db/db自发性糖尿病小鼠睾丸生殖细胞增殖及凋亡研究

目的　研究糖尿病对生殖细胞增殖和凋亡的影响。　方法　以免疫组织化学ABC法检测增殖细胞核抗原 (proliferationcellnucleusantigen ,PCNA)检测生殖细胞的增殖情况 ,电镜和脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记法 (TUNEL)检测凋亡的生殖细胞。　结果　PCNA阳性率随糖尿病病程进展 (糖尿病组 )和年龄增加 (对照组 )均呈明显下降趋势 ,但每年龄段糖尿病组PCNA阳性率均明显低于相应正常对照组。凋亡管横断面数及凋亡阳性率糖尿病组均较正常高 ,而且随糖尿病病程进展呈明显增加趋势 ,对照组中随加龄二者有增加。电镜下 ,糖尿病组小鼠睾丸精原细胞、精母细胞和精子细胞均可见到核染色质凝集、边集 ,凋亡小体形成和降解等凋亡过程。　结论　db/db自发性糖尿病小鼠睾丸生殖细胞PCNA表达减弱 ,TUNEL标记凋亡细胞增加 ,说明糖尿病使生殖细胞增殖与凋亡的平衡破坏 ,这可能是糖尿病生殖功能障碍的原因     

DahlS高血压大鼠生精细胞凋亡及Bcl-2蛋白的表达

目的　为阐明高血压导致生精障碍的确切机制。方法　应用原位末端标记法 (TUNEL)、免疫组织化学、流式细胞检测及电镜等方法 ,对DahlS高血压大鼠生精细胞凋亡的定性、定位、定量及其相关蛋白Bcl 2的表达进行了研究。结果　实验组随高血压病程延长 ,血压升高 ,生精细胞凋亡率增高 ,15、17周龄明显高于对照组 (P <0 0 5 )。Bcl 2蛋白表达降低 ,15、17周龄与对照组相比差异显著 (P <0 0 5 )。随糖尿病病变加重 ,Bcl 2表达减少 ,而且 ,糖尿病组比对照组Bcl 2表达率低。随糖尿病病程进展和年龄增加 ,表达均呈增加趋势 ,而且 ,糖尿病组比对照组Bax表达率高。结论　高血压可导致凋亡抑制基因Bcl 2表达降低 ,从而使生精细胞凋亡增加 ,这可能是高血压导致生精障碍的机制之一     

凋亡细胞在db/db自发性糖尿病小鼠颌下腺的分布

目的:观察凋亡细胞在db/db糖尿病小鼠颌下腺中的分布。方法:选取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的dh/m~(?)小鼠颌下腺,应用TUNEL标记方法染色后进行图像分析.统计凋亡细胞在颌下腺组织中分布的细胞阳性率。结果:随着糖尿病的发展,颌下腺组织出现腺体萎缩及颗粒曲管数目减少,实质细胞排列不整齐,呈簇状堆集,纤维及血管增多。凋亡细胞在对照组及糖尿病组颌下腺中均有分布,糖尿病组凋亡细胞阳性率高于对照组。糖尿病组与对照组凋亡细胞阳性率随月龄增大均呈增加趋势。结论:db/db糖尿病可导致颌下腺组织萎缩及实质细胞形态学改变;凋亡细胞阳性率在糖尿病组随疾病发展而增加显著。这与糖尿病腺体萎缩和功能受损相一致。     

甘草酸二铵对大鼠心肌缺血再灌注损伤后细胞凋亡的影响

目的 :观察甘草酸二铵 (DG)对大鼠心肌缺血再灌注 (IR)损伤后心肌细胞凋亡及凋亡相关基因表达的影响。方法 :雄性wistar大鼠2 4只 ,随机分为假手术组 ;IR组 ;DG组 ( 2 0mg/kg)。每组 8只。采用末端脱氧核苷酸转移酶介导的带荧光的dUTP缺口末端标记(TUNEL)法、westernblot法和免疫组化法检测大鼠心肌缺血 3 0min再灌注 6h心肌凋亡细胞及凋亡相关基因Bcl 2、Bax蛋白表达的变化。结果 :与假手术组比较 ,IR组心肌细胞凋亡指数 (AI)、Bax蛋白的阳性表达明显增加 (P均 <0 .0 1) ;与IR组比较 ,DG治疗后AI和Bax蛋白的阳性表达明显下降 (P <0 .0 1) ,而Bcl 2蛋白的阳性表达明显上调 (P <0 .0 1)。结论 :DG具有保护大鼠心肌缺血再灌注损伤的作用 ,其作用机制可能与其下调Bax蛋白表达 ,上调Bcl 2蛋白表达抑制心肌细胞凋亡有关     

db／db自发性糖尿病小鼠睾丸EGFR，c—Fos表达研究

目的：研究表皮生长因子受体（Epithelium growth factor receptor EGFR)和原癌基因c-Fos在db/db(diabetes obese)自发性糖尿病小鼠睾丸生殖细胞伯表达变化。方法：免疫组化ABC法。结果：EGFR在精母细胞。精子细胞、支持细胞和间质细胞均呈阳性反应。c-Fos免疫阳性细胞主要为精母细胞,圆形精子细胞和变形期精子,间质血管内皮细胞也有强阳性表达,另外,少数支持细胞,管周肌样细胞也有着色,以上两组指标。随糖尿病病程进展（糖尿病组）及年龄增加（对照组）,其阳性率呈现明显下降趋势,并且每年龄段糖尿病组阳性率明显低于相应正常对照组。结论：糖尿病睾丸中EGFR的减少例EGF与受体结合减少,第二信使cAMP和“第三信使”c-Fos均减少,以致核转录和核基因表达减弱,最终表现为糖尿病生殖细胞增殖减弱,生殖功能障碍。     

扶正解毒通络方对人肝癌HepG2 细胞增殖、凋亡的影响及其作用机制研究

目的:探讨扶正解毒通络方对人肝癌HepG2 细胞增殖、凋亡的影响及其机制。方法:MTT 法观察细胞增殖能 力;RT鄄PCR 检测细胞内Bax 和Bcl鄄2 mRNA 水平;免疫印迹(Western blot)检测细胞中Bax、Bcl-2、活化的caspase-3、SIRT3、P53 及Fas 蛋白水平。结果:扶正解毒通络方能明显诱导减少HepG2 细胞内Bcl-2 基因转录,增加Bax 基因转录;扶正解毒通络方 能明显诱导减少HepG2 胞内Bcl-2 蛋白表达,增加Bax、活化的caspase-3、SIRT3、P53 及Fas 蛋白表达。结论:与正常对照组比 较,正常血清组人肝癌细胞HepG2 细胞内Bax、Bcl-2、活化的caspase-3、基因及蛋白表达水平无明显改变;与正常血清组比较, 扶正解毒通络方低、中和高剂量含药血清和阳性对照组HepG2 细胞内Bcl鄄2 基因转录减少,蛋白表达减少;Bax 基因转录增 加,蛋白表达增加,活化的caspase鄄3 蛋白表达增加。     

小热休克蛋白家族和自噬相关蛋白 在自发性2型糖尿病db/db小鼠海马组织中的表达

目的观察自发性2型糖尿病db/db小鼠海马组织中小热休克蛋白家族(sHSPs)及自噬相关蛋白表达情况。方法正常db/m小鼠作为对照组,糖尿病db/db小鼠作为模型组,各10只。观察其体质量,空腹血糖(FBG);real-time PCR检测海马组织中小热休克蛋白家族、自噬相关基因mRNA的表达;Western blot检测HSPB8、BAG3、LC3、P62蛋白表达。结果 1)db/db小鼠体质量、空腹血糖均明显高于db/m小鼠(P0.01)。2)小热休克蛋白1-10(heat shock protein family B [small] member 1-10,HSPB 1-10)基因mRNA在小鼠海马组织中均有表达;其中HSPB1、HSPB3、HSPB5、HSPB6和HSPB8 db/db组明显低于db/m组(P0.05);HSPB2、HSPB9和HSPB10 db/db组高于db/m组(P0.05);而在db/db小鼠中LC3表达高于db/m组(P0.01),而P62反之(P0.01)。3)较db/m组,db/db组HSPB8、BAG3、LC3-Ⅱ蛋白表达均增高(P0.01),相反P62蛋白表达减低(P0.05)。结论 1)10种sHSPs均在小鼠海马组织中表达,但表达特点有所不同。2)8周龄糖尿病小鼠海马组织中存在HSPB8蛋白表达增强,且自噬激活。     

葡萄籽原花青素对糖尿病db/db小鼠肾组织细胞表型转化的影响

目的观察葡萄籽原花青素对Ⅱ型糖尿病模型db/db小鼠肾脏细胞中表型转化标志蛋白α-SMA、E-cadherin表达的影响,旨在揭示葡萄籽原花青素对db/db小鼠糖尿病肾损伤的保护机制。方法采用Ⅱ型糖尿病模型db/db小鼠为研究对象,16只雄性db/db小鼠随机分为2组:糖尿病组、糖尿病+葡萄籽原花青素灌胃组,每组各8只;8只相同周龄雄性db/m小鼠作为正常对照组及8只db/m小鼠+葡萄籽原花青素灌胃治疗对照组。以葡萄籽原花青素(5 mg/kg)灌胃,持续12周后检测Ecadherinh、α-SMA、p38MAPK和ERK1/2的蛋白表达。结果糖尿病组小鼠肾组织中α-SMA、p38MAPK和ERK1/2蛋白表达明显增加,E-cadherin表达减少,尿中8-OHd G水平增加。葡萄籽原花青素能减少糖尿病组小鼠肾组织中α-SMA、p38MAPK和ERK1/2蛋白表达,尿中8-OHd G水平也明显降低,而E-cadherin蛋白表达增高(P0.05)。结论葡萄籽原花青素抑制肾小管上皮细胞-间质转化(epithelial to mesenchymal transition,EMT)生成,可能是通过抑制活性氧簇(reactive oxygen species,ROS)生成,抑制p38MAPK和ERK1/2信号通路激活而实现的。葡萄籽原花青素对糖尿病肾病具有防治作用。     

老年鼠外周血淋巴细胞凋亡及基因调控的研究

本文采用TUNEL法及流式细胞仪观察不同鼠龄外周血淋巴细胞凋亡及凋亡发生率 ,并用免疫组织化学检测Bcl 2、Bcl xl及Bax基因表达变化。结果发现老年鼠组外周血淋巴细胞群中存有典型的凋亡细胞 ,凋亡细胞发生率明显高于青年组。老年鼠PBL的Bcl 2 ,Bcl xl表达下调 ,而Bax的表达量明显增加。提示在衰老过程中存在着免疫细胞凋亡 ,其发生与相关调控基因的表达一致。     

db/db小鼠心、肾RAS组分的分子表达

目的探讨2型糖尿病诱导心脏、肾脏肾素-血管紧张素系统(RAS)组分的表达变化。方法用db/db小鼠作为2型糖尿病动物模型,RT-PCR和蛋白免疫印迹法分别检测心脏、肾脏中RAS组分的基因和蛋白表达。结果 db/db小鼠心脏AGT mRNA表达、renin和AngⅡ蛋白表达显著升高(P0.01);而肾脏renin mRNA(P0.001)和蛋白(P0.01)表达以及AngⅡ(P0.05)的蛋白表达显著上调。结论 2型糖尿病诱导心脏、肾脏RAS组分的异常表达,最终引起RAS活性肽AngⅡ生成增加。     

糖尿病小鼠胰岛β细胞结构的光镜和电镜研究

目的观察2型糖尿病模型db/db小鼠胰岛β细胞的超微结构、胰岛素表达及数量变化,探讨β细胞的病理改变与2型糖尿病病因的关系。方法分别选取3、5、8月龄尾静脉空腹血糖高于10.1mmol/L,且肥胖的db/db自发性糖尿病小鼠,每组8只,作为糖尿病组;选取相应年龄段尾静脉空腹血糖低于6.0mmol/L,体重正常的db/+m表型正常小鼠,每组8只,作为对照组。于相应年龄段取胰尾,用于透射电镜观察、免疫组织化学观察和图像分析。结果电镜下随病情进展,db/db小鼠胰岛β细胞内的分泌颗粒数量明显减少,有的细胞甚至缺如,致密芯电子密度降低,β细胞可见凋亡的早期改变以及细胞核和细胞器的病理改变,细胞间髓样小体增多。免疫组织化学显示同月龄糖尿病组小鼠胰岛β细胞阳性率和胰岛素蛋白平均光密度值(OD值)低于相应对照组(p<0.05),且随着病程的进展,db/db小鼠胰岛β细胞阳性率和胰岛素表达呈现递减趋势(p<0.05)。结论2型糖尿病β细胞的超微结构遭到破坏,引起β细胞合成分泌胰岛素障碍和数量减少,与2型糖尿病病情的轻重有关,反映了2型糖尿病病程不同阶段的病机特点。     

NGF在db/db自发性糖尿病小鼠颌下腺的表达

目的　观察转基因糖尿病小鼠颌下腺的形态学改变以及神经生长因子 (NGF)在颌下腺表达的变化。　方法　引进日本C5 7BL ksj db m表型正常隐性基因小鼠 ,近亲交配 ,其纯合子后代 ,即为db db(单基因遗传自然发病型 )糖尿病小鼠。取 3、4、6、8、10月龄db db糖尿病小鼠及相应月龄的db m正常小鼠颌下腺。HE染色及SP免疫组织化学染色后行图像分析 ,统计NGF阳性表达的细胞数。　结果　随着糖尿病发展 ,颌下腺组织萎缩 ,细胞缩小 ,形态不规则 ,排列不整齐。不同月龄糖尿病小鼠NGF阳性细胞明显低于相应对照组 (P <0 0 1) ,且逐渐减少 ,呈下降趋势。　结论　NGF阳性细胞数的减少说明颌下腺颗粒曲管细胞合成和分泌NGF功能降低 ,而NGF缺乏与糖尿病性神经病变的发生与发展密切相关。     

Comparison of mitochondrial and macrophage content between subcutaneous and visceral fat in db/db mice

Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than peripheral (subcutaneous) obesity, however the underlying differences in morphology and pathophysiology between subcutaneous and visceral adipose are largely unknown. To evaluate the effects of diabetes and rosiglitazone (RSG) treatment, the expression of mitochondrial Hsp60, UCP-1 and F4/80 in inguinal subcutaneous (SC) fat, composed of white and brown adipose tissues, and epididymal (EP) fat, mainly white adipose tissue, were evaluated. In diabetic db/db mice, there was significant increased number of aggregated macrophage foci compared to db/+ mice, especially in EP fat. On the other hand, the expression of mitochondrial Hsp60 protein was suppressed in both SC and EP fat of db/db mice compared to db/+ mice, and the expression level of mitochondrial Hsp60 in db/+ mice was lower in EP fat compared with SC. In db/db mice, RSG suppressed the number of aggregated macrophage foci in EP fat, but not in SC fat. RSG ameliorated the mitochondrial Hsp60 expression and induced the expression of UCP-1 in both SC and EP fat. Taken together, these data suggest that differences exist in mitochondrial and macrophage content, and in the response to RSG between visceral and subcutaneous adipose tissue, and adipose type and distribution may be important for obesity-linked insulin resistance.     

Evidence for additionally increased apoptosis in the peripheral blood mononuclear cells of major depressive patients with a high risk for suicide

Several studies have suggested a pathophysiological role of blood cell apoptosis in major depressive disorder (MDD). The aim of this study was to evaluate mRNA expression levels of Bcl‐2, Bax, and Fas in peripheral blood mononuclear cells (PBMCs) of MDD patients with a high risk for suicide relative to those without a high risk for suicide as well as healthy subjects. The mRNA expression of Bcl‐2, Bax, and Fas as well as the Bcl‐2/Bax ratio was examined in the PBMCs of 30 MDD patients with a high risk for suicide, 30 MDD patients without a high risk for suicide, and 30 healthy controls. The mRNA expression of target genes was measured using real‐time quantitative Polymerase Chain Reaction (PCR). FAS mRNA expression was significantly increased, and Bcl‐2 mRNA expression and the Bcl‐2/Bax expression ratio were significantly decreased, in the PBMCs of MDD patients with or without a high risk for suicide attempts compared to healthy controls (p < .001). However, Bax mRNA expression was significantly increased only in MDD patients with a high risk for suicide. Moreover, MDD patients with a high risk for suicide had increased Bax and FAS mRNA expression and decreased Bcl‐2 and Bcl‐2/Bax ratio when compared to patients without risk for suicide (p < .001). Our findings may support the role of both internal and external apoptotic pathways in the interplay between the immune system and depressive symptoms, especially in patients with a high risk for suicide.     

Gender-dependent attenuation of cardiac potassium currents in type 2 diabetic db/db mice

Single ventricular myocytes were prepared from control db /+ and insulin-resistant diabetic db/db male mice at 6 and 12 weeks of age. Peak and sustained outward potassium currents were measured using whole-cell voltage clamp methods. At 6 weeks currents were fully developed in control and diabetic mice, with no differences in the density of either current. By 12 weeks both currents were significantly attenuated in the diabetic mice, but could be augmented by in vitro incubation with the angiotensin-converting enzyme (ACE) inhibitor quinapril (1 μ m , 5–9 h). In cells from female db/db mice (12 weeks of age), K⁺ currents were not attenuated and no effects of quinapril were observed. To investigate whether lack of insulin action accounts for these gender differences, cells were also isolated from cardiomyocte-specific insulin receptor knockout (CIRKO) mice. Both K⁺ currents were significantly attenuated in cells from male and female CIRKO mice, and action potentials were significantly prolonged. Incubation with quinapril did not augment K⁺ currents. Our results demonstrate that type 2 diabetes is associated with gender-selective attenuation of K⁺ currents in cardiomyocytes, which may underlie gender differences in the development of some cardiac arrhythmias. The mechanism for attenuation of K⁺ currents in cells from male mice is due, at least in part, to an autocrine effect resulting from activation of a cardiac renin–angiotensin system. Insulin is not involved in these gender differences, since the absence of insulin action in CIRKO mice diminishes K⁺ currents in cells from both males and females.     

Ramipril improves oxidative stress-related vascular endothelial dysfunction in db/db mice

Endothelial dysfunction often precedes Type 2 diabetes-associated cardiovascular complications. One important cause of endothelial dysfunction is oxidative stress, which can lead to reduced nitric oxide (NO) bioavailability. In this study, we examined the effects of ramipril (an angiotensin-converting enzyme inhibitor, ACEI) on reactive oxygen species (ROS) production and endothelium-dependent vasodilation using a Type 2 diabetic (db/db) murine model. Plasma concentration of 8-isoprostane ([8-isoP]) was measured and used as an indication of the amount of ROS production. Six weeks of ramipril (10 mg/kg/day) treatment significantly reduced [8-isoP] and improved acetylcholine(ACh)-induced vasodilation in db/db mice without altering responses in wild-type (WT) mice. Responsiveness of smooth muscle cells to NO, assessed by sodium nitroprusside-induced vasodilation, was not different between db/db and WT mice regardless of ramipril or vehicle treatment. Our results suggest that ramipril specifically improved endothelium-dependent vasodilation in Type 2 diabetic mice, possibly by reducing ROS levels.     

The role of interstitial macrophages in nephropathy of type 2 diabetic db/db mice

Diabetic nephropathy is associated with interstitial macrophage infiltrates, but their contribution to disease progression is unclear. We addressed this question by blockade of chemokine receptor (CCR)1 because CCR1 mediates the macrophage recruitment to the renal interstitium. In fact, when CCR1 was blocked with BL5923, a novel orally available CCR1 antagonist, the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with advanced diabetic nephropathy. Likewise, BL5923 (60 mg/kg, twice a day) orally administered from months 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, transforming growth factor-beta1, and collagen I-alpha1 when compared with untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for interstitial macrophages for tubulointerstitial injury, loss of peritubular microvasculature, interstitial inflammation, and fibrosis in type 2 diabetic db/db mice. These data identify oral treatment with the CCR1 antagonist BL5923 as a potential therapy for late-stage diabetic nephropathy.