Picric acid-sirius red staining method and application thereof in screening anti-hepatic fibrosis compound
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A technology of Sirius scarlet and picric acid, applied in the biological field, can solve the problems of unstable experimental results, difficult to achieve high-throughput screening of anti-hepatic fibrosis compounds, and long cycle.
Inactive Publication Date: 2013-08-07
JIANGNAN UNIV
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According to relevant literature reports, the picric acid Sirius red staining method is applied to the quantitative analysis of collagen fibers in the heart and kidney, the study of alcoholic liver fibrosis, and the evaluation of pancreatic fibrosis in rats, but the picric acid Sirius red staining method is mostly applied For the staining of animal tissue sections, due to the long cycle of animal experiments and the instability of experimental results due to individual differences, it is difficult to achieve the purpose of high-throughput screening of anti-hepatic fibrosis compounds in vivo experiments
Picric acid Sirius scarlet staining has not been reported yet on the use of multi-well plates for high-throughput screening of anti-hepatic fibrosis compounds at the animal cell level
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Embodiment 1
[0017] Example 1 Model establishment.
[0018] (1) Select CFSC-8B cells in the logarithmic growth phase, and adjust the concentration of the cell suspension to 8×10 with complete medium 4 Cells / mL, inoculated in 96-well plate, 100 μL per well, when the cells grew to 70-80%, cultured in complete medium containing 0.5% FBS for 24 hours to starve the cells to achieve synchronization.
[0019] (2) The experiment is divided into the following groups:
[0020] CFSC-8B cell conventional culture (control) group; CFSC-8BTGF-β1 modeling dose (0, 2.5, 5, 10ng / mL) group; time group (24h and 48h); different staining solution volume (150, 200, 250μL )Group.
[0021] (3) Piric acid Sirius scarlet staining. After culturing for 24h and 48h respectively according to step (2), rinse the cells with PBS three times, fix with Bouin's fluid at room temperature for 1h, discard the fixation solution, wash the culture plate with PBS three times (until there is no yellow color of the fixation soluti...
Embodiment 2
[0024] Example 2 Screening of anti-hepatic fibrosis compounds
[0025] (1) MTT was used to detect the survival rate of different bacterial powder extracts on cells. Select CFSC-8B cells in the logarithmic growth phase, and adjust the concentration of the cell suspension to 3×10 with complete medium 4 1 / mL, inoculated in 96-well plate, 100 μL per well, continued to culture for 24 hours, added Antrodia camphorata, Ganoderma lucidum, Cordyceps cephalosporin, Cordyceps mortierella, Gastrodia elata and Hericium erinaceus powder with different polarity extracts n-hexane, chloroform, Ethyl acetate, methanol (200μg / ml, 100, 50, 25, 12.5, 6.25, 3.125μg / mL), continue to culture for 24h and 48h, and add 10μL of MTT (5mg / ml) 4h in advance, continue to incubate for 4h, suck Discard the culture medium, add 150 μL of DMSO to each well, shake for 10 min, and measure the OD value at 570 nm with a microplate reader. The cell survival rate was calculated according to the following formula: the...
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Abstract
The invention belongs to biotechnology field and particularly relates to high throughput screening of an effective anti-hepatic fibrosis compound by means of picric acid-sirius red staining method on a collagenous fiber. The cell is hepatic stellate cell line CFSC-8B, transforming growth factor TGF-beta1 with the dose of 10ng/mL can obviously induce the CFSC-8B cell to secrete collagen when acting for 48 hours, and the secretion of the CFSC-8B cell collagen induced by the TGF-beta 1 can be effectively detected by means of picric acid-sirius red staining method. The picric acid-sirius red staining method on the collagenous fiber is applied to screening the compounds with anti-hepatic fibrosis activities in different polarity extracts of antrodia camphorata, ganoderma lucidum, cephalosporium sinensis, cordyceps mortierella, armillaria and hericium erinaceus powder. Tests prove that n-hexane and chloroform extract of the antrodia camphorata, ethyl acetate extract from the cordyceps mortierella, and n-hexane, chloroform extract and ethyl acetate extract from the armillaria can effectively inhibit the secretion of the CFSC-8B cell collagen induced by the TGF-beta 1, the above extracts have dose dependent relationship, are significant in difference, and have an anti-hepatic fibrosis function, and other extracts do not have significant inhibition effect.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to high-throughput screening of effective compounds for anti-hepatic fibrosis in vitro by using the picric acid sirius red staining method of collagen fibers. Background technique [0002] Liver fibrosis (Liver fibrosis) refers to the pathological process of excessive precipitation of diffuse extracellular matrix (ECM) in the liver caused by various pathogenic factors during the pathogenesis of many chronic liver diseases. ECM includes collagen, fiber Connexin, elastin, laminin, hyaluronic acid and proteoglycan, etc. Hepatic stellate cells (HSCs) are the main source of collagen and other extracellular matrix in the liver. Activated stellate cells express α-smooth muscle actin (alpha-smooth muscle actin, α-SMA), secrete collagen types I, III, and IV, matrix metalloproteinases (matrix metalloproteinases, MMPs) and metalloproteinase inhibitors (tissue inhibitor of metalloprot...
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