糖尿病模型胰岛细胞凋亡与多种细胞因子的相关性

目的 探讨糖尿病大鼠模型中胰岛细胞凋亡与胰腺组织多种细胞因子的相关性.方法 Wistar大鼠16只,诱发糖尿病模型,分为2组.正常组和糖尿病组大鼠各8只,各组凋亡检测为6只.统计分析各组大鼠血糖、C反应蛋白、多种细胞因子、以及胰腺组织多种细胞因子mRNA定量和蛋白表达水平的差异.分离胰岛细胞,进一步做细胞凋亡的半定量分析.Person 相关性分析胰岛细胞凋亡与细胞因子的关系.结果 糖尿病组胰腺组织中肿瘤坏死因子α(TNF-α)蛋白表达和mRNA显著升高(P＜0.05);糖尿病组与正常组的细胞凋亡率比较,差异有统计学意义[(17.52±9.95)％ vs (8.45±2.01)％,P＜0.05].糖尿病组胰腺组织TNF-α蛋白表达和mRNA含量与胰岛细胞凋亡率呈正相关(r=0.45,P=0.02;r=0.44,P=0.00).结论 糖尿病大鼠模型中胰腺组织TNF-α可能直接作用于胰岛细胞凋亡.     

糖尿病合并感染时肿瘤坏死因子α与胰岛细胞凋亡的相关性

目的探讨糖尿病（DM）合并感染动物模型肿瘤坏死因子α（TNF—α）与胰岛细胞凋亡的变化及相关性。方法在链佐菌脲霉素大鼠尾静脉注射法诱发DM模型基础上行盲肠结扎穿刺术建立动物模型，另外设立单纯DM组、感染组和正常组。采用了mRNA半定量分析；酶联免疫反应检测细胞因子蛋白水平；胆管内胶原酶消化法分离胰岛细胞；胰岛细胞进行半定量细胞凋亡率分析。结果胰腺组织中TNF-α蛋白水平在DM合并感染组较其他各组明显升高，但与正常组比较，DM组血浆TNF—α未见显著性升高；细胞凋亡率DM合并感染组也有显著性升高，两者之间Pearson correlation相关性分析具有统计学意义（P〈0．01）。结论实验结果可能反映了DM合并感染时扩大的炎症反应和胰腺损伤，可能潜在着胰岛细胞凋亡与TNF-α的正相关性。     

过量酒精摄入对大鼠细胞因子及胰岛的影响

目的研究长期酒精摄入对大鼠胰岛功能的影响,以及白细胞介素Iβ(IL-1β)、肿瘤坏死因子α(TNF-α)、白细胞介索6(IL-6)在其中的作用.方法清洁级Wistar大鼠80只(雌雄各半)给予不同浓度的酒精溶液(10%,30%,50%)灌胃.于实验第13周末,断头处死大鼠,分离血清和胰腺分别检测血清中血糖、胰岛素、IL-1β、TNF-α和IL-6以及胰腺组织中IL-1β、TNF-α和IL-6水平.结果50%酒精雌雄大鼠组血清胰岛素水平降低,空腹血糖升高,血清IL-1β、TNF-α、IL-6水平升高,与正常对照组相比均有显著性差异(P＜0.05);胰腺组织中,50%酒精雌性大鼠组IL-1β、TNF-α、IL-6和雄性大鼠IL-1β、TNF-α水平升高.雄性大鼠IL-6在各组间无显著性差异.结论IL-1β、TNF-α、IL-6可能参与了长期过量酒精摄入而引起的胰岛β细胞功能障碍.长期过量饮酒有可能与糖尿病发生相关.     

VEGF对缺血/再灌注损伤胰腺细胞凋亡的影响

目的 研究血管内皮生长因子(VEGF)对缺血/再灌注损伤胰腺组织细胞凋亡的影响.方法 将雄性sD大鼠30只随机分为3组(n=10),A组为假手术组,B组为缺血/再灌注损伤组,C组为缺血/再灌注损伤+VEGF反义寡核苷酸组.通过血管夹阻断大鼠腹腔干及肠系膜上动脉30 min,然后去除血管夹再灌注6 h,建立大鼠胰腺缺血/再灌注损伤模型.对各组胰腺组织进行VEGF免疫组化染色及TUNEL法细胞凋亡检测.结果 缺血/再灌注损伤后胰腺组织出现细胞凋亡,同时VEGF蛋白表达上调.缺血/再灌注损伤+VEGF反义寡核苷酸组的胰腺组织VEGF蛋白表达较缺血/再灌注损伤组显著减少(P<0.05),前者细胞凋亡指数较后者明显升高(P<0.05).结论 VEGF能抑制缺血/再灌注损伤胰腺细胞凋亡,可能对胰腺缺血再灌注损伤具有保护作用.     

VEGF对缺血/再灌注损伤胰腺细胞凋亡的影响

目的 研究血管内皮生长因子(VEGF)对缺血/再灌注损伤胰腺组织细胞凋亡的影响.方法 将雄性sD大鼠30只随机分为3组(n=10),A组为假手术组,B组为缺血/再灌注损伤组,C组为缺血/再灌注损伤+VEGF反义寡核苷酸组.通过血管夹阻断大鼠腹腔干及肠系膜上动脉30 min,然后去除血管夹再灌注6 h,建立大鼠胰腺缺血/再灌注损伤模型.对各组胰腺组织进行VEGF免疫组化染色及TUNEL法细胞凋亡检测.结果 缺血/再灌注损伤后胰腺组织出现细胞凋亡,同时VEGF蛋白表达上调.缺血/再灌注损伤+VEGF反义寡核苷酸组的胰腺组织VEGF蛋白表达较缺血/再灌注损伤组显著减少(P<0.05),前者细胞凋亡指数较后者明显升高(P<0.05).结论 VEGF能抑制缺血/再灌注损伤胰腺细胞凋亡,可能对胰腺缺血再灌注损伤具有保护作用.     

VEGF对缺血/再灌注损伤胰腺细胞凋亡的影响

目的 研究血管内皮生长因子(VEGF)对缺血/再灌注损伤胰腺组织细胞凋亡的影响.方法 将雄性sD大鼠30只随机分为3组(n=10),A组为假手术组,B组为缺血/再灌注损伤组,C组为缺血/再灌注损伤+VEGF反义寡核苷酸组.通过血管夹阻断大鼠腹腔干及肠系膜上动脉30 min,然后去除血管夹再灌注6 h,建立大鼠胰腺缺血/再灌注损伤模型.对各组胰腺组织进行VEGF免疫组化染色及TUNEL法细胞凋亡检测.结果 缺血/再灌注损伤后胰腺组织出现细胞凋亡,同时VEGF蛋白表达上调.缺血/再灌注损伤+VEGF反义寡核苷酸组的胰腺组织VEGF蛋白表达较缺血/再灌注损伤组显著减少(P<0.05),前者细胞凋亡指数较后者明显升高(P<0.05).结论 VEGF能抑制缺血/再灌注损伤胰腺细胞凋亡,可能对胰腺缺血再灌注损伤具有保护作用.     

VEGF对缺血/再灌注损伤胰腺细胞凋亡的影响

目的 研究血管内皮生长因子(VEGF)对缺血/再灌注损伤胰腺组织细胞凋亡的影响.方法 将雄性sD大鼠30只随机分为3组(n=10),A组为假手术组,B组为缺血/再灌注损伤组,C组为缺血/再灌注损伤+VEGF反义寡核苷酸组.通过血管夹阻断大鼠腹腔干及肠系膜上动脉30 min,然后去除血管夹再灌注6 h,建立大鼠胰腺缺血/再灌注损伤模型.对各组胰腺组织进行VEGF免疫组化染色及TUNEL法细胞凋亡检测.结果 缺血/再灌注损伤后胰腺组织出现细胞凋亡,同时VEGF蛋白表达上调.缺血/再灌注损伤+VEGF反义寡核苷酸组的胰腺组织VEGF蛋白表达较缺血/再灌注损伤组显著减少(P<0.05),前者细胞凋亡指数较后者明显升高(P<0.05).结论 VEGF能抑制缺血/再灌注损伤胰腺细胞凋亡,可能对胰腺缺血再灌注损伤具有保护作用.     

VEGF对缺血/再灌注损伤胰腺细胞凋亡的影响

目的 研究血管内皮生长因子(VEGF)对缺血/再灌注损伤胰腺组织细胞凋亡的影响.方法 将雄性sD大鼠30只随机分为3组(n=10),A组为假手术组,B组为缺血/再灌注损伤组,C组为缺血/再灌注损伤+VEGF反义寡核苷酸组.通过血管夹阻断大鼠腹腔干及肠系膜上动脉30 min,然后去除血管夹再灌注6 h,建立大鼠胰腺缺血/再灌注损伤模型.对各组胰腺组织进行VEGF免疫组化染色及TUNEL法细胞凋亡检测.结果 缺血/再灌注损伤后胰腺组织出现细胞凋亡,同时VEGF蛋白表达上调.缺血/再灌注损伤+VEGF反义寡核苷酸组的胰腺组织VEGF蛋白表达较缺血/再灌注损伤组显著减少(P<0.05),前者细胞凋亡指数较后者明显升高(P<0.05).结论 VEGF能抑制缺血/再灌注损伤胰腺细胞凋亡,可能对胰腺缺血再灌注损伤具有保护作用.     

VEGF对缺血/再灌注损伤胰腺细胞凋亡的影响

目的 研究血管内皮生长因子(VEGF)对缺血/再灌注损伤胰腺组织细胞凋亡的影响.方法 将雄性sD大鼠30只随机分为3组(n=10),A组为假手术组,B组为缺血/再灌注损伤组,C组为缺血/再灌注损伤+VEGF反义寡核苷酸组.通过血管夹阻断大鼠腹腔干及肠系膜上动脉30 min,然后去除血管夹再灌注6 h,建立大鼠胰腺缺血/再灌注损伤模型.对各组胰腺组织进行VEGF免疫组化染色及TUNEL法细胞凋亡检测.结果 缺血/再灌注损伤后胰腺组织出现细胞凋亡,同时VEGF蛋白表达上调.缺血/再灌注损伤+VEGF反义寡核苷酸组的胰腺组织VEGF蛋白表达较缺血/再灌注损伤组显著减少(P<0.05),前者细胞凋亡指数较后者明显升高(P<0.05).结论 VEGF能抑制缺血/再灌注损伤胰腺细胞凋亡,可能对胰腺缺血再灌注损伤具有保护作用.     

VEGF对缺血/再灌注损伤胰腺细胞凋亡的影响

目的 研究血管内皮生长因子(VEGF)对缺血/再灌注损伤胰腺组织细胞凋亡的影响.方法 将雄性sD大鼠30只随机分为3组(n=10),A组为假手术组,B组为缺血/再灌注损伤组,C组为缺血/再灌注损伤+VEGF反义寡核苷酸组.通过血管夹阻断大鼠腹腔干及肠系膜上动脉30 min,然后去除血管夹再灌注6 h,建立大鼠胰腺缺血/再灌注损伤模型.对各组胰腺组织进行VEGF免疫组化染色及TUNEL法细胞凋亡检测.结果 缺血/再灌注损伤后胰腺组织出现细胞凋亡,同时VEGF蛋白表达上调.缺血/再灌注损伤+VEGF反义寡核苷酸组的胰腺组织VEGF蛋白表达较缺血/再灌注损伤组显著减少(P<0.05),前者细胞凋亡指数较后者明显升高(P<0.05).结论 VEGF能抑制缺血/再灌注损伤胰腺细胞凋亡,可能对胰腺缺血再灌注损伤具有保护作用.     

胶原酶浓度对大鼠胰岛分离纯化产量的影响

目的观察胶原酶浓度对大鼠胰岛分离纯化产量的影响,筛选出获取高产量胰岛的最佳消化液的胶原酶浓度。方法 75只雄性SD大鼠按胶原酶浓度分为5组：Ⅰ组（0.5 mg/ml）、Ⅱ组（0.75 mg/ml）、Ⅲ组（1.0 mg/ml）Ⅳ组（1.2 mg/ml）和Ⅴ组（1.5 mg/ml）,每组15只。通过胆灌注胶原酶V来消化大鼠胰腺,分离胰岛,采用不连续密度梯度Ficoll离心法纯化胰岛,根据胰岛直径计数所获胰岛的数量及胰岛当量,并利用葡萄糖刺激胰岛素释放试验评估胰岛功能。结果分离的大鼠胰岛具有较好的质量,胰岛纯度为95%左右,活性率为90%左右,体外培养生长良好。随着胶原酶的浓度增加,胰岛数量和胰岛当量明显增加（P〈0.05）,并且最大直径的胰岛数量也在增加（P〈0.05）,在胶原酶浓度1.0 mg/ml时获得的胰岛产量最高,其次为1.2 mg/ml,在其他浓度时,胰岛产量都较低（P〈0.05）。在低糖和高糖刺激下胰岛素的释放分别为（1.346±0.128）ng/ml和（2.565±0.208）ng/ml,差异具有显著性意义（P〈0.05）,刺激指数为（1.908±0.152）,提示胰岛细胞功能良好,电镜照片显示胰岛生长状况良好。结论进行大鼠胰岛分离纯化时最适宜的胶原酶浓度为1.0~1.2 mg/ml,可获得稳定高产量的胰岛,过低和过高的浓度都是不适宜的。     

五指山小型猪胰岛分离和纯化的初步研究

目的探讨封闭群五指山小型猪胰岛的分离和纯化,并在体外检测分离纯化后的胰岛对高低糖刺激反应来判定胰岛功能。方法 7只雄性五指山小型猪和6只普通成年猪的胰腺分别被用于胰岛分离纯化实验研究。胰腺获取后,胰管插管并注入消化液（胶原酶V,1.8g/L）,胰腺充分灌注后,切成碎块并转入消化罐内消化,消化液过滤后并经梯度离心后,收集纯化的胰岛,经双硫宗（DTZ）染色后计数,并在不同浓度葡萄糖培养液里检测释放的胰岛素浓度来判定胰岛的活性。结果小型猪胰腺解剖上更接近人类,胰腺消化时间平均为（30±8）min,消化程度为（70±8）%,胰岛产量纯化前为（5500±1025）IEQ/g,纯化后为（4120±625）IEQ/g,纯度为85%,活力为80%;取自屠宰场猪胰岛产量纯化前为（2500±625）IEQ/g,纯化后为（1720±435）IEQ/g,活力为60%。镜下观察,小型猪胰岛镜下可被DTZ染色呈红色,纯化后的胰岛大小一致,形态完整,体外培养小型猪胰岛在低糖和高糖刺激下胰岛素释放分别为（1.346±0.128）ng/mL和（2.565±0.208）ng/mL,差异有显著性意义（P〈0.05）,刺激指数为（1.908±0.152）,而取自屠宰场猪胰岛素释放分别为（1.246±0.18）ng/mL和（1.4±0.208）ng/mL,差异无显著性。结论封闭群五指山小型猪胰腺解剖结构和人类相似,分离纯化后的胰岛产量高,功能良好,适合异种胰岛移植。     

高海拔缺氧对链脲佐菌素致大鼠胰岛损害程度的影响

[目的]通过观察西宁（低海拔）、木里（高海拔）地区链脲佐菌素（streptozotocin,STZ）糖尿病大鼠胰岛形态学特点,探讨高原缺氧环境对STZ糖尿病大鼠胰岛形态学影响。[方法]大鼠随机分成西宁、木里正常组;西宁、木里造模组。造模组大鼠空腹12h后腹腔注射STZ,以随机血糖≥16．7mmol/L为造模成功;采用免疫组织化学染色和原位末端转移酶标记技术（TdT-mediated dUTP nick end labeling,TUNEL）细胞凋亡检测分析胰岛面积、胰岛D细胞面积、平均光密度、平均累积光密度（integrated optical density,IOD）和胰岛细胞凋亡率。[结果]木里糖尿病组血糖水平低于西宁糖尿病组;糖尿病大鼠胰岛数量减少、萎缩、形状不规则,胰岛细胞变性,胰岛内β细胞分布稀少,胰岛染色颗粒较少,着色浅,凋亡细胞较正常组多;木里糖尿病组在β细胞面积、β细胞面积／胰岛面积和IOD上显著高于西宁糖尿病组。[结论]高海拔STZ糖尿病大鼠血糖较低;胰岛β细胞受损较轻;高原缺氧环境可能对STZ糖尿病大鼠胰岛β细胞有一定的保护作用。     

Dietary sucrose enhances insulin secretion of aging Fischer 344 rats.

Male Fischer 344 rats, ages 6, 12 and 26 mo, were fed a diet containing either sucrose or cornstarch (66% by weight) for 4 mo. The effects of age and dietary sucrose on glucose-stimulated insulin secretion were evaluated in whole perfused pancreases and isolated islets of Langerhans, and by intra-arterial glucose administration. In addition, glucose responsiveness of beta-cells was measured by following the rate of glucose oxidation in isolated islets. There was no significant effect of age on glucose-stimulated insulin secretion of whole perfused pancreases and islets of Langerhans. There was, however, a significant main effect of sucrose feeding on insulin secretion. That is, whole perfused pancreases and islets of Langerhans isolated from rats fed sucrose vs. starch diets secreted more insulin in response to glucose. This effect was most pronounced in the 26-mo-old rats. In general, islet glucose oxidation rates, and responses to the in vivo glucose, did not differ among the groups. We conclude that alterations in glucose-stimulated insulin secretion with age more closely reflect changes in diet rather than aging per se.     

Transplantation of the islets of Langerhans in patients with diabetes mellitus type 1

Since 1921 and until recently, insulin by injection has been the only treatment for patients with diabetes mellitus type 1. After pancreas transplantation, which became possible in 1977, the next logical step to cure patients with diabetes mellitus type 1 is the transplantation of the islets of Langerhans. In the last few years, the results of islet transplantation are markedly improved thanks to developments in the isolation technique and better immunosuppressive protocols. Ongoing problems in islet transplantation are allo-immunity, auto-immunity and the growing shortage of donor pancreases. Alternatives to pancreas donation, be it post-mortem or from a living donor, could be: sources for islets are xenotransplantation with the aid of pig islets and beta-cell neogenesis from embryonic stem cells or pancreatic duct cells.     

Dual effect of ethanol on cell death in primary culture of human and rat hepatocytes

AIMS: In-vivo and in-vitro studies have shown that ethanol induces hepatocyte damage. The aim of the present study was to evaluate the effect of a broad range of ethanol concentrations on apoptosis and necrosis in primary culture of human and rat hepatocytes. METHODS: Human and rat hepatocytes were isolated from human hepatectomies and male Wistar rats (200-250 g) using the classical collagenase perfusion method. After stabilization of cell culture, ethanol (0-10 mmol/l) was administered and the parameters were measured 24 h after ethanol addition. Apoptosis was studied by DNA fragmentation, iodide propidium-DNA staining, caspase-3 activity and annexin V binding in hepatocytes. Necrosis was evaluated by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) and GSH/GSSG were used as parameters of oxidative stress. RESULTS: Ethanol enhanced dose-dependently all the parameters associated with apoptosis in human and rat hepatocytes. Low or high ethanol concentrations induced an opposite action against cell necrosis in cultured hepatocytes. Low concentrations of ethanol (1-2 mmol/l) reduced LDH release from human and rat hepatocytes. However, the highest ethanol concentration (10 mmol/l) induced a sharp increase in cell necrosis. The effect of ethanol on cell necrosis was related to lipid peroxidation in hepatocytes. CONCLUSIONS: Ethanol differentially regulates apoptosis or necrosis in cultured hepatocytes. Although ethanol exerted a dose-dependent induction of apoptosis, low ethanol concentrations were able to reduce basal lipid peroxidation and necrosis in hepatocytes. The highest ethanol concentration (10 mmol/l) induced apoptosis and necrosis in human and rat cultured hepatocytes.     

Pancreas procurement from multiorgan donors for islet trasplantation

The outcome of human islet isolation procedures can be significantly effected by the technique used for pancreas procurement. In fact, the final step of islet purification using discontinuous density gradients requires a significant difference between the density of the islets and the density of the non-endocrine component of the gland. Therefore, any procedure during multi-organ procurement that will result in edema or degranulation of the acinar tissue will result in failure of the islet purification step. In this report a technique for combined harvesting of liver and pancreas is presented. The use of this procedure can be of assistance to avoid damage to the pancreas that could result in a compromised islet purification for improper handling of the gland even before it arrives to the isolation facility.     

Beneficial effects of oligopeptides from marine salmon skin in a rat model of type 2 diabetes

ObjectiveThis study aimed at investigating whether treatment with oligopeptides from marine salmon skin (OMSS) could modulate type 2 diabetes mellitus (T2DM)-related hyperglycemia and β-cell apoptosis in rats induced by high fat diet and low doses of streptozotocin and its therapeutic mechanisms.MethodsGroups of T2DM rats were treated with OMSS or bovine serum albumin (3.0 g/kg/d) for 4 wk and their blood samples, together with those of normal control rats, were collected before and 4 wk after treatment. The levels of fasting blood glucose (FBG) and insulin, serum superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), tumor necrosis factor-alpha (TNFα), and interferon-gamma (IFNγ) in rats were determined. The islet cell apoptosis and Fas/FasL expression were detected by TUNEL and immunohistochemistry.ResultsIn comparison with control rats, higher levels of FBG and frequency of apoptotic islet cells were detected in the bovine serum albumin group of diabetic rats, accompanied by higher levels of Fas expression in the pancreatic islets, serum TNFα, IFNγ, and MDA, but lower levels of SOD and GSH. However, the levels of FBG and frequency of apoptotic islet cells were significantly reduced in OMSS-treated rats. Lower levels of Fas expression were observed in the pancreatic islets of OMSS-treated rats. Significantly reduced levels of serum TNFα, IFNγ, and MDA, but increased levels of SOD and GSH, were detected in OMSS-treated rats.ConclusionsTreatment with OMSS significantly reduced FBG in diabetic rats. This antidiabetic activity may be mediated by down-regulating T2DM-related oxidative stress and inflammation, protecting the pancreatic β-cells from apoptosis.     

小鼠睾丸支持细胞的原代培养和鉴定

目的 建立小鼠睾丸支持细胞培养方法，为从细胞和分子水平研究环境化合物对雄性生殖系统的影响提供条件。方法 采用两步胶原酶消化，提取出生10d的ICR雄性小鼠睾丸支持细胞，用Tris-HCl处理后除去生精细胞，进一步纯化。倒置相差显微镜和苏木精-伊红（HE）染色观察细胞生长和形态。Feulgen染色法鉴定支持细胞。结果 培养的支持细胞增生活跃，纯度〉95％，Feulgen染色后明显可见支持细胞核内的双极小体。结论 酶消化法分离和Feulgen染色法鉴定大鼠支持细胞简单易行，且细胞纯度较高。为今后研究生殖毒理提供了条件和方法。     

大鼠胰岛移植后血管重建和排斥反应的实验研究

目的通过不同移植前和移植后处理,观察移植胰岛的血管重建及功能变化。方法随机将SD大鼠分成3组,未处理组:移植前后均未作处理,只作单纯胰岛移植;抗树突状细胞单克隆抗体处理组:胰岛移植前先将供体胰岛细胞用抗树突状细胞单克隆抗体和补体在体外37℃下共育45min,再移植给受体大鼠;雷帕霉素处理组:胰岛移植后给予受体大鼠1mg/kg/d的雷帕霉素。胰岛的血管密度通过Lecti nFrom Bandeiraea Simplicifolia的荧光染色来评估,胰岛功能通过测定血糖水平来评估。结果3组胰岛在移植后2d时结构松散,均未见毛细血管再生。移植后4、8、10d胰岛呈团块状和条索状,均可见微血管再生。移植后8、10d时雷帕霉素处理组的微血管密度大于抗树突状细胞单克隆抗体处理组和未处理组(P<0.05),而移植后4d时3组之间均无明显差异(P>0.05)。抗树突状细胞单克隆抗体处理组的高血糖缓解时间比未处理组延长2d。雷帕霉素处理组移植后血糖水平一直维持在低水平。结论雷帕霉素在胰岛移植早期对血管重建有着重要的临床意义,并能有效地保护胰岛细胞免受排斥反应的影响。去除供体胰岛中的树突状细胞对防止胰岛移植早期的免疫排斥有一定的...