| Mordern formula research has always been the focus of the field of Chinese medicine compound and the difficulty. The biological effect model of Chinese medicine compound acting on the human body is one of the key problems in the study of Chinese medicine compound mechanism.Taking advantage of compatibility of prescriptions of traditional Chinese medicine and its composition, multiple targets and pathways regulating characteristics, to explore traditional Chinese medicine compound effect model, and to reveal the theoretical connotation of Chinese medicine formula have important significance.The topic based on GegenQinLian decoction clinical treatment for type2diabetes experience background, and choose respectively from the four Chinese medicine (TCM) in the party involved in the four kinds of chemical constituents and their compatibility in the ejection assignment consists of characterization.We choose3T3-L1adipocyte in vitro model of IR for the tool, in observation of monomer composition of different concentration on the model cell sugar consumption or to absorb impact, on the basis of further investigation of its effect under the threshold low or very low concentration the IR resistant effect of monomer compatibility, to explore traditional Chinese medicine compound ingredients more biological effects of low concentration under the model.A literature review and experimental study included in the paper. Literature review part mainly summarizes the modern formula experimental study the clinical use of the GegenQinLian decoction and the experimental research of the party within the main ingredients or monomers and the research progress of pharmacological research, IR cell model. Experimental research part, first of all, build a stable cell line, to ensure the safety of the four kinds of traditional Chinese medicine monomer concentration effectively, then copy the3T3-L1IR model cells, probe the monomer concentration of low concentration conditions used in the model cell-effect, finally the combination of the threshold for four kinds of monomer concentration and threshold concentration under each monomer compatibility interaction research.Study one:the differentiationinduction of3T3-L1preadipocytes and four monomer effect on themThe differentiation induction of3T3-L1preadipocytes Methods: from-80℃in the refrigerator containing preadipocytescryopreserved tubes, then quickly in37℃water bath pot after melting temperature, recovery. Use3T3-L1preadipocytes, high-DMEM containing10%cow serum, culture at37℃5%CO2conditions48h. The logarithmic (index) phase offer-to cells cryopreserved. Cell inoculation on board, after waiting for it to grow and to total integration of2D, using the inducer IBMX, DEX, INS differentiation. Take3T3-L1preadipocytesof different differentiation time, microscope observe the morphological changes and oil red0of the dye eell. Results:recovery after adherent cultivation of3T3-L1preadipocytes the spindle, dispersed homogeneously distributed in the culture bottle bottom, size morphology, proliferation, steady and fast growth curve is "S" shape. During the differentiation of cells begin to gradually by spindle to oval, round and round, the cell body increases gradually, abundant cytoplasm; Oil red0staining showed cultures3T3-L1preadipocytes growing fat droplets, to D9-D10, more than90%of the cell cytoplasm of accumulating large amounts of fat droplets, fat drops of red, blue in nucleus, a large number of large lipid droplets distributed around the nucleus, forming a typical mature fat cells that form the "ring" structure. Conclusion: the preadipocytes cell differentiation success.The influence of four monomer to3T3-L1preadipocytes vitality Methods:3T3-L1preadipocytes to1×104cells per well, vaccination in a sterile96-well culture plate,200μ L per well, to be completely adherent cells, abandon the absorption solution, set up blank control group and different concentration monomer intervention group (10-3-10μM),48h after drug intervention, determined by MTT experiment testing OD value of the group. Conclusion: the four monomer low concentration range has increased the role of cell vitality.Study two:establishment of3T3-L1adipocytes IR model and the influence on the model.Establishment of3T3-L1adipocytes IR model Methods: differentiation of3T3-L1adipocytes96wells in the cell culture plate, add0.2%BSA DMEM medium after12h, divided into normal control group and the IR model group. Cultured in DMEM containing0.2%BSA medium normal group, model group with0.2%BSA,10-6m INS DMEM medium, hatch in the training after24h,48h, GOD-POD method and fluorescence (2-NBDG) marker method groups of glucose consumption and cell in cell cultures of glucose intake. Results:compared with control group, model group, 24h and48h glucose consumption significantly increase in extracellular fluid, cell glucose intake significantly lower. Conclusion:the high insulin induced24h and48h3T3-L1adipocytes, reduced intake of glucose consumption or cells has the characteristics of IR and replicate successful model.The influence on IR model cyte glucose consumption or the intake by the monomer Methods:cell culture96well board in normal con trol group and3T3-L1adipocytes IR model group, respectively with DMEM medium containing0.2%BSA and containing0.2%BSA,10-6M Ins D MEM medium for24h. Model group were divided into model control and monomer intervention groups again. In DMEM medium containing0.2%BSA normal group, model group and drug intervention groups respecti vely by DMEM medium containing10-6M Ins and contains10"6M Ins with medium of different concentrations of monomer, respectively, after incubation for24h and48h glucose GOD-POD method each cyte cultur es of consumption and/or intake. Results:compared with control gro up, model group,24h and48h of glucose in the extracellular fluid volume increased significantly; Compared with model group, Puerarin group,10-3,10-2,10-1μM24h, and10-3、10-1μM48h extracellular flu id of the glucose content were significantly reduced; Baicalein,10-4～1μM24h and10-5～1μM48h48h extracellular fluid of the amount o f glucose were significantly increased; Berberine10-2、10-1、1μM24h and10-4～μM48h extracellular fluid of the amount of glucose were significantly increased, glycyrrhizic acid10-4～1μM24h and105-1u M48h extracellular fluid of the amount of glucose were significan tly increased. Conclusion:many kinds of monomer effect of low cone entration range IR adipocytes for24h and48h have significantly in creased their consumption of glucose, have anti-IR activity.Study three:threshold concentration under four monomer compatibility effect observation of IR model cyte.The influence to the IR model by the four monomer Methods:using above threshold concentration of the monomer into compound, further diluting containing different concentrations of10-6M Ins DMEM medium. Cyte group and cultivation is same to research two. In monomer-containing medium role in24h and48h, GOD-POD method, respectively each cell cultures. Results:compared with control group, model group,24h and48h extracellular fluid glucose content increased; Compared with model group, four monomer formula in various concent rat ion (10-5μ M-0.0μ M)24h and48h extracellular fluid glucose decrease significantly. Conclusion:the four monomer concentration threshold formula for10-5μ M-0.0μ M all have anti-IR activity.Effective threshold concentrations of under4monomer compatibi lity effect on IR adipocyte model of the orthogonal experiment Met hods:take the monomer concentration of10-1,2levels orthogonal ta ble according to the four factors combined into different formula, diluted into10containing drug concentration of aboutl/4010-6M Ins DMEM medium. Cell culture is same to research two. In monomer-cont aining medium role in24h and48h,2-NBDG fluorescence labeling m ethod measuring glucose intake groups of cells, respectively. Resul ts:compared with normal group, model group,24h and48h cell gluco se intake significantly reduced; Compared with model group,4kinds of different compatibility groups of the monomers formulation for24h and48h cell sugar intake increased significantly, the24h,48h effect is better than that of four monomers used formula for24h and48h cell glucose intake were significantly higher than other g roups. Conclusion:extremely low concentrations of4kinds of ingre dients are other party compatibility has better resistance to IR ac tivity. Analysis showed that the interaction between various factor s affect the24h and48h monomer effect are the main factors of gly cyrrhizic acid and Baicalein, glycyrrhizic acid and puerarin, promp ting that glycyrrhizic acid and baicalein in all parties play an im portant role.This topic based on the complex system theory of traditional Chinese medicine compound there may be many ingredients very low concentrations of biological effect models were discussed. Theresults found on the cellular level effect under the threshold concentration (trace) in a variety of monomers formulation has obvious biological effects. So this study for "traditional Chinese medicine compound low concentrations how components interact to produce effect" of speculation to provide experimental evidence. |
PDF Full Text Request