Method for detecting influence of bilirubin stimulation on human umbilical cord mesenchymal stem cells

Abstract

The invention discloses a method for detecting the influence of bilirubin stimulation on human umbilical cord mesenchymal stem cells, and relates to the technical field of experimental medicine. Comprising the following steps: S1, culturing cells; s2, preparing a bilirubin solution; s3, detecting the cell survival rate by a CCK-8 method; s4, adipogenic differentiation induction; s5, osteogenic differentiation induction; s6, determining the concentration of cell factors through an ELISA method; and S7, detecting the expression of the apoptotic protein by using Western blotting. The invention provides a detailed and clear experimental method which is used for detecting the influence of bilirubin stimulation on human umbilical cord mesenchymal stem cells. The influence of bilirubin on hUC-MSCs can be comprehensively evaluated through the steps of detecting the cell survival rate through a CCK-8 method, inducing adipogenesis and osteogenesis differentiation, determining the cell factor concentration through an ELISA method, detecting apoptosis protein expression through Western blotting and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of experimental medicine, and particularly relates to a method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells. Background Art

[0002] Neonatal hyperbilirubinemia is a common disease in the neonatal period. If not treated in time, it can lead to acute bilirubin encephalopathy and even severe neurological sequelae. Currently, there is no effective treatment method. Mesenchymal stem cell therapy has shown significant potential in the treatment of nerve injury diseases, but whether high-concentration bilirubin will interfere with its function is still unclear.

[0003] In recent years, with the in-depth development of the stem cell research field, it has begun to be used in the treatment of various neonatal diseases and their complications, including neonatal bronchopulmonary dysplasia, neonatal hypoxic-ischemic encephalopathy, cerebral palsy, neonatal necrotizing enterocolitis and other diseases. Some diseases have started clinical treatment trials using stem cells, and the effectiveness of stem cell treatment has been proven. Research shows that these cells can be isolated from bone marrow, adipose tissue, peripheral blood, placenta, cord blood, etc., and have the potential for multi-directional differentiation such as adipogenesis, osteogenesis, chondrogenesis, and neurogenesis. Among them, perinatal umbilical cord mesenchymal stem cells (MSCs) have attracted the attention of researchers. Compared with adult stem cells, perinatal umbilical cord mesenchymal stem cells have greater clinical application value, not only with strong proliferation ability, but also can be safely used for autologous and allogeneic treatment. Research shows that in addition to its powerful differentiation effect, mesenchymal stem cells can also produce anti-inflammatory and anti-apoptotic effects through the paracrine mechanism under inflammatory conditions. For example, through mesenchymal stem cell treatment of BPD or HIE, it can be found that the paracrine function of mesenchymal stem cells plays a more obvious role in treatment than its differentiation function.

[0004] Although the current mesenchymal stem cell therapy technology is not yet mature, it provides a new direction for further exploring its application in the treatment of neonatal hyperbilirubinemia and its related encephalopathy. The purpose of the present invention is to observe the changes in the survival rate and paracrine function of mesenchymal stem cells under different bilirubin concentrations, and provide a theoretical basis for further using mesenchymal stem cells to treat bilirubin encephalopathy. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies existing in the prior art, and to propose a method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells.

[0006] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0007] A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells, comprising the following steps:

[0008] S1: Cell culture;

[0009] S2: Preparation of bilirubin solution;

[0010] S3: Detection of cell viability by CCK-8 method;

[0011] S4: Induction of adipogenic differentiation;

[0012] S5: Induction of osteogenic differentiation;

[0013] S6: Determination of cytokine concentration by ELISA method;

[0014] S7: Detection of apoptotic protein expression by Western blotting.

[0015] Preferably: The cell culture in S1 specifically includes the following steps:

[0016] S11: Inoculate hUC-MSCs in DMEM medium containing 10% FBS and double antibodies, and culture in a constant temperature incubator;

[0017] S12: Observe the cell growth condition every day, and change the medium every 3 days;

[0018] S13: Perform subculture.

[0019] Preferably: The preparation of bilirubin solution in S2 specifically includes the following steps:

[0020] S21: Dissolve bilirubin powder in DMSO in a 37°C water bath to prepare a 30 mg / dl bilirubin solution, filter it, and store it in the dark;

[0021] S22: Dilute it to the required concentration with medium before use.

[0022] Preferably: The detection of cell viability by CCK-8 method in S3 specifically includes the following steps:

[0023] S31: Take p3 generation hUC-MSCs, inoculate them, and after culturing for 24 hours, discard the medium;

[0024] S32: Add bilirubin solutions with different concentrations respectively, and continue to culture for 12 hours and 24 hours;

[0025] S33: Discard the medium, wash it with PBS, and then add medium containing 10% CCK-8 reagent to each well and incubate;

[0026] S34: Measure the absorbance once every hour, measure the absorbance of each well at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell viability.

[0027] Preferably: The induction of adipogenic differentiation in S4 specifically includes the following steps:

[0028] S41: Prepare adipogenic differentiation medium A and medium B according to the kit instructions;

[0029] S42: Coat a 6-well plate with 0.1% gelatin for 30 minutes, seed the hUC-MSCs suspension into the 6-well plate, and add 2 ml of culture medium to each well;

[0030] S43: After 24 hours, the culture medium was discarded, and the bilirubin concentration group was set and cultured for 12 hours. The bilirubin-containing culture medium was discarded and the cells were washed with PBS until no residue was left.

[0031] S44: Add 2 ml of solution A to each group. After 3 days of induction, discard solution A and add 2 ml of solution B. After 1 day, discard solution B and replace it with solution A to continue induction. Solution A and solution B are used alternately, and the cell status is observed under a microscope every day during this period.

[0032] S45: Repeat the above process until the required lipid droplets appear and prepare for staining; discard the culture medium, wash each well 3 times with PBS, add paraformaldehyde solution to fix for 30 minutes, discard the fixative, and thoroughly wash with PBS; add 2 ml of Oil Red O dye working solution to each group, stain at room temperature for 30 minutes, aspirate the staining solution, gently wash 3 times with PBS to fully wash away the staining solution, add 2 ml of PBS to each well, and observe the adipogenic differentiation staining effect under a microscope.

[0033] Preferably: in said S41, the components of culture medium A are:

[0034] Basal medium: Dulbecco's modified Eagle's medium;

[0035] Fetal bovine serum: 10%;

[0036] Double antibody: 1%;

[0037] Insulin: 10 μg / ml;

[0038] Dexamethasone: 1 μM;

[0039] IBMX: 500 μM;

[0040] Indomethacin: 100 μM;

[0041] Oil Red O dye: used for staining detection;

[0042] The preparation method is as follows:

[0043] Mix DMEM medium, FBS and double antibody evenly to prepare basal medium;

[0044] Insulin, dexamethasone, IBMX, and indomethacin were added to the basal medium and dissolved thoroughly;

[0045] 6-well plates were coated with 0.1% gelatin for 30 min to promote cell attachment;

[0046] The human umbilical cord mesenchymal stem cell suspension was seeded into a 6-well plate, and 2 ml of culture medium was added to each well.

[0047] Preferably: in said S41, the components of the culture medium B are:

[0048] Basal medium: Dulbecco's modified Eagle's medium;

[0049] Fetal bovine serum: 10%;

[0050] Double antibody: 1%;

[0051] Insulin: 10 μg / ml;

[0052] Dexamethasone: 1 μM;

[0053] Oil Red O dye: used for staining detection;

[0054] The preparation method is as follows:

[0055] Mix DMEM medium, FBS and double antibody evenly to prepare basal medium;

[0056] Add insulin and dexamethasone to the basal medium and dissolve them fully;

[0057] 6-well plates were coated with 0.1% gelatin for 30 min to promote cell attachment;

[0058] The human umbilical cord mesenchymal stem cell suspension was seeded into a 6-well plate, and 2 ml of culture medium was added to each well.

[0059] Preferably, the osteogenic differentiation induction of S5 specifically comprises the following steps:

[0060] S51: Prepare osteogenic differentiation medium according to the kit instructions and store at 4°C;

[0061] S51: After coating a 6-well plate with 0.1% gelatin, the hUC-MSCs suspension was added to the six-well plate and 2 ml of culture medium was added to each well;

[0062] S51: After 24 hours, the culture medium was discarded, and the bilirubin concentration group was set and cultured for 12 hours. The bilirubin-containing culture medium was discarded and the cells were washed with PBS until no residue was left.

[0063] S51: Add 2 ml of osteogenic differentiation medium to each group to start differentiation induction. Observe cell morphology and number changes under a microscope every day, and replace the differentiation medium every 3 days.

[0064] S51: After 4 weeks of induction, when calcium nodules appear under the microscope, prepare for staining; discard the culture medium, wash each well 3 times with PBS, add paraformaldehyde solution for fixation for 30 minutes, discard the fixing solution, and thoroughly wash with PBS; add 2 ml of alizarin red working solution to each well, stain for 15 minutes, thoroughly wash away the excess staining solution with PBS, then add PBS to each well, and observe the osteogenic differentiation staining effect under the microscope.

[0065] Preferably: The ELISA method for measuring the cytokine concentration in S6 specifically includes the following steps:

[0066] S61: Adjust the cell number in a six-well plate. After culturing for 24 hours, intervene with bilirubin at different concentrations for 12 hours;

[0067] S62: Aspirate the cell supernatant, centrifuge for 5 minutes, take the supernatant and aliquot it, and store it at -80 °C for later use;

[0068] S63: Detect the expression levels of TSG-6 and IL-6 respectively according to the ELISA kit instructions; take out the kit and cell supernatant, and equilibrate at room temperature for more than 30 minutes; set up sample, blank, and standard product wells, add the prepared standard product and cell supernatant to the corresponding wells, 100 μl per well, do not add to the blank well, seal each well with a sealing film, and incubate in a 37 °C incubator for 90 minutes;

[0069] S64: After washing the plate 4 times, add the biotinylated antibody working solution, seal the plate and incubate at 37 °C for 60 minutes; wash the plate 4 times again, add the enzyme conjugate working solution, seal the plate with a sealing film, and incubate at 37 °C for 30 minutes; wash the plate 4 times, add 100 μl of chromogenic agent to each well, and incubate at 37 °C in the dark for 15 - 20 minutes; finally, add 100 μl of stop solution to each well, and measure the absorbance of each well at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader within 15 minutes. After establishing the standard curve regression equation according to the OD values of the standard products, substitute the actual OD value of the specimen into the regression equation to obtain the concentration of the specimen.

[0070] Preferably: The Western blotting for detecting the expression of apoptotic proteins in S7 specifically includes the following steps:

[0071] S71: After stimulation with bilirubin, digest the cells, lyse the cells with RIPA on ice, centrifuge and shake once every 10 minutes, centrifuge for 10 minutes after 30 minutes, collect the supernatant, and store it at -80 °C;

[0072] S72: Detect the protein concentration with a BCA kit. Set up the standard curve wells according to the instructions, add the samples to be measured to the remaining wells, and measure the absorbance at 562 nm to calculate the protein concentration;

[0073] S73: Add 20 μl of SDS-PAGE to every 80 μl of protein, heat and denature it in the sample well at 100 °C for 5 minutes; Prepare a 10% electrophoresis gel according to the instructions. Before starting the electrophoresis, add an equal amount of protein to each lane. After the electrophoresis is completed, transfer it to a PVDF membrane;

[0074] S74: Block the PVDF membrane with a rapid blocking solution on a shaker for 20 minutes, add the primary antibody and incubate overnight at 4 °C. The next day, wash the membrane 3 times with TBST, 10 minutes each time, and then incubate with the secondary antibody for 1 hour; After washing the membrane 3 times again, prepare an ECL hypersensitive developing solution and develop the image in the dark, and analyze the bands to calculate the protein amount.

[0075] The beneficial effects of the present invention are as follows:

[0076] 1. The present invention provides a detailed and clear experimental method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells; By detecting cell viability through the CCK-8 method, inducing adipogenic and osteogenic differentiation, measuring cytokine concentrations by ELISA method, and detecting the expression of apoptotic proteins by Western blotting and other steps, the effect of bilirubin on hUC-MSCs can be comprehensively evaluated.

[0077] 2. The results of the present invention show that bilirubin has a toxic effect on human umbilical cord mesenchymal stem cells. With the increase of bilirubin concentration, its apoptosis will increase, thereby reducing the survival rate and proliferation ability; However, bilirubin does not affect the paracrine and differentiation abilities of mesenchymal stem cells; This method provides an important experimental basis for further studying the treatment of bilirubin encephalopathy. Description of the Drawings

[0078] Figure 1 It is a control schematic diagram of the survival rate of umbilical cord mesenchymal stem cells in each group with the increase of bilirubin concentration in the present invention;

[0079] Figure 2 It is a schematic diagram of the levels of IL-6 and TSG-6 after bilirubin stimulates umbilical cord mesenchymal stem cells for 12 h in the present invention;

[0080] Figure 3 It is a schematic diagram of detecting the expression of caspase 3, Bax and Bcl-2 by Western blot in the present invention. Detailed Embodiments

[0081] The technical solutions of the present invention will be further described in detail below in conjunction with the specific embodiments.

[0082] Example 1:

[0083] A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells, which is realized based on a detection experiment, and includes the following steps:

[0084] S1: Cell culture;

[0085] S2: Preparation of bilirubin solution;

[0086] S3: Detection of cell viability by CCK-8 method;

[0087] S4: Induction of adipogenic differentiation;

[0088] S5: Induction of osteogenic differentiation;

[0089] S6: Determination of cytokine concentration by ELISA method;

[0090] S7: Detection of apoptotic protein expression by Western blotting.

[0091] Among them, the cell culture in S1 specifically includes the following steps:

[0092] S11: Inoculate hUC-MSCs into DMEM medium containing 10% FBS and double antibiotics, and culture them in a constant temperature incubator at 37°C and 5% CO2;

[0093] S12: Observe the cell growth condition every day and change the medium every 3 days;

[0094] S13: When the cells occupy 80% of the field of view, perform subculture.

[0095] Among them, the preparation of bilirubin solution in S2 specifically includes the following steps:

[0096] S21: Dissolve bilirubin powder in DMSO in a 37°C water bath to prepare a 30 mg / dl bilirubin solution, filter it, and store it in the dark at -20°C in the refrigerator;

[0097] S22: Before use, dilute it to the required concentration (such as 0 mg / dl, 5 mg / dl, 10 mg / dl, 15 mg / dl) with the medium.

[0098] Among them, the detection of cell viability by CCK-8 method in S3 specifically includes the following steps:

[0099] S31: Take p3 generation hUC-MSCs, inoculate 1×10 5 cells per well into a 96-well plate. After culturing for 24 hours, discard the medium;

[0100] S32: Add bilirubin solutions with different concentrations (0 mg / dl, 5 mg / dl, 10 mg / dl, 15 mg / dl) respectively, and continue to culture for 12 hours and 24 hours;

[0101] S33: Discard the culture medium, wash with PBS, and then add 100 μl of culture medium containing 10% CCK-8 reagent to each well. Incubate at 37 °C under 5% CO2 for 4 hours;

[0102] S34: Measure the absorbance every hour. Measure the absorbance of each well at 450 nm using a microplate reader and calculate the cell survival rate.

[0103] The results showed that with the increase in bilirubin concentration, the cell survival rate decreased significantly (P < 0.001), and there was no significant difference between 12 hours and 24 hours (P > 0.05).

[0104] Among them, the adipogenic differentiation induction of S4 specifically includes the following steps:

[0105] S41: Prepare adipogenic differentiation media A and B according to the kit instructions;

[0106] S42: Coat a 6-well plate with 0.1% gelatin for 30 minutes. Suspend hUC-MSCs and inoculate them into the 6-well plate at a density of 2×10 4 cells / cm 2 Add 2 ml of culture medium to each well;

[0107] S43: After 24 hours, discard the culture medium, set up bilirubin concentration groups (0 mg / dl, 5 mg / dl, 10 mg / dl, 15 mg / dl), culture for 12 hours, discard the bilirubin-containing culture medium, and wash with PBS until there is no residue;

[0108] S44: Add 2 ml of solution A to each group. After inducing for 3 days, discard solution A, add 2 ml of solution B, discard solution B after 1 day, and replace it with solution A to continue the induction; Alternate between solution A and solution B, and observe the cell status under the microscope every day;

[0109] S45: Repeat the above process until sufficient lipid droplets of appropriate size appear and prepare for staining; Discard the culture medium, wash each well 3 times with PBS, add 2 ml of 4% paraformaldehyde solution to fix for 30 minutes, discard the fixing solution, and thoroughly wash with PBS; Add 2 ml of Oil Red O dye working solution to each group, stain at room temperature for 30 minutes, aspirate the staining solution, gently wash 3 times with PBS to fully remove the staining solution, add 2 ml of PBS to each well, and observe the adipogenic differentiation staining effect under the microscope;

[0110] The results showed that there was no obvious difference in the staining effect among groups, indicating that bilirubin had no obvious effect on adipogenic differentiation.

[0111] Among them, the osteogenic differentiation induction of S5 specifically includes the following steps:

[0112] S51: Prepare osteogenic differentiation medium according to the kit instructions and store it at 4 °C;

[0113] S51: After coating a 6-well plate with 0.1% gelatin, add the hUC-MSCs suspension into the 6-well plate at a density of 2×10 4 cells / cm 2 and add 2 ml of culture medium to each well;

[0114] S51: After 24 hours, discard the culture medium, set up bilirubin concentration groups (0 mg / dl, 5 mg / dl, 10 mg / dl, 15 mg / dl), culture for 12 hours, discard the culture medium containing bilirubin, and wash with PBS until there is no residue;

[0115] S51: Add 2 ml of osteogenic differentiation medium to each group to start induction of differentiation. Observe the cell morphology and number changes under the microscope every day, and replace the fresh induction differentiation medium every 3 days;

[0116] S51: After 4 weeks of induction, prepare for staining when calcium nodules appear under the microscope; discard the culture medium, wash each well 3 times with PBS, add 2 ml of 4% paraformaldehyde solution to fix for 30 minutes, discard the fixing solution, and wash thoroughly with PBS; add 2 ml of alizarin red working solution to each well, stain for 15 minutes, wash away the excess staining solution with PBS sufficiently, and then add PBS to each well. Observe the osteogenic differentiation staining effect under the microscope;

[0117] The results showed that there was no significant difference in the staining effect among the groups, indicating that bilirubin had no significant effect on osteogenic differentiation.

[0118] Among them, the ELISA method for measuring the cytokine concentration in S6 specifically includes the following steps:

[0119] S61: Adjust the cell number to 1×10 6 cells per well in a 6-well plate. After culturing for 24 hours, intervene with different concentrations of bilirubin (0 mg / dL, 5 mg / dL, 10 mg / dL, 15 mg / dL) for 12 hours;

[0120] S62: Aspirate the cell supernatant, centrifuge at 14,000 rpm for 5 minutes at 4°C, and then divide the supernatant into tubes of 150 μl each and store them in a -80°C refrigerator for later use;

[0121] S63: Detect the expression levels of TSG-6 and IL-6 respectively according to the ELISA kit instructions; take out the kit and the cell supernatant, and equilibrate at room temperature for more than 30 minutes; set up the sample, blank, and standard product wells, add the prepared standard product and cell supernatant to the corresponding wells, 100 μl per well, do not add to the blank well, seal each well with a sealing film, and incubate in a 37°C incubator for 90 minutes;

[0122] S64: After washing the plate 4 times, add the biotinylated antibody working solution (not added to the blank wells), seal the plate and incubate at 37 °C for 60 minutes; wash the plate 4 times again, add the enzyme conjugate working solution (not added to the blank wells), seal the plate with a sealing film, and incubate at 37 °C for 30 minutes; wash the plate 4 times, add 100 μl of chromogenic reagent to each well, and incubate at 37 °C in the dark for 15 - 20 minutes; finally, add 100 μl of stop solution to each well, and measure the absorbance (OD value) of each well at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader within 15 minutes. After establishing the standard curve regression equation based on the OD values of the standards, substitute the actual OD value of the specimen (OD value of each well - OD value of the blank well) into the regression equation to obtain the concentration of the specimen.

[0123] The results showed that the levels of IL-6 and TSG-6 were significantly increased after bilirubin stimulation (P < 0.05), but there were no significant differences among the concentration groups.

[0124] Among them, the Western blotting for detecting the expression of apoptotic proteins in S7 specifically includes the following steps:

[0125] S71: After bilirubin stimulation, digest the cells, lyse the cells with RIPA on ice, centrifuge and shake once every 10 minutes, centrifuge at 14,000 r / s at 4 °C for 10 minutes after 30 minutes, collect the supernatant, and store it in a -80 °C refrigerator;

[0126] S72: Detect the protein concentration with a BCA kit, set the standard curve wells according to the instructions, add the test samples to the remaining wells, and measure the absorbance at 562 nm to calculate the protein concentration;

[0127] S73: Add 20 μl of SDS-PAGE to every 80 μl of protein, heat and denature at 100 °C for 5 minutes in the sample wells; prepare a 10% electrophoresis gel according to the instructions. Before starting the electrophoresis, add an equal amount of protein (30 μg) to each channel, and transfer it to a PVDF membrane after the electrophoresis;

[0128] S74: Block the PVDF membrane with a rapid blocking solution on a shaker for 20 minutes, add the primary antibodies (bax, bcl-2, caspase-3 antibodies), and incubate at 4 °C overnight. The next day, wash the membrane 3 times with TBST, 10 minutes each time, and then incubate with the secondary antibody for 1 hour; after washing the membrane 3 times again, prepare the ECL hypersensitive developing solution for developing in the dark and analyze the bands to calculate the protein amount.

[0129] The results showed that the expressions of apoptotic proteins Bax, Bcl-2, and Caspase-3 in the bilirubin group were significantly increased (P < 0.05), but there were no significant differences among the intervention groups.

[0130] The present invention provides a detailed and clear experimental method for detecting the effects of bilirubin stimulation on human umbilical cord mesenchymal stem cells. By detecting cell viability through the CCK-8 method, inducing adipogenic and osteogenic differentiation, measuring cytokine concentrations by ELISA, and detecting the expression of apoptotic proteins by Western blotting, etc., the effects of bilirubin on hUC-MSCs can be comprehensively evaluated. The results of the present invention show that bilirubin has a toxic effect on human umbilical cord mesenchymal stem cells, and increasing bilirubin concentration will increase apoptosis, thereby reducing cell viability and proliferation ability; however, bilirubin does not affect the paracrine and differentiation abilities of mesenchymal stem cells. This method provides an important experimental basis for further research on the treatment of bilirubin encephalopathy.

[0131] In this embodiment:

[0132] In the detection of cell viability:

[0133] Dulbecco's Modified Eagle Medium (DMEM): provided by Gibco, USA;

[0134] Fetal Bovine Serum (FBS): provided by Gibco, USA, and the proportion added to the medium is 10%;

[0135] Double antibody (penicillin and streptomycin): provided by Gibco, USA, used to prevent microbial contamination.

[0136] In the induction of adipogenic differentiation:

[0137] Adipogenic differentiation media A and B for human umbilical cord mesenchymal stem cells: provided by Cyagen Biosciences, Guangzhou;

[0138] 0.1% gelatin: coat the 6-well plate to promote cell adhesion.

[0139] In the induction of osteogenic differentiation:

[0140] Osteogenic differentiation medium for human umbilical cord mesenchymal stem cells: provided by Cyagen Biosciences, Guangzhou, and stored at 4°C;

[0141] 0.1% gelatin: coat the 6-well plate to promote cell adhesion.

[0142] In the measurement of cytokine concentration by ELISA:

[0143] Dilution Buffer R, Wash Buffer, Standard Diluent, HRP Conjugate, Substrate Reagent Set: IL-6 ELISA kit and TNF-α ELISA kit provided by SZ SinoCare Co., Ltd., China.

[0144] In the detection of apoptotic protein expression by Western Blotting:

[0145] RIPA lysis buffer: provided by Shanghai Beyotime Biotechnology Co., Ltd., used for lysing cells to extract total protein;

[0146] BCA Protein Assay Kit: provided by Shanghai Beyotime Biotechnology Co., Ltd., used for detecting protein concentration;

[0147] SDS-PAGE Gel Preparation Kit: provided by Shanghai Yeasen Biotechnology Co., Ltd., used for preparing electrophoresis gels;

[0148] PVDF membrane: commonly used for protein transfer;

[0149] β-actin antibody: provided by Chengdu Zhengneng Biotechnology Co., Ltd., used as an internal reference;

[0150] Cck-8 Kit: provided by ApexBio Technology LLC (USA), used for detecting cell viability;

[0151] Osteogenic Induction Differentiation Kit and Adipogenic Induction Differentiation Kit for human umbilical cord mesenchymal stem cells: provided by Cyagen Biosciences Inc. (Guangzhou).

[0152] Example 2:

[0153] A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells. In adipogenic differentiation induction, the specific components and preparation methods of medium A and B are as follows:

[0154] Medium A:

[0155] Specific components:

[0156] Basal medium: Dulbecco's Modified Eagle Medium (DMEM)

[0157] Fetal Bovine Serum (FBS): 10%

[0158] Dual antibiotics (penicillin, streptomycin): 1%

[0159] Insulin: 10 μg / ml

[0160] Dexamethasone: 1 μM

[0161] IBMX: 500 μM

[0162] Indomethacin: 100 μM

[0163] Oil Red O dye: used for staining detection

[0164] Preparation method:

[0165] Mix DMEM medium with FBS and double antibiotics evenly to prepare a basal medium;

[0166] Add insulin, dexamethasone, IBMX and indomethacin to the basal medium and dissolve them thoroughly;

[0167] Coat a 6-well plate with 0.1% gelatin for 30 minutes to promote cell adhesion;

[0168] Seed the human umbilical cord mesenchymal stem cell suspension at 2×10 4 cells / cm 2 in a 6-well plate, and add 2 ml of medium to each well.

[0169] Medium B:

[0170] Specific components:

[0171] Basal medium: Dulbecco's Modified Eagle Medium (DMEM);

[0172] Fetal bovine serum (FBS): 10%;

[0173] Double antibiotics (penicillin, streptomycin): 1%;

[0174] Insulin: 10 μg / ml;

[0175] Dexamethasone: 1 μM;

[0176] Oil Red O dye: for staining detection;

[0177] Preparation method:

[0178] Mix DMEM medium with FBS and double antibiotics evenly to prepare a basal medium;

[0179] Add insulin and dexamethasone to the basal medium and dissolve them thoroughly;

[0180] Coat a 6-well plate with 0.1% gelatin for 30 minutes to promote cell adhesion;

[0181] Seed the human umbilical cord mesenchymal stem cell suspension at 2×10 4 cells / cm 2 in a 6-well plate, and add 2 ml of medium to each well.

[0182] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells, characterized in that, It includes the following steps: S1: Cell culture; S2: Preparation of bilirubin solution; S3: Detection of cell viability by CCK-8 method; S4: Induction of adipogenic differentiation; S5: Induction of osteogenic differentiation; S6: Determination of cytokine concentration by ELISA method; S7: Detection of apoptotic protein expression by Western blotting.

2. The method for detecting the influence of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 1, wherein The cell culture in S1 specifically includes the following steps: S11: Inoculate hUC-MSCs into DMEM medium containing 10% FBS and double antibiotics, and place them in a constant temperature incubator for culture; S12: Observe the cell growth condition every day and change the medium every 3 days; S13: Perform subculture.

3. The method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 2, wherein The preparation of bilirubin solution in S2 specifically includes the following steps: S21: Dissolve bilirubin powder in DMSO in a 37°C water bath to prepare a 30 mg / dl bilirubin solution, filter it and store it in the dark; S22: Dilute it to the required concentration with medium before use.

4. The method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 3, characterized in that, The detection of cell viability by CCK-8 method in S3 specifically includes the following steps: S31: Take p3 generation hUC-MSCs, inoculate them, and after culturing for 24 hours, discard the medium; S32: Add bilirubin solutions with different concentrations respectively and continue to culture for 12 hours and 24 hours; S33: Discard the medium, wash it with PBS, and then add medium containing 10% CCK-8 reagent to each well for incubation; S34: Measure the absorbance once every hour, measure the absorbance of each well at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell viability.

5. A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 4, characterized in that The induction of adipogenic differentiation in S4 specifically includes the following steps: S41: Prepare adipogenic differentiation medium A and medium B according to the kit instructions; S42: Coat a 6-well plate with 0.1% gelatin for 30 minutes, inoculate the hUC-MSCs suspension into the 6-well plate, and add 2 ml of medium to each well; S43: After 24 hours, discard the medium, set up the bilirubin concentration groups, culture for 12 hours, discard the medium containing bilirubin, and wash it with PBS until there is no residue; S44: Add 2 ml of solution A to each group. After inducing for 3 days, discard solution A, add 2 ml of solution B, and discard solution B after 1 day, then change to solution A to continue the induction; Solution A and solution B are used alternately, and the cell state is observed under the microscope every day during this period; S45: Repeat the above process until lipid droplets meeting the requirements appear and prepare for staining; Discard the medium, wash each well 3 times with PBS, add paraformaldehyde solution to fix for 30 minutes, discard the fixing solution, and wash it thoroughly with PBS; Add 2 ml of oil red O dye working solution to each group, stain at room temperature for 30 minutes, aspirate the staining solution, gently wash it 3 times with PBS to fully wash away the staining solution, add 2 ml of PBS to each well, and observe the adipogenic differentiation staining effect under the microscope.

6. A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 5, wherein In S41, the components of medium A are: Basal medium: Dulbecco's modified Eagle's medium; Fetal bovine serum: 10%; Double antibiotics: 1%; Insulin: 10 μg / ml; Dexamethasone: 1 μM; IBMX: 500 μM; Indomethacin: 100 μM; Oil red O dye: For staining detection; The preparation method is as follows: Mix DMEM medium, FBS and double antibody evenly to prepare basal medium; Insulin, dexamethasone, IBMX, and indomethacin were added to the basal medium and dissolved thoroughly; 6-well plates were coated with 0.1% gelatin for 30 min to promote cell attachment; The human umbilical cord mesenchymal stem cell suspension was seeded into a 6-well plate, and 2 ml of culture medium was added to each well.

7. A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 6, characterized in that In said S41, the components of culture medium B are: Basal medium: Dulbecco's modified Eagle's medium; Fetal bovine serum: 10%; Double antibody: 1%; Insulin: 10 μg / ml; Dexamethasone: 1 μM; Oil Red O dye: used for staining detection; The preparation method is as follows: Mix DMEM medium, FBS and double antibody evenly to prepare basal medium; Add insulin and dexamethasone to the basal medium and dissolve them fully; 6-well plates were coated with 0.1% gelatin for 30 min to promote cell attachment; The human umbilical cord mesenchymal stem cell suspension was seeded into a 6-well plate, and 2 ml of culture medium was added to each well.

8. A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 5, wherein The osteogenic differentiation induction of S5 specifically comprises the following steps: S51: Prepare osteogenic differentiation medium according to the kit instructions and store at 4°C; S51: After coating a 6-well plate with 0.1% gelatin, the hUC-MSCs suspension was added to the six-well plate and 2 ml of culture medium was added to each well; S51: After 24 hours, the culture medium was discarded, and the bilirubin concentration group was set and cultured for 12 hours. The bilirubin-containing culture medium was discarded and the cells were washed with PBS until no residue was left. S51: Add 2 ml of osteogenic differentiation medium to each group to start differentiation induction. Observe cell morphology and number changes under a microscope every day, and replace the differentiation medium every 3 days. S51: After 4 weeks of induction, calcium nodules appeared under the microscope and were ready for staining; the culture medium was discarded, each well was washed 3 times with PBS, paraformaldehyde solution was added for fixation for 30 minutes, the fixative was discarded, and the cells were thoroughly washed with PBS; 2 ml of Alizarin Red working solution was added to each well and stained for 15 minutes. After the excess staining solution was thoroughly washed away with PBS, PBS was added to each well and the osteogenic differentiation staining effect was observed under a microscope.

9. A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 8, characterized in that, The S6 ELISA method for determining cytokine concentrations specifically comprises the following steps: S61: Cell numbers were adjusted in six-well plates, and after 24 hours of culture, different concentrations of bilirubin were used for intervention for 12 hours; S62: Aspirate the cell supernatant, centrifuge for 5 minutes, take the supernatant and aliquot it, store at -80℃ for later use; S63: Detect the expression levels of TSG-6 and IL-6 according to the ELISA kit instructions; remove the kit and cell supernatant and equilibrate at room temperature for at least 30 minutes; set up sample, blank, and standard wells, add the prepared standard and cell supernatant to the corresponding wells, 100 μl per well, and leave blank wells untouched. Seal each set of wells with sealing film and incubate in a 37°C incubator for 90 minutes; S64: After washing the plate 4 times, add the biotinylated antibody working solution. After sealing the plate, incubate at 37°C for 60 minutes; then wash the plate 4 times again, add the enzyme conjugate working solution, seal the plate with a sealing film, and incubate at 37°C for 30 minutes; wash the plate 4 times, add 100 μl of chromogenic agent to each well, and incubate in the dark at 37°C for 15 - 20 minutes; finally, add 100 μl of stop solution to each well, and measure the absorbance of each well at a wavelength of 450 nm with an enzyme - linked immunosorbent assay (ELISA) reader within 15 minutes. After establishing the standard curve regression equation based on the OD values of the standard products, substitute the actual OD value of the specimen into the regression equation to obtain the concentration of the specimen.

10. A method for detecting the effect of bilirubin stimulation on human umbilical cord mesenchymal stem cells according to claim 9, characterized in that, The Western blotting for detecting the expression of apoptotic proteins in S7 specifically includes the following steps: S71: After stimulation with bilirubin, digest the cells, lyse the cells with RIPA on ice, centrifuge and shake once every 10 minutes, centrifuge for 10 minutes after 30 minutes, collect the supernatant, and store it in an environment of -80°C. S72: Detect the protein concentration using a BCA kit. Set the standard curve wells according to the instructions, and add the test samples to the remaining wells to measure the absorbance at 562 nm to calculate the protein concentration. S73: Add 20 μl of SDS - PAGE to 80 μl of protein, heat and denature at 100°C for 5 minutes in the sample wells; prepare a 10% electrophoresis gel according to the instructions. Before starting the electrophoresis, add an equal amount of protein to each lane. After the electrophoresis is completed, transfer it to a PVDF membrane. S74: Block the PVDF membrane with a rapid blocking solution on a shaker for 20 minutes, add the primary antibody, and incubate overnight at 4°C. The next day, wash the membrane 3 times with TBST, 10 minutes each time, and then incubate with the secondary antibody for 1 hour; after washing the membrane 3 times again, prepare an ECL hypersensitive developing solution for developing in the dark, and analyze the bands to calculate the protein amount.

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