【作者】 陈凯佳；

【导师】 刘小斌；

【作者基本信息】 广州中医药大学 ， 中医医史文献， 2006， 博士

【摘要】 研究目的: 对五脏相关理论进行梳理和探讨,在此基础上,对子系统“脾肾相关”理论进行更为深入的研究,提出“骨髓间充质干细胞(MSCs)为脾肾相关细胞层次表达的存在形式”的工作假说,探讨补脾益肾法对大鼠MSCs增殖和分化的影响,从微观角度和干细胞水平研究脾肾相关。 研究方法: 一、五脏相关理论探讨 (一)五脏相关学说的缘起: 1.五脏相关源流探讨:经考证,词义最接近、年代最早的文献是《素问·玉机真脏论》:“五脏相通,移皆有次”。古人通过阐述五脏功能互用和脏腑相合相通理论、气机升降出入、疾病的传变、五脏旁通之说、天人相应及药性归经理论体现五脏之气相通。 2.中医五脏相关学说的形成与发展:邓铁涛教授在几十年的临床实践中,把五行学说和脏腑学说结合起来,提出了“五脏相关”学说。 (1)理论研究继续深化:90年代末本世纪初,邓铁涛教授指导两位博士研究生赵氏、郑氏对五脏相关学说科学内涵进行探讨。认为五脏相关学说是对中医经典理论五行学说的继承和发展,具有跨学科的特点。 (2)五脏相关学说临床研究:五脏相关临床研究是目前研究的重点,主要有两个层次的研究,一是五脏相关总体层面的研究,二是深入到子系统如两脏相关的研究。面上的研究显示五脏相关理论已经广泛应用于临床各个系统疾病的治疗,与此同时,各个子系统的研究也逐渐开展。目前,以两两相关的研究较多。如脾(胃)肾相关,心脾(胃)相关,肝脾(胃)相关,胆胃相关,肺脾(胃)相关,心肺相关,心胆相关,心肾相关,肺肾相关,肝肾相关,肝肺相关等。 (3)五脏相关学说实验研究:部分学者进行了实验研究,揭示了五脏中的肝、脾(这里指空肠绒毛)、肾之间的相生、相克关系,“肺肾相关”的物质基础,中医“心肾相关”理论的科学内涵等。 (二)脾肾相关子系统研究: 中医脾肾相关子系统以阐发脾和肾相关的生理功能、病理变化机理及其证治方药为中心内容,体现了脏腑同病病机和整体观念。与传统的中医理论比较,脾肾相关吸取了五行学说中“土制水”的精神内核,并综合了历代医家对脾肾相互关系的不断发展,而将二者之间动态的、错综复杂的相互关系用“相关”来概括。近年来,国内有更多还原

【Abstract】 Objective:This topic discussed and systematized the theory of "spleen-kidney related" on the basic of literature. Which was one of subsystem of "the doctrine of the correlation of five viscera" put forward by Professor Tietao Deng, the famous TCM scientist of the contemporary. We would induce the frequent type and medication rule of Flaccidity syndrome in clinical part. While in experimental part, we approach the effect on multiplication and differentiation of Mesenchymal stem cells (MSCs) resulting from "Qiangjijianli" fluid for oral use. On the other hand, we’ d like to discuss the mechanism of action of the medicine in curing Flaccidity syndrome.Methods:1. Theory researchThe original literature of the doctrine of the correlation of five viscera is "plain questions" .The ancients explain the communication of QI of five ZANG-organs. The theory are put forward by Professor tietao deng on the basic of clinical practice, who integrate theory of five-element and zang-fu organs. The theory are developed by his students, while are widely studied on clinical application and empirical study. one of subsystem of "the doctrine of the correlation of five viscera" was the theory of spleen-kidney related.2. Empirical study: Isolation and culture of MSCsHeparinized bone marrow was obtained from SD rat. The marrow samples were diluted with Is cove’ s modified Dovecot’ s medium (IMDM) containing 5% fetal bovine serum (FBS) and 5% horse serum (HS). The mononuclear cells were prepared by gradient centrifugation at 2000rpm. The cells were washed, counted andplated at 10000 cells/75cm2 on Petri dishes in IMDM supplemental with 5% FBS and 5% HS which was selected for proper recovery and expansion of MSCs. The cells were incubated in a humidified incubator in 5% co2 at 37°C. Medium was replaced and the cells in suspension were removed at 48 hours and every three days thereafter. MSCs formed as confluent layers were detached by treatment with 0.25% trypsin(sigma) and seeded into cultural flasks. MSCs after the third subculture were used for the described experiments. Analysis of cell surface moleculesThe fixed cells were harvested from tissue culture flasks. Aliquots were incubated for one night at refrigerator 4°C with Multiclone antibody against rabbit CD44, CD54 purchased from Boston. After washing thrice in phosphate-buffered saline (PBS), cells were reacted with secondary antibodies and then coloration with Bromine chlorine benzpyrrole phosphonic /nitroblue tetrazolium (BCTP/NBT). After blocked with block fluid, cells were incubated with monoclonal antibodies Brdu for 40 minutes. Then reacted with secondary antibodies and counterstain with BAD. MTT testMSCs culture was started at 1000 cells per well in 96-well tissue culture plates. Culture medium was IMDM supplemented with "Qiangjijianli" fluid, pooled serum consist of Qiangjijianli and tortoise, tortoise plastron, 5-azacytidine or blank serum. After two days’ incubation, the MTT test was performed as routinely described. Biokinetics reader detected the values of absorbance at 490nm. All the data were expressed at mean±standard deviation (SD) . Independent-samples t test was carried out using SPSS11. 5 for windows software. Cell differentiation derivationThe differentiation ability of the MSCs was assessed at the third and tenth passages. The inducible conditions were based on the MSCs from bone marrow. The OS cells were seeded in 24-well plate (lOOOcells/well) and cultured in 5% co2 37°C for 3 ,7 or 14 days. The cell medium was discarded. The wells were washed with phosphate buffered saline (PBS), and lml serum-free medium containing medical serum at different final concentration. The samples were divided into different groups, either with serum-free medium, or with culture medium containing medicine. Cell immunohistochemistry assayThe fixed cells were harvested from tissue culture flasks. Aliquots were incubated for one night at refrigerator 4°C with m onoclonal antibodies against desmin or myosin purchased from Boston. After washing thrice in phosphate-buffered saline (PBS), cells were reacted with secondary antibodies and then coloration with BAD and counterstain with hematoxylin. Results:1. Theory researchThe study on the doctrine of "the correlation of five viscera" refered to many aspects involved in literature, clinic and experiment. As one of well-provided subsystem of "the correlation of five viscera" , predominant curative effect are showed on the treament of paralysis according the theory of "spleen-kidney related" . Internal empirical study report about the theory of "spleen-kidney reated" these year are finded one after another. But there are no study on Mesenchymal stem cells.2. Empirical study: Isolation and culture of MSCsRound cells appeared first in primary culture, then the cell were set in culture of incubation, spindle-like cells adherent to the Petri dishes appeared, followed by formation of colonies at day 3-7 and an adherent minelayer around 7-9 days. A variety of antibodies reacting with different cell lineages were used to investigate the surface antigenic markers present on MSCs. The results showed that MSCs were uniformly positive for CD44, CD54. Multiplication effect on MSCsMultiplication effect can be produced by variable dose of"Qiangjijianli" fluid, which is symbiosis with concentration. The similar result is shown on tortoise plastron. Pooled serum consists of"Qiangjijianli" and tortoise plastron have better effect on Multiplication of MSCs. Effect on differentiation of MSCs"Qiangjijianli" fluid, pooled serum consists of "Qiangjijianli" and tortoise plastron, 5-azacytidine could induce MSCs into myoblast. The differentiation of induced cells was identified by immunohistochemistry. The rate of positive cells didn’ t have strong relation with concentration.Conclusion:The doctrine of "the correlation of five viscera" is one of important ingredent of contemporary Chinese medicine academic. It showed that the therapeutic principle of Flaccidity syndrome is invigorated the spleen and kidney together. "Qiangjijianli" serum of Physiological dose can cause evident multiplication effect, the effect increasing with concentration. Pooled serum consists of "Qiangjijianli" and tortoise plastron with proper ratio have better effect on MSCs than the others. It showed that invigorate the spleen and kidney together are better than invigorate the spleen or kidney alone. "Qiangjijianli" fluid, pooled serum consists of "Qiangjijianli" and tortoise plastron, 5-azacytidine could induce MSCs into myoblast. It showed that the rat MSCss could be induced into myoblast cells by invigorating the spleen or invigorating the spleen and kidney together.更多还原