【作者】 李燕舞；

【导师】 王建华；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2005， 博士

【摘要】 胃肠激素受体及受体后信号转导与调控是在分子水平对复杂机体中单个细胞内及细胞间相互交流信号的研究,是当今生命科学研究的前沿课题。业已证实,脾虚证与胃泌素关系密切,脾虚大鼠胃粘膜及壁细胞胃泌素受体结合容量显著降低,不少调理脾胃方药对胃泌素及胃泌素受体有显著的调节作用,脾虚证很可能是胃泌素受体相关性疾病。本次实验研究应用放射配基结合法与跨膜信息转导机理研究新技术,系统研究脾虚大鼠壁细胞胃泌素受体表达方式与磷酯酰肌醇通路信号转导规律变化以及益气健脾方药党参、白术、补中益气汤的调控作用,对于探讨脾虚证与胃泌素受体的关系,阐明脾虚证病理机转规律及健脾方药药理作用均有重要意义。 1大鼠胃壁细胞的分离纯化 细胞分离的方法通常有两种:机械分离法和酶分离法。不同种属的动物及不同类型的蛋白水解酶对细胞分离效果影响迥异,胶原酶能够成功分离狗、豚鼠、兔胃的黏膜细胞,而链酶蛋白酶(Pronase)对分离大鼠胃黏膜细胞很有效,这种酶具有广泛的消化作用,与EDTA联合使用作用更加突出。本次实验采用Pronase-EDTA酶消化法分离大鼠胃壁细胞。脱颈椎处死大鼠,取胃制作翻转胃袋并注入消化酶溶液。经过消化90分钟——分散50分钟——离心收集来完成大鼠胃粘膜细胞的分离过程。大鼠胃壁细胞的纯化采用40%和60%percoll非连续密度梯度离心法。结果:即时分离的胃粘膜细胞活度经台盼兰排除实验鉴定可达到90%以上,HE染色鉴别壁细胞纯度达到70%,在0～4℃环境下22h后细胞活度仍可达70%～80%左右。大鼠壁细胞分离及纯化方法步骤明确,操作熟练,在本实验室条件下可达到较稳定的结果。 2补脾方药对脾虚大鼠壁细胞超微结构的影响更多还原

【Abstract】 Regulation of gut hormone receptor and signal transduction pathway is a previous topic of life science, which investigates the signal transduction in a single cell or between cells of organism. It has been proved that gastrin is closely related with spleen-deficiency syndrome. Our laboratory studies showed: the binding capacity of gastrin receptor markedly decreased in isolated gastric parietal cells of pi-deficiency rats, and the recipe or herbs for invigorating spleen improved the decreased binding capacity. This subject further observed the expression of gastrin receptor and the mechanism of signal transduction in isolated parietal cells from rats by some new methods for receptor, such as Radioligand Binding Assay (RBA). The effects of invigorating spleen medicines were observed too. Meanwhile we explored the essence of the syndrome of spleen deficiency from the level of gastrin receptor and molecular signal transduction in the isolated parietal cells.1 The method of isolation and purification for rat gastric parietal cellsThe method for cell isolation includes the mechanical methods and compels the use of proteolytic enzymes in conjunction with mechanical dispersion. However, methods differ depending on the species, because of variations in the histological and biochemical structures of tissue and changes in the nature of proteolytic enzymes. Thus the use of collagenaseleads to fully dispersing gastric cells in species such as dog, guinea pig and rabbit. But it has only poor dissociating power in rat. Pronase has been shown to be the most appropriate enzyme for obtaining individualized gastric cells in rat. This substance has broad substrate specific, especially in the presence of EDTA. Pronase-EDTA method was used to isolate rat gastric parietal cells in our laboratory. Rat was killed by cervical dislocation, and the stomach was removed. A small incision was made in the rumen and the fundus is everted by the use of a glass rod. The everted fundic sac was then filled withproteolytic solution. Tissue dissociation was allowed to proceed for 90 minutes. After this period, stomach was removed and cell dispersion was achieved under gentle magnetic stirring for 50 minutes. The cells were collected every 10 minutes, and they were washed by centrifugation. The parietal cell purification was achieved by nonlinear density gradient centrifugation using 40% percoll and 60% percoll. As a result, the isolated parietal cells from rat show over the viability of 90%, the purity of 70%. And the viability of parietal cells still achieve to 70-80% under 0-4X? after 22 hours. The method of isolation and purification for rat gastric parietal cells has established in our laboratory and has been mastered successfully.2 Observation of ultrastructures in rat gastric parietal cells of spleen-deficiency and the influence of invigorating medicinesThe model of spleen-deficiency was induced by herb rhubarb. Parietal cells were isolated by pronase-EDTA method and purified by nonlinear gradient centrifugation. The ultrastructures of rat parietal cells were observed by transmission electron microscope. The results show: Plenty of mitochondria and closely packed tubular cytomembranes were observed in cytoplasmic matrix of parietal cells in normal group. But in parietal cells of spleen deficiency models, a tortuous branched canaliculus filled with long villous processes is surrounded by mitochondrial clumps. The "tubulovesicular" system is diminished to the point of disappearance. Changes of the ultrastructures of parietal cells in model group showed: parietal cells of model rats were in the stimulated state, which may increase the sensibility of gastric mucosa to the attack factors. The ultrastructures of rat parietal cells in treatment group by usinginvigorating spleen herbs such as Dangshen, Baizhu and recipe Buzhong Yiqi decoction show a recovery status. The cellular canaliculus collapsed, and the microvilli lining canalicular lumen became short because of the endocytosis of the membrane of canalicular. Meanwhile the images of electron dense appeared in the cytoplasm such as horseshoe shape or oval-shape or round shape. The microvilli turned to irregularity in this status. As a result, the resorption of the membrane of canalicular would deduce the obliteration.3 Influence of invigorating spleen medicines on the gastrin receptor in spleen-deficiency rat parietal cellsOn the base of the methods of isolation and purification for rat gastric parietal cells, RBA was used to observe the binding capacity of gastrin receptor in parietal cells of spleen-deficiency model group and other treatment groups, in order to further search the essence of pi-deficiency syndrome and the mechanisms of the herbs or recipe for invigorating spleen. The results showed that the binding capacity of gastrin receptor markedly decreased in parietal cells of pi-deficiency rat than normal group, and increased after treatment by Dangshen, Baizhu and Buzhong Yiqi decoction. The experiment above mentioned demonstrates a certain relationship between gastin receptor and pi-deficiency syndrome. So we deduce that the decreased number of gastrin receptor might be due to pathology of pi-deficiency syndrome, and the effect of herbs or recipe for invigorating spleen might partly be regulating gastrin receptor.4 Effect of medicines for invigorating spleen on Inositol 1,4.5-Triphosphate (IPi) in rat parietal cells of pi-deficiencyInositide messenger system is a classic signal transduction pathway of gastrin receptor. Once stimulated by gastrin on rat parietal cells, phospholipase C is activated and two messengers such as IP3 and DG are produced. IP3 dispersed into cells because of water-soluble, then calcium channel activated combined with IP3 receptor in endoplasmic reticulum. So IP3 play an important role as a messenger in the signal transduction pathway. Based on the experiments of gastrin receptor binding capacity, we further observed the changes of IP3 in parietal cells of pi-deficiencyrat and the effects of medicines for invigorating spleen. The results showed as following: the contents of IP3 in parietal cells of pi-deficiency rat obviously decreased. We deduced the deseased synthesis of IP3 perhaps related with the decreased of gastrin receptor capacity. After treatment by medicines for invigorating spleen such as Dangshen, Baizhu and Buzhong Yiqi decoction, the contents of IP3 in parietal cells increased. So we suppose that the mechanism of medicines for invigorating spleen partly act through the regulation of the receptor and the intracellular messenger.5 Effect of medicines for invigorating spleen on [Ca2*] i in parietal eel Is rat of pi-deficiency ratCalcium is a basic substance for physiologic function in cell. From the extracellular signal to the formation of intracellular substance, it takes an important role as a messenger. Gastrin receptor is a kind of transmembrane receptor in parietal cell. When gastrin combinated with the receptor, gastric parietal cells [Ca2+]i changes after the intracellular protein activation and hydrolization. This process may deduce the physiological or pathological changes in cell. Our study has showed that the binding capacity of gastrin receptor decreased obviously in parietal cell of pi-deficiency rat, and herb Dangshen, Baizhu and Buzhong Yiqi decoction could upgrade the decreased capacity. The changes of [Ca2+]i in rat parietal cells were measured by using Fura-2/AM fluorescence indicator. The results showed: the resting [Ca^i of parietal cells in spleen-deficiency rats were significantly lower than that of the normal rats. But the elevating percentage of [Ca2+Ji stimulated by [Leul5]-gastrin (0.1 u raol/L) were higher markedly than that of the normal group. The herbs or recipe for invigorating spleen had some effects of regulating the disorder conditions. That mean the parietal cells of pi-deficiency rat might be in a kind of subnormal metabolism associated with pi-deficiency syndrome. But the reactivity increased to stimulation hint the hypersensibility in the pi-deficiency model induced by Dahuang. And enhanced sensitivity of mucosa to attack factors might produce the vulnerability of gastric mucosa in pi-deficiency syndrome.6 Influence of medicines for invigorating spleen on calmodul in (CaM) and Ca27CaM-dependent-proteinkinase II (CaVCaM-PK II) in parietal eel Is of pi-deficiency ratCaM is a receptor of intracellular Ca2* and which transfers the information to regulate the cell function. One CaM can bind four calcium ions and the compound then turned to biological activity. CaJ+/CaM-PK II can be activated by CaM. So these are key signal substances in parietal cell. On the base of our laboratory experiment, we further observe the activity of CaM and Ca27CaM-PK II in parietal cells of every group rat. We found that the activity of CaM increased obviously in parietal cells of pi-deficiency rat, and herbs Dangshen, Baizhu and Buzhong Yiqi decoction for invigorating spleen decreased the enhanced activity. The activity of Ca2+/CaM-PK II increased markedly in pi-deficiency rat parietal cells than normal rat, and decreased after treatment by Dangshen, Baizhu and Buzhong Yiqi decoction. So we deduced that the parietal cell might be in a high stringent state of pi-deficiency rat. Once stimulated by receptor, the intracellular signal substances produced, then the activity increased and which deduced the enhanced biological effects. The result conformed to the observation of the increased gastric acid secretion of pi-deficiency rat after stimulation by histamin in vivo.Summarized the documents and our research results, we found that there is a close relationship between pi-deficiency syndrome and gastrin. Under the status of pi-deficiency syndrome, the protecting function of gastric mucosa decreased and the vulnerability increased. The ultrastructures of parietal cells in Pi-deficiency model deduced by herb Dahuang appeared the stringent state; the gastrin receptor binding capacity decreased, the resting [Ca2t]i decreased in parietal cell of pi-deficiency rat and increased markedly at the stimulation of gastrin; the activity of CaM and Ca2+/CaM-PK II increased. So we deduced that the expression of gastrin receptor was low at the condition of pi-deficiency syndrome. The parietal cells stayed in hypometabolism status, but the response to gastrin stimulation increased which due to the abnormality function of parietal cells. So the reason of high vulnerability of gastric mucosa in pi-deficiency rat might be the hypersensitivity to attack factors.In all, our experiments explored the essence of spleen-deficiency更多还原