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

高胃泌素血症常见于Zollinger-Ellison(ZE)肿瘤和萎缩性胃炎患者。众所周知，ZE肿瘤产生高水平的胃泌素，刺激壁细胞和肠嗜铬细胞(ECL)，这两种细胞都表达胃泌素受体。在胃泌素的刺激下，壁细胞产生胃酸，ECL细胞产生组胺，组胺是壁细胞上一种有效的酸性促分泌剂。在胃泌素和组胺的协同作用下，壁细胞产生大量的胃酸，胃粘膜肥大的机制尚不清楚。高胃泌素血症的另一个原因是萎缩性胃炎，它被称为癌前状态。在萎缩性胃炎中，由于胃酸缺乏，胃液变得中性，因为壁细胞经常受到自身免疫机制或幽门螺杆菌毒素的损害。结果，胃窦G细胞的胃泌素产生因缺乏抑制信息Ac…而上调。为了阐明胃泌素和转化生长因子α促进胃粘膜肥大生长的机制，我们试图建立胃泌素或转化生长因子α过表达的转基因小鼠。幸运的是，合作研究人员H.Takagi之前已经制造出胃粘膜中过表达TGFα的转基因小鼠。因此，我们致力于制备胃泌素过表达转基因小鼠。胃泌素是作为前体孕激素合成的。这一前体需要一系列翻译后加工反应才能成为具有生物活性的胃泌素，包括在成对的碱性残基上进行二碱基切割，随后被羧基肽酶H去除，并通过酰胺化酶-肽甘氨酸α-酰胺化单加氧酶(PAM)的作用形成羧基(C-)末端的酰胺部分。由此形成的酰胺化胃泌素(G17-NH_2)的胃酸分泌活性比甘氨酸延伸的胃泌素(G17-Gly)高三个数量级。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基因，该基因在多种组织中都有强表达。我们使用的另一种装置是原蛋白加工酶呋喃的裂解位点，它几乎分布在所有组织中。通过在胃泌素-17之前创建一个呋喃酶切位点，并删除C-末端延伸肽成为甘氨酸作为有效的酰胺化反应的最后一个氨基酸，我们成功地从两个常规培养细胞系CHO和COS-7中生产了具有生物活性的胃泌素。然后，我们将胃泌素表达单位DNA注射到大鼠卵子中，以产生转基因小鼠。我们获得了5只血清胃泌素浓度为400-500pg/ml的高胃泌素血症小鼠。通过杂交这些杂合子小鼠，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，高胃泌素血症小鼠胃粘膜肥大，表面粘液细胞层沿胃窝延长高度。虽然胃腺单位高度伸展，但其结构排列正常，表面粘液细胞看起来正常。与之相反，过度表达TGFα的胃粘膜表现为粘液上皮细胞增大，并伴有增生性、异型和囊性改变。胃腺结构类似于人类的梅内特里尔病，这是一种胃癌的癌前状态。由于胃泌素受体定位于壁细胞和ECL细胞，胃泌素对粘膜的生长作用被认为是由壁细胞中未知的因素介导的。这些因素的候选者之一是TGFpha。然而，考虑到胃泌素和转化生长因子α的组织学变化的不同，胃泌素似乎直接作用于胃表面粘液细胞。我们的初步数据表明，胃表面粘液细胞可能表达其质膜上的胃泌素受体。正如预期的那样，高胃泌素血症小鼠出现胃黏膜肥大，表面粘液细胞层沿胃窝延长高度。虽然胃腺单位高度伸展，但其结构排列正常，表面粘液细胞看起来正常。与之相反，过度表达TGFα的胃粘膜表现为粘液上皮细胞增大，并伴有增生性、异型和囊性改变。胃腺结构类似于人类的梅内特里尔病，这是一种胃癌的癌前状态。由于胃泌素受体定位于壁细胞和ECL细胞，胃泌素对粘膜的生长作用被认为是由壁细胞中未知的因素介导的。这些因素的候选者之一是TGFpha。然而，考虑到胃泌素和转化生长因子α的组织学变化的不同，胃泌素似乎直接作用于胃表面粘液细胞。我们的初步数据表明，胃表面黏液细胞可能在其质膜上表达胃泌素受体。长期以来，胃泌素一直被认为是胃癌的促进剂。但是，我们的胃泌素过表达模型不支持这种可能性，尽管转化生长因子α似乎参与了胃粘膜癌前病变的形成。较少

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