脾虚大鼠壁细胞胃泌素受体及黄芪的调控作用研究
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摘要
胃泌素以其重要而广泛的生理作用成为研究得最多的胃肠激素，并
被最早应用于中医领域的研究。胃泌素除具有传统的促进胃酸分泌作用
外，还是胃肠道和其他组织的生长因子，现在更倾向于认为刺激生长是
胃肠道胃泌素的最重要功能。已有的资料表明，胃泌素与脾虚证关系密
切，脾虚病理过程中胃泌素水平处于紊乱状态。我们在国内首次建立了
大鼠胃壁细胞分离方法，建立了壁细胞胃泌素受体放射配基结合法，尝
试从壁细胞表面胃泌素受体、胞内第二信使水平入手探讨脾虚证的本质
以及益气健脾中药的细胞水平的作用机制。
1.大鼠胃壁细胞分离方法的建立
利用Pronase-EDTA消化翻转的大鼠胃袋以游离胃粘膜细胞，利用
Percoll线性密度梯度作为介质以富集壁细胞，所得壁细胞纯度在65％以
上，台盼蓝排除法测得细胞活率大于92％。首次在国内分离到纯度较高的
大鼠胃壁细胞，为壁细胞相关研究提供了较理想的实验工具。
2.大鼠壁细胞胃泌素受体放射配基结合法的建立与方法学考察
以~(125)I-[Leu~(15)]-gastrin17-I为标记配基，以大鼠胃壁细胞悬液为受
体制备，在国内首次建立了大鼠胃壁细胞胃泌素受体放射配基结合实验
歪克：脾出大鼠同区蛔胞同泌月受体及中殇黄苗的词控作用研究
方法．反应体系总体积为 200ul，37T恒温水陇荡孵育 30min．yX 49
型玻璃纤维滤膜分离结合和游离配基．经 L i gand浮序进行非线性拟合，
Scatchard作困求出胃泌素受体结合饨Bmax回4．4604 x 10－“M，Ld吕1．249
x 10－‘M．单点法求出特异结合位点数 703．＂ites／cell．细胞浓度在
0．25刁 x 10七 ＊／L范围内，与标记配基特异性结合容量成正比．同一
样品特异结合量测定方法变异系数为7．04儿符合受体结合实验方法学要
求。竞争抑制实验证明“订Gastrin与大鼠胃壁细胞受体制备结合特异
性良好。
3．m大瓦＠细胞胃况受体及黄茂的调节作用船
利用我们建丘的壁细胞胃泌素受体放射配基结合法，研究发现大黄
脾虚模型大鼠壁细胞胃泌十受体结合容量明显低于正常（P硼．01）；经
中药黄芭治疗后，胃泌素受体结合位点数显天回升，与模型组比较有非
常显著差异（P北．01）。椎测胃泌素受体可能参与了脾虚证的病理过程，
调节胃泌素受体结合容量可能是中药黄氏发挥健脾益气作用的细胞水平
的新机制。
4．m大鼠羹k砧浆游离匆离子水早o茂的调节作用研大
采用Fura刁／AM ＆光分光先度法检测壁细胞静息状态下和胃泌素刺
激下胞内第H信使钙离子水平（［Ca“］i），尝试从胞内信使水平阐释脾
虚证的本质以及健脾益气方药的作用机理．研究发现，大黄脾虚模型大
鼠壁细胞静息状态下［CS‘”］i 明显低于正常大鼠（P＜0．01），胃泌素刺激后
K‘”＊升高幅度亦明显低于正常组＊d．01）和模型组Od．01八 黄蔑治
疗组大鼠静息状态下壁细胞［Ca‘“］i 比模型组略有升高，但无统计学意义
O刊．05厂 这可能与我们造模程度较重有关．胃泌素刺激下黄蔑治疗组
壁细胞K‘“D明显升高，其数值及升高幅度明显高于模型组O＜0．05，
一2 一
广州中医药大学2000届博士学位论文
P刊．01人 其升高幅度与正常组比较无明显差异＊＞O．05L腑大鼠胃壁
细胞静息状态下和胃泌素刺漱下Ka‘”D aP显低于正常大鼠，说明在脾虚
证的病理过程中壁细胞功能处于一种低代谢状态，推测胞内钙库容量可
能降低，而且壁细胞表面胃泌素受体结合位点数下调或／和功能下降，不
能正常介导胞外信号。健脾益气中药黄茁能够使低下的胞内代谢水平有
一定程度恢复，胞内钙库客量有一定增大，并能使胃泌素受体数上调和／
或功能加强，这可能是黄蔑健脾益气作用的一个细胞水平的新机制。
综合文献资料和我们的研究结果，我们推测壁细胞胃泌素受体可能
参与了脾虚证的病理过程，脾虚证很可能是一种胃泌素受体相关性疾病；
调节壁细胞胃泌素受体及胞内第二信使水平，可能是黄蔑发挥益气健脾
作用的细胞水平的新机制。我们设想，通过进一步的临床和实验研究，
有可能通过受体调节开辟一个治疗脾胃病证的新的途径和方法，同时也
会为从中药中开发受体激动剂和桔抗剂提供新的思路．这对于中医药理
论的发展和指导临床实践，以及中药的现代化都将会产生深远影响．
1. Isolation of Gastric Parietal Cells from Rat
Pronase-EDTA method was used to isolate gastric mucosal cells from everted gastric sacs of rat, Percoll linear density gradient to enrich parietal cells. The purity of parietal cells was over 65%, the viability over 92% excluded by trypan blue test. Purified gastric parietal cells from rat were obtained first time in China, which will be useful experimented models for parietal cells-related studies.
2.Establishment of Radioligand Binding Assay of Gash-in Receptors in Rat Gastric Parietal Cells
Using 125I-labeled [Leu15]-gastrin-17-I, radioligand binding assay of gastrin receptor was established in rat gastric parietal cell preparations after an incubation period of 30min at 37癈(pH7.4) with a cell concentration of 1.8 x 105cells/L per assay tube. In the range of the study, a Scatchard plot of the binding was made by Ligand program
with an equilibrium Kd of approximately 1.249 x 10'10M and a maximum binding capacity of 4.4604x 10~12M. The amount of gastrin binding was strongly associated with the cell concentration of rat gastric parietal cell preparations ranged from 0.25 x 109 to 2 x 109cells/L. The variant coefficent of the binding assay was 7.04% within the same sample. The competitive inhibiting analyses showed that 125I-gastrin was bound to gastric parietal cell preparations with a high specificity. These results suggested that the radioligand binding assay of gastrin receptor in rat gastric parietal cell preparations meets the criteria for establishing receptor binding assay.
S.Measurement of Gastrin Receptor Binding Capacity of Parietal Cells of Spleen-Deficency Model in Rat and the Regulating Effects of Herbal Huangqi
The results showed that the gastrin receptor binding capacity was markedly decreased in isolated gastric parietal cell of spleen-deficency rat induced by administration of herbal medicine Dahuang (vs. the normal rat, P<0.01). After the treatmeat with Hangqi, which functions are replenishing Qi and invigorating spleen, the gastrin receptor binding capacity was increased significantly (vs. the model rat, P<0.01). The present study confirmed our previous results using rat gastric mucosa as receptor preparation. We speculate that the spleen-deficiency syndrome might be a gastrin receptor related disease, and receptor-regulation might be a new method to treat this syndrome. 4. Measurement of Intracellular Calcium Level of Gastric Parietal Cells of Spleen-Deficiency Model in Rats and the Regulating Effects of Huangqi
Gastric parietal cells [Ca2+]i of spleen-deficiency model in rat was measured using fura-2/ AM fluorescent indicator. (1) The resting [Ca2+]i
of the model and its elevating percentage under gastrin stimulation were significantly lower than those of normal rats (P<0.01); (2) The resting
[Ca2+]i of treating group treated with Huangqi was increased in a degree, but had no statistical significance compared with that of the model. Stimulating by gastrin, the percentage of increased [Ca2+]i of the treating group was significantly higher than that of the model (P<0.01), and recovered to the normal level (P>0.05). These results indicated that gastric parietal cells might be in a lower metabolic condition when the body was suffered with spleen-deficiency syndrome. We speculates that calcium pool volume of parietal cell might be decreased, or gastrin receptor sites and/or their function were decreased which could not mediate extracellular signals. The Chinese herbal Huangqi, which function is replenishing Qi and invigorating spleen, can elevate intracellular calcium pool volume or upregulate gastrin receptor sites of gastric parietal cells, these might be the cellular mechanism of Huangqi's function.
被最早应用于中医领域的研究。胃泌素除具有传统的促进胃酸分泌作用
外，还是胃肠道和其他组织的生长因子，现在更倾向于认为刺激生长是
胃肠道胃泌素的最重要功能。已有的资料表明，胃泌素与脾虚证关系密
切，脾虚病理过程中胃泌素水平处于紊乱状态。我们在国内首次建立了
大鼠胃壁细胞分离方法，建立了壁细胞胃泌素受体放射配基结合法，尝
试从壁细胞表面胃泌素受体、胞内第二信使水平入手探讨脾虚证的本质
以及益气健脾中药的细胞水平的作用机制。
1.大鼠胃壁细胞分离方法的建立
利用Pronase-EDTA消化翻转的大鼠胃袋以游离胃粘膜细胞，利用
Percoll线性密度梯度作为介质以富集壁细胞，所得壁细胞纯度在65％以
上，台盼蓝排除法测得细胞活率大于92％。首次在国内分离到纯度较高的
大鼠胃壁细胞，为壁细胞相关研究提供了较理想的实验工具。
2.大鼠壁细胞胃泌素受体放射配基结合法的建立与方法学考察
以~(125)I-[Leu~(15)]-gastrin17-I为标记配基，以大鼠胃壁细胞悬液为受
体制备，在国内首次建立了大鼠胃壁细胞胃泌素受体放射配基结合实验
歪克：脾出大鼠同区蛔胞同泌月受体及中殇黄苗的词控作用研究
方法．反应体系总体积为 200ul，37T恒温水陇荡孵育 30min．yX 49
型玻璃纤维滤膜分离结合和游离配基．经 L i gand浮序进行非线性拟合，
Scatchard作困求出胃泌素受体结合饨Bmax回4．4604 x 10－“M，Ld吕1．249
x 10－‘M．单点法求出特异结合位点数 703．＂ites／cell．细胞浓度在
0．25刁 x 10七 ＊／L范围内，与标记配基特异性结合容量成正比．同一
样品特异结合量测定方法变异系数为7．04儿符合受体结合实验方法学要
求。竞争抑制实验证明“订Gastrin与大鼠胃壁细胞受体制备结合特异
性良好。
3．m大瓦＠细胞胃况受体及黄茂的调节作用船
利用我们建丘的壁细胞胃泌素受体放射配基结合法，研究发现大黄
脾虚模型大鼠壁细胞胃泌十受体结合容量明显低于正常（P硼．01）；经
中药黄芭治疗后，胃泌素受体结合位点数显天回升，与模型组比较有非
常显著差异（P北．01）。椎测胃泌素受体可能参与了脾虚证的病理过程，
调节胃泌素受体结合容量可能是中药黄氏发挥健脾益气作用的细胞水平
的新机制。
4．m大鼠羹k砧浆游离匆离子水早o茂的调节作用研大
采用Fura刁／AM ＆光分光先度法检测壁细胞静息状态下和胃泌素刺
激下胞内第H信使钙离子水平（［Ca“］i），尝试从胞内信使水平阐释脾
虚证的本质以及健脾益气方药的作用机理．研究发现，大黄脾虚模型大
鼠壁细胞静息状态下［CS‘”］i 明显低于正常大鼠（P＜0．01），胃泌素刺激后
K‘”＊升高幅度亦明显低于正常组＊d．01）和模型组Od．01八 黄蔑治
疗组大鼠静息状态下壁细胞［Ca‘“］i 比模型组略有升高，但无统计学意义
O刊．05厂 这可能与我们造模程度较重有关．胃泌素刺激下黄蔑治疗组
壁细胞K‘“D明显升高，其数值及升高幅度明显高于模型组O＜0．05，
一2 一
广州中医药大学2000届博士学位论文
P刊．01人 其升高幅度与正常组比较无明显差异＊＞O．05L腑大鼠胃壁
细胞静息状态下和胃泌素刺漱下Ka‘”D aP显低于正常大鼠，说明在脾虚
证的病理过程中壁细胞功能处于一种低代谢状态，推测胞内钙库容量可
能降低，而且壁细胞表面胃泌素受体结合位点数下调或／和功能下降，不
能正常介导胞外信号。健脾益气中药黄茁能够使低下的胞内代谢水平有
一定程度恢复，胞内钙库客量有一定增大，并能使胃泌素受体数上调和／
或功能加强，这可能是黄蔑健脾益气作用的一个细胞水平的新机制。
综合文献资料和我们的研究结果，我们推测壁细胞胃泌素受体可能
参与了脾虚证的病理过程，脾虚证很可能是一种胃泌素受体相关性疾病；
调节壁细胞胃泌素受体及胞内第二信使水平，可能是黄蔑发挥益气健脾
作用的细胞水平的新机制。我们设想，通过进一步的临床和实验研究，
有可能通过受体调节开辟一个治疗脾胃病证的新的途径和方法，同时也
会为从中药中开发受体激动剂和桔抗剂提供新的思路．这对于中医药理
论的发展和指导临床实践，以及中药的现代化都将会产生深远影响．
1. Isolation of Gastric Parietal Cells from Rat
Pronase-EDTA method was used to isolate gastric mucosal cells from everted gastric sacs of rat, Percoll linear density gradient to enrich parietal cells. The purity of parietal cells was over 65%, the viability over 92% excluded by trypan blue test. Purified gastric parietal cells from rat were obtained first time in China, which will be useful experimented models for parietal cells-related studies.
2.Establishment of Radioligand Binding Assay of Gash-in Receptors in Rat Gastric Parietal Cells
Using 125I-labeled [Leu15]-gastrin-17-I, radioligand binding assay of gastrin receptor was established in rat gastric parietal cell preparations after an incubation period of 30min at 37癈(pH7.4) with a cell concentration of 1.8 x 105cells/L per assay tube. In the range of the study, a Scatchard plot of the binding was made by Ligand program
with an equilibrium Kd of approximately 1.249 x 10'10M and a maximum binding capacity of 4.4604x 10~12M. The amount of gastrin binding was strongly associated with the cell concentration of rat gastric parietal cell preparations ranged from 0.25 x 109 to 2 x 109cells/L. The variant coefficent of the binding assay was 7.04% within the same sample. The competitive inhibiting analyses showed that 125I-gastrin was bound to gastric parietal cell preparations with a high specificity. These results suggested that the radioligand binding assay of gastrin receptor in rat gastric parietal cell preparations meets the criteria for establishing receptor binding assay.
S.Measurement of Gastrin Receptor Binding Capacity of Parietal Cells of Spleen-Deficency Model in Rat and the Regulating Effects of Herbal Huangqi
The results showed that the gastrin receptor binding capacity was markedly decreased in isolated gastric parietal cell of spleen-deficency rat induced by administration of herbal medicine Dahuang (vs. the normal rat, P<0.01). After the treatmeat with Hangqi, which functions are replenishing Qi and invigorating spleen, the gastrin receptor binding capacity was increased significantly (vs. the model rat, P<0.01). The present study confirmed our previous results using rat gastric mucosa as receptor preparation. We speculate that the spleen-deficiency syndrome might be a gastrin receptor related disease, and receptor-regulation might be a new method to treat this syndrome. 4. Measurement of Intracellular Calcium Level of Gastric Parietal Cells of Spleen-Deficiency Model in Rats and the Regulating Effects of Huangqi
Gastric parietal cells [Ca2+]i of spleen-deficiency model in rat was measured using fura-2/ AM fluorescent indicator. (1) The resting [Ca2+]i
of the model and its elevating percentage under gastrin stimulation were significantly lower than those of normal rats (P<0.01); (2) The resting
[Ca2+]i of treating group treated with Huangqi was increased in a degree, but had no statistical significance compared with that of the model. Stimulating by gastrin, the percentage of increased [Ca2+]i of the treating group was significantly higher than that of the model (P<0.01), and recovered to the normal level (P>0.05). These results indicated that gastric parietal cells might be in a lower metabolic condition when the body was suffered with spleen-deficiency syndrome. We speculates that calcium pool volume of parietal cell might be decreased, or gastrin receptor sites and/or their function were decreased which could not mediate extracellular signals. The Chinese herbal Huangqi, which function is replenishing Qi and invigorating spleen, can elevate intracellular calcium pool volume or upregulate gastrin receptor sites of gastric parietal cells, these might be the cellular mechanism of Huangqi's function.
引文
1． Chew. CS. Parietal cell culture: New models and Directions. Annu. Rev. Phsiol. 1994,56: 445-61
2． Soll. AH. The Actions of Secretagogue on oxygen uptake by isolated mammalian parietal cells . J. Clin .Invest. 1978,61:370-80．
3． Lewin. MJM, Cheret. AM, Soumarmon. A, et al. Methode pour I'isolement et le tri des cdllules de la mugueuse fundique de rat . Biol. Gastroenterol (paris) 1974, 7, 139-44．
4． Sewing.KF, Harms. P, Schultz.G,etal. Effect of substituted benzimidazoles on acid secretion in isolated and enriched guinea pig parietal cells. Gut, 1983, 24:557-60．
5． Mardh. S, Norberg. L, Ljungstrom. M,et a1． Preparation of cells from pig gastric mucosa. Isolation of parietal cells by isopycnic centrifugation on linear density gradients of Percoll . Acta Physiol Scand ,1984,122:607-13．
6． Fryklund. J, Wallmark. B, Larsson. H, et al. Effect of omeprazole on gastric secretion in H+, K+-ATPase and in pepsinogen-riched cell fractions from rabbit
gastric mucosa. Biochem Pharmacol, 1984, 33: 273-80
7. Soll. AH, Wollin. A. Histamine and cyclic AMP in isolated canine parietal cells. Am. J. Physiol. 1979, 237: E444-50.
8. Kajimura. M, Rruber. MA, Sachs. G. The muscarinc receptor gene expressed in rabbit parietal cells is the M_3 subtype. Gastroenterology, 1992, 103: 870-875.
9. Kopin. AS, Lee. YM and Beinborn. M. Expression cloning and characterization of the canine parietal cell gastrin receptor. Proc Natl Acad Sci USA. 1992, 89: 3605-9.
10. Zdon. MJ, Zucker. KA, Adrian. TE. Somatostatin analogue inhibition of isolated parietal cell secretion. Surgery, 1987, 102: 967-73.
11. Kajita. H, Kotera. T, Shirakata. Y, et al. A maxi Cl-channel coupled to endothelin B receptors in thc basolateral membrane of guinea-pig parietal cells. J. Physiol. 1995, 488: 65-75.
12. Ding. M, Kinoshita. Y, Kishi. K, et al. Distribution of prostaglandin E receptors in the rat gastrointestinal tract. Prostaglandins, 1997, Mar, 53(3): 199-216.
13. Joshi. V, Ray. GS, Goldenring. JR. Inhibition of parietal cell acid secretion is mediated by the classical epidermal growth factor receptor. Dig. Dis. Sci. 1997, Jun, 42(6): 1194-8.
14. Schepp. W, Dehne. K, Herrmuth. H, et al. Identification and functional importance of IL-1 receptors on rat parietal cells. Am. J. Physiol. 1998, 275: G1094-G1105.
15．李晓波，钱家鸣，陈元方。壁细胞的淘洗分离与应用。胃肠病学和肝病学杂志。1996，5(4)：254-6。
16. Lewin. MJM, Cheret. AM and Sachs. G. Separation of Individual cells from the fundic gastric mucosa. In: Cell separation methods and selected applications. Vol. 1. Edited by PretlowII TG and Pretloq TP. Academic Press, New York, 1982. pp223-6.
17. Agnihotri. N, Kaur. U, Dhawan. V, et al. Extrahepatic portal hypertensive gastropathy in Wistar rats: Modulation of acid secretion in isolated parietal cells. Dig. Dis. Sci. 1998, 43(1): 56-66.
18．杨景山主编。医学细胞化学与细胞生物技术。北京：北京医科大学中国协和医科大学联合出版社，1990年第一版：119。
19. Black. EW, Strada. SJ and Thompson. W. Relationship of secretagogue-induced cAMP accumulation and acid secretion in elutriated rat gastric parietal cells. J Pharmac Methods 1988, 20: 57-78.
20. Gibson D'RE and D'ambrosio SM. A new method for the isolation of high viable singoe cells from human tissues. In Vitro, 1985, 21(3), part2: 45A.
21. Scott D, Reuben M, Zampighi G, et al. Cell idolation and genotoxicity assessment in gastric mucosa. Dig Dis Sci, 1990, 35(10): 1217-25.
22．周柔丽。细胞与亚细胞结构的分离。见：刘鼎新，吕证宝主编。细胞生物学研究方法与技术。北京：北京医科大学中国协和医科大学联合出版社。1990年第一版：122-155。
23. Berglindh T, Chrink K J. A method for preparing isolated glands from the rabbit gastric mucosa. Acta Physical Scand, 1976, 96: 150-159.
24. Soll AH, Berglindh T. Receptors that regulate gastric acid secretory function. In: Physiology of the gastrointestinal tract. Third edition, edited by Johnson LR. Raven Press, New York, 1994. pp1139-1169.
25. McEwan CR, Stallard RW, Julos ET. Separation of biological particles by centrifugal elutriation. Anal Biochem, 1968, 23: 369-77.
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