NDRG2在人胚胎组织中的表达分布特点

本文旨在研究NDRG2在不同胎龄人胚胎组织中的表达水平及细胞定位。利用RT-PCR和Western blot研究NDRG2 mRNA和蛋白在胎心、肺、肝和肾中的表达水平,免疫组织化学分析NDRG2蛋白在多种胚胎组织中的分布特点。结果表明,NDRG2在胚胎组织中的表达随胚龄的延长而增加。NDRG2 mRNA和蛋白在胎心和肺中的变化一致;在胎肝中mRNA表达低而蛋白表达高,在胎肾中则相反。NDRG2蛋白阳性反应产物存在于细胞胞浆,见于小肠绒毛上皮细胞、结肠上皮细胞、皮肤表层细胞及毛囊、肺内小气道内衬上皮细胞、肝细胞、心肌细胞、胸腺小体、肾小管上皮细胞。结果提示,NDRG2蛋白可能不是一个组织特异性蛋白,并在组织和器官的形成中起作用。     

神经元限制性沉默因子与胰岛素在成年小鼠胰腺中的表达

目的:观察神经元限制性沉默因子(NRSF)在正常成年小鼠胰腺组织中的表达情况。方法:以6～8周BALB/c小鼠胰腺为实验材料,制备冰冻切片,与地高辛标记的NRSF cDNA探针进行原位杂交,观察mRNA表达,并结合免疫组织化学方法检测NRSF和胰岛素的表达。结果:原位杂交显示,NRSF mRNA仅表达于胰腺组织外分泌部腺泡腺细胞中,胞浆呈蓝紫色,与免疫荧光组织化学检测NRSF蛋白表达的部位一致,而胰岛细胞中无NRSF mRNA及蛋白的表达。免疫酶组织化学染色显示,胰岛大部分细胞中表达胰岛素,胞浆染成黄棕色,而腺泡腺细胞则不表达胰岛素。结论:NRSF与胰岛素不存在共定位关系,即成年小鼠胰岛细胞不表达NRSF,而表达胰岛素。提示NRSF蛋白表达的消失可能是建立完全分化成熟、具有完好分泌反应的胰岛细胞所必需的。     

NDRG2在人胚胎呼吸系统的表达特点

目的该实验通过对16-28周人胚胎呼吸系统NDRG2表达的研究,旨在阐明NDRG2在16-28周人胚胎呼吸系统中的表达规律,为进一步明确新的发育相关基因ndrg2的功能提供依据。方法搜集16-28周胎儿呼吸系统的肺和气管组织,制成石蜡切片,用抗NDRG2单克隆抗体,行免疫组化染色(ABC法),从蛋白质水平观察NDRG2表达情况。统计阳性细胞数,利用统计学方法,判断不同胎龄的肺和气管间NDRG2表达有无差别。结果免疫组化染色表明,NDRG2在16-28周胚胎呼吸系统中有广泛的表达,阳性产物主要位于上皮细胞的胞浆中,但是不同胎龄之间NDRG2的表达未见显著差别。结论NDRG2在16-28周胚胎呼吸系统中有广泛的表达,提示NDRG2在早期胚胎的呼吸系统上皮细胞的生长与发育过程中起一定作用。而阳性产物主要表达在上皮细胞的胞浆中,说明NDRG2可能是一种胞浆蛋白。     

口服AdipoRon对2型糖尿病小鼠脾脏和胰腺功能的影响

目的:观察口服AdipoRon对2型糖尿病小鼠脾脏和胰腺功能的影响,为AdipoRon的临床应用提供基础资料。方法:将40只C57/BL6雄性小鼠随机分为正常对照组(NC,n=10)和造模组(n=30),并分别给予普通饲料和高脂高糖饲料喂养。4周后,造模组小鼠腹腔注射链脲佐菌素(STZ,40 mg/kg)以诱导建立2型糖尿病模型。造模成功后将糖尿病模型小鼠随机分为糖尿病模型组(DM)、高剂量AdipoRon(50 mg/kg)(DM+H)组、低剂量AdipoRon(20 mg/kg)(DM+L)组,每组10只。DM+L组和DM+H组灌胃相应浓度的AdipoRon(使用去离子水溶解AdipoRon),NC组和DM组灌胃等体积去离子水,每日灌胃1次,灌胃10 d。末次干预后禁食12 h,处死小鼠取血液、胰腺和脾脏。HE染色光镜下观察胰腺的病理改变; ELISA法检测小鼠胰腺和脾脏组织中胰岛素受体(INSR)、胰岛素受体底物1(IRS-1)和肿瘤坏死因子-α(TNF-α)蛋白质含量;小鼠脾脏系数; Western blot法检测胰腺组织中pIRS-1蛋白质水平;实时荧光定量PCR检测胰腺组织中insulin mRNA表达。结果:光镜下可见正常组小鼠胰腺组织排列紧密、饱满、胰岛体积大,DM组小鼠胰腺组织排列较为疏散、胰岛体积较小,口服AdipoRon组小鼠胰腺组织基本紧密、饱满、胰岛体积略小。与NC比较,DM组小鼠胰腺和脾脏TNF-α水平明显升高,INSR、IRS-1水平均降低,脾脏系数、胰腺p-IRS-1蛋白质水平和insulin mRNA表达均降低,均具有统计学意义(P<0.05);与DM组比较,口服AdipoRon组小鼠胰腺和脾脏TNF-α水平明显下降,INSR和IRS-1水平均升高,脾脏系数升高,DM+H组胰腺p-IRS-1蛋白质水平和insulin mRNA表达均升高(P<0.05);与DM+L组比较,DM+H组小鼠TNF-α水平明显下降,INSR和IRS-1水平均升高(P<0.05)。结论:口服AdipoRon可通过减弱糖尿病小鼠炎症反应,上调INSR表达、提高p-IRS-1水平,从而对糖尿病小鼠脾脏和胰腺组织有一定的保护作用。     

LIGHT在自发Ⅰ型糖尿病小鼠中表达的分析

目的建立I型糖尿病NOD小鼠模型,探讨LIGHT在I型糖尿病模型NOD小鼠中的表达及意义。方法监测雌性NOD小鼠血糖、胰岛素水平;NOD小鼠分为4组,即未发病5周龄及11周龄组、发病1 w和2 w组。采用qRT-PCR、Western blot、免疫组化技术分析NOD小鼠胰腺组织LIGHT的mRNA、蛋白表达变化;ELISA法检测血清胰岛素、可溶性LIGHT及Th1细胞因子IFN-γ含量;HE染色检测胰岛炎,对胰岛炎评分,并对血清LIGHT含量与IFN-γ含量及胰岛炎积分进行相关性分析。结果糖尿病发病组NOD小鼠胰腺组织LIGHT基因的mRNA和蛋白表达及血清LIGHT浓度均明显高于未发病的5周龄组(P0.05),血清中Th1细胞因子IFN-γ分泌显著增加。血清LIGHT含量与IFN-γ水平及胰岛炎积分呈明显正相关(r1=0.52,r2=0.87,P0.01)。结论 LIGHT在NOD小鼠胰腺组织中的表达上调,可能影响Th1细胞因子IFN-γ的分泌及胰岛炎程度,血清LIGHT能反映I型糖尿病胰岛炎的严重程度。     

游离锌离子和锌转运体-8在小鼠胰岛β细胞中的定位

目的观察游离锌离子和锌转运体-8(zinc transporter-8,ZNT-8)在小鼠胰腺定位,探讨游离锌离子和ZNT-8与胰岛素分泌的关系。方法应用金属自显影(AMG)染色技术显示小鼠胰腺中游离锌离子的定位,应用RT-PCR和免疫组织化学ABC法分别在mRNA水平和蛋白水平检测ZNT-8在小鼠胰腺内的表达,应用免疫荧光双标技术证明ZNT-8在小鼠胰岛β细胞内与胰岛素的共存。结果小鼠胰腺外分泌组织和胰岛均含有游离锌离子;在胰岛中,游离锌离子均匀分布在包括β细胞分布区在内的各个区域。胰腺组织表达ZNT-8 mRNA,ZNT-8主要表达于胰腺内分泌部胰岛中;在胰岛β细胞中,ZNT-8与胰岛素共存。结论游离锌离子在小鼠胰岛β细胞的存在及ZNT-8在小鼠胰岛β细胞中与胰岛素的共存提示ZNT-8可能通过参与胰岛β细胞内游离锌离子的转运而调节胰岛素的分泌。     

TGF-βⅡ型受体在实验性IgA肾病肾小管的表达

用免疫组织化学方法研究了ＴＧＦ－βⅡ型受体在实验性ＩｇＡ肾病及正常小鼠肾内的表达。结果表明ＴＧＦ－βⅡ型受体在实验性ＩｇＡ肾病及正常小鼠肾内均有表达,但最明显的是ＩｇＡ肾病的肾小管。该结果提示ＩｇＡ肾病时,肾小管有可能在ＴＧＦ－β的作用下增加细胞外基质的合成,从而导致肾小管间质的纤维化     

过敏毒素受体(C3aR)在db/db糖尿病肾病小鼠肾脏中的表达及病理意义分析

利用半定量RT-PCR、免疫组化和Western blotting的方法,同时从mRNA水平和蛋白质水平对过敏毒素受体(C3aR)在不同病理阶段的2型糖尿病肾病模型小鼠——db/db小鼠肾脏中的表达情况进行了较为系统的分析．结果发现：a．在糖尿病前的db/db小鼠(4周龄的db/db小鼠),C3aR与作为正常对照的db/m小鼠相比没有明显差异．随着肥胖的加剧,高血糖、蛋白尿的发生和发展,C3aR在db/db小鼠肾脏中的表达显著升高．b．免疫组化分析显示,C3aR广泛地表达于db/m和db/db小鼠肾脏的皮质和髓质,分布于肾脏的上皮细胞中(包括肾小管上皮细胞、肾小球中的脏层上皮细胞(足细胞)和壁层上皮细胞)．从部位来看,皮髓交界处的肾小管中C3aR表达量明显要比其他部位的多．在肾小球,C3aR特异地存在于足细胞部位．在db/m小鼠,不同周龄小鼠肾脏中C3aR的表达量并没有明显变化,但在db/db小鼠,从8周龄开始,分布在db/db小鼠肾小管上皮细胞和小球足细胞中的C3aR均随小鼠周龄的增加而增加,至少在时间上,与小鼠糖尿病肾病的发生发展相关,其中尤以足细胞中和皮髓交界处肾小管上皮细胞中的变化最为明显． c．在糖尿病肾病小鼠中高表达C3aR的肾小管上皮细胞常有空泡变性的情况．上述工作印证了先前对2型糖尿病肾病患者肾小球基因表达谱的分析结果,更加明确了C3aR与糖尿病肾病的相关性,同时揭示了C3aR在正常小鼠和糖尿病肾病小鼠肾脏中的表达、分布和变化规律,有利于进一步揭示C3aR的功能及其在糖尿病肾病发生、发展过程中的可能作用,探讨糖尿病肾病的分子机制．     

热休克反应中小鼠肾HSP70和乳酸脱氢酶同工酶表达变化的研究

用免疫组织化学和聚丙烯酰胺凝胶同工酶电泳方法研究了小鼠肾在热休克(46℃,30分钟)恢复期(4h和12h)HSP70的表达和乳酸脱氢酶(LDH)同工酶的变化。结果表明:(1)HSP70主要定位于肾小管上皮细胞胞质中,细胞核内未见表达;(2)HSP70免疫阳性反应在肾髓质较肾皮质强,肾小管较肾小球强;(3)热休克诱导小鼠肾LDH同工酶活性增强。提示:LDH同工酶可能对细胞热耐受性的建立有重要作用。     

人胰腺干细胞建系及移植诱导胰岛治疗大鼠糖尿病

供体不足已成为移植胰岛治疗Ⅰ型和部分Ⅱ型糖尿病的主要障碍,分离克隆胰腺干细胞作为种子细胞并诱导其分化为功能性胰岛可提供丰富的移植资源．本研究从人流产胎儿胰腺组织分离获得1例单克隆胰腺干细胞系．无菌取流产胎儿胰腺组织,0．1％Ⅳ型胶原酶消化分离为单个细胞和细胞团．低糖DMEM＋10％FBS培养,单个细胞和细胞团贴壁,原代上皮样胰腺干细胞克隆性生长．0．25％胰蛋白酶＋0．04％已二胺四乙酸（EDTA）消化传代,成纤维样细胞和其他细胞逐渐被消除,上皮样胰腺干细胞逐渐被纯化．克隆环筛选,获得单克隆人胰腺千细胞．在培养液中添加10ng／mL表皮生长因子（EGF）,单克隆人胰腺干细胞快速生长至单层,呈铺路石样．继续传代培养,1例来源于4月龄男性流产胎儿胰腺干细胞已传50代．液氮冷冻保存细胞1×10^9个以上．染色体核型分析,该干细胞系为正常的二倍体细胞．免疫组织化学反应,共表达pdx1,glucagon,nestin及CK19蛋白,不表达insulin,CD34,CD44及CD45．RT-PCR检测,转录pdx1,glucagon,nestin及CK19的mRNA,不转录insulin．β-巯基乙醇诱导,分化为神经细胞,免疫组织化学反应表达NF蛋白．烟酰胺诱导,分化为DTZ染色阳性,转录表达insulin,分泌insulin和C肽的功能性类胰岛．将单克隆人胰腺干细胞体外诱导胰岛移植在STZ制备的糖尿病大鼠肾囊内,能降低糖尿病大鼠血糖水平,延长寿命．     

Effect of short- and long-term diabetes on carnitine and myo-inositol in rats.

1. The effect of short- (2 wk) and long-term (20 wk) streptozotocin diabetes was studied on urine, blood, liver, heart, brain, skeletal muscle, pancreas and kidney concentrations of acid-soluble carnitine and free myo-inositol. 2. Short-term diabetic rats excreted significantly higher concentrations of carnitine as well as myoinositol than normal rats. Blood carnitine and myo-inositol were not different between normal and diabetic rats. Diabetes caused a decrease in liver, brain and pancreatic carnitine, but not in heart, skeletal muscle and kidney. Myo-inositol concentration was decreased in liver, heart and kidney but not in brain, pancreas and skeletal muscle. 3. Long-term diabetic rats had higher urinary excretions of both carnitine and myo-inositol. Blood carnitine did not change; however, myo-inositol was higher in diabetic than in normal rats. Diabetes caused a significant increase in liver and a decrease in heart, brain, skeletal muscle and pancreatic content of carnitine; no difference in kidney carnitine was noted. Myo-inositol content was elevated only in liver of diabetic rats. 4. We suggest that carnitine and myo-inositol concentrations are influenced both by short- and long-term diabetes through changes in tissue metabolism.     

NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia

The N-myc downstream-regulated gene (NDRG) family consists of four related proteins, NDRG1-NDRG4, in mammals. We previously generated NDRG1-deficient mice that were unable to maintain myelin sheaths in peripheral nerves. This condition was consistent with human hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D, caused by a nonsense mutation of NDRG1. In contrast, the effects of genetic defects of the other NDRG members remain unknown. In this study, we focused on NDRG4, which is specifically expressed in the brain and heart. In situ mRNA hybridization on the brain revealed that NDRG4 was expressed in neurons of various areas. We generated NDRG4-deficient mice that were born normally with the expected Mendelian frequency. Immunochemical analysis demonstrated that the cortex of the NDRG4-deficient mice contained decreased levels of brain-derived neurotrophic factor (BDNF) and normal levels of glial cell line-derived neurotrophic factor, NGF, neurotrophin-3, and TGF-β1. Consistent with BDNF reduction, NDRG4-deficient mice had impaired spatial learning and memory but normal motor function in the Morris water maze test. When temporary focal ischemia of the brain was induced, the sizes of the infarct lesions were larger, and the neurological deficits were more severe in NDRG4-deficient mice compared with the control mice. These findings indicate that NDRG4 contributes to the maintenance of intracerebral BDNF levels within the normal range, which is necessary for the preservation of spatial learning and the resistance to neuronal cell death caused by ischemic stress.     

Lipid peroxidation and activity of antioxidant enzymes in diabetic rats

We hypothesized that oxygen free radicals (OFRs) may be involved in pathogenesis of diabetic complications. We therefore investigated the levels of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS) and activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] in tissues and blood of streptozotocin (STZ)-induced diabetic rats. The animals were divided into two groups: control and diabetic. After 10 weeks (wks) of diabetes the animals were sacrificed and liver, heart, pancreas, kidney and blood were collected for measurement of various biochemical parameters. Diabetes was associated with a significant increase in TBARS in pancreas, heart and blood. The activity of CAT increased in liver, heart and blood but decreased in kidney. GSH-Px activity increased in pancreas and kidney while SOD activity increased in liver, heart and pancreas. Our findings suggest that oxidative stress occurs in diabetic state and that oxidative damage to tissues may be a contributory factor in complications associated with diabetes.     

Carbohydrate contents, and glycosidase and glycosyl transferase activities in tissues from streptozotocin diabetic mice

The contents of hexoses and hexosamines in brain, liver, and kidney of streptozotocin diabetic mice are significantly increased in comparison to the controls. These differences for hexoses contents in the heart are not significant. N-acetyl-beta-D-glucosaminidase and beta-D-glucosidase activities in brain, liver and kidney of diabetic mice are significantly higher when compared to the controls. However, beta-D-galactosidase activity is significantly lower in brain, liver, spleen and kidney of the diabetic mice, in comparison to the controls and similar in heart. alpha-D-Mannosidase activity of diabetic mice is significantly increased in spleen and heart and significantly decreased in liver and kidney. alpha-L-Fucosidase of diabetic mice shows higher activities, with significant differences, in liver and spleen; however, in heart and kidney the activities are significantly lower. Brain sialyltransferase and galactosyltransferase activities are significantly increased in diabetic mice; but for heart and kidney these differences are not significant. The activity for brain and kidney fucosyltransferase is not significant and that for the other assayed organs is significantly higher in comparison to the controls.     

Effect of sophora japonica total flavonoids on pancreas,kidney tissue morphology of streptozotocin-induced diabetic mice model

To observe effect of sophora japonica total flavonoids on pancreas, kidney tissue morphology of streptozotocin-induced diabetic mice model. Mice received tail vein injection of streptozotocin (60 mg/kg) for diabetes modeling. The model mice were divided into five groups, to be respectively fed with high, middle and small doses of sophora japonica total flavonoids solution, metformin solution and saline of the same volume. Another blank control group was set to be fed with saline of the same volume. The mice were administered once a day for 30 consecutive days, to be euthanatized after fasting blood glucose level testing on 30th day with pancreas, kidney taken out for pathological section and microscopic examination. The mice chain streptozotocin diabetes modeling was successful, with significant pathological changes (P < 0.01) in pancreas, kidney. Compared with model group, high, middle and small doses of sophora japonica total flavonoids could significantly alleviate streptozotocin-induced pancreas, kidney damage (P < 0.01). Conclusion: Sophora japonica total flavonoids can effectively alleviate pancreas, kidney injury of streptozotocin-induced diabetic mice model.     

Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.     

Parenteral zinc and tissue metallothionein in normal and diabetic rats

The effect of parenteral zinc on tissue metallothionein (MT) was studied in normal and streptozotocin-induced diabetic rats. The accumulation of Zn-MT in liver and pancreas of normal and diabetic rats following the administration (ip) of various amounts of zinc was not different. Renal Zn-MT was higher in the diabetic group, and this was not changed by zinc injection. Although diabetic rats, relative to normal, possessed a markedly higher concentration of Cu-MT in kidney initially, this difference decreased considerably after zinc injection. The ratio of Cu-MT to cytosolic Cu in kidney was not affected by parenteral zinc and was highest in diabetic rats. Zinc injection markedly reduced food intake, water consumption, and urine output in both normal and diabetic rats. Blood glucose of diabetic rats also decreased 24 h after zinc administration. Our results indicate that relative to normal, MT and zinc metabolism are different in kidney, and to some extent liver, but not different in the pancreas of the chemically induced diabetic rat.     

Tissue norepinephrine concentration in spontaneously diabetic Chinese hamsters

There is some controversy concerning a possible effect of diabetes mellitus on the sympathetic nervous system in humans with spontaneous diabetes mellitus and in animals with experimental diabetes mellitus. In this study we compared the tissue norepinephrine (NE) concentration of normal and diabetic Chinese hamsters in the untreated state and after treatment with insulin. Diabetes resulted in a 128% increase in the NE concentration of the kidney in female but not male hamsters. The NE concentration was increased in the liver (133%) and in the cerebral cortex (118%) of both male and female hamsters. There was no significant increase in the NE concentration of hypothalamus, acinar pancreas, pancreatic islets, or heart of diabetic hamsters. Three days of insulin therapy reduced the elevated NE concentration in kidney, liver and cerebral cortex of diabetic hamsters to the levels found in normal hamsters. However, insulin therapy of normal hamsters did not reduce the tissue NE concentration of the kidney, liver, and cerebral cortex below the normal levels found in these animals. Insulin therapy reduced the hypothalamic concentration of NE in both diabetic and normal hamsters. The increase in kidney NE concentration in female diabetic hamsters was not due to a reduction in renal size, for the kidneys of both female and male diabetic hamsters were larger than those of normal hamsters. When synthesis of NE was inhibited with alpha-methyltyrosine, there was a comparable rate of fall in the tissue NE concentration in the four experimental groups, suggesting that the increased tissue NE concentration in the tissues of diabetic hamsters was not due to a decreased rate of disappearance of this compound.(ABSTRACT TRUNCATED AT 250 WORDS)