高脂喂养大鼠肾小管上皮细胞SREBP-1、TGF-β1、α-SMA的表达和细胞外基质沉积

目的:探讨高脂喂养对大鼠肾脏小管上皮细胞SREBP-1、TGF-β1、α-SMA表达和细胞外基质(ECM)的影响。方法:高脂饲料喂养大鼠12周后,油红O检测肾脏脂质沉积,Masson染色检测肾小管间质细胞外基质沉积,免疫组化、Western blot和原位杂交检测SREBP-1、TGF-β1、α-SMA和FN的表达。结果:高脂喂养后大鼠体重明显增加,血糖、甘油三酯和胰岛素均升高,油红O检测显示大鼠肾小管上皮细胞内出现明显脂滴。SREBP-1蛋白和mRNA在肾小管上皮细胞内表达,高脂组高于正常对照组,分别是正常组的1.88倍和1.85倍;TGF-β1和α-SMA也定位于肾小管上皮细胞胞浆并出现上调。Masson染色显示高脂喂养大鼠肾间质ECM沉积增多,纤维粘连蛋白FN检测也显示模型组表达强于对照组。结论:高脂饮食喂养可能通过上调肾脏小管上皮细胞SREBP-1表达使细胞内脂滴沉积,并进一步诱导TGF-β1、α-SMA合成而导致细胞外基质堆积。     

雌激素对SD大鼠肾脏缺血再灌注肾小管上皮细胞Cx43蛋白表达的影响

为了探讨雌激素（estrogen,E₂）对SD大鼠肾脏缺血再灌注（ischemia-reperfusion,I/R）肾小管上皮细胞Cx43蛋白表达的影响,选取32只健康雌性SD大鼠作为实验材料,所有雌性SD大鼠均先实施去卵巢（ovariectomized,OVX）处理,之后32只雌性OVX SD大鼠随机分为OVX组、OVX+假手术组、OVX+I/R组、OVX+I/R+E₂组,每组8只。去卵巢2周后,OVX组不进行任何处理,OVX+Sham组进行假手术处理,OVX+I/R组进行缺血再灌注处理,OVX+I/R+E₂组在进行缺血再灌注处理前,连续3 d予以雌激素处理。通过比较各组SD大鼠血肌酐水平、尿素氮水平、肾脏HE染色及Paller评分,评价肾脏组织损伤程度,并通过免疫组化和蛋白印迹法分析各组SD大鼠肾小管上皮细胞中Cx43蛋白的表达位置及水平。与OVX组比较,OVX+I/R组血清BUN、SCr水平明显升高（P<0.05）;与OVX+I/R组比较,OVX+I/R+E₂组血清BUN、SCr水平明显降低（P<0.05）。HE染色观察发现,与OVX组相比,OVX+I/R组肾小管扩张明显,部分细胞碎裂坏死,Paller评分明显升高（P<0.05）;与OVX+I/R组相比,OVX+I/R+E2组可见肾小管轻度扩张,细胞结构较为完整,Paller评分明显下降（P<0.05）。通过免疫组化发现,OVX+I/R组Cx43蛋白在肾小管上皮细胞中阳性表达高于OVX组及OVX+Sham组（P<0.05）,OVX+I/R+E₂组Cx43蛋白表达低于OVX+I/R组（P<0.05）。Western blotting结果显示,与OVX组相比,OVX+I/R组Cx43蛋白在肾小管上皮细胞中表达量增加,肾脏损伤后肾小管上皮细胞Cx43表达明显上调（P<0.05）;与OVX+I/R组相比,OVX+I/R+E₂组经过雌激素干预后,肾脏功能损伤减轻,肾小管上皮细胞Cx43的蛋白表达量下调（P<0.05）。结果表明雌激素可减轻肾缺血再灌注损伤造成的肾脏损伤,其可能是通过调节肾小管上皮细胞中Cx43的表达实现的。研究结果为临床治疗肾脏缺血再灌注损伤提供了新的思路。     

干预脂毒性改善糖尿病大鼠胰岛素分泌及氧化应激的损害

目的探讨干预脂毒性改善糖尿病大鼠胰岛分泌功能及氧化应激损害的机制。方法将大鼠分为4组①正常组（NC）,全程普通饲料喂养;②高脂组（HF）,全程高脂饲料喂养。糖尿病组,高脂饲料喂养8周后腹腔注射低剂量STZ（30mg/kg）,48h后行OGTT试验判断成模情况后分组。③糖尿病对照组（DM）,不给予药物干预;④血脂干预组（SIM）,灌胃辛伐他汀5mg/（kg.d）4周干预脂毒性。通过免疫组化染色观察胰岛B、A细胞形态学特点,RT-PCR测定胰腺内胰岛素原mRNA表达水平,DHE荧光染色检测胰岛中活性氧化产物ROS水平。结果与糖尿病对照组相比,干预脂毒性4周后血清胆固醇（TC）和甘油三酯（TG）水平分别下降了22.9%（P〈0.01）和57.0%（P〈0.05）。OGTT血糖水平均显著下降（P〈0.01）。胰岛中B细胞相对量是对照组的2.6倍（P〈0.01）,B细胞胞质内胰岛素水平增加了26.5%（P〈0.05）,胰岛素原mRNA表达升高18.3%（P〈0.01）;A细胞相对量减少了50%（P〈0.01）。血清丙二醛（MDA）水平和胰腺中ROS表达显著下降。结论辛伐他汀干预脂毒性4周可以显著改善糖尿病大鼠胰岛分泌功能和氧化应激损害。     

高脂喂养联合链脲佐菌素注射的糖尿病大鼠模型特征

目的观察高脂喂养联合低剂量STZ注射的SpragueDawley(SD)大鼠2型糖尿病模型的代谢特征、病理学以及胰岛分子生物学变化。方法4周龄雄性SD大鼠36只随机分为三组(1)正常对照组(Control)9只,普通饲料喂养。(2)高脂组(HighFatchow,HE)9只,高脂饲料喂养,为普通饲料中添加20%脂肪(猪油和蛋黄粉各50%)和20%蔗糖。(3)糖尿病组(DM)18只。喂养4周后腹腔注射STZ(40mg/kg)。所有大鼠做灌胃葡萄糖耐量(OGTT)试验。放免法测定血清胰岛素,免疫组化染色观察胰岛β细胞的形态学特点,彩色图像分析系统测定胰岛素表达量,RT-PCR测定胰腺β细胞胰岛素mRNA表达水平。结果糖尿病大鼠空腹血糖(FBG)、胰岛素水平(FINS)显著高于Control组和HE组大鼠(P<0.01),空腹血清甘油三酯(TG)和游离脂肪酸(FFA)水平显著高于Control组(P<0.05);胰岛β细胞吸光度(A)显著低于高脂组大鼠(P<0.05),降低11.6%。胰岛素免疫反应阳性区占胰岛百分比显著低于Control组和HE组,分别下降31.9%(P<0.05)和43.1%(P<0.01)。胰岛素mRNA表达水平显著低于HE组(P<0.05)。STZ注射后48h(基线值)大鼠FBG水平的分布情况为A组(FBG<10.0mmol/L)占7/18;B组(FBG10~19.9mmol/L)占5/18;C组(FBG≥20mmol/L)占6/18。STZ注射后9d的OGTT结果与基线值相比,B组OGTT值总体变化最小,A组FBG的变异最大,达到25%。结论高脂喂养联合低剂量STZ注射的糖尿病大鼠模型模拟2型糖尿病发生的主要病理生理过程,具有高血糖、高胰岛素血症以及血脂异常等基本特征。     

性别对小鼠高脂血症动物模型的影响

目的观察性别对建立小鼠高脂血症动物模型的影响。方法健康成年昆明种小鼠40只,雌雄各半,随机分4组：雄性对照组、雌性对照组、雄性高脂组、雌性高脂组,按各组要求分别给予正常饲料和高脂饲料喂养。连续2周,取血测定实验小鼠血中总胆固醇（total cholesterol,TC）、甘油三酯（triglyceride,TG）、低密度脂蛋白（low density lipoprotein,LDL）、高密度脂蛋白胆固醇（high density lipoprotein cholesterol,HDL-C）含量。结果雌性对照组血脂四项指标与雄性对照组差异不显著,高脂饮食使小鼠血中TC、LDL水平明显增高（P〈0.05）,雌雄组之间差异不显著。结论性别对实验小鼠高脂血症模型的建立及效果无明显影响。     

大剂量维生素D3补充对糖尿病大鼠胰岛及肌肉细胞组织学的影响

目的：探讨大剂量维生素D3补充对糖尿病（DM）大鼠Lees指数及胰岛、肌肉细胞组织学的影响。方法：高糖高脂喂养结合注射链脲佐菌素的方法制备DM模型,不同浓度的维生素D3灌胃6w,HE染色观察胰岛和肌肉形态。结果：125倍和200倍维生素D3组大鼠Lees指数比DM组明显升高。各维生素D干预组胰岛和骨骼肌组织学形态明显改善。结论：适量维生素D3补充能增加DM大鼠Lees指数,恢复异常的胰岛和肌肉形态。     

HFD对不同性别SD大鼠肠道菌群及室旁核小胶质细胞激活的影响

【目的】通过建立雌、雄性SD (Sprague-Dawley)大鼠模型,研究高脂饮食(high fat diet,HFD)对不同性别大鼠肠道菌群及室旁核(hypothalamic paraventricular nucleus,PVN)区小胶质细胞激活的影响。【方法】选取24只3周龄的SD大鼠,雌雄各半,随机分成雄性对照组(CM)、雌性对照组(CF),雄性高脂组(HM)、雌性高脂组(HF),共4组,每组6只。喂养至10周龄,收集大鼠新鲜粪便并提取细菌总基因组DNA,通过PCR扩增16SrDNA的V3+V4区域,进行高通量测序;采用气相色谱法分析大鼠盲肠内容物中短链脂肪酸(short chain fatty acids,SCFAs)含量;以real-time PCR技术分析PVN区小胶质细胞激活标记物CD11b和炎性细胞因子TNF-α、IL-6的m RNA相对表达水平。【结果】雌、雄高脂组分别与对照组相比,HM组大鼠肠道菌群总数及菌群多样性明显减少(P<0.05),HF组大鼠肠道菌群丰度明显减少(P<0.05)。与CM组相比,HM组大鼠盲肠中乙酸含量显著下降(P<0.0...     

深黄被孢霉脂质提取物改善大鼠脂质肾损害

目的：探讨深黄被孢霉脂质提取物能否改善脂质肾损害模型大鼠血脂异常及肾脏损伤。方法：雄性Wis-tar大鼠随机分为4组（n=8）：①对照（N）组;②高脂模型（M）组;③深黄被孢霉脂提物干预（T）组;④辛伐他汀治疗（S）组。N组饲以普通饲料,M组给予高脂饲料喂养,两组均每日灌服生理盐水;T组和S组在高脂饲料喂养同时每日分别灌服深黄被孢霉脂提物和辛伐他汀。于实验前、第4、8、12周检测24h尿蛋白含量,12周时检测血清白蛋白、总蛋白、甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白（LDL）、尿素氮（BUN）、肌酐（Scr）水平;检测肾组织TG、TC、LDL含量;观察肾组织病理及超微结构改变、脂质沉积。结果：实验第12周,T组和S组24h尿蛋白定量、血清及肾组织TC、LDL明显低于模型组,血清白蛋白、总蛋白显著高于模型组,T和S组之间差异无显著性。病理改变显示：M组肾小管间质大量炎症细胞浸润,管腔内有蛋白管型,肾小球系膜细胞轻度增生、系膜基质增宽,T组与S组炎症细胞浸润明显减少,未见蛋白管型及系膜区病理改变;M组肾小球及肾小管内大量脂肪沉积,足细胞足突融合消失;T组与S组未见脂肪沉积;足细胞仅部分足突融合,小管上皮细胞内仅少量脂滴。结论：深黄被孢霉脂质提取物能改善高脂模型大鼠的高胆固醇血症,减少肾组织中胆固醇的沉积、炎症细胞的浸润及足细胞的损伤,减少尿蛋白漏出,对脂质肾损害大鼠肾脏具有保护作用。     

2型糖尿病大鼠模型制备的影响因素及其特点

目的探讨高脂喂养联合低剂量链脲佐菌素(Streptozotocin,STZ)制备2型糖尿病大鼠模型的造模方法和影响因素。方法4周龄雄性Sprague Dawley(SD)大鼠45只随机分为三组:(1)正常组(normal control,NC),9只,普通饲料喂养。(2)高脂组(high fat,HF),9只,高脂饲料喂养。(3)糖尿病模型组,根据高脂喂养时间差异和STZ剂量不同设计了3种模型制备方法:A组,9只,高脂喂养满4周,注射STZ 30 mg/kg;B组,9只,高脂喂养满8周,注射STZ 20 mg/kg;C组,9只,高脂喂养满8周,注射STZ 30 mg/kg。所有大鼠于48h、2周和4周后行灌胃葡萄糖耐量试验(OGTT)评价成模率和血糖波动情况。实验结束时测定血清胰岛素、甘油三酯(TG)和胆固醇(TC),RT-PCR测定胰腺内胰岛素mRNA表达水平,免疫组化染色观察胰岛细胞形态学特点,用彩色图像分析系统进行定量比较。结果糖尿病C组血糖显著升高,成模后2周血糖下降,4周后又上升到基线水平,成模率100%。糖尿病A组、B组在4周后血糖逐渐降低到接近正常水平,成模率分别为55.6%、11.1%。C组与HF组相比,胰岛素敏感性显著下降(P<0.01)。β细胞内胰岛素水平下降39.3%(P<0.01),胰岛内β细胞所占比例下降了79.2%(P<0.01),胰腺内胰岛素mRNA表达水平减少19.2%(P<0.01),α细胞升高了1倍(P<0.01)。结论高脂喂养8周后腹腔注射低剂量STZ(30 mg/kg)制备的2型糖尿病大鼠模型,成模率高,模型稳定。     

儿童单纯性肥胖小鼠模型的制作

目的通过调整窝仔数的方法建立幼儿单纯性肥胖模型,并与高脂饲料制备模型进行比较。方法48只雌性KM小鼠产仔鼠后,一半仔鼠数目为14～16只,一半仔鼠调整为6只（雌雄各3只）。仔鼠离乳时或第9周时仔鼠分别饲喂普通饲料或高脂饲料。仔鼠在15周后处死,称重,测量体长、腰围,生殖器重、脂肪重（肾周和生殖器周脂肪）,计算体脂比。结果①BD2组常规饲料饲喂至15周结束后,无论雌雄仔鼠,其体重与BDl组比较差异均有显著性（P〈0．05）,且BD2组体重超过BD1组体重雌性为26．3％,雄性为20．0％。同一处理方式,雌性仔鼠体脂比均高于雄性。②不同时间饲喂高脂饲料,无论调整窝仔数与否,高脂饲料各组仔鼠,无论雌雄,其各组仔鼠体重均比BD1组高（P均〈0．05）;HFD4组雌性和雄性仔鼠体重与BD2组相比差异均存在显著性（P〈0．05）。结论通过调整窝仔数的方法可以成功制备儿童单纯性肥胖模型。在儿童早期肥胖的情况下,成年后过度高脂饮食会导致机体储存更多的脂肪。     

间隙连接蛋白Cx43在人胚肺和肺癌细胞表达的研究

细胞与细胞之间通过细胞膜上的间隙连接通道交换小分子和离子进行细胞间通讯,对细胞增殖分化调控和机体内环境稳定有重要作用。用间隙连接蛋白Ｃｘ４３ｃＤＮＡ探针Ｎｏｒｔｈｅｒｎ印迹杂交,Ｃｘ４３抗体免疫荧光染色和罗氏黄荧光染料传输方法检查,正常人胚肺细胞的Ｃｘ４３在ｍＲＮＡ和蛋白水平有高表达,Ｃｘ４３蛋白免疫荧光分布在间隙连接的部位,细胞间隙连接通讯功能明显。与正常相反,人肺癌ＰＧ系细胞Ｃｋ４３无论在ｍＲＮＡ或蛋白质水平都无表达,细胞通讯功能缺陷。结果表明Ｃｘ４３在培养的人胚肺细胞有功能性表达。人肺癌ＰＧ细胞通讯功能缺陷与Ｃｘ４３基因转录抑制有关。对Ｃｘ基因的抑癌基因性质进行讨论。     

Connexin40 and connexin43 in mouse aortic endothelium: evidence for coordinated regulation

In the vessel wall, endothelial cells are metabolically and electrically coupled to each other and to the adjacent smooth muscle cells by gap junctions composed of connexins. Gap junctions may be formed from combinations of several different connexin proteins, and deletion of one connexin can lead to modification of the expression of another. To reveal a possible interaction between connexin40 (Cx40) and connexin43 (Cx43) in endothelium, we studied their distribution in vessels from C57Bl/6 and Cx40 knockout mice (Cx40-/-) using immunoblots and immunocytochemistry on aortic cross sections and en face whole mounts. En face preparations from C57Bl/6 mice revealed two distinct pools of Cx43, one pericellular and the other intracellular. Cx40 was largely restricted to the periphery of the cells, and in Cx40-/- mice it was, as expected, undetectable. In the Cx40-/- mice, total Cx43 protein was also modestly reduced (immunoblots), but there was a major redistribution of the protein within the cell. The pericellular component of Cx43 was rendered virtually undetectable, and the intracellular compartments were normal or even slightly elevated. Smooth muscle Cx43 was also reduced in the Cx40-/- animals. These findings indicate that the cellular distribution of Cx43 is dependent on the presence of Cx40, and in view of the profound effects on the pericellular pool of the Cx43, the findings suggest that interactions between Cx40 and Cx43 regulate communication between endothelial cells and perhaps between smooth muscle and endothelial cells as well.     

间隙连接蛋白基因connexin43、ER、PR的表达在子宫平滑肌瘤中相关性研究

研究细胞间隙连接蛋白基因43（connexin43,CX43）及其蛋白、雌激素受体（estrogen receptor,ER）、孕激素受体（progesterone receptor,PR）在子宫平滑肌瘤中的表达,从核酸及蛋白水平探讨在子宫平滑肌瘤发生中的相关关系。应用核酸原位杂交技术和SP免疫组织化学法,研究37例子宫平滑肌瘤、20例正常子宫平滑肌组织中cx43mRNA及其蛋白、ER、PR的表达规律。结果显示,cx43mRNA及其蛋白、ER、PR在子宫平滑肌瘤中的表达明显高于在子宫平滑肌组织中的水平,差异有显著性（P＜0.05）。cx43mRNA及其蛋白在子宫平滑肌瘤中的过度表达,是子宫平滑肌瘤发生过程的重要事件,与ER、PR水平升高呈现一致性,对进一步揭示子宫平滑肌瘤的复杂分子机制、寻求可靠的早期标志有重要意义。     

Electrophysiology of Single and Aggregate Cx43 Hemichannels

Connexin43 (Cx43) is the most ubiquitous gap junction protein in the human body and is essential for cell-to-cell communication in a variety of organs and organ systems. As a result, Cx43 is responsible for mediating both electrical and chemical signals, passing dissolved solutes and small signaling molecules between cells in a coordinated fashion. Here, we explore the electrophysiological properties of hemichannels formed from Cx43 and Cx43 fused to eGFP (Cx43eGFP) and their interactions in a planar lipid membrane (BLM). Unlike in vivo patch clamp experiments, Cx43 was purified and isolated from other membrane constituents allowing elucidation of individual protein responses to various electrical and chemical stimuli. Using this system, we examined hemichannel electrophysiology and the roles of several well-known gap junction blockers, namely: lanthanum, heptanol, carbenoxalone and lindane. We also observed a critical number of hemichannels required for an accelerated conductance increase, an emergent electrical signature indicative of plaque formation.     

Overexpression of the gap junction protein Cx43 as found in diabetic foot ulcers can retard fibroblast migration

Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.     

Comparative immunohistochemical distribution of connexin 37 and connexin 43 throughout folliculogenesis in the bovine ovary

Among gap junctional proteins previously identified in the mouse ovary, connexins (Cx) Cx37 and Cx43 appeared to be essential for normal follicular growth. The aim of this work was to detect Cx37 expression in the bovine ovary, then to quantify and compare its follicular distribution pattern with that of Cx43 using quantitative analysis of immunofluorescently labeled ovary sections viewed with a confocal laser scanning microscope. Cx37 immunoreactivity was detected in bovine ovarian follicles and was predominantly localized at preantral stages. Unlike follicular Cx43 expression which was restricted to granulosa cells, Cx37 staining was observed in both oocyte and granulosa cell compartments. While no changes were seen during early follicular growth, the level of Cx37 expression decreased significantly at the onset of antral cavity formation (P 相似文献   

Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping.

We have used low stringency hybridization and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides to identify four new members of the rat connexin gene family. On the basis of their predicted molecular mass, these proteins have been designated connexin (Cx) 40 (Cx40), Cx37, Cx33, and Cx31.1. The new connexins exhibit all of the conserved structural features of the connexin family, including highly similar extracellular and transmembrane domains but divergent major cytoplasmic domains. On the basis of primary sequence similarity, the connexin family may be divided into two classes. Cx40, Cx37, and Cx33 are similar to the previously characterized Cx43 and Cx46. Cx31.1 is similar to Cx26, Cx31, and Cx32. Cx37 and Cx40 mRNAs are expressed in a wide variety of adult organs and tissues, with particular abundance in lung. However, their relative levels are different in many organs and thus their distribution is not completely coincident. Cx33 and Cx31.1 genes exhibit a much more restricted pattern of expression; mRNAs are detected only in testes and skin, respectively. Chromosomal mapping studies indicate that Cx26 and Cx46 are tightly linked on chromosome 14, and Cx37 and Cx31.1 are linked on chromosome 4, while the rest of the connexin genes are dispersed.