CN103234928A - Picric acid-sirius red staining method and application thereof in screening anti-hepatic fibrosis compound - Google Patents
Picric acid-sirius red staining method and application thereof in screening anti-hepatic fibrosis compound Download PDFInfo
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- CN103234928A CN103234928A CN201310143535XA CN201310143535A CN103234928A CN 103234928 A CN103234928 A CN 103234928A CN 201310143535X A CN201310143535X A CN 201310143535XA CN 201310143535 A CN201310143535 A CN 201310143535A CN 103234928 A CN103234928 A CN 103234928A
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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
The invention belongs to biological technical field, be specifically related to utilize the picric acid sky scarlet decoration method of wolf of collagenous fibres to carry out the active compound of external high flux screening anti-hepatic fibrosis.
Background technology
Liver fibrosis (Liver fibrosis) refers in many chronic hepatic diseases pathogenic processes by diffusivity extracellular matrix (Extracellular matrix in the liver due to the various virulence factors, ECM) pathologic process that excessively precipitates, wherein ECM comprises collagen, fibronectin, elastin laminin, laminin, hyaluronic acid and proteoglycans etc.(hepatic stellate cells HSCs) is the main source of the interior collagen of liver and other extracellular matrix to HSCs.Sternzellen express alpha-smooth muscle actin (alpha-smooth muscle actin of activation, α-SMA), secretion I, III, IV Collagen Type VI, matrix metalloproteinase (matrix metalloproteinase, MMPs) and metal protease inhibitors (tissue inhibitor of metalloproteinase, the short fiberization factor such as TIMP) plays an important role to generation and the development of liver fibrosis.The vital role in the liver fibrosis forming process in view of HSCs and function thereof, important goal of following anti-hepatic fibrosis is exactly the direct targeting of secreting superfluous ECM at the HSCs that activates, and is expected to improve curative effect, suppresses fiberization formation, reduces toxic and side effect.TGF β 1 is synthetic by the HSC of Kupffer Cell, lymphocyte, hole endothelial cell and activation etc., is the cell factor that a kind of cell growth, differentiation and multiple physiology, pathologic process play important regulating action, and TGF β 1 signal mainly promotes the generation of ECM.Liver fibrosis is further development of cirrhosis, and hepatic failure often needs liver transfer operation to effect a radical cure, because the organ origin critical shortage, many patients are dead in waiting for the organ process.In the majority with chronic hepatitis B in China's chronic liver disease, due to illness the continuation of poison exists, and can cause pathological changes such as liver parenchyma is inflamed, necrosis, causes the continual fibroplasia of liver and forms liver fibrosis gradually.Along with the raising of living standards of the people, the incidence of disease of China's AML also constantly rises, and excessive drinking causes fat hepatitis, is further development of liver fibrosis.Because its number of patients is many, the mortality ratio height, social economy's burden is heavy, has become important public health problem of China.
The method that present existing mensuration fibroblast produces collagen mainly is some physico-chemical processes, comprise chromogenic reaction (specific dyestuff), molecular weight ratio is (agarose gel electrophoresis and chromatography), immune response (specific antigen-antibody response) and radioaction (labelled with radioisotope).Wherein the chromogenic reaction cost is low, and is nontoxic, easy, can be in the purpose of external realization high flux screening anti-hepatic fibrosis compound by porous plate.It wolf is scarlet to be negative ion strong acid dyestuff, easily with collagenous fibres in basic group react.So the picric acid sky scarlet dye liquor of wolf can make collagenous fibres specifically dye and be scarlet, non-collagenous fibres partly lose the scarlet background.Tool pertinent literature report, the picric acid sky scarlet decoration method of wolf is applied to the quantitative test of heart collagenous fibres, kidney collagenous fibres, the research of alcoholic fibrosis and evaluation pancreas in rat fiberization, but the scarlet dyeing of picric acid sky wolf majority is the dyeing that is applied to animal tissue's section, because it is long that experiment has the cycle in the animal body, because individual difference causes the shortcoming of experimental result instability etc., so in vivo studies is difficult to realize the purpose of high flux screening anti-hepatic fibrosis compound.The scarlet dyeing of picric acid sky wolf utilizes porous plate to carry out high flux screening in the zooblast level to select the research of anti-hepatic fibrosis compound to yet there are no report.
Summary of the invention
The objective of the invention is to by collagen and the specific scarlet F3B chromogenic reaction of dyestuff sky wolf, providing a kind of utilizes the scarlet dyeing of picric acid sky wolf of collagenous fibres to measure the method that HSCs is secreted collagen content, this method has need more a spot of testing compound sample, can be at the active compound of external high flux screening anti-hepatic fibrosis, characteristics such as fast, economical comparatively.Used cell is that HSCs is CFSC-B.
The method of HSCs secretion collagen content is measured in the scarlet dyeing of picric acid provided by the invention sky wolf, and emphasis has carried out correlative study to HSCs CFSC-8B secretion collagen content.
Use the method that HSCs CFSC-8B secretion collagen content is measured in the scarlet dyeing of wolf of picric acid sky, its step is as follows:
(1) cell is cultivated.Rats'liver astrocyte CFSC-8B is available from the refined centralab in Central South University Hunan.Cell culture fluid is the RPMI-1640 nutrient culture media, includes 10%FBS, and penicillin 100IU/mL and streptomysin 100 μ g/Ml cultivate in 37 ℃, the incubator of 5%CO2, and are long after suitable density (80-90% fusion) when cell, go down to posterity by 1:5.
(2) modeling experiment.Choose the CFSC-8B cell of exponential phase, with complete medium the concentration of cell suspension is transferred to 8 * 10
4Individual/m L is inoculated in 96 orifice plates, and every hole 100 μ L when treating that cell grows to 70-80%, cultivate 24h with the complete medium that contains 0.5%FBS and make cell hunger with after reaching synchronization, and experiment is divided into following several groups:
The CFSC-8B cell routine is cultivated (contrast) group; CFSC-8B TGF-β 1 modeling dosage (0,2.5,5,10ng/mL) group; Time group (24h and 48h); Coloured differently liquid long-pending (150,200,250 μ L) group.
(3) the scarlet dyeing of picric acid sky wolf.After cultivating 24h and 48h respectively according to step (2), with PBS rinse cell three times, Bouin ' s fluid room temperature is 1h fixedly, inhales and abandons immobile liquid, and culture plate cleans (up to the color of the immobile liquid that does not have yellow), aseptic air drying three times with PBS; Add day scarlet dyeing liquor of wolf, 1h(26 ℃ of shaking table concussion dyeing, 100rpm); Dyeing liquor is abandoned in suction, and 0.01NHCL rinse cell three times is to remove the dyestuff of not being combined with cell collagen, and the dyestuff with 0.1N NaOH dissolving is combined with cell dissolves 30min(26 ℃ in the shaking table concussion, 100rpm), detects OD with microplate reader
540nm, return to zero with 0.1N NaOH.
Being formulated as of step (3) the described picric acid scarlet dyeing liquor of sky wolf wherein: 0. 1 g days wolf is scarlet, be dissolved in the 100 mL picric acid saturated aqueous solutions, composition and the proportioning of Bouin ' s fluid are: the saturated picric acid solution of Bouin ' s fluid:15mL, 5mL formaldehyde, the 1mL glacial acetic acid.
The picric acid sky scarlet decoration method of wolf of collagenous fibres of the present invention can be measured the content of sternzellen secretion collagen, utilizes 96 orifice plates can be at the compound of external high flux screening anti-hepatic fibrosis by this method, and is comparatively quick and economical.
Description of drawings
Fig. 1 is the dose study of the scarlet decoration method model of picric acid of the present invention sky wolf.
Fig. 2 is research action time of the scarlet decoration method model of picric acid of the present invention sky wolf.
Fig. 3 is the dyeing liquor volume research of the scarlet decoration method model of picric acid of the present invention sky wolf.
Embodiment
The present invention may be better understood by the following example, and they just explain the present invention, and it are not limited.
(1) chooses the CFSC-8B cell of exponential phase, with complete medium the concentration of cell suspension is transferred to 8 * 10
4Individual/mL, be inoculated in 96 orifice plates, every hole 100 μ L when treating that cell grows to 70-80%, cultivate 24h with the complete medium that contains 0.5%FBS and make cell hunger to reach synchronization.
(2) experiment is divided into following several groups:
The CFSC-8B cell routine is cultivated (contrast) group; CFSC-8BTGF-β 1 modeling dosage (0,2.5,5,10ng/mL) group; Time group (24h and 48h); Coloured differently liquid long-pending (150,200,250 μ L) group.
(3) the scarlet dyeing of picric acid sky wolf.After cultivating 24h and 48h respectively according to step (2), with PBS rinse cell three times, Bouin ' s fluid room temperature is 1h fixedly, inhales and abandons immobile liquid, and culture plate cleans (up to the color of the immobile liquid that does not have yellow), aseptic air drying three times with PBS; Add day scarlet dyeing liquor of wolf, (26 ℃ of shaking table concussion dyeing 1h, 100rpm), dyeing liquor is abandoned in suction, 0.01NHCL rinse cell three times is to remove the dyestuff of not being combined with cell collagen, the dyestuff with 0.1N NaOH dissolving is combined with cell dissolves 30min(26 ℃ in the shaking table concussion, 100rpm), detect OD with microplate reader
540nm, return to zero with 0.1N NaOH.
(4) being formulated as of step (3) the described picric acid scarlet dyeing liquor of sky wolf wherein: 0. 1 g days wolf is scarlet, be dissolved in the 100 mL picric acid saturated aqueous solutions, composition and the proportioning of Bouin ' s fluid are: the saturated picric acid solution of Bouin ' s fluid:15mL, 5mL formaldehyde, the 1mL glacial acetic acid.
Experimental result: through the scarlet dyeing of picric acid sky wolf, the normal groups of cells collagenous fibres color of microscopically TGF-β 1 stimulating group is especially bright-coloured.Dosage effect the results are shown in Figure 1, and time effect the results are shown in Figure 2, and the dyeing liquor volume the results are shown in Figure 3.
The screening of embodiment 2 anti-hepatic fibrosis compounds
(1) MTT detects different bacterium powder extracts to the survival rate of cell.Choose the CFSC-8B cell of exponential phase, with complete medium the concentration of cell suspension is transferred to 3 * 10
4Individual/mL, be inoculated in 96 orifice plates, every hole 100 μ L, after continuing to cultivate 24h, add the camphor tree sesame, glossy ganoderma, the Chinese caterpillar fungus cephalo, Chinese caterpillar fungus is by spore, rhizoma Gastrodiae honey ring and Hericium erinaceus powder opposed polarity extract normal hexane, chloroform, ethyl acetate, methyl alcohol (200 μ g/ml, 100,50,25,12.5,6.25,3.125 μ g/mL), continue to cultivate 24h and 48h, and 4h adds MTT (5mg/ml) 10 μ L in advance, after continuing to hatch 4h, nutrient solution is abandoned in suction, every hole adds DMSO150 μ L, and concussion 10min is with the OD value of microplate reader survey 570nm.Cell survival rate calculates according to following formula: the average OD value of control group * 100% of the average OD value of administration group/not dosing, find best drug effect concentration.(2) with the 0.5%FBS nutrient solution with the CFSC-8B cell synchronization after, experiment is divided into following several groups: the CFSC-8B cell routine is cultivated (contrast) and is organized; CFSC-8B TGF-β 1 modeling (model) group; TGF-β 1 modeling CFSC-8B cell adds the different extract groups of various bacterium powder.
(3) after cell is cultivated 24h and 48h respectively, press the scarlet decoration method of step (3) picric acid sky wolf among the embodiment 1 from the camphor tree sesame, glossy ganoderma, Chinese caterpillar fungus cephalo, Chinese caterpillar fungus is by spore, and screening has the compound of anti-hepatic fibrosis in rhizoma Gastrodiae honey ring and the Hericium erinaceus powder opposed polarity extract.
Claims (2)
1. scarlet decoration method of picric acid sky wolf, its feature is as follows: may further comprise the steps:
(1) will be seeded to 96 orifice plates behind the passage, when treating that cell grows to 70-80%, 0.5%FBS carries out synchronization process 24h with cell, and experiment is divided into control group and TGF-β 1 stimulating group, acts on the different time;
(2) use PBS rinse cell three times, Bouin ' s fluid room temperature is 1h fixedly, inhales and abandons immobile liquid, and culture plate cleans (up to the color of not having yellow immobile liquid), aseptic air drying three times with PBS;
(3) add day scarlet dyeing liquor of wolf, at shaking table concussion dyeing 1h, 26 ℃, 100rpm;
(4) inhale and to abandon dyeing liquor, 0.01N HCL rinse cell three times is to remove not and the cell collagen binder;
(5) dyestuff of being combined with cell with 0.1N NaOH dissolving is at shaking table concussion dissolving 30min, 26 ℃, 100rpm;
(6) detect OD with microplate reader
540nm, return to zero with 0.1N NaOH.
2. the application process of the described method of claim 1 in screening anti-hepatic fibrosis compound, used TGF-β 1 dosage of described TGF-β 1 stimulating group is 0,2.5,5,10ng/mL, described effect different time is 24h and 48h, and the volume of the scarlet dyeing liquor of described adding picric acid sky wolf is respectively 150 μ L, 200 μ L, 250 μ L; Test shows that the optimal dose of TGF-β 1 is 10ng/mL, and the best use of time is 48h, and the volume of the scarlet dyeing liquor of best picric acid sky wolf is 200 μ L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103776778A (en) * | 2014-01-10 | 2014-05-07 | 华南理工大学 | Quantitative determination method in extraction process of fish collagen and application of quantitative determination method |
| CN104232588A (en) * | 2014-09-12 | 2014-12-24 | 中国医学科学院医药生物技术研究所 | Construction and application of anti-hepatic fibrosis drug high-throughput screening cell model |
| CN111631188A (en) * | 2019-03-01 | 2020-09-08 | 广西中医药大学 | Method for rapidly inducing alcoholic fatty liver model by single factor |
-
2013
- 2013-04-24 CN CN201310143535XA patent/CN103234928A/en active Pending
Non-Patent Citations (6)
| Title |
|---|
| MARY H 等: "TGF-β and fibrosis", 《MICROBES AND INFECTION》 * |
| TOLOSA MJ 等: "La diabetes altera el potencial osteogénico de células progenitoras de médula ósea. Efectos del tratamiento con metformina", 《RAEM》 * |
| 戴晴 等: "丹参素对肝星状细胞TGF-β信号转导的影响", 《中国病理生理杂志》 * |
| 王淳 主编: "《基础医学实验技术》", 30 September 2009, 中国医药科技出版社 * |
| 解克芳 等: "葛根素对人胚肺成纤维细胞增殖及细胞外基质合成的影响", 《中国中医药信息杂志》 * |
| 郑伟: "苦味酸天狼猩红染色在心脏胶原纤维定量分析中的应用", 《福建医科大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103776778A (en) * | 2014-01-10 | 2014-05-07 | 华南理工大学 | Quantitative determination method in extraction process of fish collagen and application of quantitative determination method |
| CN104232588A (en) * | 2014-09-12 | 2014-12-24 | 中国医学科学院医药生物技术研究所 | Construction and application of anti-hepatic fibrosis drug high-throughput screening cell model |
| CN111631188A (en) * | 2019-03-01 | 2020-09-08 | 广西中医药大学 | Method for rapidly inducing alcoholic fatty liver model by single factor |
| CN111631188B (en) * | 2019-03-01 | 2022-04-22 | 广西中医药大学 | Method for rapidly inducing alcoholic fatty liver model by single factor |
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Application publication date: 20130807 |