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胚胎及新生期大鼠胃肠道胰岛淀粉样多肽与生长抑素的免疫组织化学定位
http://www.100md.com 《解剖学报》 1998年第4期
     作者:梁文妹 石爱荣*

    单位:贵阳医学院组织学胚胎学教研室,贵阳 550004;*北京医科大学组织学胚胎学系

    关键词:胰岛淀粉样多肽;生长抑素;胃肠道;胚胎和新生大鼠

    解剖学报/980421 摘 要 为探讨胚胎期及新生期大鼠胃肠道的胰岛淀粉样多肽(IAPP)与其他生物活性物质的关系,用免疫组织化学PAP法,在相邻切片上分别显示第15~21d大鼠胚胎及新生大鼠胃肠道的IAPP免疫反应性(IR)细胞和生长抑素(SS)IR细胞,观察了IAPP与SS在胚胎及新生大鼠胃肠道的定位分布。结果表明:第15d胚胎,胃肠道内未见IAPP-和SS-IR细胞。第17d胚胎,IAPP-与SS-IR细胞均很少,免疫染色较弱,着色较浅,免疫反应性细胞散布于分化未完全的肠上皮细胞间。胚胎19d,IAPP-和SS-IR细胞出现于胃和小肠,以十二指肠为多。胚胎第21d及新生期,胃肠道各段均可见到IAPP-和SS-IR细胞。经邻片比较证明,胚胎第19d始至新生期,十二指肠和空肠中部分IAPP-IR细胞与部分SS-IR细胞的定位相同,表明IAPP与SS在十二指肠和空肠D细胞中有共存。胃、回肠、结肠和直肠未见到IAPP与SS有细胞内共存的现象。本文对上述结果的意义进行了讨论。
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    胰岛淀粉样多肽(islet amyloid polypeptide, IAPP)是一种主要分布于胰腺的多肽激素,由37个氨基酸残基组成[1,2]。目前关于IAPP在胰腺外定位的报道仍然很少。我们曾报道IAPP免疫反应性细胞在大鼠胚胎期、新生期及生后发育期胃肠道的个体发生和定位分布[3,4]。已有实验证明,IAPP在胰腺内与胰岛素(insulin,Ins)和生长抑素(somatostatin, SS)均有细胞内共存的关系[5~7],但IAPP在胃肠道内与SS在D细胞中有无共存以及相互关系如何目前尚未见报道。

    本研究用邻片单染免疫组织化学PAP法,观察了IAPP、SS在大鼠胃肠道的分布及其在D细胞中是否共存,将为进一步了解胃肠内分泌细胞中生物活性物质间的相互关系提供形态学依据。

    材料和方法

    1. 动物分组与标本制备
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    正常Wistar大鼠胚胎20只,新生Wistar大鼠5只,雌雄不拘。胚胎大鼠以查见母鼠有阴栓之日起计算受孕日期为0d,分为胚胎第15、17、19、21d组和新生期组,每组5只。孕鼠经三氯甲烷吸入麻醉后,打开腹腔取出胎鼠。将第15、17d组胃肠道全长一起取材。第19d组按胃至十二指肠及其余肠道两部分取材。第21d胎鼠及新生大鼠分别取胃体、胃窦、十二指肠、空肠、回肠、结肠和直肠。组织块入Bouin液固定,石蜡包埋,连续切片,厚4μm。将相同胎龄组同一组织的切片各取1片,裱于同一载片上,按顺序编号,每例观察切片3张以上,切片间隔36μm。

    2. 免疫组织化学方法

    取相邻组织切片,用Sternberger PAP法分别显示IAPP-及SS-IR细胞。切片常规脱蜡至水,用甲醇-H2O2封闭后,正常羊血清(1∶50)室温30min;兔IAPP抗血清(1∶4 000,Peninsula Lab, USA产品),兔SS抗血清(1∶1 000,第二军医大学提供),于4℃过夜;羊抗兔IgG(1∶50)37℃45min;兔PAP复合物(1∶100)37℃45min;DAB-H2O2液显色:苏木精复染胞核,树胶封片。
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    方法对照:以PBS分别代替IAPP和SS抗血清,其余步骤同上述。

    结 果

    用PAP法显示的IAPP-和SS-IR细胞,其反应产物为棕褐色细颗粒状,存在于胞质内。方法对照均为阴性。

    1. IAPP-IR细胞和SS-IR细胞的定位

    第15d胚胎,胃肠道未见IAPP-和SS-IR细胞。第17d胚胎,胃肠道免疫反应性细胞很少,散在分布于分化未完全的上皮细胞间。第19d胚胎,IAPP-IR细胞仍少,见于胃和小肠,以十二指肠稍多,形状多为锥形和柱状。邻片显示的SS-IR细胞也见于胃和小肠,在胃中单个散在,小肠中也以十二指肠为多,存在于绒毛上皮及肠腺上皮细胞间,此时SS-IR胞体较小,多呈卵圆形,反应颗粒位于细胞的基底部(图1)。第21d胚胎及新生期,胃肠道各段均可见到IAPP-及SS-IR细胞,两种细胞仍以十二指肠为多。胃粘膜的SS-IR细胞仍较少,分布于上皮细胞基部间,靠近上皮,胞体较小,胞体卵圆形(图2)。在小肠,SS-IR细胞较多,形态多样,有圆、卵圆、锥形等(图3,5,7)。偶于绒毛固有层结缔组织中可见到SS-IR细胞。结肠和直肠中也见到SS-IR细胞,数量较少,分散存在于粘膜上皮内。
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    图1~7免疫组织化学PAP法,苏木精复染胞核

    图1 第19d胚胎大鼠小肠。(↑)示位于小肠绒毛上皮细胞间的SS-IR细胞。 ×268

    图2 第21d胚胎大鼠胃。(↑)示位于胃粘膜上皮细胞间的SS-IR细胞。 ×268

    图3 新生大鼠十二指肠。(↑)示位于肠绒毛上皮细胞间的SS-IR细胞。 ×268

    图4 第21d胚胎大鼠十二指肠。(↑)示肠绒毛上皮内的IAPP-IR细胞。 ×268

    图5 为图4的邻片。(↑)示与IAPP-IR细胞定位相同的SS-IR细胞。 ×268

    图6 新生大鼠十二指肠。(↑)示肠绒毛上皮内的IAPP-IR细胞。 ×134
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    图7 为图6的邻片。(↑)示与IAPP-IR细胞定位相同的SS-IR细胞。 ×134

    Fig.1~7 Immunohistochemical PAP method. Hematoxylin counterstain.

    Fig.1 The small intestine of a rat embryo on the 19th day.↑ showing the SS-IR cell located between epithelial cells of a intestinal villus. ×268

    Fig.2 The stomach of a rat embryo on the 21st day. ↑ showing the SS-IR cell between gastric epithelium cells. ×268
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    Fig.3 The duodenum of a newborn rat.↑ showing the SS-IR cell between epithelial cells of a intestinal villus. ×268

    Fig.4 The duodenum of a rat embryo on the 21st day. showing the IAPP-IR cell (↑) in the epithelium of a intestinal villus. ×268

    Fig.5 The adjacent section of Fig 4.↑ showing the SS-IR cell had the same location as the one of IAPP-IR cells. ×268

    Fig.6 The duodonum of a newborn rat.↑ showing the IAPP-IR cell in the epithelial of a intestinal villus. ×134
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    Fig.7 The adjacent section of Fig 6 showing the SS-IR cell (↑) had a same location with IAPP-IR cells. ×134

    在胚胎和新生期大鼠胃肠道,IAPP-IR细胞与SS-IR细胞的发育比较相似。经邻片观察,在各发育时间IAPP-IR细胞的数量均少于SS-IR细胞,免疫染色强度也较SS-IR细胞弱些,细胞形态也有不同,IAPP-IR细胞以锥形为多,而SS-IR细胞则以卵圆形为多。

    2. IAPP与SS共存的形态学观察

    经相邻切片观察比较,第17d胚胎,胃肠道上皮分别有较少的IAPP-和SS-IR细胞,未见IAPP与SS在同一细胞内有共存。第19d胚胎,发现十二指肠有少数细胞内IAPP与SS定位相同。第21d胚胎至新生期大鼠,于十二指肠和空肠可见一些细胞既呈IAPP-IR又呈SS-IR,其中以十二指肠多见,多位于绒毛上皮内(图4~7),但免疫染色强度有一定差异,即IAPP-IR细胞较SS-IR的染色弱些。在胃、回肠、结肠和直肠,则未见有上述现象。
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    讨 论

    IAPP与Ins共存于胰岛B细胞已为较多实验所证实[2,5]。De Vroede等[6]曾在分离的大鼠胰岛细胞中发现SS-IR细胞也显示IAPP-IR。Ahren等[7]也在大鼠胰腺发现,除胰岛B细胞外,D细胞也显示IAPP免疫反应性。关于IAPP与SS是否也共存于胃肠道同一细胞,目前尚未见报道。本实验观察到从第19d胚胎至新生期大鼠十二指肠和空肠中部分IAPP-IR细胞与相邻切片中部分SS-IR细胞定位一致,表明IAPP与SS在胚胎及新生大鼠胃肠道的D细胞中有共存现象。这一结果为拓宽IAPP的研究提供了新的形态学资料。

    已有实验证实,IAPP主要与糖稳态调节有关[8,9]。氨基酸序列分析显示,IAPP与降钙素基因相关肽(calcitonin gene-related peptide, CGRP)有约50%的同源性,CGRP和IAPP都可抑制B细胞释放Ins并对Ins分泌起着局部旁分泌的调节作用[10]。病理学研究证明,IAPP与非胰岛素依赖型糖尿病的发病密切相关[1,2],一些神经内分泌肿瘤如类癌等也显示IAPP免疫反应性[11]。早有文献报道,生长抑素细胞可释放CGRP[12]。本实验结果发现IAPP与SS也共存于肠道D细胞,表明D细胞与IAPP和CGRP的释放可能均有关系。Dharmsathaphorn等[13]曾报道SS可缓解类癌综合症引起的分泌性腹泻,Davis等[14]也有类似观察。有实验证明,在糖的刺激下IAPP和Ins同时释放,但IAPP的释放又为SS所抑制[8]。因此我们推测,IAPP和SS在D细胞中有共存,有可能是通过自分泌的方式相互进行调节,在功能上可能相互影响。但对于在肠道D细胞中IAPP与SS共存究竟发挥何种生物学反应及其调控机制,有待进一步研究。
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    收稿 1997-09 修回 1997-12

    参考文献

    [1]Westermark P, Wernstedt C, O'Brien TD, et al. Islet amylois in type 2 human diabetes mellitus and adult diabetic cats in composed of a novel putative polypeptide hormone. Am J Pathol, 1987, 127(3):414

    [2]Sanke T, Nishi M, Nanjo K. Role of IAPP in the pathogenesis and development of NIDDM. Nippon Rinsho, 1994, 52(10):2737

    [3]梁文妹,李占淳,石爱荣.胚胎及新生大鼠胃肠道IAPP免疫反应细胞的个体发生.解剖学报,1994,25(4):433
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    [4]梁文妹,石爱荣,李占淳.大鼠生后发育期间胃肠道IAPP免疫反应细胞的分布.解剖学报,1994,25(4):413

    [5]梁文妹,黄 岩,石爱荣.人胎胰腺内胰岛淀粉样多肽和胰岛素免疫反应细胞的个体发生.解剖学报,1997,28(3):329

    [6]De Vroede M, Foriers A, Van de winkel M, et al. Presence of islet amyloid polypeptide in the rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content. Biochem Biophys Res Commun, 1992, 182(2):886

, 百拇医药     [7]Ahren B, Sundle F. Localization of calcitonin gene related peptide and islet amyloid polypeptide in the rat and mouse pancreas. Cell Tissue Res, 1992, 269(2):315

    [8]Mitsukawa T, Takemura J, Asai J, et al. Islet amyloid polypeptide response to glucose, insulin and somatostatin analogue administration. Diabetes, 1990, 39(5):639

    [9]Bretherton- Watt D, Ghatei MA, Jamal H, et al. Altered islet amyloid polypeptide (amylin) gene expression in rat models of diabetes. Diabetologia, 1989, 32(12):881
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    [10]Parlapiano C, Minniti G, Primi F, et al. Calcitonin gene-related peptide in diabetes mellitus type 2:A possible eliopathogenetic role. Riv Eur Sci Med Farmacol, 1995,17(1):35

    [11]Bretherton- Watt D, Chatel MA, Suda K, et al. IAPP(Amylin) in normal human tissues and neuroendocrine tumours. J Endocrinol, 1990, 124(suppl):109

    [12]潘国宗.胃肠激素研究的进展.生理科学进展,1989,20(2):182

    [13]Dharmsathaphorn K.Sherwin RS,Cataland S,et al.Somatostatin inhibits diarrhea in the carciniod syndrome.Ann Int Med,1980,92(1):68
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    [14]Davis GR,Camp RC,Raskin P,et al.Effect of somatostatin infusion on jejunal water and electrolyte transport in a patient with secretory diarrhea due to malignant carcinoid syndrome.Gastroenterology,1980,78(2):346

    IMMUNOHISTOCHEMICAL LOCALIZATION OF IAPP

    AND SS IN THE GASTROINTESTINAL TRACT OF

    FETAL AND NEWBORN RATS

    AND SS IN THE GASTROINTESTINAL TRACT OF
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    FETAL AND NEWBORN RATS

    Liang WenmeiΔ Shi Airong

    (Department of Histology and Embryology, Guiyang Medical College, Guiyang;

    Department of Histology and Embryology, Beijing Medical University, Beijing)

    In order to explore the relationship between islet amyloid polypeptide (IAPP) and classical transmitters in the gastrointestinal tract of fetal and newborn rat, the distribution of IAPP immunoreactive (IR) cells and somatostatin (SS)-IR cells were shown on serial adjacent sections separately by use of the immunohistochemical PAP method in this study. The coexistence of IAPP and SS in the gastrointestinal tract of fetal and newborn rat was examinaed. The result showed that no IAPP-or SS-IR cell was found in the gastrointestinal tract on the 15th day. On the 17th day, a few IAPP-and SS-IR cells could be observed in the differentiating intestinal epithelium. On the 19th day, IAPP-and SS-IR cells were found in the gastric and small intestinal epithelium and the cell number was more in the duodenum. The cells containing IAPP-and SS-IR were present in the whole gut from day 21 of fetal life to newborn. The co-localization of IAPP and SS in some cells of duodenum and jejunum was proved as compared with adjacent sections. The results suggested that IAPP co-existed with SS in D cells of the duodenum and jejunum epithelium. The co-existence of IAPP and SS was not detected in the stomach, ilium, colon and rectum. The possible function on the co-existence of IAPP and SS in the gastrointestinal tract of fetal and newborn rats was discussed in this paper.

    KEY WORDS Islet amyloid polypeptide; Somatostatin; Gastrointestinal tract; Fetus; Newborn; Rats

    △Department of Histology and Embryology, Guiyang Medical College, Guiyang 550004, China, http://www.100md.com(梁文妹 石爱荣*)