Medicinal composition for treating diabetes hepatic fibrosis
A liver fibrosis and composition technology, which is applied in the field of traditional Chinese medicine or medicinal chemistry, can solve the problems of compound medicines with many flavors, complex components, influence on application, etc., and achieves the effect of good inhibition effect and proliferation inhibition effect.
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Embodiment 1
[0055] Example 1: Establishment of primary rat hepatic stellate cell proliferation model
[0056] 1. Reagents and Cells
[0057] Male Wistar rats, about 400 g, were provided by the Experimental Animal Center of the Academy of Military Medical Sciences.
[0058] 96-well plate, Corning; Type Ⅳ collagenase, sigma company; Pronase E, Roche company; Dnase Ⅰ, Roche company; DMEM medium, Tianrun Shanda Biotechnology Co., Ltd.; Heparin sodium, Sinopharm Chemical Reagent Co., Ltd.; PDGF-BB, Peprotech Corporation.
[0059] Peristaltic pump, Baoding Lange Constant Flow Pump Co., Ltd.; multi-head cell sample collector, Zhejiang Shaoxing ZT-2 type; liquid scintillation counter, PerkinElmer Company, model: 1450-024.
[0060] 2. Experimental steps
[0061] Primary rat hepatic stellate cells were isolated by in situ perfusion (Jiang Tao, Liang Liwu, Guo Shungen, et al. Isolation and culture of hepatic stellate cells. Journal of Armed Police Medical College, 2001,11(1):7.), according to ...
Embodiment 2
[0067] Example 2: Toxic effect of ursolic acid and Schisandrin B alone on primary hepatocytes
[0068] 1. Experimental materials
[0069] Thiazolyl blue tetrazolium bromide (MTT), Amreaco Company; Ensipire multifunctional microplate reader, PerkinElmer, model: 2300; other reagent sources are the same as in Example 1.
[0070] Ursolic acid and Schizandrin B can be purchased commercially (for example, purchased from Shanghai Tongtian Biotechnology Co., Ltd., the article numbers are E-0076 and E-0133, and the purity is greater than 97%), or refer to the methods of the following examples 8-9 preparation.
[0071] 2. Experimental steps
[0072]The toxic effects of ursolic acid and schisandrin B alone on primary hepatocytes were observed by MTT method. Separation of primary rat hepatocytes by in situ perfusion (Lao Quanlin, Jiang Qingyan, Gao Shujing, etc. Separation of rat hepatocytes by in situ perfusion. Journal of South China Agricultural University (Natural Science Edition...
Embodiment 3
[0076] Embodiment 3: Ursolic acid and Schizandrin B are used alone and in combination on primary HSC Effects on DNA Synthesis
[0077] 1. Reagents and Cells
[0078] Ursolic acid and Schizandrin B can be purchased commercially (for example, purchased from Shanghai Tongtian Biotechnology Co., Ltd., the article numbers are E-0076 and E-0133, and the purity is greater than 97%), or refer to the methods of the following examples 8-9 preparation.
[0079] 2. Experimental method and steps
[0080] use 3 The effects of ursolic acid and schisandrin B on DNA synthesis in hepatic stellate cells were observed by H-TdR incorporation. Primary rat hepatic stellate cells were isolated by in situ perfusion method, and the ratio of 1×10 5 Inoculate in a 96-well plate at a density of 1 / ml, and the medium is DMEM high-glucose medium containing 20% FBS, 37°C, 5% CO 2 Cultivate in the incubator for 24 hours, replace with DMEM high-glucose medium containing 0.5% FBS, 37°C, 5% CO 2 After...
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