内质网分子伴侣糖调节蛋白94在大鼠酒精性肝损伤中的高表达和意义

目的检测酒精性肝损伤大鼠肝组织内质网分子伴侣糖调节蛋白94的表达情况,探讨酒精性肝损伤的发病机制.方法24只SD雌性大鼠分成对照组和模型组.模型组大鼠给予乙醇加鱼油灌胃配合高脂饮食8周,建立酒精性肝损伤模型,应用半定量逆转录聚合酶链反应(RT-PCR)法和免疫组化法检测大鼠肝组织内质网分子伴侣糖调节蛋白94 mRNA和蛋白水平.结果与正常组比较,模型组大鼠肝组织糖调节蛋白94 mRNA和蛋白表达明显增强(P＜0.01).结论酒精性肝损伤大鼠存在糖调节蛋白94高表达,这可能是该病的发病机制之一.     

大鼠非酒精性脂肪肝中UCP2的动态表达

目的探讨解偶联蛋-2(uncoupling protein-2,UCP2)在大鼠非酒精性脂肪肝发病机理中的作用.方法Wistar大鼠64只,随机分为高脂饮食诱导脂肪肝组和正常对照组,用免疫组织化学和Western blot技术检测肝组织中UCP2表达变化,生化检测大鼠血清甘油三酯(TG)、游离脂肪酸(FAA)和丙氨酸氨基转移酶(ALT)含量.结果大鼠非酒精性脂肪形成过程中UCP2阳性细胞数和表达强度逐渐增加,血清TG、FAA和ALT含量较对照组增高,以高脂饮食喂养8、12周为著.结论随着非酒精性脂肪的形成和程度加重,UCP2表达逐渐增强,其介导的酶活性显著增高,启动脂质过氧化反应,促进脂肪肝的形成和发展.     

肝细胞L-FABP、FATP4在大鼠非酒精性脂肪性肝病形成中的表达及意义

目的研究肝脏型脂肪酸结合蛋白（L—FABP）、4型脂肪酸转运蛋白（FATP4）在大鼠非酒精性脂肪性肝病（NAFLD）发病机制中的表达及意义。方法建立高脂饮食脂肪肝模型，用定量逆转录聚合酶链反应与半定量聚丙烯凝胶蛋白电泳方法测定脂肪肝肝组织中L—FABP、FATP4表达变化。结果高脂饮食脂肪肝大鼠肝脏中L—FABP于2周时其mRNA及蛋白表达增强，12周时表达最为明显，与正常组比较，差异有统计学意义；FATP4 mRNA水平及蛋白表达趋势同L—FABP，结果基本相近似（L—FABP mRNA，F=124．9；蛋白表达，F=92．6；FATP4 mRNA，F=602．9；蛋白表达，F=108．8，JD值均〈0．05）。结论高脂饮食引起L—FABP、FATP4表达增强，最初是一种适应性反应，随着L—FABP、FATP4表达进一步增强，导致脂肪酸代谢失衡，引起脂肪肝的发生。     

Cystatin C在大鼠酒精性肝病中的表达及意义

目的探讨胱蛋白酶抑制剂C（Cystatin C,Cys C）在大鼠酒精性肝病中的表达及意义。方法酒精灌胃法制备大鼠酒精性肝病模型。12周末处死大鼠,测定大鼠肝指数,检测血清天门冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）含量,HE染色观察肝脏病理学改变,免疫组化SAB法比较正常组和模型组Cys C蛋白的表达,RT-PCR法比较两组肝组织Cys C mRNA的表达变化。结果模型组肝指数较正常组显著升高（P〈0.01）;HE染色观察模型组肝细胞肿胀,包浆内可见大小不等的脂肪空泡及炎性细胞浸润;模型组血清ALT和AST较正常组明显升高（P〈0.05）;免疫组化及RT-PCR显示,肝组织中Cys C蛋白及mRNA表达模型组较正常组显著上调（P〈0.01）。结论肝组织Cys C表达上调可能参与了酒精性肝病的发生、发展过程。     

非酒精性脂肪肝大鼠肝细胞色素P450 2E1基因及表达变化的意义

细胞色素P450 2E1(cytochrome P450 2E1,CYP 2E1),是细胞色素P450的乙醇诱导形式,它在非乙醇脱氢酶氧化途径中起重要作用,为酒精性肝病的主要发病机制。CYP 2E1基因及多型性与酒精性肝病的发生有密切关系,通过高脂饲料诱导的大鼠脂肪肝模型,探讨在脂肪肝的发展过程中肝CYP 2E1基因及表达变化规律,为脂肪肝的发生和治疗提供理论基础。     

多重耐药蛋白-1在胃癌中的表达及意义

目的:研究胃癌组织中多重耐药蛋白(MRP)-1的表达规律.方法:用免疫组织化学染色(SP法)检测30例慢性浅表性胃炎(CSG)、30例胃黏膜肠上皮化生(IM)、45例异型增生(Dys)和65例胃癌(GC)组织标本中MRP-1阳性表达情况,RTPCR技术检测MRP-1 mRNA的表达情况.均数的比较采用t检验,样本率的比较采用χ2检验,两因素的相关分析用直线相关分析.结果:在CSG→IM→Dys→GC的过程中,MRP-1的表达呈逐步递增趋势,MRP-1的阳性表达率GC组与Dys,IM组,均具有显著性差异(43/65 vs 19/45,6/30,P＜0.05),GC组MRP-1以及MRP-1 mRNA阳性表达率均高于相应癌旁组,差异具有非常显著性(66.2% vs 33.3%,63.1% vs 28.9%,P＜0.01);MRP-1与MRP-1mRNA表达强度呈正相关(r=0.598,P＜0.05).结论:胃癌形成过程中,MRP-1与MRP-1mRNA表达逐渐上调.     

解偶联蛋白2在非酒精性脂肪肝中的作用探讨

随着生活水平的不断提高,饮食结构和生活方式的改变,脂肪肝已成为仅次于病毒性肝炎的第二大肝病。脂肪肝的发病机制目前尚不十分清楚,多种因素、多种细胞因子参与其发病;解偶联蛋白2(UCP_2)是近年发现的一种线粒体内膜上的载体蛋白,它与线粒体ATP合成、脂质代谢等密切相关。现对UCP_2与肥胖、游离脂肪酸(FFA)、活性氧(ROS)、脂质过氧化物(LPO)、胰岛素、瘦素以及肿瘤坏死因子(TNFα)等因素的关系作一阐述,以探讨UCP_2在非酒精性脂肪肝(NAFLD)发病中的可能作用。     

解偶联蛋白2在非酒精性脂肪肝中的作用探讨

随着生活水平的不断提高，饮食结构和生活方式的改变，脂肪肝已成为仅次于病毒性肝炎的第二大肝病。脂肪肝的发病机制目前尚不十分清楚，多种因素、多种细胞因子参与其发病；解偶联蛋白2(UCP2)是近年发现的一种线粒体内膜上的载体蛋白，它与线粒体ATP合成、脂质代谢等密切相关。现对UCP2与肥胖、游离脂肪酸(FFA)、活性氧(ROS)、脂质过氧化物(LPO)、胰岛素、瘦素以及肿瘤坏死因子(TNFα)等因素的美系作一阐述．以探讨UCP2在非酒精性脂肪肝(NAFLD)发病中的可能作用。     

Syndecan-1蛋白及mRNA在肺鳞癌中的表达及意义

目的探讨Syndecan-1蛋白及mRNA在肺鳞癌组织中的表达及其与临床病理特征的关系。方法应用免疫组化催化放大法（CSA）及RT-PCR法检测Syndecan-1蛋白及mRNA在30例肺鳞癌组织、30例癌旁正常肺组织中的表达。结果 Syndecan-1蛋白及mRNA在肺鳞癌组织中表达低于癌旁正常肺组织（P〈0.05）。Syndecan-1蛋白及mRNA低表达与肿瘤分化程度、淋巴结转移正相关（P〈0.05）,与年龄、性别及TNM分期无关（P〉0.05）。结论 Syndecan-1蛋白及mRNA在肺鳞癌中表达减低,并与肺鳞癌发生发展及浸润转移相关。     

腺苷酸环化酶相关蛋白2在肝癌组织中的表达及其意义

有关腺苷酸环化酶相关蛋白(adenylate cyclase-associated protein,CAP)2在肝癌中的生物学作用机制尚不明确.本研究应用逆转录聚合酶链反应(RT-PCR)及免疫组织化学技术检测CAP2在人正常肝脏、肝硬化和肝癌组织中的表达,探讨CAP2在肝癌发生发展过程中的作用.     

内质网应激在非酒精性脂肪性肝病发病机制中的作用

非酒精性脂肪性肝病(NAFLD)的发病率在全球范围内迅速增加,但其发病的确切机制并不十分清楚。一方面探讨内质网应激(ERS)可以通过未折叠氮蛋白反应(URP)促进NAFLD的形成及进展为非酒精性脂肪性肝炎(NASH),另一方面探讨NAFLD的脂质环境,尤其是脂肪酸,又可诱发ERS的发生。从而指出ERS相关信号转导在NAFLD发病机制中的作用,为NAFLD的治疗提供新的思路。     

Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease(NAFLD)has emerged as a common public health problem in recent decades.However,the underlying mechanisms leading to the development of NAFLD are not fully understood.The endoplasmic reticulum(ER)stress response has recently been proposed to play a crucial role in both the development of steatosis and progression to nonalcoholic steatohepatitis.ER stress is activated to regulate protein synthesis and restore homeostatic equilibrium when the cell is stressed due to the accumulation of unfolded or misfolded proteins.However,delayed or insufficient responses to ER stress may turn physiological mechanisms into pathological consequences,including fat accumulation,insulin resistance,inflammation,and apoptosis,all of which play important roles in the pathogenesis of NAFLD.Therefore,understanding the role of ER stress in the pathogenesis of NAFLD has become a topic of intense investigation.This review highlights the recent findings linking ER stress signaling pathways to the pathogenesis of NAFLD.     

The endoplasmic reticulum chaperone BiP is a closure-accelerating cochaperone of Grp94

Hsp70 and Hsp90 chaperones provide protein quality control to the cytoplasm, endoplasmic reticulum (ER), and mitochondria. Hsp90 activity is often enhanced by cochaperones that drive conformational changes needed for ATP-dependent closure and capture of client proteins. Hsp90 activity is also enhanced when working with Hsp70, but, in this case, the underlying mechanistic explanation is poorly understood. Here we examine the ER-specific Hsp70/Hsp90 paralogs (BiP/Grp94) and discover that BiP itself acts as a cochaperone that accelerates Grp94 closure. The BiP nucleotide binding domain, which interacts with the Grp94 middle domain, is responsible for Grp94 closure acceleration. A client protein initiates a coordinated progression of steps for the BiP/Grp94 system, in which client binding to BiP causes a conformational change that enables BiP to bind to Grp94 and accelerate its ATP-dependent closure. Single-molecule fluorescence resonance energy transfer measurements show that BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that otherwise acts as an energetic barrier to closure. These findings provide an explanation for enhanced activity of BiP and Grp94 when working as a pair, and demonstrate the importance of a high-energy conformational state in controlling the timing of the Grp94 conformational cycle. Given the high conservation of the Hsp70/Hsp90 system, other Hsp70s may also serve dual roles as both chaperones and closure-accelerating cochaperones to their Hsp90 counterparts.

Heat shock proteins maintain protein folding quality control under nonstress conditions, and their function is essential for survival under stress conditions (1). Hsp70 and Hsp90 family chaperones are conserved components of folding quality control from bacteria to humans, and serve critical roles in the endoplasmic reticulum (ER) and mitochondria. Hsp90 often exhibits a minimal capacity to assist client folding when working alone, while becoming functional when operating with Hsp70 (2–4), and the underlying mechanistic explanation is an active area of research (5, 6).BiP and Grp94 (Fig. 1 A and B) aid in the folding of ER-specific clients ranging from immunoglobulins (7, 8) to insulin-like growth factors (9–11). BiP and Grp94 associate only when BiP adopts the “ADP conformation” (Fig. 1C) (12), a state in which BiP can trap client proteins (13–15). The BiP nucleotide binding domain (NBD) interacts with the Grp94 middle domain (MD) through stabilizing salt bridges that are conserved from bacteria to humans (12, 16, 17). Despite core similarities, Hsp70/Hsp90 systems differ in their regulation. For example, eukaryotic Hsp70/Hsp90 systems in the cytosol can be further regulated via Hop (a cochaperone that bridges Hsp70 and Hsp90). Mechanistic dissection of the Hsp70/Hop/Hsp90 system is complicated by conformational and compositional heterogeneity (18, 19). No Hop equivalent is expressed in the ER, which makes the BiP/Grp94 system a relatively simple system for mechanistic analysis.Open in a separate windowFig. 1.Overview of BiP and Grp94 conformational cycles. (A) Conformations of BiP in the presence of ATP (Left, PDB ID code 5E84) and ADP (Right, modeled from PDB ID code 2KHO). (B) Conformations of Grp94 in the presence of ADP (Left, open state, PDB ID code 2O1V) and ATP (Right, closed state, PDB ID code 5ULS). (C) Conformation-specific binding between BiP and Grp94.Grp94, as with other Hsp90 family members, is a homodimer with three domains on each arm: N-terminal domain (NTD), MD, and C-terminal domain (CTD) (Fig. 1B). When ADP is bound, Grp94 adopts an open conformation with an exposed client binding pocket. Upon ATP binding, Grp94 can close, and potentially trap a client between the dimer arms (20). However, Grp94 closure occurs slowly [on the order of 0.1 to 1 per minute (21–23)], suggesting that regulated closure acceleration is necessary. Extensive cochaperone regulation is a hallmark of the cytosol-specific Hsp90 (24), although, in many cases, these cochaperones appear to suppress Hsp90 client folding activity rather than enhance it (25). In the case of Grp94, much less is known about cochaperones; only two have been identified (26, 27), and these appear to specifically recruit certain clients to Grp94 rather than act generally on the Grp94 conformational cycle. A key feature of such cochaperones is their targeted influence on accelerating or decelerating specific steps of the Hsp90 conformational cycle (28–30).Hsp90 family conformational dynamics are more complex than a simple cycling between one closed and one open state. For example, single-molecule fluorescence resonance energy transfer (smFRET) measurements have identified a Grp94 conformational intermediate (termed C′) populated upon Grp94 opening from the closed state (21). The C′ state is observed in the closed→open half-cycle but is not significantly populated during the open→closed half-cycle. Similarly, two closed conformations have been detected for cytosolic Hsp90 from bulk FRET, smFRET, and electron paramagnetic resonance measurements (31–33). These alternative closed structures are not known with certainty, but one has been proposed from smFRET measurements (34), and a recent report shows cytosolic Hsp90 in a semiclosed conformation when in complex with Hsp70, Hop, and the glucocorticoid receptor (16). Structural studies of Trap1 (the mitochondria-specific Hsp90 paralog) have also identified different closed conformations that are distinct from the well-established closed conformations of cytosolic Hsp90 and Grp94. Trap1 can adopt an asymmetric closed conformation (35) in which the two arms adopt different conformations at their MD/CTD interface region, while their NTD region resembles the closed conformation of cytosolic Hsp90 and Grp94. Trap1 can also adopt a “coiled-coil” conformation in which the N-terminal α-helix adopts a cross-monomer configuration that differs from both the open and closed states (36). The functional roles of these alternative Hsp90 closed conformations are unclear at present. Here we show that BiP is a powerful closure-accelerating cochaperone of Grp94. BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that, our data suggest, resembles the coiled-coil conformation of Trap1.     

大鼠非酒精性脂肪性肝病与内质网应激关系的研究

目的 通过建立大鼠非酒精性脂肪性肝病(NAFLD)模型,探讨内质网应激(ERS)在NAFLD中的作用.方法 36只雄性SD大鼠分为对照1组(n=8)、对照2组(n=8)、模型1组(n=10)和模型2组(n=10).对照组大鼠给予普通饲料,模型组大鼠则喂饲高脂饲料.分别于第12周末处死对照1组和模型1组,第20周末处死对照2组和模型2组大鼠.测定血清丙氨酸转氨酶(ALT)、天冬氨酸转氮酶(AST)、γ-谷氨酰转移酶(GGT)、碱性磷酸酶(ALP)、总蛋白(TP)、三酰甘油(TG)、总胆固醇(TC)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)水平.H-E和Masson染色观察肝组织脂肪变、炎症和纤维化的变化.实时定量PCR测定肝组织内GRP78、CHOP及proeaspase-12 mRNA表达情况.Western印迹法检测肝组织内procaspase-12和caspase-12蛋白的变化.结果 第12周末.模型1组大鼠ALT、AST、ALP、TC及LDL水平均较对照1组显著增高(P<0.01),而HDL水平显著降低(P<0.01);第20周末模型2组大鼠TC和LDL水平较模型1组显著增加(P<0.05).与对照1组相比,模型1组肝组织的脂肪变和炎症程度均明显增加(P值均<0.01);模型2组肝组织的脂肪变、炎症程度和纤维化分期较对照2组均明显增加(P值均<0.01),炎症程度和纤维化分期较模型1组明显加重(P值分别<0.05和0.01).模型组大鼠肝组织GRP78、CHOP及procaspase-12 mRNA水平在第12周末及第20周末时与对照组比较差异均无统计学意义(P值均>0.05).模型组caspase-12及其前体蛋白水平与对照组相比也无显著变化(P值均>0.05).结论 成功建立高脂饮食大鼠NAFLD模型.研究中未发现ERS,提示此模型中ERS诱导的肝脏损伤可能未参与NAFLD的发病机制.     

内质网应激与非酒精性脂肪性肝病

近年来对内质网应激在代谢综合征等方面的作用研究十分广泛,而非酒精性脂肪性肝病的发病机制与代谢综合征密切相关,现就内质网应激在非酒精性脂肪性肝病发生、发展中的作用和意义作一综述。     

内质网应激相关蛋白在阿霉素诱导的急性心肌损伤中的表达及意义

目的探讨内质网应激(ERS)相关葡萄糖调节蛋白78(GRP78)和增强子结合蛋白环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)在阿霉素诱导的急性心肌损伤中的表达及意义。方法将8~10周雄性野生型C57BL/6J小鼠随机分为对照组与阿霉素组,每组20只,阿霉素组小鼠腹腔注射大剂量阿霉素(20mg/kg)诱导急性心肌损伤模型,对照组小鼠给予腹腔注射等量生理盐水。检测2组小鼠LVEF以及左心室短轴缩短率(LVFS)等心功能指标,记录体质量、心质量,心肌组织行苏木精-伊红染色。采用Real time-PCR和Western blotting法检测小鼠心肌组织GRP78、CHOP、Bax及Bcl-2mRNA和蛋白表达。结果与对照组相比,阿霉素组小鼠体质量、心脏质量、LVEF及LVFS明显降低,差异有统计学意义[(19.92±0.80)g vs(24.34±1.29)g,(96.58±8.37)mg vs(122.37±6.72)mg,(68.67±3.39)%vs(84.20±2.91)%,(27.10±1.24)%vs(39.96±3.53)%,P0.01]。组织病理结果显示阿霉素组小鼠心肌细胞排列紊乱,细胞质空泡化,心肌纤维扭曲,断裂。与对照组比较,阿霉素组小鼠心肌组织中GRP78、CHOP及Bax表达明显上调(P0.05),Bcl-2表达明显降低(P0.05),Bax/Bcl-2显著升高(P0.05)。结论 ERS可能通过调控心肌细胞凋亡,参与阿霉素诱导的急性心肌损伤过程。     

非酒精性脂肪肝患者血清瘦素水平检测的临床意义

研究非酒精性脂肪肝患者血清瘦素水平检测的临床意义。选择门诊或住院患者非酒精性脂肪肝30例,正常对照30例,采用放射免疫法检测血清瘦素及胰岛素水平。结果发现,脂肪肝患者血清瘦素(23.7±13.1)μg/ml,胰岛素水平、(16.7±4.9)μg/ml显著高于正常对照组(4.7±3.2)μg/ml及(9.7±2.6)μg/ml,P<0.001。无论脂肪肝组还是正常对照组,血清瘦素水平升高存在性别差异,女性明显高于男性。脂肪肝患者存在瘦素抵抗和胰岛素抵抗,早期检测血清瘦素水平,为早期干预治疗脂肪肝提供了可能的途径。