钙化大鼠主动脉钙调神经磷酸酶的表达及活性

目的探讨钙调神经磷酸酶是否参与血管钙化的调节过程。方法4周龄SD雄性大鼠36只,随机分为对照组、钙化组和环孢霉素A组,每组12只。采用华法林和维生素D,建立大鼠主动脉钙化模型,免疫组织化学方法测定主动脉组织钙调神经磷酸酶蛋白,比色法测定组织钙调神经磷酸酶活性,比较各组间钙调神经磷酸酶蛋白表达及活性是否有差别。结果钙化组大鼠主动脉钙调神经磷酸酶蛋白表达较对照组明显增加（P〈0．01）,钙化组大鼠主动脉钙调神经磷酸酶活性较对照组明显增强（P〈0．01）;环孢霉素A组大鼠主动脉钙调神经磷酸酶蛋白表达较对照组明显增加（P〈0．01）,较钙化组表达减少（P〈0．05）,环孢霉素A组大鼠主动脉钙调神经磷酸酶活性较对照组明显增加（P〈0．01）,与钙化组比较无显著性差异（P〉0．05）。结论钙调神经磷酸酶很可能参与血管钙化的调节过程。     

伊贝沙坦和培垛普利对大鼠压力负荷性心肌肥厚的影响

目的:探讨钙调神经磷酸酶和钙泵活性在大鼠压力负荷性心肌肥厚时的变化及伊贝沙坦和培垛普利对它们的影响。方法:40只雄性SD大鼠随机分为5组。除假手术组外,其余4组大鼠采用腹主动脉部分结扎法造成压力负荷性心肌肥厚模型,术后1周分别用下列药物开始灌胃:假手术组和对照组生理盐水2mL·kg^-1·d^-1,伊贝沙坦组20mg·kg^-1·d^-1,培垛普利组2mg·kg^-1·d^-1及联合用药组(培垛普利2mg·kg^-1·d^-1,伊贝沙坦20mg·kg^-1·d^-1)。用药6周后检测左室质量指数、心肌细胞横径、心肌钙调神经磷酸酶及钙泵活性,免疫组化测定心肌钙调神经磷酸酶的表达。结果:联合用药组LVMI显著低于对照组及单用伊贝沙坦或培垛普利药组,各用药组TDM及钙调神经磷酸酶活性显著低于对照组,对照组心肌肌浆网钙泵活性显著低于其他各组,联合用药组钙泵活性明显高于单独用药组。免疫组化显示对照组心肌组织钙调神经磷酸酶表达显著高于其他各组。相关分析显示LVMI与TDM、CaN均呈显著正相关,与钙泵活性呈负相关。结论:伊贝沙坦和培垛普利可抑制钙调神经磷酸酶活性,增加心肌肌浆网钙泵活性,联合应用对减轻心肌肥厚有协同作用。     

狼疮样肾炎小鼠脾、肾组织中钙调磷酸酶活性的检测及其意义

为检测狼疮样肾炎小鼠脾、肾组织中钙调磷酸酶 (CaN )活性 ,以探讨其与肾炎小鼠肾组织RANTES表达及肾组织病理改变的关系。采用慢性移植物抗宿主小鼠 (GVHD )狼疮样肾炎模型 ,CaN活性检测采用发色底物法。结果 :(1) 12周模型组肾小球硬化指数及小管间质损伤总积分显著高于 8周模型组 (P <0 0 1) ;(2 ) 12周模型组RANTES蛋白及mRNA表达显著高于 8周模型组 (P <0 0 1) ,FK5 0 6显著抑制狼疮样肾炎小鼠肾组织RANTES蛋白及mRNA表达 ;(3)模型组脾、肾组织中CaN活性显著增强 ,与正常对照组脾、肾组织CaN活性有显著差异 (P <0 0 1) ,FK5 0 6组中CaN活性显著下降 (P <0 0 5 ) ;(4 )狼疮样肾炎小鼠脾组织中CaN活性与血清抗dsDNA抗体水平呈正相关关系 (r1=0 92 5 ,P <0 0 1) ;(5 )狼疮样肾炎小鼠肾组织中CaN活性与肾小球硬化指数及小管间质损伤总积分呈正相关关系 (r2 =0 6 2 6 ,P <0 0 1;r3 =0 772 ,P <0 0 5 ) ,与RANTES蛋白阳性小管数呈正相关关系 (r4=0 6 2 5 ,P <0 0 5 ) ,狼疮样肾炎小鼠肾组织中CaN活性与RANTESmRNA表达呈正相关关系 (r5=0 714 ,P <0 0 5 )。表明狼疮样肾炎小鼠脾细胞存在CaN异常活化 ,肾组织中异常活化的CaN参与了肾脏病理损伤     

钙调神经磷酸酶在哮喘豚鼠气道重塑中的作用

目的:观察钙调神经磷酸酶(CaN)在哮喘豚鼠气道重塑中的作用。方法:实验分3组:对照组、哮喘组及CaN抑制剂环孢霉素(CsA)组,测定指标包括:①支气管肺泡灌洗液(BALF)蛋白含量、细胞计数及分类;②大气道平滑肌[³H]-TdR掺入量;③肺组织切片中小气道壁厚度及气道平滑肌厚度;④气管和肺组织CaN活性。结果:①BALF:CsA组蛋白含量、细胞计数及嗜酸粒细胞分别较哮喘组少46%、51%及60%(P＜0.01);②大气道平滑肌[³H]-TdR掺入量:CsA组较哮喘组低22%(P＜0.05);③小气道壁厚度:CsA组较哮喘组少34%(P＜0.01);气道平滑肌厚度:CsA组较哮喘组少37%(P＜0.01);④肺组织CaN活性:CsA组较哮喘组低52%(P＜0.01);气管CaN活性:CsA组较哮喘组低44%(P＜0.01)。结论:CsA可减轻哮喘豚鼠气道重塑,推测CaN参与气道重塑过程。     

大鼠肢体缺血再灌注后肺组织一氧化氮合酶的变化

目的:研究正常大鼠肺组织内一氧化氮合酶(NOS)的分布及肢体缺血再灌注(LIR)后肺组织内NOS分布及活性的变化。方法:用止血带复制肢体缺血再灌注模型,利用β-NADPH-d组织化学方法、计算机图像分析系统及分光光度法,观察对照组大鼠肺内NOS的分布及LIR组肺内NOS分布及活性的变化。结果:组织学上显示,对照组大鼠呼吸道包括支气管、细支气管、终末细支气管、肺泡管的上皮细胞和血管内皮细胞NOS表达均阳性,肺泡上皮细胞NOS表达阴性;LIR组上述肺组织阳性部位NOS表达增强,且出现血管平滑肌细胞、肺泡上皮细胞NOS表达阳性;生化测定结果显示,LIR组与对照组比较,NOS活性增强,NO₂^-/NO₃^-水平增多。结论:一氧化氮不仅参与肺的生理过程,而且在LIR后急性肺损伤(ALI)病理生理过程中可能发挥重要作用。     

骨骼肌重塑中的钙调神经磷酸酶信号转导通路

背景：骨骼肌重塑是骨骼肌对多种刺激因素所产生的形态结构与代谢机能的适应性变化。近几年关于钙调神经磷酸酶(CaN )/ (NFATS)在骨骼肌重塑中的作用备受关注。 目的：探讨钙调神经磷酸酶在骨骼肌从无氧转向有氧的代谢重塑、肌纤维类型转化以及在骨骼肌肥大过程中的信号转导作用。 方法：由第一作者用计算机检索ISI Web of knowledge数据库(1998/2010),检索词为 “calcineurin,skeletal muscle,hypertrophy,NFAT,myofiber type”,语言设定为英文。从钙调神经磷酸酶信号系统在骨骼肌代谢、肌纤维类型转换和肌肉肥大中的作用方面进行总结,对其调节机制、肌肉重塑等方面进行介绍。 结果与结论：共检索到186篇文章,按纳入和排除标准对文献进行筛选,共纳入33篇文章。结果表明CaN/NFATS信号激活有助于Ⅰ型肌纤维分化,提高线粒体有氧代谢能力,但骨骼肌对耐力运动的适应并不绝对依赖CaN。CaN/NFATS转导通路有可能通过转录激活utrophin A来调控骨骼肌的肥大反应。由此可知钙调神经磷酸酶参与骨骼肌代谢、纤维转化和肥大的重塑过程,调节骨骼肌对刺激产生适应性应答反应。     

钙调神经磷酸酶在血管紧张素Ⅱ刺激的大鼠心肌细胞肥大中的作用及其活性调节

目的:观察钙调神经磷酸酶(CaN)在血管紧张素Ⅱ(AngⅡ)刺激的大鼠心肌细胞肥大中的作用及其活性调节。方法:建立AngⅡ诱导的大鼠心肌细胞肥大模型,观察CaN抑制剂对AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入的影响,以及各种因素对心肌细胞CaN酶活性的影响。结果:10、 100、 1000 nmol·L^-1的AngⅡ作用12 h分别使心肌细胞的CaN活性增加了13%、 57%(P＜0.05)、 228%(P＜0.01)。AngⅡ(10 nmol·L^-1)刺激心肌细胞2 h内,CaN活性与对照组无明显差异(P＜0.05);AngⅡ刺激心肌细胞12 h以上,CaN活性才明显增高(P＜0.05)。Losartan(50 μmol·L^-1)、H₇(50 μmol·L^-1)及Fura-2/AM(4 μmol·L^-1)可明显抑制AngⅡ刺激的心肌细胞CaN活性;而PD98059(50 μmol·L^-1)对AngⅡ刺激的心肌细胞CaN活性无明显影响。AngⅡ(10^-7mol/L)刺激的大鼠心肌细胞 [³H]-亮氨酸掺入明显高于对照组(P＜0.01),而CaN特异性抑制剂-环孢素A(0.5~5 μg/mL)可以明显抑制AngⅡ刺激的心肌细胞 [³H]-亮氨酸掺入。结论:依赖Ca²⁺/CaM活化的CaN可能在AngⅡ刺激的心肌细胞肥大中起重要作用;CaN的活化可能有赖于胞内Ca²⁺水平的持续升高,另外,CaN的活性还可能受到蛋白激酶C等信号分子的磷酸化调节。     

慢性心力衰竭大鼠肾组织中血小板源性生长因子及结缔组织生长因子的表达

目的:探讨血小板源性生长因子BB(PDGF-BB)及结缔组织生长因子(CTGF)在慢性心力衰竭大鼠肾组织的表达及意义。方法:采用肾上腹主动脉缩窄法制作Wistar大鼠慢性心力衰竭模型,随机分为假手术组,心衰10 d、20 d、30 d组。观察及比较各组大鼠的血流动力学数值;Masson染色方法观察大鼠慢性心衰时肾组织结构变化;免疫组织化学方法检测各组大鼠肾组织的PDGF-BB及CTGF的表达部位;免疫印迹半定量检测各组大鼠肾组织的PDGF-BB、CTGF的表达水平。结果:心衰模型各组大鼠PDGF-BB、CTGF的蛋白表达均较假手术组高,且随心衰程度的加重而表达升高。结论:慢性心衰可引起肾间质纤维化,其发生发展与心衰的严重程度密切相关。     

葛根素对糖尿病肾病大鼠肾组织中一氧化氮和诱导型一氧化氮合酶水平的影响

目的：探讨葛根素（Pur）对糖尿病（DM）大鼠肾组织中一氧化氮（NO）含量、诱导型一氧化氮合酶（iNOS）表达的影响。方法:一次性腹腔注射链脲佐菌素（STZ）复制糖尿病大鼠模型。每日腹腔注射Pur注射液,共16周,收集24 h尿液测定尿蛋白（Upro）;腹主动脉取血分离血清测定血糖（Glu）和血尿素氮（BUN）。光镜下观察肾组织病理形态学改变;采用免疫组织化学技术观察肾组织中的iNOS定位与分布。制备组织匀浆,以检测肾组织中的NO活性。结果:与对照组相比,DM组出现肾功能下降、肾小球及肾小管肥大等异常表现,同时,iNOS表达及NO含量明显增加;应用Pur后肾功能及肾组织病理变化有明显改善,且iNOS表达及NO含量显著降低。结论:Pur可降低肾组织iNOS表达及NO含量从而减轻肾组织损伤,这可能是其减轻DM所致大鼠肾脏功能及结构异常的作用机制之一。     

环孢素A对神经肽Y刺激的大鼠血管平滑肌细胞增殖的影响

目的:观察环孢素A(CsA)对神经肽Y(NPY)诱导的大鼠主动脉平滑肌细胞增殖的影响,以探讨钙调神经磷酸酶(CaN)信号通路在血管平滑肌细胞增殖中的作用。方法:体外培养的大鼠主动脉平滑肌细胞分为3组:(1)NPY组,(2)CsA+NPY组,(3)对照组。检测CaN活性(定磷法)、细胞增殖活度(MTT法)的变化,观察增殖细胞核抗原(PCNA)的表达水平(免疫组化定量技术)。结果:NPY组CaN活性、血管平滑肌细胞增殖活度(吸光度表示),PCNA表达水平(光密度值表示)明显高于对照组(P＜0.01或P＜0.05),CsA+NPY组各项指标明显低于NPY组(P＜0.01或P＜0.05)。结论:环孢素A可显著阻滞神经肽Y刺激的血管平滑肌细胞增殖,这种作用可能通过抑制CaN信号通路所致。     

The distribution of carnosine and related dipeptides in rat and human tissues

Carnosine and anserine are present in high concentrations in most skeletal muscles. In addition, carnosine and homocarnosine have been detected in brain and cardiac muscle. Other tissues have been found to be devoid of these histidine-containing dipeptides. However, Flancbaum et al. [5] reported that carnosine was present in every rodent and human tissue analyzed. These authors postulated that carnosine serves as a non-mast cell reservoir for histidine which becomes available for histamine synthesis during periods of physiological stress. We have analyzed many rat and human tissues using an immunohistochemical procedure. Carnosine and related dipeptides were detected in skeletal muscle, cardiac muscle and brain, but not in kidney, liver, lung or several other organs. These negative results seem valid since the immunoassay gave positive staining in the tissues generally known to contain carnosine.accepted by W. Lorenz     

Distribution of the thrombomodulin antigen in the rabbit vasculature

The purpose of this study was to determine the distribution of the thrombomodulin (TM) antigen in the rabbit vasculature in vivo. Acetone-fixed, frozen sections of various tissues (brain, heart, aorta, lung, liver, spleen, kidney, small intestine, large intestine, skeletal muscle, bone, and skin) were stained by indirect immunofluorescence with goat affinity-purified antibodies to TM. Both large and small vessels (including capillaries) had demonstrable TM antigen localized to the endothelium while vascular smooth muscle and connective tissue cells were negative. The parenchyma of the organs examined were also negative. Based on antibody dilutions and brightness of staining, there is an indication that lung, followed by heart and intestinal vessels, has the highest concentration of antigen; kidney glomerular capillary loops have the least. These results also indicate that TM is an excellent cell type-specific marker of endothelial cells as the endothelium of all vessels and vascular beds was positive.     

Short‐term distribution of 134Cs in relation to 51Cr‐EDTA after intravenous dose in goats

Aim: The aim of this study was to gather information about the short‐term rate of caesium uptake (incorporation) in different animal tissues and explain them with known physiological mechanisms affecting ion distribution. Methods: Six goats were given an intravenous bolus containing ¹³⁴Cs as a tracer and ⁵¹Cr‐EDTA as an extracellular marker. After 30 min, the animals were killed and the activity concentration of radioactive isotopes in different tissues and fluid compartments were measured. Results: The highest relative activity concentration of ¹³⁴Cs was found in kidney cortex, with a tissue/plasma‐ratio around 50. In urine, the ratio varied between 5 and 28. In the salivary gland, cardiac muscle and small intestine the ratio was around 11, 7 and 6, respectively. The contents of small intestine had an average activity concentration five times that of plasma. In skeletal muscle the terminal activity concentration was surprisingly low, with a tissue/plasma ratio mostly far less than unity. Even in connective tissue and cartilage the terminal activity concentration was generally higher than in skeletal muscle. Conclusion: The rate of uptake of caesium varies widely from tissue to tissue. Many of these differences can be explained with differences in Na,K‐ATPase activity. Also, perfusion and accessibility play a role in some tissues, like brain and possibly part of the skeletal muscles. The short‐term distribution of caesium differs distinctly from the long‐term distribution reported in literature.     

Tissue distribution and pharmacokinetics of3H-butylated hydroxyanisole in female mice

Butylated hydroxyanisole (BHA), a commonly used food additive, is a phenolic antioxidant. While metabolism of BHA has been studied in dog, man, rabbit and rat, information on tissue distribution is limited to rat and dog. Time dependent distribution and elimination of³H-BHA following a single oral dose of 50 mg/kg body weight in female Swiss-Webster mice were investigated. Animals were sacrificed at time intervals up to 168 h after dosing. Levels of³H were determined by liquid scintillation counting in the following tissues: brain, blood, serum, fat, heart, kidney, liver, lung, skeletal muscle and spleen. In most tissues the decline of BHA (log) was nonlinear with respect to time; a biphasic, post-absorptive aspect was observed. The highest³H activity was noted in kidney and liver 15 min after dosing. The lowest activity was in fat. In addition, a peak³H activity was recorded at 3 and 10 h after dosing for the blood and brain, respectively. Distribution of the³H-BHA in mouse organs is similar to that in rat.     

Expression of metalloprotease insulin-degrading enzyme insulysin in normal and malignant human tissues

Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3.4.22.11), a thiol metalloendopeptidase, is involved in intracellular degradation of insulin, thereby inhibiting its translocation and accumulation to the nucleus. Recently, protein expression of IDE has been demonstrated in the epithelial ducts of normal breast and breast cancer tissue. Utilizing four different antibodies generated against different epitopes of the IDE molecule, we performed Western blot analysis and immunohistochemical staining on several normal human tissues, on a plethora of tumor cell lines of different tissue origin, and on malignant breast and ovarian tissue. Applying the four IDE-directed antibodies, we demonstrated IDE expression at the protein level, by means of immunoblotting and immunocytochemistry, in each of the tumor cell lines analyzed. Insulin-degrading enzyme protein expression was found in normal tissues of the kidney, liver, lung, brain, breast and skeletal muscle, as well as in breast and ovarian cancer tissues. Immunohistochemical visualization of IDE indicated cytoplasmic localization of IDE in each of the cell lines and tissues assessed. In conclusion, we performed for the first time a wide-ranging survey on IDE protein expression in normal and malignant tissues and cells thus extending our knowledge on the cellular and tissue distribution of IDE, an enzyme which to date has mainly been studied in connection with Alzheimer's disease and diabetes but not in cancer.     

败血症休克大鼠组织中性内肽酶与肾上腺髓质素的相关关系研究

目的:观察败血症休克大鼠组织肾上腺髓质素(ADM)含量和中性内肽酶(NEP)的活性及其表达水平,以探讨NEP的变化在休克时组织ADM水平变化中的意义。方法:盲肠结扎穿孔方法复制大鼠败血症性休克模型,采用放射免疫方法和荧光分光光度法分别检测血浆和组织的ADM含量和NEP的活性,用半定量RT-PCR和免疫组织化学法分别检测NEP的mRNA及其蛋白的组织分布。结果: ADM和NEP广泛分布于大鼠的血浆和组织。休克早期,大鼠各组织ADM含量普遍高于对照组,NEP活性在心脏和小肠低于对照组,在血浆中高于对照组,在肺、肾和主动脉无明显变化。免疫组化结果显示,休克早期NEP在大鼠心脏、主动脉内膜和中膜的分布低于对照组。休克晚期,除大鼠小肠的ADM含量低于休克早期,血浆和其余组织的ADM含量均高于休克早期;与休克早期比较, NEP的活性除在大鼠肾脏无明显变化外,在血浆和其余组织均明显降低。NEP的免疫组化结果显示,休克晚期,NEP在大鼠心脏、主动脉内膜和中膜、肺和肾的分布弱于休克早期,在主动脉外膜无明显变化。RT-PCR结果显示,休克晚期大鼠心脏、主动脉、肺脏和小肠的NEP mRNA 表达均显著低于对照组。 结论:NEP在大鼠败血症休克的不同时期和不同组织中的变化与ADM含量的变化不一致,提示休克过程中,不同组织局部的NEP对ADM含量的影响不同。     

Lipopolysaccharide induction of tissue factor expression in rabbits

Tissue factor (TF) is the major activator of the coagulation protease cascade and contributes to lethality in sepsis. Despite several studies analyzing TF expression in animal models of endotoxemia, there remains debate about the cell types that are induced to express TF in different tissues. In this study, we performed a detailed analysis of the induction of TF mRNA and protein expression in two rabbit models of endotoxemia to better understand the cell types that may contribute to local fibrin deposition and disseminated intravascular coagulation. Northern blot analysis demonstrated that lipopolysaccharide (LPS) increased TF expression in the brain, lung, and kidney. In situ hybridization showed that TF mRNA expression was increased in cells identified morphologically as epithelial cells in the lung and as astrocytes in the brain. In the kidney, in situ hybridization experiments and immunohistochemical analysis showed that TF mRNA and protein expression was increased in renal glomeruli and induced in tubular epithelium. Dual staining for TF and vWF failed to demonstrate TF expression in endothelial cells in LPS-treated animals. These results demonstrate that TF expression is induced in many different cell types in LPS-treated rabbits, which may contribute to local fibrin deposition and tissue injury during endotoxemia.     

Redefinition of Dystrophin Isoform Distribution in Mouse Tissue by RT-PCR Implies Role in Nonmuscle Manifestations of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by a defect in a 427-kDa membrane-associated protein: dystrophin. The DMD gene also encodes several shorter isoforms which are believed to participate in nonmuscle manifestations of DMD, including abnormal retinal electrophysiology, dilated cardiomyopathy, mental retardation, and hearing defects. The purpose of this work was to determine the normal tissue expression of full-length dystrophin (Dp427) and the dystrophin isoforms Dp260, Dp140, Dp116, and Dp71, to aid in understanding what roles these isoforms might play in DMD nonmuscle manifestations. RT-PCR was performed on mRNA isolated from wild-type C57BL/6J mouse tissues, including brain, cardiac muscle, eye, intestine, kidney, liver, lung, skeletal muscle, spleen, stomach, testis, thymus, and uterus. RT-PCR amplification demonstrated that the isoforms were in a number of tissues which had not been revealed by previous Western and Northern blot analyses. Dp427 was expressed at equal levels in all tissues. Dp260 and Dp140 were present in all tissues tested, but the levels of expression varied. Dp116 was expressed in a subset of tissues and levels of expression varied. Dp71 was constitutively expressed in all tissues, suggesting that this isoform plays a basic role in normal tissue function. The expanded tissue distribution supports the hypothesis that dystrophin isoforms serve essential and unique functions, necessitating further investigation into their potential roles in DMD nonmuscle manifestations.