脾阴虚证大鼠胃动素、胃蛋白酶、水通道蛋白4及蛋白质组学改变的实验研究

【作者】 宋雪娇；

【导师】 王彩霞；

【作者基本信息】 辽宁中医药大学 ， 中医基础理论， 2011， 硕士

【摘要】 中医学中认为脾为后天之本,气血生化之源泉,生命体离开母体之后独立生存,身体生命物质的获得主要依赖于脾。结合现代医学研究发现,不单整个消化系统功能有脾的参与,脾且作用于内分泌、自主神经、免疫以及血液等其他系统的代谢。中医学认为,脾阴为后天阴液之本,主濡养,主散精,参与运化,参与统血。近年来,学术界在血清、器官组织及应用蛋白质组学技术方面对脾阴虚证的探讨日益加深。本研究以脾阴虚证为模型,采用健康组为对照,探讨脾阴虚证发生发展过程中的现代医学基础。目的：本实验通过观察脾阴虚证模型大鼠胃动素(MTL)、胃蛋白酶、水通道蛋白4(APQ4)、及蛋白质组学方面的改变,探讨它们在脾阴虚证时出现异常改变的作用机制,为揭示脾阴虚证本质及采用中医药治疗脾阴虚证提供了科学的实验依据。材料与方法：采用饮食不节加劳倦过度结合耗伤阴液的方法塑造脾阴虚证大鼠模型。16天后,观察各组大鼠生理状态。并以光镜下观察各组大鼠胃底组织与回肠组织的形态学改变；采用免疫组化方法,测得各组大鼠回肠组织APQ4的表达差异；采用酶联免疫吸附双抗体夹心法(ELISA法),检测各组大鼠血清中MTL含量的表达差异；采用比色法,测定各组大鼠十二指肠胃蛋白酶活性的表达差异；应用蛋白质组学技术,鉴定健康对照组与实验组回肠组织蛋白表达差异。结果：1.胃动素的ELISA检测结果：与健康对照组比较,脾阴虚实验组和脾阴虚中药反证组MTL的表达均出现明显降低(P<0.01)；与实验组比较,脾阴虚中药反证组MTL的表达提高(P<0.05)。2.胃蛋白酶的比色法检测结果：与健康对照组比较,脾阴虚实验组胃蛋白酶的表达显著降低(P<0.01),脾阴虚中药反证组胃蛋白酶的表达降低(P<0.05)；脾阴虚实验组与脾阴虚中药反证组之间,胃蛋白酶的表达无统计学意义。3.水通道蛋白4的免疫组化检测结果：光镜下观察,健康对照组阳性颗粒的表达在上皮细胞中呈现较深的棕色,数量较多；与健康对照组比较,脾阴虚实验组中阳性颗粒的表达明显减少,并呈现浅淡的棕色；脾阴虚中药反证组较实验组阳性颗粒表达显著增多,颜色偏于较深的棕色。4.蛋白质组学的质谱鉴定结果：与健康对照组比较,脾阴虚实验组发现8个差异表达蛋白点。结论：塑造脾阴虚证大鼠模型,造模后血清中MTL含量下降,回肠APQ4阳性细胞表达减弱,十二指肠胃蛋白酶活性降低,它们的异常改变可能与脾阴虚证发生的病理学机制有关。在蛋白质组学方面,实验组回肠鉴定出蛋白差异点,这些蛋白表达的异常可能与脾阴虚证发生的分子生物学机制有关。参与细胞信号转导的热休克蛋白90,其作用于端粒酶而影响端粒的长度,基于蛋白质组学理论的端粒长度缩短可能是脾阴虚证发生的分子生物学机制之一。更多还原

【Abstract】 Traditional Chinese Medical(TCM) science holds that spleen is acquired foundetion and origin of producing qi and blood.The obtainance of living substances mainly relies on spleen after birth. The spleen participates in the whole digestive syetem function as well as the endocrine system, autonomic system, immune system and metabolic blood system, combing with the modern medical. TCM consideres that the spleen Yin is the foundation of acquired Yin, which govers nourishing, dispersing the essence to the whole body and participates transformation and transportation and controlling blood. Recently, the academic more and more pay attention to discuss on serum, organs and proteomics technology of spleen Yin deficiency syndrome. This study is to establish the model of spleen Yin deficiency syndrome and explore the modern medical basis on occurrence and development of spleen Yin deficiency syndrome, comparing to control group.Purpose:This supplement of science evidence is to explore the mechanism of spleen Yin deficiency syndrome by observing the change of motilin (MTL)、aquaporin4 (APQ4)、pepsin and proteomics index of model rats, in order to reveal the essence of spleen Yin deficiency syndrome and treatment of spleen Yin deficiency syndrome with Chinese drugs.Materials and methods:Rats model of spleen Yin deficiency syndrome was established by the improper diet, overstrain and consumption of Yin fluid. These physiological conditions of each rat were observed after 16 days. The changes in stomach bottom tissue and ileum tissue of each rat were observed by microscope.The changes of expression in ileum tissue of APQ4 were detected by immunohistochemistry method. The changes of expression in serum of MTL were detected by enzyme-linked immunosorbent double antibody clip method (ELISA). The changes of expression in duodenal of activity of Pepsin by colorimetric method. The changes of expression in proteomics were idengtified between the contol and the experimental group by proteomics technology.Result:1. The result of MTL by ELISA:Compareing with control, the expression of MTL signicantly decreased (P<0.01) in experimental group and the TCM proof group Compareing with experimental group, the expression of MTL improved in TCM proof group (P<0.05)2. The result of Pepsin by colorimetric method:compareing with control group, the expression of Pepsin decreased signicantly (P<0.01) in spleen Yin deficiency experimental group, and the expression of Pepsin decreased (P<0.05) in TCM proof group. The expression of Pepsin had no any change between spleen Yin deficiency experimental group and spleen Yin deficiency TCM proof group.3.The result of APQ4 by immunohistochemistry:The expression of positive particles in the epithelial cells of the control is marked deeper brown and larger quantities by microscope. Compared with the control group, the expression of positive particles in spleen Yin deficiency experimental group is marked lighter brown and less quantities by microscope. The expression of positive particles in Spleen Yin deficiency TCM proof group is marked deeper brown and larger quantities by microscope.4. The result of Proteomics by mass spectrometry:compareing with control group, the different expression of proteins spots were 8 in spleen Yin deficiency experimental group.Conclusions:After the establishment of the model of spleen Yin deficiency syndrome, the content of MTL in serum, the expression of positive cells of APQ4 in ileum, the activity of pepsin in duodenal all decreased, which may be related to pathological mechanism of spleen Yin deficiency syndrome. In proteomics, there are diferences in the expression of ileum proteins, which may be related to molecular biological mechanism of spleen Yin deficiency syndrome. Heat shock protein 90 which affects on telomerase and telomere length, which may be related to one of molecular biological mechanism of spleen Yin deficiency syndrome.更多还原