人红细胞RhD血型抗原的甲氧基聚乙二醇修饰研究

目的：探讨甲氧基聚乙二醇（mPEG）遮蔽修饰的红细胞RhD血型抗原的有效性和稳定性以及被修饰红细胞结构和功能的变化。方法：用mPEG-SPA修饰RhD血型阳性人红细胞,观测修饰和未修饰红细胞的抗-D凝集反应性、mPEG结合产物稳定性、电镜下细胞形态、变形指数、渗透脆性、自身溶血率、2,3-二磷酸甘油酸含量、膜Na^＋,K^＋ATP酶、乙酰胆碱酯酶活性和胆固醇含量。结果：0．5mmol／L、1．0mmol／L、2．Ommol／L、2．5mmol／L和5．0mmol／L的mPEG-SPA修饰红细胞与抗D的凝集反应情况依次为50％凝集、可见凝集、无凝集、无凝集、无凝集,在4℃保存14d和在37℃保存2d的修饰红细胞与抗D均无凝集反应,而末修饰红细胞则全凝集。电镜下修饰和未修饰红细胞均为双凹圆盘状形态．细胞大小均一。修饰和未修饰红细胞的各观测指标分别为：2,3-二磷酸甘油酸含量（71．00±12．88）mmol／L和（65．13±13．98）mmol／L,红细胞沉降率（2．75±2．05）mmjh和（8．00±3．82）mm／h,细胞变形指数（0．98土0．18）和（0．98士0．29）,自身溶血率（2．27±0．28）％和（1．32±0．32）％,渗透脆性（0．44±0．06）％和（0．44±0．03）％,膜胆同醇含量（0．10±0．03）g／i．和（0．10±0．06）g／L,Na^-,K^-ATP酶活性（4．834±1．27）U／mg和（5．41±1．32）U／mg,乙酰胆碱酯酶活性（27．88±5．09）U／mg和（29．68±4．165U／mg。修饰红细胞的沉降速率低于未修饰红细胞,自身溶血率高于未修饰红细胞（P＜0．05）,但是修饰红细胞沉降速率和自身溶血率都在参考值范围。结论：2．0mmol／L的mPEG-SPA能够有效并且稳定地遮蔽红细胞RhD血型抗原。修饰红细胞具有未修饰红细胞的双凹圆盘状形态、细胞膜结构、变形性和运送氧气能力．     

大鼠胰岛与睾丸细胞共移植诱导同种胰岛移植免疫豁免

目的　探究大鼠胰岛与睾丸细胞共移植后睾丸细胞Fas配体表达能否对胰岛移植物提供免疫豁免作用。方法　将同种大鼠胰岛及睾丸细胞移植于糖尿病受体,观察移植物存活情况,并体外检测睾丸Sertoli细胞对活化淋巴细胞的抑制作用以及移植物内淋巴细胞凋亡情况。结果　单纯移植胰岛组移植胰岛平均存活期为(6±1)天。睾丸细胞和胰岛细胞共移植,当睾丸细胞数量为5×10     

胰岛移植与免疫

糖尿病是当前影响人类健康的常见病、多发病之一。其发病率约为3%~5%。自1922年胰岛素问世以来,糖尿病患者的死亡率明显下降。但是,时至今日尚无根治糖尿病的手段。而胰岛移植被认为是当今最有希望治愈这一顽疾的方法之一,它具有安全、简单、不良反应轻等优点。尤其是近年来人胰岛移植取得令世人瞩目的成功,使得移植技术再次成为国内外研究的热点。一、胰岛移植的发展历史1966年,美国明尼苏达大学首次完成了世界上第一例胰腺移植手术。然而,胰腺移植手术创伤大、并发症多,难以成为治疗糖尿病的常规手段。1973年,Ballinger等首次报道了大鼠…     

糖尿病小鼠腹腔内移植免疫隔离膜包裹大鼠胰岛长期疗效的观察

作者观察免疫隔离膜包裹大鼠胰岛移植至糖尿病小鼠腹腔内的长期疗效。移植后有效率为91％,11只中有4只完全缓解,缓解持续时间最长一只达360天。而未包膜胰岛异种移植均无效。对长期保持完全缓解的小鼠作胰腺病理学检查,显示胰岛萎缩,因此证实异种胰岛移植的有效是由于免疫隔离膜起到预期的抗排异反应。     

免疫隔离胰岛移植的研究进展

免疫隔离胰岛移植的研究进展周茂华，陈东明，夏兆骥免疫隔离技术是通过一个人造屏障将移植物与受体的免疫系统隔离开来，这一技术允许使用从动物胰腺分离出的胰岛，因而解决了从人类获取胰岛困难的问题。胰岛细胞被包裹在人工合成的具有选择通透性的膜中，此膜可以阻止受...     

人工合成多肽（P—15）对单层培养胰岛细胞的作用

观察人工合成多肽（Ｐ－１５）的新生大鼠单层培养胰岛细胞素分泌和胰岛素表达的作用。含有１６．７ｍｍｏｌ／Ｌ葡萄糖的ＴＣＭ１９９培养基培养２４小时的Ｗｉｓｔａｒ大鼠单层胰岛细胞被Ｐ－１５刺激３０、９０分钟。结果表明，短时期内Ｐ－１５对胰岛细胞产生刺激分泌效果，长时间７．２μｍｏｌ／ＬＰ－１５既增加胰岛素的分泌，也刺激胰岛素的基因表达，高浓度却丧失了这种刺激效果。     

糖尿病Wistar大鼠腹腔内移植免疫隔离膜包囊S—D仔鼠胰岛研究

作者为解决组织移植中发生排异反应,研制了一种新的免疫隔离膜,在体外培养中,胰岛组织不断繁殖并分泌胰岛素。本文是在活体内考核其抗排异反应。在四氧嘧啶所引起的22只糖尿病Wistar大鼠腹腔内移植包囊sprague-Dawley仔鼠胰岛,观察其效果,其中20只完全缓解,完全缓解率达90％。有6只已观察至一年,血糖仍保持正常,上述效果比未包膜胰岛移植为好,在两种不同纯系大鼠间进行胰岛移植获得了较理想的效果。     

胰腺胰岛肿的免疫组织化学研究

本研究对最常见的胰岛素瘤和非功能性内分泌肿瘤进行正分泌激素 ,异位激素等的免疫组织化学研究 ,以探讨其免疫组化特征及其意义和免疫组化在诊断胰腺内分泌肿瘤(ｐａｎｃｒｅａｔｉｃｅｎｄｏｃｒｉｎｅｔｕｍｏｒ,ＰＥＴ)中的作用。　　一、对象和方法1.对象 :17例胰腺肿瘤均为上海市第六人民医院 1976年 1月至 1997年 12月收治的手术病例。男 8,女 9,2 4～ 70岁 ,平均 46 .2岁 ,病程 1个月～ 10年。临床表现为 9例反复发作的低血糖性昏迷 ,血糖 0 .5～ 2 .2ｍｍｏｌ/Ｌ ,4例血清胰岛素 2 5～ 36ｍＵ/Ｌ ,临床诊断胰岛细胞瘤。 8例非内分…     

胰岛移植中免疫抑制剂的应用进展

胰岛移植为需终生注射外源性胰岛素的1型糖尿病患者提供了一种新的选择,但移植后排斥反应、终生应用免疫抑制剂及其不良反应仍然限制着胰岛移植的广泛开展.应用Edmonton方案前胰岛移植1年后脱离外源胰岛素的患者仅占8%.Edmonton方案的应用使移植1年后不依赖外源性胰岛素患者的比例达80%(2年后36例接受胰岛移植的受者仅5例不需要胰岛素而且血糖控制良好),该方案是以白细胞介素_2受体单克隆抗体诱导,雷帕霉素、小剂量他克莫司为基础的无激素免疫抑制方案.近来新型免疫抑制剂的出现使胰岛移植的免疫抑制方案更趋安全、有效.本文综述了胰岛移植开展以来免疫抑制剂的应用和进展.     

Insulin secretion by a transplantable rat islet cell tumour

Summary Investigation of the subcellular and molecular components of insulin secretion has been made difficult by the small quantities of material available. The recent development of a transplantable rat islet cell tumour of high insulin content and state of differentiation suggested a system more amenable to analysis. To validate the tumour as a model of secretion we have studied its release of insulin. In acute experiments in vitro immunoreactive insulin release was increased by leucine, glucagon, theophylline and dibutyryl cyclic AMP, though not by glucose. Leucine (20mmol/l) plus theophylline (5 mmol/l) caused an abrupt, sustained and rapidly reversible stimulation of two- to fivefold. The response was inhibited by antagonists of cellular oxidative phosphorylation (cyanide, 2,4-dinitrophenol, antimycin A), calcium flux (EGTA, verapamil, Mg²⁺), calmodulin (trifluoperazine), microtubules (vinblastine, colchicine) and by adrenaline and somatostatin. These findings suggest that the tumour secretes insulin by an exocytotic mechanism similar to that of normal islet tissue.     

大鼠胰岛细胞与睾丸支持细胞联合微囊化的体外功能研究

将大鼠胰岛细胞和Sertoli细胞联合微囊化后培养11天，胰岛细胞和Sertoli联合微囊化组的胰岛功能明显好于胰岛Sertoli细胞分别微囊化的共同培养组及胰岛单独微囊组（P〈0．05）。     

碘克沙醇及Ficoll-400分离纯化大鼠胰岛细胞的效果对比

目的:分别使用碘克沙醇和Ficoll-400密度梯度液纯化大鼠胰岛细胞,比较两者分离纯化大鼠胰岛细胞的收获量、纯度、活性和功能.方法:将20只SD大鼠随机分为碘克沙醇纯化组和Ficoll-400纯化组,以胶原酶P消化分离SD大鼠胰腺,分别采用碘克沙醇及Ficoll-400密度梯度液来纯化胰岛细胞.通过DTZ染色,计数胰岛细胞的数量和纯度,用胰岛素释放试验和胰岛移植评估胰岛细胞的功能.结果:碘克沙醇-HCA液纯化组的胰岛细胞收获量及纯度与Ficoll-HCA无明显差别,碘克沙醇-HCA组纯化的胰岛细胞活率则显著高于Ficoll-HCA组(93.3%±3.5% vs 84.8%±3.8%,P<0.01),同时胰岛细胞逆转糖尿病大鼠高血糖状态的时间显著延长(11.4±2.1 vs 7.0±1.6 d,P<0.05).结论:与Ficoll-400相比,使用碘克沙醇纯化大鼠胰岛可提高胰岛细胞的活率及功能.     

Macrophage cytotoxicity towards isolated rat islet cells: neither lysis nor its protection by nicotinamide are Beta-cell specific

Summary In animal models of Type 1 (insulin-dependent) diabetes mellitus macrophages were shown to be the first immunocytes that infiltrate the pancreatic Langerhans islets in the autoimmune process. We now show direct macrophage cytotoxicity against isolated rat islet cells in an electron microscopical study, which permits investigation of the specificity of this process. Freshly isolated islet cells were co-incubated with syngeneic peritoneal macrophages at a targeteffector-cell ratio of 12. After various time periods, the cells were directly fixed and embedded; the ratio of live and dead cells was evaluated by electron microscopy. Our results demonstrate that activated but not resident macrophages lyse islet cells in a time-dependent manner. After 15 h of co-incubation lysis of islet cells is complete. No islet cell-macrophage contacts and no differences between the lysis of Beta cells or non-Beta cells were observed during the observation period. Islet cells encapsulated in alginate were also lysed by macrophages as a direct proof for soluble mediator(s) of cytotoxicity. Nicotinamide protected islet cells from lysis in a dosedependent manner. As a result of this electron microscopic study we conclude that even at very low targeteffector ratios, activated macrophages lyse syngeneic islet cells regardless of islet cell type via secretion of humoral mediator(s).     

Bcl-X基因转录后剪切方式改变在大鼠胰岛细胞凋亡中的作用

目的　探明 Bcl- x基因在大鼠胰岛细胞凋亡时剪切方式的变化及其该基因在胰岛细胞凋亡中的作用。方法　应用 TUNEL法检测长期高糖培养时大鼠胰岛细胞凋亡 ,应用半定量多重 RT- PCR(SQ- RT- PCR)检测了胰岛细胞凋亡时 Bcl- x基因 m RNA的表达变化情况 ;同时观察小剂量 STZ所致的糖尿病大鼠胰岛 Bcl- x基因 m RNA的表达变化。结果　 2 5 .5 m M葡萄糖浓度体外培养 14天较 5 .5 m M和 11.1m M葡萄糖浓度培养 ,胰岛细胞凋亡百分率明显增加 ,分别为 31.5± 4 .5 % ,15 .4± 3.0 % ,17.4± 4 .8% ;P <0 .0 1。细胞凋亡百分率变化的同时伴有 Bcl- x基因转录后剪切方式的改变 ,表现为 Bcl- x S m RNA表达明显增加 ;低剂量 STZ所致的糖尿病大鼠胰岛 Bcl- x S m RNA表达明显增加 ,Bcl-x L与 Bcl- x S比率明显减少。结论　长期高糖可以诱导大鼠胰岛细胞凋亡 ,Bcl- x基因剪切方式的改变所致的 Bcl- x L/Bcl- x S比率的变化在高糖和 STZ所致的胰岛细胞凋亡中起重要作用     

Insulin secretion by rat islet isografts of a defined endocrine volume after transplantation to three different sites

Summary We have analysed the graft function of rat islet isografts of identical and well-defined endocrine volumes after transplantation to three different sites (kidney, liver and spleen). Graft endocrine mass was determined by measuring the total islet volume prior to transplantation and was chosen to be similar to the endocrine volume in the normal adult rat pancreas. Graft function was tested in unanaesthetized, unstressed rats by the responses to glucose infusion and to a meal. All transplanted animals returned to normoglycaemia within one week after transplantation. At one month, basal glucose and insulin levels were similar to controls in rats with grafts to the spleen, but higher in rats with grafts to the kidney or liver. Irrespective of the transplantation site, recipients had higher glucose and lower insulin levels than controls in response to glucose infusion, but in response to a meal these differences from normal were less obvious. Finally, recipients showed both an acute insulin response to glucose infusion as well as a pre-absorptive insulin release after food ingestion, irrespective of the transplantation site. Our findings indicate that the insulin response to glucose infusion and to a meal is quantitatively reduced, but qualitatively intact after transplantation to the kidney, liver or spleen.     

GLP-1对大鼠胰岛细胞增殖及凋亡的影响

目的 观察胰升糖素样肽1（GLP-1）对大鼠胰岛细胞增殖及凋亡的影响，并探讨其作用机制。方法 （1）GLP-1与大鼠胰岛细胞瘤细胞株RINm5f共育，观察其增殖情况；（2）检测GLP-1对IL-1D诱导的RINm5f细胞凋亡的保护作用；（3）GLP-1与原代培养大鼠胰岛细胞共育1、3、5天后，检测BAX及Bcl-2基因的表达。结果（1）随着GLP-1浓度增高及刺激时间延长，RINm5f细胞A值呈剂量及时间依赖性增加；（2）GLP-1组与对照组相比RINm5f细胞凋亡率显著下降（P〈0．05）；（3）与GLP-1共育后，大鼠胰岛BAX基因的表达量无明显变化，对照组BAX基因的表达逐渐增加（P〈0．05）；而Bcl-2基因的表达量随着与GLP-1共育时间的延长呈时间依赖性增加。结论GLP-1对大鼠胰岛细胞增殖有明显促进作用；同时对胰岛细胞的凋亡有保护作用。     

Oxygen free radical scavengers protect rat islet cells from damage by cytokines

Summary A possible role for oxygen free radicals in mediating the cytotoxic effects of cytokines in islets was sought by the use of agents that scavenge free radicals. Rat islet cell monolayer cultures were incubated for 6 days with t-butylhy-droperoxide, alloxan, streptozotocin, or the cytokines, interleukin 1, tumour necrosis factor, and interferon gamma, without and together with the oxygen free radical scavenger combination of dimethylthiourea and citiolone, and islet cell lysis was measured in a ¹⁵chromium cytotoxicity assay. The free radical scavengers significantly inhibited the islet cell cytotoxic effects of t-butylhydroperoxide and alloxan, but not streptozotocin. Similarly, the cytotoxic effects of the cytokine combinations of interleukin 1 plus tumour necrosis factor, interferon gamma plus tumour necrosis factor, and interferon gamma plus interleukin 1 were significantly inhibited by the free radical scavenger combination of dimethylthiourea and citiolone. These results suggest that the cytokine products of macrophages and lymphocytes infiltrating islets in Type 1 (insulin-dependent) diabetes may contribute to B-cell damage by inducing the production of oxygen free radicals in the islet cells.