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Isolation of the Islets of Langerhans for Transplantation
D. R. Thomas et al.
Editor’s Note
This paper describes a method for isolating rat islets of Langerhans as a potential transplant source for diabetes. Attempts to treat diabetes by transplanting a whole pancreas had met with limited success, and efforts to isolate the component insulin-producing islets of Langerhans were hampered by methodological problems and low viability. The revised method, by D. R. Thomas and colleagues at Sheffield in England, includes digestion, washing and separation steps and yielded large numbers of viable islets of Langerhans. Islet cell transplantation is currently being assessed as a potential treatment for type I diabetes mellitus, where the immune system destroys insulin-producing beta cells in the islets of Langerhans. Successful transplants have reduced or removed the need for insulin therapy. 中文
PANCREATIC transplantation with the aim of treating diabetes mellitus has so far met with little success. Of 23 patients thus treated and reported to the Transplant Registry in 1971 1 , 15 died within 3 months and the longest survived one year. One of the major problems has been to overcome pancreatic exocrine digestion, and pancreatic duct ligation (“Banting pancreas”) before transplantation has been performed. It was shown by Dragstedt 2 , however, that dogs treated in this way often became diabetic or showed a diabetic glucose tolerance test after several months, probably due to fibrosis and consequent ischaemia of the Islets. Transplantation of the whole gland with its vascular supply is a major undertaking and the problems of thrombosis, leakage and digestion, coupled with immunological rejection, have prevented success so far. 中文
Attempts have been made to isolate the Islets of Langerhans from the pancreas in order to study glucose metabolism. A micro-dissection technique was described by Hellerstrom 3 but this was tedious and produced only small numbers of Islet clumps. Subsequently Moskalewski 4 described a method of collagenase digestion which was successful in the rabbit and improved the yield, and Lacy and Kostianovski 5 modified the method for the rat. There were still difficulties in harvesting the liberated Islets, however, even with methods of separation such as zonal centrifugation and density gradients, and there were also problems with viability of the cells. 中文
We have developed a technique by which large numbers of Islets of Langerhans are prepared consistently from the rat pancreas for purposes of transplantation. Viability was confirmed by transplantation. 中文
Initially, albino outbred rats and subsequently young adult inbred hooded rats (strain PVG/C) were used. Following killing by cervical dislocation, the ventral surface of the animal was shaved, prepared with chlorhexidine in spirit and the abdomen opened with a midline incision. Using a dissecting microscope, magnification×10, a fine polyethylene catheter (“Intracath”—B. R. Bard, London) was introduced into the common bile duct and secured with a 2-0 linen thread ligature. The lower end of the duct was occluded with an artery forceps just before its entry into the duodenum. The pancreas was distended by injection of 10 ml. Hanks’ solution containing bovine albumen (fraction V), 2 mg·ml. -1 . It was found that after some practice this procedure could be completed within 5 min of the death of the animal. The pancreas was removed, transferred to a glass Petri dish, cut into small pieces with scissors and any excess fat removed. It was then transferred to a tube to which a further 10 ml. of Hanks’ solution was added. The pancreatic tissue sank to the bottom and any remaining fat floated on the surface and was readily aspirated and discarded. The prepared tissue was transferred to a small conical flask together with 2 ml. Hanks’ solution containing glucose 0.6 mg ml. –1 and collagenase type 1 (Sigma Chemicals, London). The stoppered flask was placed in a shaking water bath at 37℃ for about 30 min. The exact time for separation was determined by frequent sampling and examination under the dissecting microscope. 中文
The digested pancreas was transferred to a tube and diluted with further cold Hanks’ solution containing glucose in the same concentration as before and gently centrifuged for 1 min. The supernatant was discarded and fresh medium added, and the resultant suspension filtered into a Petri dish with a blackened base for examination under the dissecting microscope at×10 magnification. 中文
Initially the view was obscured by fine fragments of acinar tissue, which were removed by gently agitating the Petri dish and allowing them to become suspended in the Hanks’ solution, then aspirated and discarded, the Islets remaining on the bottom of the vessel. They could then be seen clearly (Fig. 1) by the aid of dark ground illumination as yellowish white domes and were picked out with a finely drawn Pasteur pipette. Fig. 2 shows the histological appearance of an isolated pancreatic Islet. Its architecture and cells appear normal. 中文
Fig. 1. Islet cell tissue seen after isolation from the pancreas using dark ground illumination. Dissecting microscope, ×5.
Fig. 2. Isolated pancreatic Islet. Normal architecture and appearance of cells. Haematoxylin and eosin, ×300.
Yields of up to 350 Islets per rat pancreas have been achieved using this method. To obtain larger quantities, rat pancreases have been processed in batches of four. 中文
Viability of the isolated cells was confirmed by transplantation beneath the renal capsule and into the testis of isogeneic rats. The longest period of follow-up was one month, when viable looking Islet cells containing beta cell granules staining with aldehyde-fuchsin were seen. A similar method has been successfully applied in the rabbit. 中文
The relationship of the Islets of Langerhans to diabetes mellitus was established in 1889 by Von Mering and Minkowski 6 , and in 1892 Hedon 7 demonstrated that subcutaneous implantation of a small piece of pancreas could delay the appearance of diabetes in an animal that had undergone pancreatectomy. The concept of Islet cell grafting appears to be neglected in the extensive literature on pancreatic transplantation, although one early report suggests that transplanted Islets may modify alloxan diabetes in the rat 8 . 中文
We have now established a successful method of isolation of Islets of Langerhans in an animal strain in which inbred immunologically isogeneic lines are available, making possible transplantation studies uncomplicated by rejection problems. These cells can be grafted and survive for appreciable periods of time as shown by the viability studies. Histological and functional studies of Islet cell transplantation will be reported elsewhere. 中文
We acknowledge a grant from the Endowment Fund of the United Sheffield Hospitals and from the Medical Research Council. We thank Dr. Laurence Henry for his help with the histology and Mr A. Tunstill and his staff for photography. 中文
( 242 , 258-260; 1973)
D. R. Thomas, M. Fox and A. A. Grieve
Royal Hospital, Sheffield
Received October 12, 1972. Requests for reprints to M. F.
References:
用于移植的朗格汉斯岛（胰岛）的分离
托马斯等
编者按
本文介绍了一种用于分离大鼠朗格汉斯岛（胰岛）的方法，以用作治疗糖尿病的潜在移植来源。通过移植整个胰腺治疗糖尿病的尝试收效甚微，而分离产生胰岛素的胰岛的尝试受到了方法学问题和低生存力的限制。由托马斯和他的同事在英国谢菲尔德所改良的方法，获得了大量可存活的胰岛，这种方法包含消化、洗涤和分离几个步骤。现在胰岛细胞移植正被评估是否可作为一种治疗I型糖尿病的潜在方法，在I型糖尿病中，免疫系统会破坏产生胰岛素的胰岛β细胞。成功的移植可以减少或消除对胰岛素治疗的依赖。 英文
迄今为止，胰腺移植用于治疗糖尿病成效甚微。1971年，经过这样治疗并在移植登记处登记备案的23例患者中 [1] ，有15例在3个月内死亡，生存时间最长的患者也只有1年。一个主要的问题就是患者在移植前需要克服胰腺外分泌消化和胰管结扎（“班廷胰腺”）。但是，德拉格施泰特 [2] 发现，经过这样治疗的狗在数月后会患糖尿病或者表现出糖尿病糖耐量试验的结果，这很可能是由于胰岛的纤维化以及继发的贫血造成的。伴血液供应的全腺体移植是目前的主流，但是伴随免疫排斥发生的血栓、渗漏和消化等问题使得该方法到目前为止也没能取得成功。 英文
人们试图从胰腺中分离出胰岛以研究葡萄糖代谢。赫勒斯特伦 [3] 描述了一种显微解剖技术，但是这种方法非常繁冗，而且只能得到少数的胰岛细胞团。此后，莫斯卡勒夫斯基 [4] 描述了一种胶原酶消化法，该法已经在兔子中取得成功，而且分离出的胰岛数量也得到提高，后来莱西和科斯蒂安诺夫斯基 [5] 在大鼠中对这个方法进行了改进。但是，即便使用了诸如区带离心和密度梯度离心的分离方法，仍然很难获得游离的胰岛，而且这些细胞的生存能力也是一个大问题。 英文
我们建立了一种方法，并用此法从大鼠胰腺中稳定制备了大量用于移植的胰岛，并且通过移植确认了细胞的生存能力。 英文
起初使用的是白化的远交系大鼠，后来使用年轻成年的头部有斑点的顶罩大鼠（PVG/C系）的近交系。颈椎脱臼法处死大鼠后，刮除腹面毛发，用溶于酒精的氯己定处理后选取正中线切口开腹。使用解剖显微镜放大10倍，将一条细小的聚乙烯导管（“Intracath”（译者注：一种留置针）—巴德，伦敦）插入胆总管，并用2–0的亚麻缝合线固定。在导管恰好进入十二指肠前，用动脉钳夹闭其下端。注射10 ml含有2 mg·ml –1 牛血清蛋白（第五组分）的汉克氏液（译者注：一种用于细胞培养的平衡盐溶液）使胰腺膨胀。经过一些实践之后我们发现，这些步骤可以在动物死后5分钟之内完成。将胰腺摘除并转移到一个玻璃培养皿内，用剪刀将其剪碎并去除所有多余的脂肪。然后将其转移到一个试管内，并加入10 ml的汉克氏液。胰腺组织沉到管底，所有残存的脂肪都会漂浮在表面，将它们吸出并丢弃。将制备好的组织转移到一个小的锥形瓶内，该瓶内装有2 ml含0.6 mg·ml –1 葡萄糖和I型胶原酶（西格玛化学公司，伦敦）的汉克氏液。把盖好的瓶子置于37℃水浴摇床内约30分钟。分离过程的限速步骤是解剖显微镜下的频繁取样和检查。 英文
将消化好的胰腺转移到一个试管内，用含有相同浓度葡萄糖的冷的汉克氏液稀释，温和离心1分钟。弃上清液并加入新鲜的培养液（汉克氏液），得到的悬浮液过滤到培养皿内；该培养皿的底为暗色，以便于在放大10倍的解剖显微镜下进行观察。 英文
起初，许多细小的腺泡组织碎片使得视野很模糊；轻轻地摇动培养皿使其悬浮在汉克氏液中，然后将其吸出并丢弃，而胰岛则存留在容器底部。通过暗场照明能够清晰地看到它们呈黄白色的圆顶状（图1），用尖端拉长的巴斯德吸管将其移出。图2显示了分离出来的胰岛的组织学形态。其结构和细胞表现正常。 英文
图1. 通过暗场照明观察到的从胰腺中分离出来的胰岛细胞组织。解剖显微镜下放大5倍。
图2. 分离出的胰岛。正常的结构和细胞外观。苏木精—伊红染色，放大300倍。
用这种方法能够在每个大鼠胰腺中得到多达350个胰岛。为了获得更多胰岛，大鼠胰腺以四个为一组一起处理。 英文
将分离的胰岛细胞移植到同种大鼠的肾小囊内或者睾丸内以确认其生存能力。最长的随访期是1个月，此时可以观察到成活的胰岛细胞含有能被醛复红染色的β细胞颗粒。同样的方法在兔子中也获得了成功。 英文
1889年，冯·梅灵和明科夫斯基阐述了胰岛和糖尿病之间的关系 [6] 。1892年，埃东 [7] 证明了移植到皮下的一小块胰腺可以延缓进行胰腺切除术后的动物出现糖尿病症状。在大量有关胰腺移植的文献中，人们都忽略了胰岛细胞移植的想法，尽管有一篇早期报道提出移植的胰岛可能会减轻在大鼠中由四氧嘧啶诱导的糖尿病 [8] 。 英文
现在，我们已经在近交的免疫同系的动物品系中成功地建立了一种分离胰岛的方法，这使得移植研究摆脱排斥问题的困扰成为可能。正如在对胰岛细胞的生存力研究中所证实的，这些细胞能够被移植并能生存相当长的时间。胰岛细胞移植的组织学和功能方面的研究将另作报道。 英文
我们感谢联合谢菲尔德医院的捐助基金和医学研究理事会的资金。我们感谢劳伦斯·亨利博士帮助进行组织学检查，感谢滕斯蒂尔先生及其职员在成像方面的帮助。 英文
（毛晨晖 翻译；王敏康 审稿）