of IL-6 transsignaling by the administration of soluble gp130 (sgp130) receptor to capture the IL-6/soluble IL-6R complex has shown promise for the treatment of rheumatoid arthritis (RA). However, enhancing endogenous sgp130 via alternative splicing of the gp130 gene has not yet been tested. We found that epigallocatechin-3-gallate (EGCG), an anti-inflammatory compound found in green tea, inhibits IL-1β–induced IL-6 production and transsignaling in RA synovial fibroblasts by inducing alternative splicing of gp130 mRNA, resulting in enhanced sgp130 production. Results from in vivo studies using a rat adjuvant-induced arthritis model showed specific inhibition of IL-6 levels in the serum and joints of EGCG-treated rats by 28% and 40%, respectively, with concomitant amelioration of rat adjuvant-induced arthritis. We also observed a marked decrease in membrane-bound gp130 protein expression in the joint homogenates of the EGCG-treated group. In contrast, quantitative RT-PCR showed that the gp130/IL-6Rα mRNA ratio increased by ~2-fold, suggesting a possible mechanism of sgp130 activation by EGCG. Gelatin zymography results showed EGCG inhibits IL-6/soluble IL-6R–induced matrix metalloproteinase-2 activity in RA synovial fibroblasts and in joint homogenates, possibly via up-regulation of sgp130 synthesis. The results of these studies provide previously undescribed evidence of IL-6 synthesis and transsignaling inhibition by EGCG with a unique mechanism of sgp130 up-regulation, and thus hold promise as a potential therapeutic agent for RA.     

Preconditioning-induced protection of photoreceptors requires activation of the signal-transducing receptor gp130 in photoreceptors

Retinal degenerations are a class of neurodegenerative disorders that ultimately lead to blindness due to the death of retinal photoreceptors. In most cases, death is the result of long-term exposure to environmental, inflammatory, and genetic insults. In age-related macular degeneration, significant vision loss may take up to 70–80 years to develop. The protracted time to develop blindness suggests that retinal neurons have an endogenous mechanism for protection from chronic injury. Previous studies have shown that endogenous protective mechanisms can be induced by preconditioning animals with sublethal bright cyclic light. Such preconditioning can protect photoreceptors from a subsequent damaging insult and is thought to be accomplished through induced expression of protective factors. Some of the factors shown to be associated with protection bind and activate the signal transducing receptor gp130. To determine whether stress-induced endogenous protection of photoreceptors requires gp130, we generated conditional gp130 knockout (KO) mice with the Cre/lox system and used light-preconditioning to induce neuroprotection in these mice. Functional and morphological analyses demonstrated that the retina-specific gp130 KO impaired preconditioning-induced endogenous protection. Photoreceptor-specific gp130 KO mice had reduced protection, although the Müller cell KO mice did not, thus gp130-induced protection was restricted to photoreceptors. Using an animal model of retinitis pigmentosa, we found that the photoreceptor-specific gp130 KO increased sensitivity to genetically induced photoreceptor cell death, demonstrating that gp130 activation in photoreceptors had a general protective role independent of whether stress was caused by light or genetic mutations.     

An essential role for gp130 in neointima formation following arterial injury

Interleukin (IL)-6 induced vascular smooth muscle cell (VSMC) motility in a dose-dependent manner. In addition, IL-6 stimulated tyrosine phosphorylation of gp130, resulting in the recruitment and activation of STAT-3. IL-6-induced VSMC motility was found to be dependent on activation of gp130/STAT-3 signaling. IL-6 also induced cyclin D1 expression in a time- and gp130/STAT-3-dependent manner in VSMCs. Suppression of cyclin D1 levels via the use of its small interfering RNA molecules inhibited IL-6-induced VSMC motility. Furthermore, balloon injury induced IL-6 expression both at mRNA and protein levels in rat carotid artery. Balloon injury also caused increased STAT-3 phosphorylation and cyclin D1 expression, leading to smooth muscle cell migration from the media to the intimal region. Blockade of gp130/STAT-3 signaling via adenovirus-mediated expression of dngp130 or dnSTAT-3 attenuated balloon injury-induced STAT-3 phosphorylation and cyclin D1 induction, resulting in reduced smooth muscle cell migration from media to intima and decreased neointima formation. Together, these observations for the first time suggest that IL-6/gp130/STAT-3 signaling plays an important role in vascular wall remodeling particularly in the settings of postangioplasty and thereby in neointima formation.     

Requirement of gp130 signaling for the AGM hematopoiesis

OBJECTIVE: Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region during mouse development and remarkably expands in the liver at a later stage of ontogeny. gp130 is a signal transducing receptor component shared by all the IL-6 family cytokines, whose gene ablation in mouse results in the significant reduction in the fetal liver hematopoiesis. The present study aims to evaluate the role of gp130 signaling in the fetal mouse AGM hematopoiesis. METHODS AND MATERIALS: Mouse AGM regions from the wild-type and gp130-deficient mice on embryonic day 11.5 were dissociated and cultured with a mixture of cytokines, including one which activates gp130. Wild-type human gp130 and its mutant constructs were introduced into cultured gp130-deficient AGM cells using retrovirus system. To further analyze gp130 downstream signaling, a dominant-negative mutant of STAT3 was also introduced. RESULTS: The gp130 deficiency in the culture of fetal mouse AGM cells resulted in the failure of the expansion of the c-kit(+), Sca-1(+), and lineage markers(-) population. Such failure was rescued by introduction of a wild-type gp130 expression construct but not its mutant constructs having no ability to activate STAT3. In the normal AGM cell culture, introduction of a dominant-negative form of STAT3 in which Y(705) was changed to phenylalanine suppressed the expansion of hematopoietic cell colonies. CONCLUSION: gp130 plays an indispensable role in the expansion of hematopoietic precursor cells in the fetal mouse AGM. In particular, the activation of STAT3 by gp130 is found to be important in this process.