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Study on the precancerous gastric mucosa using gastrin-overexpressing transgenic mice

胃泌素过表达转基因小鼠胃粘膜癌前病变的研究

基本信息

批准号：
06454255
负责人：
TAKEUCHI Toshiyuki
金额：
$ 4.48万
依托单位：
Gunma University, Institute for Molecular and Cellular Regulation
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454255/
关键词：
Gastrin TGFalpha Gastric mucosa Hypertrophic growth Transgenic mouse 高ガストリン血症プロセシング

项目摘要

Hypergastrinemia is frequently observed in patients with Zollinger-Ellison (ZE) tumor and with atrophic gastritis. ZE tumor is known to produce a high level of gastrin, which stimulates parietal cells and enterochromaffin-like (ECL) cells, both of which are known to express a gastrin receptor. Upon gastrin stimulation parietal cells produce gastric acid, and ECL cells produce histamine, a potent acid secretagogue on parietal cells. With a synergistic effect of gastrin and histamine, parietal cells produce a plentiful of gastric acid, and gastric mucosa becomes hypertrophic by yet unknown mechanisms. Another cause of hypergastrinemia is atrophic gastritis that is known as a precancerous state. In atrophic gastritis, gastric fluid becomes neutral due to deficiency of gastric acid because parietal cells are often impaired by autoimmune mechanisms or toxins from Helicobacter pylori. Resultantly, gastrin production is up-regulated from antral G cells by the lack of an inhibitory message, ac … More idity in gastric fluid.To elucidate the mechanism of hypertrophic growth of gastric mucosa by gastrin and TGFalpha, We attempted to produce transgenic mice with overexpression of gastrin or TGFalpha in gastric mucosa. Fortunately, co-investigator, H.Takagi, has already made transgenic mice with overexpressing TGFalpha in the gastric mucosa before. Thus, we focused on the production of gastrin-overexpressing transgenic mice. Gastrin is synthesized as a precursor preprogastrin. This precursor requires a series of post-translational processing reactions to become bioactive gastrin, which include dibasic cleavage at paired basic residues, their subsequent removal by carboxypeptidase H,and formation of a carboxyl (C-) terminal amide moiety via the action of the amidation enzyme, peptidyl-glycine alpha-amidating mono-oxygenase (PAM). The amidated gastrin (G17-NH_2) thus formed, exhibits gastric acid-secreting activity three orders of magnitude higher than does glycine-extended a gastrin (G17-Gly). To attain hypergastrinemia in tansgenic mice, we expressed a gastrin cDNA under the control of a beta-actin promoter, which exhibits strong expression in a variety of tissues. Another devise we used is the cleavage site for proprotein-processing enzyme furin, which distributes in virtually all tissues. By creating a furin creavable site before gastrin-17 and deleting the C-terminal extension peptide to become glycine as a last amino acid for efficient amidation reaction, we succeeded in producing bioactive gastrin from two conventional culture cell lines CHO and COS-7. Then, we injected the gastrin expression uint DNA into a rat ovum for producing transgenic mice. We obtained five hypergastrinemic mice with 400-500 pg/ml serum gastrin. By cross-mating these heterozygous mice, we obtained homozygous mice with a 600-1500 pg/ml serum gastrin level. As expected, hypergastrinemic mice had hypertrophic gastric mucosa with an extended hight of a surface mucous cell layr along a gastric pit. Although a gastric gland unit is highly extended, its structure was normally arranged and surface mucous cells appear to be normal. In contrast, TGFalpha-overexpressing gastric mucosa exhibited enlarged mucous epithelia with hyperplastic, dysplastic and cystic changes. Gastric glandular structure was similar to Menetrier's disease in humans, which is a precancerous state for gastric cancer. Since gastrin receptors are localized in parietal cells and ECL cells, the growth of mucosa by gastrin is thought to be mediated by unknown factors from parietal cells. One of the candidates of these factors was TGFalpha. However, considering the difference in histological changes between gastrin and TGFalpha, gastrin appears to exert its asction directly onto gastric surface mucous cells. Our preliminary data suggests that gastric surface mucous cells may express gastrin receptors on their plasma membrane.As expected, hypergastrinemic mice had hypertrophic gastric mucosa with an extended hight of a surface mucous cell layr along a gastric pit. Although a gastric gland unit is highly extended, its structure was normally arranged and surface mucous cells appear to be normal. In contrast, TGFalpha-overexpressing gastric mucosa exhibited enlarged mucous epithelia with hyperplastic, dysplastic and cystic changes. Gastric glandular structure was similar to Menetrier's disease in humans, which is a precancerous state for gastric cancer. Since gastrin receptors are localized in parietal cells and ECL cells, the growth of mucosa by gastrin is thought to be mediated by unknown factors from parietal cells. One of the candidates of these factors was TGFalpha. However, considering the difference in histological changes between gastrin and TGFalpha, gastrin appears to exert its asction directly onto gastric surface mucous cells. Our preliminary data suggests that gastric surface mucous cells may express gastrin receptors on their plasma membrane.Gastrin has long been thought to act as a tumor promoter for gastic cancer. But, our gastrin-overexpressing model dose not support this possibility although TGFalpha appears to be involved in the formation of precancerous changes in the gastric mucosa. Less

高胃泌素血症常见于Zollinger-Ellison（ZE）肿瘤和萎缩性胃炎患者。已知ZE肿瘤产生高水平的胃泌素，其刺激壁细胞和肠嗜铬样（ECL）细胞，已知这两者都表达胃泌素受体。在胃泌素刺激时，壁细胞产生胃酸，ECL细胞产生组胺，一种对壁细胞有效的酸性促分泌素。在胃泌素和组胺的协同作用下，壁细胞产生大量胃酸，胃粘膜通过未知的机制变得肥大。高胃泌素血症的另一个原因是萎缩性胃炎，被称为癌前状态。在萎缩性胃炎中，由于壁细胞经常被自身免疫机制或幽门螺杆菌毒素损害，胃酸缺乏导致胃液变为中性。结果，胃窦G细胞分泌的胃泌素由于缺乏抑制信息而上调， ...更多信息为了阐明胃泌素和TGF α引起胃粘膜肥大的机制，我们尝试在胃粘膜中过表达胃泌素或TGF α的转基因小鼠。幸运的是，合作研究者H.Takagi之前已经制造了胃粘膜中过度表达TGF α的转基因小鼠。因此，我们专注于生产胃泌素过表达转基因小鼠。胃泌素是作为前前前胃泌素合成的。该前体需要一系列翻译后加工反应以成为生物活性胃泌素，其包括在成对碱性残基处的二元裂解，随后通过羧肽酶H去除，以及通过酰胺化酶肽基-甘氨酸α-酰胺化单加氧酶（PAM）的作用形成羧基（C-）末端酰胺部分。由此形成的酰胺化胃泌素（G_（17）-NH_2）比甘氨酸延伸的α-胃泌素（G_（17）-Gly）具有高三个数量级的胃酸分泌活性。为了在转基因小鼠中获得高胃泌素血症，我们在β-肌动蛋白启动子的控制下表达了胃泌素cDNA，其在多种组织中表现出强表达。我们使用的另一个设计是前蛋白加工酶弗林蛋白酶的切割位点，它分布在几乎所有的组织中。通过在胃泌素-17之前创建弗林蛋白酶可切割位点并删除C-末端延伸肽以成为甘氨酸作为有效酰胺化反应的最后一个氨基酸，我们成功地从两种常规培养细胞系CHO和COS-7中产生生物活性胃泌素。然后，我们将胃泌素表达单位的DNA注射到大鼠卵细胞中，制备转基因小鼠。我们获得了5只血清胃泌素为400-500 pg/ml的高胃泌素血症小鼠。通过将这些杂合子小鼠交叉交配，我们获得了血清胃泌素水平为600-1500 pg/ml的纯合子小鼠。正如预期的那样，高胃泌素血症小鼠具有肥大的胃粘膜，其表面粘液细胞层沿着胃小凹延伸。虽然胃腺单位高度延伸，但其结构正常排列，表面粘液细胞似乎正常。与此相反，TGF α过表达的胃粘膜表现出粘膜上皮增生，异型增生和囊性变化的扩大。胃腺结构类似于人类的Menetrier病，这是胃癌的癌前状态。由于胃泌素受体位于壁细胞和ECL细胞中，因此认为胃泌素引起的粘膜生长由壁细胞的未知因子介导。这些因子的候选者之一是TGF α。然而，考虑到胃泌素和TGF α之间的组织学变化的差异，胃泌素似乎直接对胃表面粘液细胞施加其吸收。我们的初步研究结果提示，胃粘膜细胞可能在其质膜上表达胃泌素受体，高胃泌素血症小鼠胃粘膜肥厚，粘膜细胞层沿胃小凹沿着延伸。虽然胃腺单位高度延伸，但其结构正常排列，表面粘液细胞似乎正常。与此相反，TGF α过表达的胃粘膜表现出粘膜上皮增生，异型增生和囊性变化的扩大。胃腺结构类似于人类的Menetrier病，这是胃癌的癌前状态。由于胃泌素受体位于壁细胞和ECL细胞中，因此认为胃泌素引起的粘膜生长由壁细胞的未知因子介导。这些因子的候选者之一是TGF α。然而，考虑到胃泌素和TGF α之间的组织学变化的差异，胃泌素似乎直接对胃表面粘液细胞施加其吸收。我们的初步研究结果提示，胃粘膜细胞可能在其细胞膜上表达胃泌素受体，而胃泌素一直被认为是胃癌的促癌因子。但是，我们的胃泌素过表达模型不支持这种可能性，尽管TGF α似乎参与了胃粘膜癌前病变的形成。少