亨廷顿蛋白相关蛋白1基因沉默对小鼠胰岛β细胞系NIT细胞凋亡的影响

目的探讨沉默亨廷顿蛋白相关蛋白1(HAP1)基因表达对小鼠胰岛β细胞株-NIT细胞凋亡的影响。方法化学合成针对小鼠HAP1基因的siRNA,转染NIT细胞,观察干扰效果;通过膜联蛋白V/碘化丙啶(AnnexinⅤ/PI)染色和原位末端核苷酸标记法(TUNEL),检测沉默HAP1表达后NIT细胞的凋亡;通过AnnexinⅤ/PI染色检测沉默HAP1表达后,链脲佐菌素(STZ)所诱导的NIT细胞凋亡。结果靶向HAP1的siRNA能有效抑制NIT细胞HAP1的表达;HAP1 siRNA实验组,NIT细胞凋亡数增多,凋亡率显著高于空白对照组(P0.01);STZ可明显诱导NIT细胞的凋亡,沉默HAP1的表达能增加STZ所诱导的NIT细胞凋亡。结论沉默HAP1的表达可以增加小鼠胰岛β细胞株NIT细胞的凋亡,同时也能促进凋亡诱导剂(STZ)所诱导的胰岛NIT细胞的凋亡。     

侧脑室注射链脲佐菌素对大鼠海马突触相关蛋白PSD-95和Shank 1表达的影响

目的 观察侧脑室注射链脲佐菌素对大鼠脑PSD-95和Shank1表达的影响.方法 将大鼠随机分为正常对照组和模型组,模型组大鼠双侧侧脑室注射链脲佐菌素3mg/kg,第3d重复此剂量;对照组以人工脑脊液代替链脲佐菌素.21d后,取大鼠海马,用免疫组织化学和Western blotting方法观察突触相关蛋白PSD-95和Shank1的表达.结果 与对照组相比,模型组大鼠海马PSD-95和Shankl阳性细胞明显减少,PSD95和Shank1蛋白表达降低.结论 侧脑室注射链脲佐菌素使海马PSD-95和Shank1表达减少,干扰了神经元突触信号传导.     

脑室注射链脲佐菌素对大鼠海马神经元凋亡相关蛋白表达的影响

目的 观察App17肽对脑室注射链脲佐菌素的大鼠海马神经元凋亡相关蛋白表达的影响,探讨胰岛素信号转导通路障碍对神经元存活的作用.方法脑室注射链脲佐菌素(STZ)制作大鼠痴呆模型.3周后,皮下注射App17肽.4周后取脑组织做Bcl-2、Bax、CytoC免疫组织化学染色及Western blotting半定量分析.结果 模型组大鼠海马内Bax、CytoC阳性反应神经元细胞数目多,胞质深染,细胞计数与正常组及治疗组有显著性差异(P＜0.01);模型组大鼠海马内Bcl-2阳性细胞数目少,胞质染色淡,细胞计数与正常组及治疗组有显著性差异(P＜0.01).Western blotting半定量分析可见、Bcl-2、Bax、CytoC出现清晰的条带,组间可见较明显的差异(P＜0.01).结论 脑室注射STZ的大鼠海马内促进凋亡的Bax、CytoC表达增加;抑制凋亡的Bcl-2表达降低.App17肽可影响上述蛋白的表达,使之接近正常.胰岛素信号转导通路对神经元存活有一定作用.     

大鼠胰腺促甲状腺激素释放激素的免疫细胞化学观察

随着免疫化学技术的发展,已证实促甲状腺激素释放激素(TRH)广泛分布于生物体内。Morely等(1977)采用放射免疫测定法首先发现大鼠消化管和胰腺有TRH;Engler等(1981)报导尤其是新生大农,胰腺TRH含量高出成年大鼠丘脑下部TRH的一倍;Nielsen等(1982)通过组织培养发现胰岛能产生和释放TRH;Kazarmi等(1982)用选择性破坏胰岛p细胞的药物链脲佐菌素(Streptozotocin)注射大鼠,发现能显著降低胰岛TRH和升高血糖,若同时合用链脲佐菌素的拮抗剂烟酰胺,则能阻断这种变化,从而认为胰岛的     

1型糖尿病小鼠胰岛CD3、CD57阳性细胞表达改变的研究

目的探讨1型糖尿病(type 1 diabetes mellitus,T1DM)小鼠胰岛CD3、CD57阳性细胞的表达变化及意义。方法正常雄性C57BL/6J小鼠104只,随机分为实验组、盐水对照组和正常对照组。模型诱导采用连续多次小剂量链脲佐菌素给药法(multiple low-dose of streptozotocin,MLDSTZ)。分别于注射后第3、7、10、14、21及28天测空腹血糖、取胰组织,应用免疫组织化学SABC单染法、图像分析及形态计量法进行研究。结果 1)CD3阳性细胞散在分布于胰岛内,实验3 d开始面数密度(NA)逐渐增大,以14 d组最大,之后有所减小,除28 d组外,均大于对照组,比较有显著性差异(P0.01)。2)正常及盐水对照组CD57阳性细胞主要分布于胰岛周边,实验组CD57阳性细胞除分布于胰岛周边,还可见于胰岛中央。NA从实验3 d开始增大,以14 d组最大,之后有所减小,均高于对照组,比较有显著性差异(P0.01)。结论实验组CD3、CD57阳性细胞数量增多,变化趋势相似,提示T1DM初期,T淋巴细胞和NK细胞可能同时浸润胰岛,协同作用胰岛发生自身免疫反应,破坏胰岛B细胞,产生T1DM;随着胰岛B细胞进行性受损,数量减少,胰岛自身免疫反应相对减弱,淋巴细胞也相应减少。     

血小板反应蛋白和纤维连接蛋白在糖尿病大鼠肾的表达

目的:研究血小板反应蛋白(TSP-1)、纤维连接蛋白(FN)在链尿佐菌素诱导的糖尿病模型大鼠肾组织的表达改变.方法:选取成年SD雄性大鼠随机分为2组,实验组用链脲佐菌素诱导产生糖尿病模型,正常对照组注射等量枸橼酸钠缓冲液.成模后6周,H-E染色观察肾组织形态学变化,免疫组织化学和实时荧光定量PCR观察各组大鼠肾TSP-1和FN的蛋白和mRNA的表达情况.结果:大鼠成功建立糖尿病模型后6周,免疫组织化学显示糖尿病大鼠肾组织中表达TSP-1和FN蛋白数量较正常对照组上升,差异有统计学意义.同时,实时荧光定量PCR观察到糖尿病大鼠肾组织中TSP-1的mRNA较正常对照组上升,差异有统计学意义,而糖尿病大鼠肾组织中FN的mRNA较正常对照组也有上升,但差异无统计学意义.结论:高血糖状态下,糖尿病大鼠肾组织TSP-1和FN的表达增加,可能是糖尿病肾病发生、发展的一个重要因素.     

寒冷应激对大鼠肾上腺髓质亨廷顿蛋白相关蛋白1表达的影响

目的 探讨亨廷顿蛋白相关蛋白1(HAP1)在大鼠肾上腺髓质的超微结构定位,以及寒冷应激对大鼠肾上腺髓质HAP1表达的影响. 方法 成年雄性Wistar大鼠14只,2只用于免疫电镜研究,12只用于寒冷实验研究.寒冷实验中,将动物随机分为对照组和寒冷组,每组6只,寒冷组动物放置4℃环境下,12h后用免疫组织化学和Western blotting方法 检测大鼠肾上腺髓质HAP1表达的变化. 结果 免疫电镜结果 显示,HAP1免疫反应产物分布在肾上腺髓质细胞分泌颗粒外膜及分泌颗粒间的膜性细胞器上.寒冷组大鼠肾上腺髓质HAP1的表达明显减少,和对照组比较有显著性差异( P <0.01). 结论 HAP1可能与肾上腺髓质细胞内分泌颗粒及位于分泌颗粒内的肾上腺素/去甲肾上腺素的运输和释放有关.     

丝胶对Ⅱ型糖尿病大鼠胰岛细胞的保护作用

目的:观察丝胶对Ⅱ型糖尿病大鼠胰岛细胞的保护作用.方法:SD大鼠随机分为正常组、模型组、丝胶组和二甲双胍组.模型组、丝胶组和二甲双胍组大鼠均建立链脲佐菌素致Ⅱ型糖尿病模型.待模型成功建立后,丝胶组和二甲双胍组大鼠分别给予丝胶和二甲双胍灌胃35d.分别检测各组大鼠的血糖;免疫印迹法检测各组大鼠胰腺Bax和Bcl-2蛋白、免疫组织化学显色观察各组大鼠胰岛细胞胰岛素和神经肽Y (NPY)蛋白的表达.结果:与模型大鼠相比,丝胶组和二甲双胍组大鼠的血糖、胰Bax和NPY蛋白的表达明显降低,Bcl-2和胰岛素蛋白的表达明显升高;且两组比较无明显差别.结论:丝胶对Ⅱ型糖尿病胰岛损伤具有保护作用;且作用与二甲双胍相当.     

腺病毒介导鼠胰岛素样生长因子1基因对胰岛β细胞的保护作用**

背景：研究表明自身高表达胰岛素样生长因子1的小鼠对药物诱导糖尿病有一定的预防作用。 目的：观察腺病毒介导鼠胰岛素样生长因子1基因对胰岛β细胞损伤的保护作用。 方法：①体外实验：以Ad-rIGF-1、Ad-eGFP直接感染鼠胰岛β细胞标准细胞株-R1Nm5F细胞,然后两组再分别加入0,1.5 mmol/L链脲佐菌素。②体内预防实验：将60只昆明小鼠随机分为空白对照组(不做任何处理)、糖尿病对照组(制作糖尿病模型)、空载体对照组(仅腹腔注射Ad-eGFP重组腺病毒液)、Ad-rIGF-1组(于糖尿病模型制作前2周腹腔注射Ad-rIGF-1重组腺病毒液)。③体内治疗实验：将75只昆明小鼠随机分组：空白对照组(不做任何处理)、糖尿病对照组、胰岛素组、Ad-rIGF-1组、Ad-rIGF-1联合胰岛素组。后3组制作糖尿病模型后给予相应干预。 结果与结论：①鼠胰岛素样生长因子1在胰岛β细胞有效表达,并具有抑制链脲佐菌素诱导胰岛β细胞凋亡的作用。②Ad-rIGF-1组糖尿病发病率低,平均血糖水平低,胰腺炎症浸润程度轻。③与糖尿病对照组、胰岛素组相比,Ad-rIGF-1组和Ad-rIGF-1联合胰岛素组胰腺炎症浸润程度轻,鼠胰岛素样生长因子1在胰岛局部高表达;胰岛素组、Ad-rIGF-1组、Ad-rIGF-1联合胰岛素组血清C-肽水平低,组间比较无明显差别(P > 0.05)。表明胰岛β细胞局部表达鼠胰岛素样生长因子1能够保护胰岛β细胞功能,提高细胞存活率,预防和减轻链脲佐菌素诱导的昆明小鼠1型糖尿病。但鼠胰岛素样生长因子1基因转导与胰岛素皮下注射联合应用对糖尿病早期残存胰岛细胞功能的保护效果不明显。      

丝胶对Ⅱ型糖尿病大鼠肝胰岛素受体和胰岛素受体底物-2的调控作用

目的:研究丝胶对Ⅱ型糖尿病大鼠肝细胞胰岛素受体(IR)及胰岛素受体底物-2 (IRS-2)表达的影响.方法:SD大鼠随机分为5组,分别为正常对照组、糖尿病模型组、丝胶治疗组、阳性对照组、丝胶预防组.采用链脲佐菌素连续腹腔注射法制作Ⅱ型糖尿病大鼠模型.丝胶治疗组大鼠给予丝胶(2.4g·kg-1·d-1)灌胃35 d,阳性对照组大鼠给予二甲双胍(55.33 mg·kg-1·d-1)灌胃35 d,丝胶预防组大鼠于2％链脲佐菌素(25 mg/kg)连续腹腔注射之前给予丝胶(2.4g·kg-1·d-1)灌胃35d.采用葡萄糖氧化酶法检测各组大鼠的空腹血糖;SP免疫组织化学显色、蛋白免疫印迹和RT-PCR检测肝细胞中IR和IRS2的表达.结果:丝胶可明显上调糖尿病大鼠肝胰岛素受体和胰岛素受体底物-2mRNA表达,显著增加IR和IRS-2蛋白的表达.结论:丝胶可通过上调糖尿病肝IR和IRS-2的表达,改善胰岛素抵抗,起到降低血糖的作用.     

DEGRANULATION AND REGRANULATION OF PANCREATIC ISLET CELLS OF ALLOXAN DIABETIC RATS: I. HISTOCHEMICAL STUDY

The process of degranulation and regranulation of pancreatic islet cells was followed in the male rats given one intramuscular or intraperitoneal injection of alloxan (200 mg/Kg). The serum glucose showed a typical triphasic curve, reaching permanent hyperglycemia 24 hours after injection. The insulin levels of both serum and pancreas have been subnormal for the first 24 hours and then marked decrease of insulin levels was observed on the third day thereafter. In accord with subnormal pancreatic insulin levels, B granules have been present in the necrotic cells up to the first 24 hours, then complete disappearance was observed on the third day. Histochemistry of zinc was parallel to the preservation of B cells. Reactive zinc decreased from the center of the islets In 2–4 hours after alloxan, and after 24 hours zinc reaction was positive only around the periphery of the islets. Regranulated islet cells were observed from 7 days through 28 days in the presence of permanent diabetes with decreased population of B cells.     

Insulin-like growth factor I in the pancreas of normal and diabetic adult rats

Insulin-like growth factor I (IGF-I, somatomedin C) was mapped by immunocytochemistry in the pancreas of normal and experimentally influenced rats. The polyclonal IGF-I antiserum K 37 was characterized and demonstrated to be specific. In the exocrine pancreas some duct cells showed IGF-I immunoreactivity, other components being negative. The three main endocrine cell types in the islets of Langerhans were IGF-I immunoreactive, most strikingly the D cells. Hypophysectomy resulted in loss of IGF-I immunoreactivity in all three endocrine cell types, i.e. D, A and B cells, while the levels of somatostatin, glucagon and insulin, respectively, remained unchanged. Starvation seemed to increase and feeding to decrease the IGF-I immunoreactivity in the B cells. Cysteamine pre-treatment reduced the normally intense IGF-I and somatostatin immunoreactivities in the D cells. In rats made diabetic with alloxan or streptozotocin, the B cells were irreversibly damaged and lost both their insulin and IGF-I immunoreactivities, while the IGF-I immunoreactivity was increased in A cells; the D cells remained unchanged. The concentrations of IGF-I mRNA in the pancreas were almost equal in normal and alloxan diabetic rats as were the concentrations of extractable IGF-I. We conclude that IGF-I immunoreactive material can be demonstrated in adult animals in all endocrine islet cells, most prominently in the D cells. The expression of IGF-I immunoreactivity is in part under pituitary control. In the adult rat only one islet cell type synthesizes IGF-I immunoreactive material, i.e. the D cells, while, in contrast, the B cells are likely to be a major IGF-I source in fetal and neonatal islets.     

Changes in the islets of langerhans in the obese Zucker rat.

Pancreatic islet tissue from lean and obese Zucker rats was investigated with histologic and immunocytochemical techniques, and the changes in cytologic composition were correlated with levels of serum glucose, lipids, and insulin. The insular changes in obese rats progressed in severity with increasing age. Changes consisted of pronounced insulin cell hyperplasia, disarrangement of islet architecture, and disappearance (degranulation?) of a new islet cell type, the pancreatic polypeptide cell. High levels of free fatty acids, triglyceride, and insulin were detected in serum of obese rats. Upon diet restriction, these parameters decreased and islet morphology became normalized. When obese Zucker rats were treated with streptozotocin, high levels of serum free fatty acids and triglycerides remained but there was a great reduction in serum insulin levels to near normal levels. Pancreatic polypeptide cells werenot found in the islets. It is suggested that high free fatty acid and triglyceride levels in obese rats may be related to the inability to demonstrate pancreatic polypeptide cells in the islets of 100- to 300-day-old obese Zucker rats.     

Dissociation between pancreatic islet blood flow and insulin release in the rat

The blood flow to the pancreatic islets was estimated with the aid of microspheres in fed or starved (72 h) rats. The total pancreatic blood flow (PBF) in fed animals was 0.55 +/- 0.04 ml X min-1 X g pancreas and in the starved animals 0.30 +/- 0.04 ml X min-1 X g pancreas (P less than 0.001), and the corresponding islet blood flow (IBF) 82.0 +/- 12.4 and 50.5 +/- 9.7 microliter min-1 X g pancreas respectively (P greater than 0.05). Intraperitoneal injection of 2 ml of a 30% glucose solution caused a marked increase in IBF in both fed (P less than 0.05) and starved (P less than 0.01) animals to approximately the same level. The circulating insulin concentration remained unaffected by glucose in the starved rats but increased (P less than 0.001) in the fed rats, indicating that insulin release does not necessarily rise in parallel with an elevated IBF. Intraperitoneal injection of 2 ml of a 30% solution of mannoheptulose, an inhibitor of islet glucose metabolism, decreased the serum insulin concentrations although the serum glucose concentrations rose significantly in both fed (P less than 0.001) and starved (P less than 0.001) animals. This treatment, however, caused both IBF and PBF to increase significantly in both groups. The data support the view that islet blood flow is not necessarily related to the metabolic status of the islet cells or to the insulin release.     

Immunolocalization of 8-OHdG and OGG1 in pancreatic islets of streptozotocin-induced diabetic rats

This study examined whether oxidative DNA damage and its repair system contribute to the occurrence of diabetes in an experimental rat model. The changed morphological findings of the 8-hydroxydeoxyguanosine (8-OHdG) and 8-oxoG-DNA glycosylase (OGG1) were examined in the pancreatic islets in streptozotocin-induced diabetic rats (60 mg/kg, i.p.). The patterns of immunolocalization were mainly observed in the periphery of the normal pancreatic islet: 8-OHdG in the nucleus and OGG1 in the cytoplasm. The altered immunolocalization of 8-OHdG and OGG1 were greatest in the first hours after streptozotocin injection, and then declined in parallel with the morphological observations of pancreatic beta cell destruction. These results suggested that increased oxidative DNA damage might play a role as the inducer of diabetes and that OGG1 may not successfully mediate DNA repair in streptozotocin-induced diabetic rat pancreas.     

Morphological Study of Pancreatic Endocrine in an Experimental Chronic Pancreatitis with Diabetes Induced by Stress and Cerulein

The purpose of this study was to investigate the morphological changes in the islets observed in a new chronic pancreatitis model with diabetes induced by repetition of cerulein injection plus water-immersion stress in rats. The rats of this model were treated with water-immersion stress for 5 h and two intraperitoneal injections of 20 mug/kg body weight of cerulein once a week for 16 weeks. In the stress and cerulein group, 62% of the islets exhibited infiltration of mononucleated cells, and/or peri- and intrainsular fibrosis. On immunohistochemical study, some islets showed reduced density of the insulin immunoreactivity. The glucagon-producing cells decreased in number. With electron microscopy, various endocrine changes were observed, mainly in the B cells. The changes included scattered debris damage with reduction of secretary granules, and vesiculation of the endoplasmic reticulum. Numerous fibroblasts clustered around the islets, and proliferating collagen fibers invaded the islets. The microvascular changes consisted of bleeding and damage to the endothels. In the pancreas treated with stress alone or cerulein alone, significant endocrine damage was not observed. In conclusion, chronic repetitive treatment with stress and cerulein, together with poor islet circulation due to fibrosis and vascular changes, resulted in the endocrine cellular damage.     

Morphometric analysis of the endocrine cell composition of rat pancreas following treatment with streptozotocin and nicotinamide

Using immunohistochemistry and linear scanning, a morphometric analysis was made of the composition of the rat endocrine pancreas at sequential intervals after combined injections of streptozotocin (SZ) and nicotinamide (NA). One week after treatment, the volume of islet tissue was significantly higher than that of the corresponding, saline-injected controls, probably as the result of acute hyperplasia of insulin- and somatostatin-positive cells. However, at all time periods thereafter (6, 20, and 36 weeks), the drug-treated rats showed decreased islet volumes compared to controls. Analysis of aggregate (total) volumes of hormone producing cells at various time periods after drug treatment indicated that decreases in insulin (B-cell) volumes only partially accounted for the observed changes in total islet volume. There were, in addition, early decreases in glucagon (A-cell) and increases in somatostatin (D-cell) volumes. The results suggest that SZ/NA treatment caused limited islet B-cell destruction and transient changes in the proportions of islet A and D cells. Microscopic endocrine tumors were observed at 20 weeks, and both gross and microscopic tumors were observed 36 weeks after SZ/NA treatment. When islet and tumor tissues were included in computation, aggregate volumes of insulin and somatostatin-positive cells were markedly increased, with no significant changes in glucagon-positive cell volumes compared to controls, indicating that the tumors were rich in B and D cells, but poor in A cells. These results are discussed in relation to changes in glucose tolerance and serum insulin levels, and to islet cell volumes following treatment with a diabetogenic dose of streptozotocin alone.     

胎鼠胰腺干细胞与胰岛联合移植保护胰岛

背景：胰腺干细胞可在体外维持胰岛的结构,减少胰岛细胞坏死及凋亡,延长胰岛的体外存活时间,保护胰岛的活性。 目的：探索胎鼠胰腺干细胞与胰岛共移植体内保护移植胰岛,提高胰岛移植疗效的可行性。 方法：将成年大鼠35只随机等分为联合移植组、单独胰岛移植组、单纯胰腺干细胞移植组、模型组及对照组,前4组均腹腔注射链脲佐菌素-柠檬酸盐缓冲液建立糖尿病模型。联合移植组、单独胰岛移植组、单纯胰腺干细胞大鼠分别在左侧肾包膜下移植分离纯化孕16 d SD大鼠胎鼠胰腺干细胞和/或成年SD大鼠胰岛。 结果与结论：联合移植组大鼠移植后5 d内血糖可降至正常,血浆胰岛素达到正常水平,胰岛存活时间(18.2±2.4) d;单独移植组大鼠血糖可于移植后1周内降至正常,胰岛存活时间(14.4±2.1) d;两组胰岛存活时间比较差异有显著性意义(P < 0.05)。而其他组糖尿病大鼠血糖均未能降至正常范围。说明胎鼠胰腺干细胞与胰岛共移植可延长胰岛体内存活时间,保护胰岛功能,提高移植疗效。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Intraperitoneal transplantation of pancreatic anlagen

To determine whether embryonic pancreatic anlagen transplanted to an intraperitoneal site in adult hosts grow, differentiate, and function, we implanted pancreas from embryonic day (E) 12.5 Lewis rat embryos into the omentum of adult Lewis rats or C57Bl/6J mice. E12.5 pancreatic anlagen were relatively undifferentiated except for the presence of condensing tubuloacinar cords. By 2 weeks after implantation, pancreatic anlagen transplanted into rats had enlarged and differentiated such that islets of Langerhans that stained positive for insulin could be delineated. Continued differentiation, as reflected by the presence of "ductal" islets connected to the duct epithelium, was observed at 6 weeks after implantation. At 15 weeks after implantation, "mature" islets had separated from the ducts. Electron microscopy showed eccentric dense bodies within clear vacuoles consistent with insulin granules. Little or no acinar tissue was present in developed anlagen. Within 5 weeks of pancreatic anlagen transplantation, levels of glucose in rats rendered diabetic by an injection of streptozotocin were normalized compared with levels in nontransplanted diabetic controls. Rat pancreatic anlagen underwent growth and development in the peritoneum of C57Bl/61 mice that received costimulatory blocking agents but not in the absence of costimulatory blockade. We concluded that whole E12.5 pancreatic anlagen undergo growth, differentiation, and function after intraperitoneal placement. Implantation of the embryonic pancreas, a "cellular" transplant, is followed by selective differentiation of islet compared with acinar components.