Method for detecting influence of medicated serum of Sijunzi decoction on hepatic stem cells

Abstract

The invention provides a method for detecting the influence of drug-containing serum of Sijunzi decoction on hepatic stem cells. The method comprises the steps of cell culture, drug-containing serum preparation, cell proliferation experiment, cell damage model establishment and treatment, oxidative stress related index detection, protein expression analysis and the like. According to the method, cell activity is detected through a CCK-8 method, cell nucleus morphological change is observed through Hoechst 33342 staining, the apoptosis rate is detected through Annexin V / PI double staining in combination with flow cytometry, apoptosis-related protein expression is detected through Western blot, and the action mechanism of the drug-containing serum is comprehensively evaluated. Experimental results show that the drug serum of the Sijunzi decoction can promote proliferation of liver stem cells, reduce oxidative stress injury induced by H2O2, improve cell viability and oxidation resistance, reduce cell apoptosis rate, up-regulate expression of anti-apoptotic protein Bcl-2 and inhibit expression of pro-apoptotic protein Bax and Cleaved Caspase-3 at the same time.

Description

Technical Field

[0001] The present invention relates to a detection method, and more particularly to a detection method based on the influence of Sijunzi decoction-containing serum on liver stem cells. Background Art

[0002] As a cell type with multidirectional differentiation potential, liver stem cells play an important role in liver damage repair and regeneration. Studies have shown that promoting the proliferation and differentiation of liver stem cells and resisting oxidative stress-induced damage are important strategies for the treatment of liver diseases. In recent years, the role of Chinese herbal compound and its metabolites in liver protection has received increasing attention. Among them, Sijunzi Decoction has been widely used in the field of traditional Chinese medicine due to its characteristics of strengthening the body.

[0003] Traditional methods for detecting liver stem cell proliferation and anti-oxidative damage mainly rely on the single effect of chemical drugs or direct separation of components, which often cannot fully reveal the synergistic mechanism of multiple active ingredients and their metabolites in the compound. At the same time, oxidative stress is one of the important pathogenic mechanisms of liver disease. Using model cells to detect the anti-damage effect of Chinese medicine-containing serum on liver stem cells can be closer to the real physiological environment and more effectively evaluate the efficacy mechanism of Chinese medicine compound. At present, there is no clear standardized method for using Sijunzi Decoction-containing serum to detect liver stem cell proliferation and anti-oxidative damage. Summary of the invention

[0004] The present invention combines modern cell culture technology, flow cytometry and molecular biology methods to comprehensively evaluate multiple indicators such as proliferation, apoptosis and oxidative stress of liver stem cells, filling this research gap. This method not only provides a scientific basis for the efficacy research of Sijunzi Decoction, but also provides a reference for the research of other Chinese medicine compounds. The specific scheme is as follows:

[0005] A detection method based on the effect of Sijunzi Decoction-containing serum on liver stem cells, the detection method comprising the following steps:

[0006] S1. Cell culture: Rat liver stem cells WB-F344 were cultured in DMEM medium containing 10% newborn calf serum at 37°C and 5% CO2. They were allowed to grow adherently and were passaged every 2-3 days. Cells in the logarithmic growth phase were used for the experiment.

[0007] S2. Preparation of drug-containing serum: Rats were gavaged with Sijunzi decoction for 7 consecutive days. Serum was collected 2 hours after the last administration, sterilized with a 0.22 μm microporous filter membrane, and stored at -80°C for later use;

[0008] S3. Cell proliferation assay: 4×10 4The cells were seeded in 96-well plates at a cell density of 100 cells / mL, and three concentrations of drug-containing serum were added in sequence. After culturing for 24 hours, the cell activity was determined by the CCK-8 method;

[0009] S4. Establishment and treatment of cell injury model: cells were treated with 100 μmol / L H2O2 for 40 minutes, followed by addition of 10% drug-containing serum for 24 hours, and cell viability and apoptosis were detected by CCK-8 method, Hoechst 33342 staining, and Annexin V / PI staining;

[0010] S5. Detection of oxidative stress related indicators: The total SOD activity of cells was detected by SOD kit, and the MDA content of cells was detected by MDA kit.

[0011] S6. Protein expression detection: Cell proteins were extracted and the expression levels of apoptosis-related proteins Bax, Bcl-2 and caspase-3 were detected by Western blot.

[0012] Furthermore, when the CCK-8 method is used for cell viability detection: 10 μL of CCK-8 reagent is added to each well and incubated at 37° C. for 2.5 hours; the absorbance value is measured at a wavelength of 450 nm using an enzyme reader to calculate cell proliferation or activity.

[0013] Further, it is characterized in that the Hoechst 33342 staining experiment comprises the following steps:

[0014] Add 10 μg / mL Hoechst 33342 dye to each well and incubate in the dark for 10 minutes; use a fluorescence microscope to observe changes in nuclear morphology to identify apoptotic cells.

[0015] Furthermore, when SOD and MDA were used to detect oxidative stress related indicators: SOD activity was calculated by colorimetry using an enzyme reader, and the result was expressed in U / mgprot; MDA content was calculated by colorimetry, and the result was expressed in nmol / mgprot.

[0016] Furthermore, in Western blot detection: the electrophoresis conditions were 12.5% ​​SDS polyacrylamide gel electrophoresis, the stacking gel voltage was 80V, and the separation gel voltage was 100V; the transfer conditions were 50V constant voltage transfer for 4 hours, and the blocking solution was a mixture of 1g skim milk powder and 0.2g BSA; the primary antibodies included Bax, Bcl-2 and caspase-3 antibodies, and the dilution ratio was 1:4000.

[0017] Furthermore, the concentration of drug-containing serum added to S3 was 5%, 10% and 20%.

[0018] Furthermore, the concentration of the Sijunzi Decoction decoction in S2 is 1 mL / 100 g.

[0019] Beneficial effects:

[0020] The present invention provides a method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells, which has the following beneficial effects:

[0021] (1) The present invention systematically combines cell culture, flow cytometry, and molecular biology techniques to comprehensively evaluate the effects of Sijunzi Decoction-containing serum on the proliferation, anti-oxidative damage, and apoptosis of liver stem cells, filling the gap in the research field of the mechanism of action of Chinese herbal compound-containing serum, and providing a scientific basis for the efficacy research of Sijunzi Decoction.

[0022] (2) The present invention detects cell activity, apoptosis rate and antioxidant capacity from multiple angles through multiple methods (CCK-8 method, Hoechst 33342 staining, Annexin V / PI staining, SOD, MDA detection, Western blot analysis), ensuring the comprehensiveness and scientificity of the experimental data and accurately revealing the mechanism of action of the drug-containing serum.

[0023] (3) Using drug-containing serum as the source of experimental drugs to simulate the drug state after metabolism in vivo, avoiding the limitations of the action of a single compound, being closer to actual clinical application, and providing more reliable experimental results. The experimental results showed that 10% concentration of Sijunzi Decoction drug-containing serum can significantly increase the activity of liver stem cells and promote their proliferation; at the same time, it can reduce cell damage induced by H2O2, reduce MDA levels, increase SOD activity, and improve the cells' ability to resist oxidative stress. At the same time, Sijunzi Decoction drug-containing serum can reduce the expression of pro-apoptotic protein Bax, inhibit the activation of Cleaved Caspase-3, and upregulate the expression of anti-apoptotic protein Bcl-2, thereby reducing the proportion of early and late apoptosis of cells and protecting liver stem cells from oxidative stress damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram illustrating the effect of drug-containing serum on WB-F344 cell activity in the examples.

[0025] Figure 2 It is the inhibition rate of cells after treating cells with different concentrations of H2O2 for 20min, 40min and 60min in the embodiment.

[0026] Figure 3 This is a diagram showing the toxic effects of different concentrations of H2O2 on cells.

[0027] Figure 4 This is a diagram showing the protective effect of 10% drug-containing serum on WB-F344 cell damage caused by 100 μmol / L H2O2.

[0028] Figure 5 This is a diagram illustrating the effect of 10% drug-containing serum on SOD and MDA in WB-F344 cells damaged by H2O2.

[0029] Figure 6 Figure 1: Hoechst 33342 staining method to detect changes in cell nuclear morphology.

[0030] Figure 7 The legend shows that 10% drug-containing serum reduces the apoptosis rate of cells.

[0031] Figure 8 This is a legend that 10% drug-containing serum promotes the expression of Bcl-2 protein and reduces the expression of cellular Bax and Cleaved Caspase-3 proteins. DETAILED DESCRIPTION

[0032] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0033] Example:

[0034] A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells, specifically comprising the following steps:

[0035] 1. Experimental materials:

[0036] ① Cell lines

[0037] Hepatic stem cell line: Rat (Fischer344) hepatic stem cell WB-F344 was kindly donated by Professor Li Yan and Teacher Chen Hui from the Institute of Materia Medica, Chinese Academy of Medical Sciences. Rat hepatocytes: purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.

[0038] ②Drugs and reagents

[0039] DMEM medium: 10.4g dissolved in 1000ml distilled water, add 3.7g NaHCO3, 2.385g Hepes sodium salt, MEMMEM / 1640 medium: 10.4g dissolved in 1000ml distilled water, add 2.2g NaHCO3, 4.8g Hepes sodium salt. Each culture medium needs to add 100,000 units of penicillin and streptomycin, adjust the pH to 7.2 with HCl, add newborn calf / fetal bovine serum to adjust the concentration to 10%, filter sterilize, and store in a refrigerator at 4℃;

[0040] 0.25% trypsin: Amresco, USA. Dissolve 0.25 g trypsin powder and 0.037 g EDTA·2Na in 100 mL D-hanks solution, then filter through a 0.22 μm microporous filter to sterilize, and store in a refrigerator at 4°C.

[0041] Newborn / fetal bovine serum: product of Hangzhou Sijiqing Bioengineering Co., Ltd., China, stored in a -20℃ refrigerator;

[0042] 10×PBS: 80g NaCl, 2g KCl, 21.6g Na2HPO4·12H2O, 2g KH2PO4, add 800mL of distilled water, mix well, add distilled water to 1000mL, store in a refrigerator at 4℃, and dilute with double distilled water when needed;

[0043] 10×D-hanks solution: 40 g NaCl, 2 g KCl, 0.67 g Na2HPO4·12H2O, 0.3 g KH2PO4, 1.75 g NaHCO3, add 500 mL double distilled water, mix well, and store in a refrigerator at 4°C;

[0044] 1.5M Tris-HCl buffer: Dissolve 18.15g Tris base in 80mL distilled water, add concentrated hydrochloric acid to adjust the pH to 8.8, and add water to make up to 100mL;

[0045] 0.5M Tris-HCl buffer: Dissolve 6 g Tris base in 60 mL distilled water, add concentrated hydrochloric acid to adjust the pH to 6.8, and add water to make up to 100 mL;

[0046] 5×SDS gel electrophoresis buffer: 15.15 g Tris base, 72 g glycine, 5 g SDS, dissolved in 800 mL distilled water, adjusted to pH 8.3, fixed to 1000 mL, stored at room temperature;

[0047] Transfer buffer: 5.8 g Tris base, 2.9 g glycine, 200 ml methanol, dilute to 1000 mL, store at room temperature;

[0048] Western blocking solution: 1g skim milk powder, 0.2g BSA dissolved in 20mL TBST solution, prepared before use;

[0049] Antibody diluent: TBST solution. The amount of antibody is added according to the antibody dilution ratio. For example, if the antibody dilution concentration is 1:4000, add 0.5 μL of antibody stock solution to 2 mL of PBST solution.

[0050] TBST solution: Dissolve 0.5 mL of Tween20 in 1000 mL of TBS solution;

[0051] 30% polyacrylamide: 29 g acrylamide, 1 g N-N'methylenebisacrylamide, dissolved in 100 mL distilled water;

[0052] Ammonium persulfate: AMRESCO imported and packaged in the United States; Dissolve 0.1g of ammonium persulfate in 1mL of distilled water to make 10% ammonium persulfate, and use it immediately after preparation;

[0053] 6× SDS-PAGE protein loading buffer: Biotech Biotechnology Co., Ltd., China;

[0054] Hepes: Promega Corporation, United States;

[0055] Acrylamide: analytical grade, imported and packaged by BBI;

[0056] Tris(hydroxymethyl)aminomethane: imported and packaged by AMRESCO, USA;

[0057] PVDF membrane: BIO-RAD, USA;

[0058] ECL superluminescent liquid, Millipore, USA;

[0059] BCA protein concentration assay kit: Beyotime Biotechnology Co., Ltd., China;

[0060] Cell counting kit CCK-8 (cell counting-kit8): purchased from Dojindo Company, Japan;

[0061] Total SOD assay kit: Nanjing Jiancheng Bioengineering Institute, China;

[0062] MDA assay kit: Nanjing Jiancheng Bioengineering Institute, China;

[0063] Hoechst 33342: Sigma, USA. Dissolve Hoechst 33342 powder in double distilled water to a final concentration of 10 mg / mL. Dilute to 10 μg / mL with PBS before use.

[0064] Annexin V / PI apoptosis detection kit: Molecular Probes, USA.

[0065] Mouse anti-mouse caspase-3: Abcam, UK;

[0066] Mouse anti-rat Bax: Santa Cruz Company, USA;

[0067] Rabbit anti-mouse Bcl-2: Santa Cruz Company, USA.

[0068] ③Experimental instruments

[0069] HealForce CO2 incubator, USA;

[0070] TS100 Nikon inverted phase contrast microscope, Nikon Corporation, Japan;

[0071] KT-30L fully automatic high pressure sterilizer, TOKYO Co., Ltd., Japan;

[0072] Ultra-low temperature refrigerator, SANYO Co., Japan;

[0073] TE601-L electronic balance, BS124S analytical balance, Beijing Sartorius Instrument Systems Co., Ltd., China;

[0074] Sigma 1-13 high-speed desktop centrifuge, Sigma, Germany;

[0075] Spectra Max 190 microplate reader, molecular device company, USA;

[0076] Clean bench, Suzhou Fengqiao Purification Equipment Co., Ltd., China;

[0077] Eupu ultrapure water machine, Chengdu Ultrapure Technology Co., Ltd., China;

[0078] XK02-201 Yaxi brand liquid nitrogen biological container, China;

[0079] FACS Calibur flow cytometer, BD, USA;

[0080] Ti-E live cell workstation microscope, Nikon Corporation, Japan.

[0081] 2. Experimental Methods

[0082] ① Cell culture

[0083] Adherent growth WB-F344 cells were cultured in DMEM containing 10% inactivated newborn calf serum and penicillin and streptomycin (1 million U / L each) and placed in a carbon dioxide incubator at 37°C, 5% CO2, and saturated humidity. The cells adhered to the wall and were passaged once every 2 to 3 days. During passage, the culture medium was first poured out, washed twice with PBS, and after trypsin digestion, fresh culture medium was added and blown evenly, and the cells were adjusted to an appropriate concentration and transferred to a new culture bottle. The culture medium was added to an appropriate amount, and the cells in the logarithmic growth phase were used for the experiment.

[0084] ②CCK-8 assay to detect cell viability

[0085] Experimental method of drug-containing serum on cell proliferation: cells in logarithmic growth phase were counted at 4×10 4 / ml density was inoculated in a 96-well culture plate, 100μL per well. After 24 hours of culture, the cells were divided into a normal group and a drug-containing serum treatment group, and the cells were treated with 5%, 10%, and 20% normal rat serum and 5%, 10%, and 20% drug-containing serum, respectively. Five replicate wells were set for each concentration in the experimental group, and five blank zero wells were set at the same time, and culture medium was added. After 24, 48, and 72 hours of drug action, the supernatant was removed, 10μL CCK-8 was added to each well, oscillated and mixed, and culture was continued for 2.5 hours. The absorbance value was measured at 450nm using an enzyme marker.

[0086] Establishment of H2O2-induced cellular oxidative stress injury model: cells in logarithmic growth phase were incubated at 4×10 4 / ml density was inoculated in a 96-well culture plate, 100 μL per well. After 24 hours of culture, H2O2 was added to a final concentration of 12.5, 25, 50, 100, 200, 400, 800 μmol / L. After 20, 40, 60 minutes, 10 μL CCK-8 was added to each well, and culture was continued for 2.5 hours. The absorbance value was measured at 450 nm using an enzyme reader.

[0087] Experimental method of drug-containing serum treatment: cells in logarithmic growth phase were taken, digested, counted, and 4×10 4 / ml density was inoculated in a 96-well culture plate, 100μl per well. After culturing for 24 hours, 100μmol / L H2O2 was added to act on liver stem cells for 40 minutes, and then 10% normal and drug-containing serum were added for another 24 hours, and the cell activity was detected by CCK-8 method.

[0088] ③Hoechst 33342 staining to detect nuclear morphology

[0089] Experimental method: Take cells in the logarithmic growth phase, digest and count them, and use 4×10 4 / mL density was inoculated in a 24-well culture plate, 400μL per well. After culturing for 24h, the medium was aspirated, 100μmol / LH2O2 was added to act on liver stem cells for 40min, and then 10% normal and drug-containing serum were added for another 24h, washed twice with PBS, 300μL of 10μg / mL Hoechst 33342 dye solution was added, incubated for 10 minutes in the dark, the dye solution was aspirated, washed twice with PBS, and nuclear changes were observed under a fluorescence microscope.

[0090] ④Annexin V / PI staining method to detect cell apoptosis

[0091] Experimental method: Take cells in the logarithmic growth phase, digest and count them, and use 1×10 5 / mL was inoculated in a 6-well culture plate, 2mL per well. After culturing for 24 hours, the medium was removed, 100μmol / L H2O2 was added to act on liver stem cells for 40 minutes, and then 10% normal and drug-containing serum were added for another 24 hours, washed twice with PBS, and 1×10 6 Cells were washed twice with pre-cooled PBS, 400 μL binding buffer was added, 5 μL Annexin V-FITC and 5 μL PI were added after gently mixing the cells, and the apoptosis rate of cells was detected by flow cytometry after incubation in the dark for 15 min. In the double staining image of flow cytometry detection: the cell group in the lower left corner is normal cells, the lower right corner is early apoptotic cells, the upper right corner is late apoptotic cells, and the upper left corner is necrotic or mechanically damaged cells.

[0092] ③Detection of SOD and MDA content

[0093] Detection of total SOD

[0094] Experimental method: WB-F344 cells in the logarithmic growth phase were taken at 1×10 5 / mL density was inoculated in a 6-well culture plate, 2mL per well. After culturing for 24 hours, the culture medium was removed, 100μmol / L H2O2 was added to act on liver stem cells for 40 minutes, and then 10% normal and drug-containing serum were added for another 24 hours. After the cultured cells were broken, the cell SOD activity was determined according to the steps in the kit instructions. The microplate method was used for analysis with an ELISA reader. SOD activity (U / mgprot) = (OD of the assay tube - OD of the blank tube) / (OD of the standard tube - OD of the blank tube) × concentration of the standard tube ÷ protein concentration of the sample to be tested.

[0095] Determination of MDA content

[0096] Experimental principle: Malondialdehyde (MDA) in the degradation products of lipid peroxidation combines with thiobarbituric acid to form a red product with a maximum absorption peak at 532nm.

[0097] Experimental method: WB-F344 cells in the logarithmic growth phase were taken at 1×10 5 / mL density was inoculated in a 6-well culture plate, 2mL per well. After culturing for 24 hours, the culture medium was aspirated, 100μmol / L H2O2 was added to act on liver stem cells for 40 minutes, and then 10% normal and drug-containing serum were added for another 24 hours. After the cultured cells were broken, the MDA content was determined according to the instructions of the kit, and the microplate method was used for analysis with an enzyme reader. MDA content (nmol / mgprot) = (measurement tube OD-blank tube OD) / (standard tube OD-blank tube OD) × standard tube concentration ÷ test sample protein concentration.

[0098] Western blot detection of protein expression

[0099] Experimental principle: The protein sample separated by SDS polyacrylamide gel electrophoresis is transferred to a PVDF membrane. The primary antibody and the secondary antibody are successively bound to the target protein through antigen-antibody specific reaction. Finally, the chemiluminescent color development reaction between the enzyme labeled with the secondary antibody and the substrate is used to display the location and content of the target antigen.

[0100] Experimental methods:

[0101] 1. Protein extraction: After treating each group of cells with drug-containing serum for a specific period of time, collect the cells, centrifuge, wash twice with PBS, add a certain amount of cell lysis buffer, and lyse on ice for 20 minutes. Transfer the lysate into an eppendorf tube, centrifuge at 12000g for 2 minutes, and aspirate the supernatant. Take 2-5μL for protein quantification using the BCA protein concentration assay kit, and dissolve the remaining samples in 6×SDS gel loading buffer according to the proportion and boil for 5 minutes.

[0102] 2. SDS gel electrophoresis: Take 20 μg of protein sample and perform 12.5% ​​SDS polyacrylamide gel electrophoresis; under constant voltage conditions, the electrophoresis voltage in the stacking gel is 80V and the separation gel voltage is 100V.

[0103] 3. Western blot: Place the gel in the transfer system under a constant voltage of 50V for 4 hours to transfer the protein to the PVDF membrane; place the membrane in the blocking solution for 2 hours; after a simple wash with PBST, place the membrane in the primary antibody dilution at 4°C overnight; then wash with PBST 6 times, 10 minutes each time; place the membrane in the secondary antibody dilution and incubate at room temperature for 2 hours; then wash with PBST 6 times, 10 minutes each time; ECL luminescence for 3-5 minutes, and X-ray film exposure, development, fixation, and observation in a dark room.

[0104] Statistical analysis

[0105] SAS statistical software was used for ANOVA analysis of variance, and the data were expressed as mean ± SD, with P < 0.05 as the significance standard. Each experiment was repeated at least three times.

[0106] 3. Experimental results:

[0107] ① The serum containing Sijunzi Decoction has the effect of promoting the proliferation of liver stem cells

[0108] Figure 1 This is a diagram showing the effect of the drug-containing serum on the activity of WB-F344 cells in the example, as shown in the figure:

[0109] Compared with the normal serum group, different concentrations of drug-containing serum had no toxic effect on WB-F344 cells, and had a certain effect of promoting proliferation at different time points. The cell activity was higher after 72 hours of drug serum than that at 24 and 48 hours, and the cell activity of 10% drug-containing serum group was higher than that of 5% and 20% groups, indicating that 10% drug-containing serum has a better effect in promoting the proliferation of WB-F344 cells.

[0110] Figure 2 It is the inhibition rate of cells after treating cells with different concentrations of H2O2 for 20min, 40min and 60min in the embodiment. Figure 3 This is a diagram showing the toxic effects of different concentrations of H2O2 on cells. Figure 2 As shown:

[0111] The cells were treated with different concentrations of H2O2 for 20min, 40min, and 60min. As the action time prolonged, the inhibition rate of the cells continued to increase. When the action time was the same, as the concentration of H2O2 increased, the inhibition rate of the cells also gradually increased. Among them, when the drug acted for 40 and 60min, the inhibition rate of 50μmol / L to 100μmol / L H2O2 on the cells was relatively stable. At 40min, the inhibition rate of the two concentrations of H2O2 on the cells was less than 50%. The experiment chose to treat the cells with 100μmol / L H2O2 for 40min to induce cell oxidative stress damage. The results of cell morphology showed that the number of cells decreased significantly after the concentration was greater than 100μmol / L, and the morphology was wrinkled and rounded.

[0112] Figure 4 This is a diagram showing the protective effect of 10% drug-containing serum on WB-F344 cell damage caused by 100 μmol / L H2O2. Figure 4 As shown:

[0113] Compared with the control group, H2O2 significantly induced cell damage and reduced cell activity (P<0.05). After treatment with drug-containing serum, cell activity increased by 43.56%, but there was still a significant difference compared with the control group (P<0.05). Cell morphology results showed that the H2O2 group had obvious wrinkling and rounding, and after treatment with drug serum, the morphology did not improve significantly, indicating that Sijunzi Decoction-containing serum improved the H2O2-induced cell activity reduction to a certain extent.

[0114] Figure 5 This is a diagram showing the effect of 10% drug-containing serum on SOD and MDA in WB-F344 cells damaged by H2O2. Figure 5 As shown:

[0115] The MDA content of cells treated with H2O2 increased significantly (P<0.05), and the SOD activity also tended to increase. The drug serum could significantly reduce the MDA level of cells, increase the SOD activity, scavenge free radicals, improve the antioxidant capacity, and repair the damaged liver stem cells.

[0116] Figure 6 1The Hoechst 33342 staining method is used to detect changes in cell nuclear morphology. Figure 7 The legend is that 10% drug-containing serum reduces the apoptosis rate of cells. Figure 8 This is a diagram showing that 10% drug-containing serum promotes the expression of Bcl-2 protein and reduces the expression of cell Bax and CleavedCaspase-3 protein, as shown in the figure:

[0117] After 100μmol / L H2O2 caused liver stem cell damage, the drug serum acted for 24 hours. The results were as follows Figure 6 Compared with the control group, 100 μmol / L H2O2 can induce typical apoptosis-like changes in cells, namely, nuclear condensation, chromatin aggregation and fragmentation. Compared with the injury group, 10% drug-containing serum treatment can improve the above changes in the nucleus. Flow cytometry results show that the proportion of early apoptotic cells (Annexin V(+) / PI(-) in the lower right corner) decreased from 6% to 3.5%, while the proportion of late apoptotic cells (Annexin V(+) / PI(+) in the upper right corner) decreased from 10% to 7.7%. For the statistics of apoptosis rate, see Figure 8 Western blot results showed that compared with the injury group, 10% drug-containing serum could reduce the expression of activated Caspase-3, decrease the level of pro-apoptotic protein Bax, and increase the level of anti-apoptotic protein Bcl-2.

[0118] In this embodiment, after different concentrations of Sijunzi Decoction-containing serum were used to act on WB-F344 cells, the serum with different drug concentrations had no toxic effect on liver stem cells. The cell activity was detected by CCK-8 method, and it was found that 10% medicated serum had a good effect of promoting cell proliferation. Then, 10% Sijunzi Decoction-containing serum was used to study its anti-hydrogen peroxide damage effect in vitro. The results showed that the medicated serum could resist the cell damage caused by hydrogen peroxide, increase the activity of damaged cells and the level of cell SOD, reduce the level of MDA in damaged cells, reduce the cell apoptosis rate, reduce the activation of Capase-3, reduce the expression of pro-apoptotic protein Bax, and increase the expression of anti-apoptotic protein Bcl-2, indicating that Sijunzi Decoction-containing serum can protect liver stem cells from the aspects of promoting liver oval cell proliferation and resisting cell damage.

[0119] As a further improvement, the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells, characterized in that: The detection method comprises the following steps: S1. Cell culture: Rat liver stem cells WB-F344 were cultured in DMEM medium containing 10% newborn calf serum at 37°C and 5% CO2. They were allowed to grow adherently and were passaged every 2-3 days. Cells in the logarithmic growth phase were used for the experiment. S2. Preparation of drug-containing serum: Rats were gavaged with Sijunzi decoction for 7 consecutive days. Serum was collected 2 hours after the last administration, sterilized with a 0.22 μm microporous filter membrane, and stored at -80°C for later use; S3. Cell proliferation assay: 4×10 4 The cells were seeded in 96-well plates at a cell density of 100 cells / mL, and three concentrations of drug-containing serum were added in sequence. After culturing for 24 hours, the cell activity was determined by the CCK-8 method; S4. Establishment and treatment of cell injury model: cells were treated with 100 μmol / L H2O2 for 40 minutes, followed by addition of 10% drug-containing serum for 24 hours, and cell viability and apoptosis were detected by CCK-8 method, Hoechst 33342 staining, and Annexin V / PI staining; S5. Detection of oxidative stress related indicators: The total SOD activity of cells was detected by SOD kit, and the MDA content of cells was detected by MDA kit; S6. Protein expression detection: Cell proteins were extracted and the expression levels of apoptosis-related proteins Bax, Bcl-2 and caspase-3 were detected by Western blot.

2. A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells according to claim 1, characterized in that: When the CCK-8 method is used for cell viability detection: 10 μL of CCK-8 reagent is added to each well and incubated at 37° C. for 2.5 hours; the absorbance value is measured at a wavelength of 450 nm using an enzyme marker to calculate cell proliferation or activity.

3. A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells according to claim 1, characterized in that: The Hoechst 33342 staining experiment includes the following steps: Add 10 μg / mL Hoechst 33342 dye to each well and incubate in the dark for 10 minutes; use a fluorescence microscope to observe changes in nuclear morphology to identify apoptotic cells.

4. A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells according to claim 1, characterized in that: When detecting oxidative stress related indicators by SOD and MDA: SOD activity was calculated by colorimetry using an enzyme reader, and the result was expressed in U / mgprot; MDA content was calculated by colorimetry, and the result was expressed in nmol / mgprot.

5. A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells according to claim 1, characterized in that: In Western blot detection: the electrophoresis conditions were 12.5% ​​SDS polyacrylamide gel electrophoresis, the stacking gel voltage was 80V, and the separation gel voltage was 100V; the transfer conditions were 50V constant voltage transfer for 4 hours, and the blocking solution was a mixture of 1g skim milk powder and 0.2g BSA; the primary antibodies included Bax, Bcl-2 and caspase-3 antibodies, and the dilution ratio was 1:4000.

6. A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells according to claim 1, characterized in that: The concentrations of drug-containing serum added to S3 were 5%, 10% and 20%.

7. A method for detecting the effect of Sijunzi Decoction-containing serum on liver stem cells according to claim 1, characterized in that: The concentration of the Sijunzi Decoction decoction in S2 was 1 mL / 100 g.

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