慢性胃炎肝郁证大鼠模型表征与胃黏膜细胞凋亡及Bax、Bcl-2表达的相关研究
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摘要
现代社会生活节奏不断加快,精神压力随之增高,情志失常导致的疾病日渐增多。慢性胃炎是临床常见的消化系统疾病,其发病多与情志失常有关。目的:采用改良设计的病证结合大鼠模型,探讨慢性胃炎肝郁证胃黏膜病理形态、细胞凋亡及Bax、Bcl-2的变化机理,为揭示慢性胃炎肝郁证的发病机理提供依据。
方法:综合病、证两种造模方法,酌加改良,建立慢性胃炎肝郁证病证结合大鼠模型,设立正常组、慢性胃炎组、肝郁组予以对照,分别在9周、11周、13周动态观察记录各组大鼠表征,检测大鼠血清一氧化氮(NO)、内皮素(ET)的变化,进行HE染色观察胃黏膜组织形态改变,显微镜下观察胃黏膜的炎症程度,组织切片TUNEL法染色观察大鼠细胞凋亡情况。采用石蜡包埋的免疫组织化学技术,对各组胃黏膜切片进行Bax、Bcl- 2免疫组织化学染色,光学显微镜下观察胃黏膜组织中的表达情况。
结果:慢性胃炎肝郁证大鼠(病证组)出现:体重增长迟缓,皮毛蓬松、枯黄,目暗、眼眯、溜边、扎堆、粪便偏软等特征。胃黏膜病理:具有黏膜色苍白,胃壁弹性较差,皱襞平坦,黏液较少,黏膜有出血点等特征;血清NO、ET水平升高。慢性胃炎肝郁证大鼠模型表征、胃黏膜病理表现与血清NO、ET的变化趋势一致。胃黏膜中较多细胞出现凋亡及Bc1-2、Bax的表达。显微镜下观察,凋亡细胞见于上皮细胞、壁细胞、主细胞;Bcl-2蛋白表达于壁细胞、主细胞、上皮细胞的胞浆中。病证组血清NO、ET、细胞凋亡及Bc1-2、Bax的表达均大于胃炎组、肝郁组和正常组(P<0.05);而且与同组中上一时间段相比有显著差异,提示病证组各指标有动态变化的趋势。
结论:(1)利用氨水与束缚筒相结合的方法,单因素、分时段影响大鼠饮食活动,建立的慢性胃炎肝郁证大鼠模型符合预期设想,造模方法可行。(2)慢性胃炎肝郁证模型大鼠外在表征与胃黏膜的病理变化趋势相同,可以反映病情的轻重变化;慢性胃炎在肝郁因素的影响下胃黏膜的病理表现明显加重,单纯肝郁造模组后期也出现胃黏膜病理表现,提示肝郁因素与慢性胃炎存在相关性。(3)慢性胃炎肝郁证模型大鼠血清NO、ET的变化可能参与了慢性胃炎肝郁证胃黏膜的病理变化机制。(4)慢性胃炎肝郁证胃黏膜的病理变化与细胞凋亡及Bc1-2、Bax表达失衡有关。其中,Bcl-2表达下调以及Bax表达上调加重了慢性胃炎肝郁证胃黏膜细胞凋亡的程度。
With quick life rhythm of people,weighing heavily on the spirit has been a gradual growth.Disease caused by undesirable mood for a long time is on the increase. Chronic gastritis is a common diseases of digestive system. The cause of chronic gastritis is correlated with Overstrain and emotional maladjustment
Purpose:Study of the relationship between chronic gastritis liver-qi stagnation syndrome and pathological change of stomach mucosal biopsy in rat models of syndrome and disease with chronic gastritis. Discussion for apoptosis in stomach mucosal cell and the expression of apoptosis-related genes Bcl-2 and Bax in rat models syndrome and disease.
Methods: The method and the design about CG Model combining syndrome with disease, establish a model of chronic gastritis liver-qi stagnation syndrome in rats.The normal group, the CG group, the liver-qi stagnation group as control. In 9 weeks, 11 weeks and 12weeks, follows up the changes of exterior signs in the circadian activity rhythm of rats, to test the serum level of NO by colorimetry and ET by ELISA,take stomach for HE dye, observe the histology and morphology under stomach mucosal. The stomach mucosal specimens from executing the treated rats in different group were randomly collected and fixed in formalin and embedded with paraffin. The samples were examined with HE stain,immunocytochemistry techniqu(Bcl-2,Bax)and TUNEL dyeing.After executing the treated rats,the samples were examined with HE stain, immunocytochemistry techniqu(Bcl-2,Bax)and TUNEL dyeing.
Results: The exterior signs of chronic gastritis liver-qi stagnation group had body weight of slow grow, dry and thin hair, lost lustre and slitting its eyes ,crawled alongside or into one place , loose bowel movements , etc. Pathological investigation: the stomach mucosa had distinctive feature, such as look pale, elasticity of stomach lining poorly, plica flat, the secretion of mucus decreased, bleeder on the stomach mucosa. The serum level of NO and ET increased. The exterior signs of rats had a similar trend to that of the stomach mucosa and the serum level of NO and ET. Apoptosis and the expression of genes Bcl-2 and Bax were observed in the stomach mucosa. Microscopically, the apoptosis was mainly located in epithelial cells, parietal cells and chief cells. Bcl-2 protein was expressed in the cytoplasm of parietal cells ,chief cells and epithelial cells.Results showed that the serum level of NO and ET,apoptosis and the expression of genes Bcl-2 and Bax in the rats CG group of syndrome and disease were greater than those in the normal group , the CG group and the liver-qi stagnation group (P<0.05); In addition, remarkable differences were also found in the same group at different time points (P<0.05). It was now clear that the main outcome measures of the syndrome and disease group had a dynamic changes of trend.
Conclusions:(1) Building a chronic gastritis liver-qi stagnation rat model which effected the daily life of rats by combining ammonia and restrained bottle is identical with expectation. This method is found to be quite practicable(.2) The same change trend with the exterior signs and the pathological change of stomach mucosa of chronic gastritis liver-qi stagnation rats can reflect the progress of disease. In the function of liver-qi stagnation, the pathological change of CG stomach mucosa got worse.It is showed that there is a close correlation between the relationship between liver-qi stagnation and chronic gastritis for the pathological change of stomach mucosa of the liver-qi stagnation group in the terminal modeling period.(3)The change in chronic gastritis liver-qi stagnation rat model of the serum level of NO and ET may be involved in the pathological changes and mechanism of stomach mucosa.(4) The change in chronic gastritis liver-qi stagnation rat model is related to the unbalance of apoptosis and the expression of genes Bcl-2 and Bax. Includeing, the level of apoptosis increased progressively along with up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.
方法:综合病、证两种造模方法,酌加改良,建立慢性胃炎肝郁证病证结合大鼠模型,设立正常组、慢性胃炎组、肝郁组予以对照,分别在9周、11周、13周动态观察记录各组大鼠表征,检测大鼠血清一氧化氮(NO)、内皮素(ET)的变化,进行HE染色观察胃黏膜组织形态改变,显微镜下观察胃黏膜的炎症程度,组织切片TUNEL法染色观察大鼠细胞凋亡情况。采用石蜡包埋的免疫组织化学技术,对各组胃黏膜切片进行Bax、Bcl- 2免疫组织化学染色,光学显微镜下观察胃黏膜组织中的表达情况。
结果:慢性胃炎肝郁证大鼠(病证组)出现:体重增长迟缓,皮毛蓬松、枯黄,目暗、眼眯、溜边、扎堆、粪便偏软等特征。胃黏膜病理:具有黏膜色苍白,胃壁弹性较差,皱襞平坦,黏液较少,黏膜有出血点等特征;血清NO、ET水平升高。慢性胃炎肝郁证大鼠模型表征、胃黏膜病理表现与血清NO、ET的变化趋势一致。胃黏膜中较多细胞出现凋亡及Bc1-2、Bax的表达。显微镜下观察,凋亡细胞见于上皮细胞、壁细胞、主细胞;Bcl-2蛋白表达于壁细胞、主细胞、上皮细胞的胞浆中。病证组血清NO、ET、细胞凋亡及Bc1-2、Bax的表达均大于胃炎组、肝郁组和正常组(P<0.05);而且与同组中上一时间段相比有显著差异,提示病证组各指标有动态变化的趋势。
结论:(1)利用氨水与束缚筒相结合的方法,单因素、分时段影响大鼠饮食活动,建立的慢性胃炎肝郁证大鼠模型符合预期设想,造模方法可行。(2)慢性胃炎肝郁证模型大鼠外在表征与胃黏膜的病理变化趋势相同,可以反映病情的轻重变化;慢性胃炎在肝郁因素的影响下胃黏膜的病理表现明显加重,单纯肝郁造模组后期也出现胃黏膜病理表现,提示肝郁因素与慢性胃炎存在相关性。(3)慢性胃炎肝郁证模型大鼠血清NO、ET的变化可能参与了慢性胃炎肝郁证胃黏膜的病理变化机制。(4)慢性胃炎肝郁证胃黏膜的病理变化与细胞凋亡及Bc1-2、Bax表达失衡有关。其中,Bcl-2表达下调以及Bax表达上调加重了慢性胃炎肝郁证胃黏膜细胞凋亡的程度。
With quick life rhythm of people,weighing heavily on the spirit has been a gradual growth.Disease caused by undesirable mood for a long time is on the increase. Chronic gastritis is a common diseases of digestive system. The cause of chronic gastritis is correlated with Overstrain and emotional maladjustment
Purpose:Study of the relationship between chronic gastritis liver-qi stagnation syndrome and pathological change of stomach mucosal biopsy in rat models of syndrome and disease with chronic gastritis. Discussion for apoptosis in stomach mucosal cell and the expression of apoptosis-related genes Bcl-2 and Bax in rat models syndrome and disease.
Methods: The method and the design about CG Model combining syndrome with disease, establish a model of chronic gastritis liver-qi stagnation syndrome in rats.The normal group, the CG group, the liver-qi stagnation group as control. In 9 weeks, 11 weeks and 12weeks, follows up the changes of exterior signs in the circadian activity rhythm of rats, to test the serum level of NO by colorimetry and ET by ELISA,take stomach for HE dye, observe the histology and morphology under stomach mucosal. The stomach mucosal specimens from executing the treated rats in different group were randomly collected and fixed in formalin and embedded with paraffin. The samples were examined with HE stain,immunocytochemistry techniqu(Bcl-2,Bax)and TUNEL dyeing.After executing the treated rats,the samples were examined with HE stain, immunocytochemistry techniqu(Bcl-2,Bax)and TUNEL dyeing.
Results: The exterior signs of chronic gastritis liver-qi stagnation group had body weight of slow grow, dry and thin hair, lost lustre and slitting its eyes ,crawled alongside or into one place , loose bowel movements , etc. Pathological investigation: the stomach mucosa had distinctive feature, such as look pale, elasticity of stomach lining poorly, plica flat, the secretion of mucus decreased, bleeder on the stomach mucosa. The serum level of NO and ET increased. The exterior signs of rats had a similar trend to that of the stomach mucosa and the serum level of NO and ET. Apoptosis and the expression of genes Bcl-2 and Bax were observed in the stomach mucosa. Microscopically, the apoptosis was mainly located in epithelial cells, parietal cells and chief cells. Bcl-2 protein was expressed in the cytoplasm of parietal cells ,chief cells and epithelial cells.Results showed that the serum level of NO and ET,apoptosis and the expression of genes Bcl-2 and Bax in the rats CG group of syndrome and disease were greater than those in the normal group , the CG group and the liver-qi stagnation group (P<0.05); In addition, remarkable differences were also found in the same group at different time points (P<0.05). It was now clear that the main outcome measures of the syndrome and disease group had a dynamic changes of trend.
Conclusions:(1) Building a chronic gastritis liver-qi stagnation rat model which effected the daily life of rats by combining ammonia and restrained bottle is identical with expectation. This method is found to be quite practicable(.2) The same change trend with the exterior signs and the pathological change of stomach mucosa of chronic gastritis liver-qi stagnation rats can reflect the progress of disease. In the function of liver-qi stagnation, the pathological change of CG stomach mucosa got worse.It is showed that there is a close correlation between the relationship between liver-qi stagnation and chronic gastritis for the pathological change of stomach mucosa of the liver-qi stagnation group in the terminal modeling period.(3)The change in chronic gastritis liver-qi stagnation rat model of the serum level of NO and ET may be involved in the pathological changes and mechanism of stomach mucosa.(4) The change in chronic gastritis liver-qi stagnation rat model is related to the unbalance of apoptosis and the expression of genes Bcl-2 and Bax. Includeing, the level of apoptosis increased progressively along with up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.
引文
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