啮齿类动物胰岛的分离与纯化

啮齿类动物胰岛分离的应用广泛,其方法也早已建立,但影响胰岛分离的因素较多,使获得足量、纯净、存活和功能良好的胰岛常较困难,且胰岛分离的稳定性和一致性亦难令人满意。因此,针对分离技术、纯化方法、分离剂、消化酶及消化条件等诸方面进行了广泛的改进,胰岛的收获量、纯度及活力等均有显著的提高。     

成人胰岛分离及纯化的研究进展

复习了成人胰岛分离及纯化的研究进展，主要在自动消化装置、分离纯化技术、胶原酶品种及胰腺保存诸方面已有较大改进，促胰岛的得量、纯度及活力显著提高。     

啮齿类动物胰岛的分离与纯化

啮齿类动物胰岛分离的应用广泛,其方法也早已建立,但影响胰岛分离的因素较多,使获得足量、纯净、存活和功能良好的胰岛常较困难,且胰岛分离的稳定性和一致性亦难令人满意。因此,针对分离技术、纯化方法、分离剂、消化酶及消化条件等诸方面进行了广泛的改进,胰岛的收获量、纯度及活力等均有显著的提高。     

成人胰岛分离及纯化的研究进展

复习了成人胰岛分离及纯化的研究进展，主要在自动消化装置、分离纯化技术、胶原酶品种及胰腺保存诸方面已有较大改进，使胰岛的得量、纯度及活力显著提高。     

胰岛β细胞的分离纯化与原代培养技术

胰岛β细胞是糖尿病研究的中心环节之一。目前体外研究胰岛β细胞的模型系统有两类，一是从胰岛β细胞瘤组织克隆产生的β细胞株，二是从活体胰岛组织分离纯化获取胰岛β细胞。国内利用细胞株作为研究模型者较多，而β细胞的体外原代培养尚未见研究报道。     

成人胰岛分离纯化的初步研究

目的初步研究成人胰岛的分离、纯化方法,为同种异体胰岛移植治疗1型糖尿病进行前期准备。方法采用改良的Ricordi技术消化成人尸体胰腺,然后用连续性密度梯度离心法纯化胰岛。胰岛收获量以国际标准的胰岛当量（islet equivalent,IEQ）表示。结果完成10例成人胰岛分离和纯化,其中5例完成了胰岛当量的统计,胰岛收获量为6367～108725IEQ/胰腺,平均为47678.8IEQ/胰腺,平均每克组织收获2055IEQ。结论采用改进的人胰岛分离方法,可以获得较大产量的有活性的胰岛。     

我国恒河猴粪便腺病毒聚合酶基因片段的序列分析

目的研究非人灵长类动物猴子腺病毒的分子多样性。方法呆集圈养的恒河猴粪便样本,以腺病毒聚合酶基因为目的基因,用PCR3-法进行扩增,对PCR阳性产物进行克隆、测序,并进行系统进化分析。结果57只恒河猴粪便样本有12份存在腺病毒DNA,系统进化分析提示这些序列主要可分为两大组：类SAdV6组（2个非重复序列）和类SAdV一7组（9个非重复序列）。此外,有3个克隆,其最相似序列分别为：SAdV一1,SAdv_3和HAdV一52。结论研究证实腺病毒在非人灵长类粪便中普遍存在,揭示这些动物肠道内腺病毒的多样性以及系统进化情况。辇铺{司．帽河准．獬早限病毒：其闵相多样忡     

恒河猴人工感染BRUCELLA OVIS后 病理学观察及病原回收

选用健康公恒河猴6只分3组每组2只,以Brucella ovis进行人工1亿、0.1亿、0.01亿菌量感染后35天再分别以10亿、5亿和1亿菌感染。结果在180天开始1、2组猴出现明显临床症状第3组则症状轻微。300天扑杀后可见附睾,睾丸、精索、淋巴结、脾、肝、肾、肺等器官和组织有典型病变;并在病变器官中分离到B.ovis培养物。     

Experimental immune mediated arthritis in rhesus monkeys

Summary The induction of experimental arthritis in rhesus monkeys was studied by intradermal immunization of bovine type II collagen and antigens derived fromMycobacterium tuberculosis, Streptococcus pyogenes, andEubacterium aerofaciens. The tested bacterial antigens proved to be not arthritogenic. Bovine type II collagen induced clinical arthritis in 50% of the rhesus monkeys. Type II collagen induced arthritis in rhesus monkeys proved to be a potential model to study clinical, serological, histological, genetic, and immunologic features associated with human RA.     

Longitudinal analysis of early stage sarcopenia in aging rhesus monkeys

We present a longitudinal study using the rhesus monkey to determine biochemical and histological changes in vastus lateralis (VL) muscle fibers and whether these changes correlate with muscle mass loss. Dual-energy X-ray absorptiometry (DXA) was used to determine body weight, body fat and to estimate upper leg muscle mass in 12 adult male rhesus monkeys over 12 years. Muscle mass (MM) was evaluated at years 6, 9 and 12 of the study. Concurrently, VL muscle biopsy samples were collected. Muscle tissue was sectioned, stained and individual muscle fibers were analyzed for fiber type, cross-sectional area (CSA) and mitochondrial electron transport system (ETS) enzyme abnormalities. The animals' body weight did not change over time, however a significant increase in DXA-measured percent body fat was observed. Significant MM loss occurred in the upper leg over 12 years. A reduction in muscle fiber CSA significantly contributed to the MM loss observed in the VL of middle-aged rhesus monkeys. An age-dependent increase in muscle fibers developing mitochondrial enzyme abnormalities due to mitochondrial DNA deletion mutations was observed. The longitudinal approach of this study demonstrated that significant muscle changes occurred during middle age in a cohort of aging rhesus monkeys.     

Human islet purification: a prospective comparison of Euro-Ficoll and bovine serum albumin density gradients

Euro-Ficoll (EF) and bovine serum albumin (BSA) are the two most commonly used media for the density gradient purification of human pancreatic islets. The aim of this study was to compare these two media with respect to the efficiency of human islet isolation. Ten human pancreata were collagenase-digested, and samples of digest were separated on either a continuous linear density gradient of BSA or a discontinuous gradient of EF (1.108/1.096/1.037/Euro-Collins). Efficiency of islet purification was assessed by insulin and amylase assay of aliquots aspirated from the BSA gradients, and from the interfaces of the EF gradients. Islets were obtained from two interfaces in the EF gradients. Islet yield from the upper interface was generally poor (median 28% of total insulin; range 2–71%), but purity was better than for an equivalent yield using BSA [1% (0–3%) amylase contamination for EF versus 6% (0–37%) for BSA;P=0.013]. Pooling both EF interfaces increased yield to 66% (17–81%) but markedly reduced purity [46% (0–50%) amylase for EF versus 31% (0–52%) for BSA]. In conclusion, the efficiency of human islet purification is similar, though disappointingly low, with BSA and with EF. Considerable scope exists, therefore, for improvement in the density gradient purification of human islets.     

Ultrastructure of monolayer cultures of pancreatic islet cells

Summary Isolated islet cells from obese-hyperglycemic mice and normal guinea-pigs were culturedin vitro for periods of up to 7 days. Ultrastructural investigation showed that the cultured cells retained the morphologic characteristics of islet cellsin vivo. ₂-cells and -cells could be identified in explants from both obese-hyperglycemic mice and normal guinea-pigs. The most distinctive feature of the cultured islet cells was a marked degranulation of the -cells of both species. The degree of degranulation was not correlated with the glucose concentration in the incubation medium.This work was supported by the Swedish Medical Research Council (B69-12X-2297-02 and B69-12X-109-05), the United States Public Health Service (AM-12535) and the Medical Faculty, University of Uppsala.     

脂联素受体在胰岛细胞表达,脂联素促进胰岛素的分泌

目的　检测脂联素受体(AR)在大鼠胰岛细胞的表达和脂联素对体外胰岛细胞分泌胰岛素的影响。方法　RT PCR和免疫细胞化学方法检测AR1、AR2的mRNA和蛋白表达;并在体外用脂联素(100μg/L)和不同浓度葡萄糖(3. 3, 5. 6, 16. 7mmol/L)处理胰岛细胞,放免法测定上清液的胰岛素浓度。结果　RT PCR扩增出胰岛AR1和AR2基因,并经直接和亚克隆测序证实;胰岛免疫细胞化学荧光染色AR1和AR2呈阳性;经脂联素处理后的胰岛细胞,在高糖(16. 7mmol/)培养 6~24h,其胰岛素分泌持续增加(均P<0. 05)。结论　胰岛细胞上存在AR1和AR2,以前者为主。在高糖情况下,一定浓度的脂联素可在体外促进胰岛细胞的胰岛素分泌和释放。     

CD4+CD25+T细胞延长同种异体胰岛移植物存活

探讨CD4+ CD25+调节性T细胞作为细胞疫苗抑制小鼠同种异体胰岛移植物排斥反应的作用,采用免疫磁珠分离技术分选CD4+ CD25+调节性T细胞联合BALB/cByJ小鼠同种异体胰岛移植.结果 显示,CD4+ CD25+调节性T细胞可明显延长同种异体移植物的存活时间.     

他汀类调脂药对大鼠胰岛胰岛素分泌的影响及机制研究

目的 观察不同他汀类药物对大鼠胰岛葡萄糖刺激的胰岛素分泌(GSIS)的抑制作用及机制.方法 新鲜分离或经24 h培养的胰岛均匀分为对照组、阿托伐他汀组、氟伐他汀组和普伐他汀组,对照组给予Kreb-Ringer碳酸氢盐缓冲液,他汀类药物组分别给予100μmol/L阿托伐他汀、氟伐他汀和普伐他汀,水浴30 min或过夜培养24 h.各组经2.8、5.5、11.1、16.7、25.0 mmol/L葡萄糖刺激后,37℃水浴法测定胰岛GSIS变化,生物化学发光法测定三磷酸腺苷(ATP)含量.结果 100μmol/L阿托伐他汀水浴30 min后,在16.7 mmol/L葡萄糖刺激下,与相应对照组比较,ATP含量[(9.54±1.64)pmol/胰岛比(12.33±1.89)pmol/胰岛]及胰岛素分泌(1.60±0.21比2.39±0.30)均下降(P＜0.05);100 μmol/L氟伐他汀过夜培养24 h后,在16.7 mmol/L葡萄糖刺激下,与相应对照组比较,ATP含量[(10.24±2.01)pmol/胰岛比(12.31±2.16)pmol/胰岛]及胰岛素分泌(3.12±0.32比4.17±0.37)也均下降(P＜0.05).结论 阿托伐他汀、氟伐他汀通过抑制胰岛ATP的生成而抑制GSIS,抑制程度与其脂溶性强弱有关.

Abstract：

Objective To evaluate the inhibitory effect of statins on glucose-stimulated insulin secretion (GSIS) of pancreatic islet in rat and to explore its mechanisms. Methods According to the average volume, freshly isolated or 24-hour cultured pancreatic islets were randomly divided into control group( incubated with Kreb-Ringer bicarbonate buffer), the atorvastatin group( incubated with 100 μ mol/L atorvastatin), the fluvastatin group (incubated with 100 μ mol/L fluvastatin)and the pravastatin group (incubated with 100 μ mol/L pravastatin). Stimulated by 2. 8,5. 5,11.1,16. 7 mmol/L and 25.0 mmol/L glucose respectively, the effect of 100 μ mol/L statins on ATP content and GSIS was compared in the four groups. GSIS was performed by the 37℃ bath incubation method and ATP content was measured by chemiluminescence method. Results Incubated with 100 μ mol/L atorvastatin for 30 minutes, in the presence of 16. 7 mmol/L glucose, the ATP content [(9. 54 ± 1. 64) pmol/islet vs ( 12. 33 ± 1.89) pmol/islet] and GSIS (1.60 ± 0. 21 vs 2. 39 ± 0. 30) were significantly reduced in comparison with the control group (P＜0. 05). Cultured with 100 μmol/L fluvastatin for 24 hours, the ATP content [( 10. 24 ±2.01 )pmol/islet vs (12. 31 ±2. 16) pmol/islet] and GSIS (3. 12 ± 0. 32 vs 4. 17 ±0. 37 ) were all significantly decreased at the higher glucose concentration of 16. 7 mmol/L ( P ＜ 0. 05). Conclusion Atorvastatin and fluvastatin may inhibit GSIS by decreasing ATP content in pancreatic islet and the inhibitory effect is related to the strength of its lipophilicity.     

Ibrolipim对糖尿病小型猪胰岛β细胞的保护作用

目的 通过观察Ibrolipim对贵州小型猪（小型猪）的胰岛结构和功能的影响，探讨其调节糖代谢的作用机制。方法 小型猪15头，随机分为3组。分别喂普通猪饲料、高脂高糖饲料（HFSD）和HFSD加1％Ibrolipim。每月末测定血糖（PG）和胰岛素（Ins）浓度。第6个月末作OGTT和胰岛素敏感性试验。第8个月末处死动物，取胰腺标本，作胰岛素含量测定。用HE染色、免疫组化和透射电镜观察口细胞结构的变化。结果 HFSD组小型猪PG和Ins水平增高，以及加重胰岛素抵抗（IR）和口细胞损害。HFSD＋1％Ibrolipim组FPG显著降低，PG清除率以及胰岛素分泌急性相都明显改善，p细胞的结构和功能受到保护。结论 Ibrolipim有降低PG，减轻IR和保护口细胞的作用。     

Changes in food intake during menstrual cycles and pregnancy of normal and diabetic rhesus monkeys

Summary Food intake of control and streptozotocin-diabetic rhesus monkeys was measured during menstrual cycles and pregnancy. Intake of control monkeys was lower at the time of ovulation than during other phases of the menstrual cycle. Intake of control monkeys was also low during most of pregnancy, but this was accompanied by normal fetal growth and net maternal weight gain. Diabetic monkeys ate more than controls in all conditions and their intake did not vary reliably according to reproductive status. It is suggested that (1) oestrogen normally inhibits food intake during menstrual cycles and pregnancy, (2) food energy is utilized more efficiently during pregnancy than during non-pregnant states, and (3) the influence of oestrogen on food intake is either attenuated by insulinopenia or is obscured by the hyperphagia typically exhibited by the diabetic monkeys.