Verification method for gene interference regulation and control of bovine adipocyte proliferation and differentiation

Abstract

The invention discloses a verification method for regulating and controlling proliferation and differentiation of bovine adipocytes by gene interference, which comprises the following steps: cutting bovine adipocyte tissues into small blocks, directly pasting the small blocks in a culture dish for culture, and promoting cell growth and proliferation by using nutrition and growth factors of the tissues; a ZNF423 gene is targeted by adopting an siRNA technology, siRNA is transfected into bovine fat cells by utilizing a transfection reagent, and the siRNA is an interference fragment containing si-ZNF423; the method comprises the following steps: respectively extracting total RNA (Ribonucleic Acid) and total protein of bovine adipocytes, and detecting by utilizing qPCR (Quantitative Polymerase Chain Reaction) quantitative analysis, a WB (White Batch) technology, EdU dyeing, flow cytometry detection, oil red O, BODIPY (Boron Dipyrromethene), Nile Red dyeing, TG (Triglyceride) content determination and the like. According to the invention, ZNF423 gene expression is interfered by the interference fragment containing si-ZNF423, and the effect of inhibiting proliferation and differentiation of bovine adipocytes is detected by various methods, so that the effect of the interference fragment in the bovine adipocytes is known conveniently, and regulation and control of proliferation and differentiation of the bovine adipocytes are realized by interfering gene expression by the interference fragment. The beef quality can be improved; the beef cattle breeding can be optimized.

Description

Technical Field

[0001] The invention belongs to the technical field of cell experiments and relates to a verification method for regulating the proliferation and differentiation of bovine adipocytes through gene interference. Background Art

[0002] In recent years, with the growing market demand for high-quality beef, the optimization of fat deposition, the improvement of meat quality and the control of fat content have become one of the key areas of beef cattle breeding research. The proliferation, differentiation and lipid metabolism of adipocytes are regulated by multiple genes and complex signaling networks, which have a direct impact on beef quality.

[0003] The differentiation and regulation process of adipocytes involves multiple key genes and transcription factors. In the prior art, it has been clarified that transcription factors such as peroxisome proliferator-activated receptor γ (PPAR γ), CCAAT / enhancer binding protein α (C / EBP α) and sterol regulatory element binding protein-1c play a key role in adipogenesis. These factors regulate the expression of adipogenic genes by binding to specific DNA sequences. In the regulatory network of adipogenesis, ZNF423 (zinc finger protein 423) is considered to be an important transcriptional regulatory factor. ZNF423 contains 30 C2H2-type zinc finger domains, which can initiate the transcriptional cascade reaction of adipocyte differentiation by inducing the expression of master regulatory factors such as PPAR γ and C / EBP.

[0004] However, the existing technology lacks transfection methods with strong specificity and stable interference effects, which makes it difficult to achieve ideal effects in regulating the proliferation and differentiation of adipocytes, and makes the role and specific mechanism of ZNF423 regulation in bovine adipocytes unclear. Summary of the invention

[0005] In view of the above problems, the present invention proposes a verification method for gene interference regulation of bovine adipocyte proliferation and differentiation, which well solves the problems in the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A verification method for gene interference regulation of bovine adipocyte proliferation and differentiation comprises the following steps:

[0008] S1. Cut the bovine fat cell tissue into small pieces and directly place them in a culture dish for culture, using the tissue's own nutrients and growth factors to promote cell growth and proliferation;

[0009] S2, constructing an interference fragment containing si-ZNF423, using siRNA technology to target the ZNF423 gene, using a transfection reagent to transfect siRNA into bovine adipocytes, and setting up a control group without an interference fragment;

[0010] S3, extract the total RNA and total protein of bovine adipocytes, and detect the ZNF423 gene interference efficiency by qPCR and WB;

[0011] S4. EdU staining, flow cytometry, and qPCR techniques were used to detect the regulatory effects of the interference fragments on bovine adipocyte proliferation;

[0012] S5. Oil Red 0 staining, BODIPY, Nile Red staining, TG content determination, qPCR and WB were used to detect the regulatory effect of the interference fragment on bovine adipocyte differentiation.

[0013] Optionally, the interference fragment is specifically: forward: GCACCAACUCUCCCAUCUUTT, reverse: AAGAUGGAGAGUGUGCTT.

[0014] Optionally, the step S1 includes:

[0015] Adipose tissue pretreatment: The collected adipose tissue was washed several times with PBS solution containing 1% penicillin / streptomycin in the cell chamber, and the non-adipose tissue on the adipose tissue was removed using sterile instruments;

[0016] Tissue block preparation: After washing the adipose tissue three times again, the adipose tissue was cut into small pieces of 3 × 3 nm and inoculated into collagen-treated culture dishes;

[0017] Initial culture: Place the culture dish in a cell culture incubator at 37°C and 5% CO2, invert and culture for 6 hours, then add high-glucose DMEM medium containing 10% FBS;

[0018] Cell expansion: After a large number of fat cells grow around the tissue block, remove the tissue block and continue culturing until the cell confluence reaches 80-90%, then expand the culture and cryopreservation of the cells;

[0019] Cell differentiation induction: When the adipocyte confluence reached 100% and cell growth was inhibited, the medium was replaced with differentiation medium;

[0020] Differentiation maintenance: After 2 days of differentiation induction, the culture medium was replaced with maintenance medium. Thereafter, the culture medium was replaced every two days to maintain the differentiation state.

[0021] Optionally, step S2 includes:

[0022] Transfection pretreatment: Bovine adipocytes were seeded into 6-well culture dishes and 2 mL of growth medium without antibiotics was added. During transfection, the cells reached a seeding density of 70-80% of 3.0x10^5 cells / well;

[0023] Preparation of transfection complex: dilute the control group and si-ZNF423 validation group solutions and transfection reagent solution with Opti-MEM medium, mix thoroughly using a vortex mixer, and incubate the mixture at room temperature for 10-15 minutes;

[0024] Add transfection complexes to cells: Add 135 μL of the incubated complexes to a culture dish containing bovine adipocytes. At this time, replace the base medium with 1.5 ml of serum-free Opti-MEM culture medium to starve the cells.

[0025] Culture and observation: The culture dish was placed in an incubator at 37°C and 5% CO2. Six hours after transfection, the culture medium was replaced with complete growth medium.

[0026] Compared with the prior art, the present invention has the following beneficial effects: interfering with ZNF423 gene expression through an interfering fragment containing si-ZNF423, and detecting the effect of inhibiting the proliferation and differentiation of bovine adipocytes through a variety of detection methods, thereby facilitating understanding the role of the interfering fragment in bovine adipocytes, and interfering with ZNF423 gene expression through the interfering fragment to achieve regulation of bovine adipocyte proliferation and differentiation, which is beneficial to improving beef quality and optimizing beef cattle breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a picture of bovine primary adipocytes isolated and cultured using the tissue explant method;

[0028] Figure 2 This is a graph showing the mRNA expression of ZNF423 in bovine adipocytes after interference with ZNF423;

[0029] Figure 3 This is the protein expression result graph of ZNF423 in bovine adipocytes after interfering with ZNF423 and the gray value quantification result graph;

[0030] Figure 4 This is the EdU staining result and quantification result of bovine adipocytes after interference with ZNF423;

[0031] Figure 5 This is a graph showing the cell cycle and quantification results of bovine adipocytes after interfering with ZNF423;

[0032] Figure 6 This is the result diagram of apoptosis of bovine adipocytes after interfering with ZNF423 and the quantitative result diagram;

[0033] Figure 7 This is a graph showing the mRNA expression of proliferation-related genes PCNA and CCNB1 in bovine adipocytes after interference with ZNF423;

[0034] Figure 8 This is the Oil Red 0 staining result and quantification result of bovine adipocytes after interference with ZNF423;

[0035] Fig. 9 This is the result of BODIPY staining and Nile Red of bovine adipocytes after interference with ZNF423;

[0036] Fig.10 This is the result of TG content in bovine adipocytes after interfering with ZNF423;

[0037] Fig.11 This is a graph showing the mRNA expression results of adipogenic differentiation-related genes PPARγ, C / EBPβ, SCD1 and FABP4 in bovine adipocytes after interference with ZNF423;

[0038] Fig.12 This is the protein expression result graph of adipogenic differentiation-related proteins PPARγ and FABP4 in bovine adipocytes after interference with ZNF423, as well as the gray value quantification result graph. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] See also Figure 1 , which is a verification method for gene interference regulation of bovine adipocyte proliferation and differentiation disclosed in an embodiment of the present invention, comprising the following steps:

[0041] S1. Cut the bovine fat cell tissue into small pieces and directly place them in a culture dish for culture, using the tissue's own nutrients and growth factors to promote cell growth and proliferation;

[0042] S2, constructing an interference fragment containing si-ZNF423, using siRNA technology to target the ZNF423 gene, using a transfection reagent to transfect siRNA into bovine adipocytes, and setting up a control group without an interference fragment;

[0043] S3, extract the total RNA and total protein of bovine adipocytes, and detect the ZNF423 gene interference efficiency by qPCR and WB;

[0044] S4. EdU staining, flow cytometry, and qPCR techniques were used to detect the regulatory effects of the interference fragments on bovine adipocyte proliferation;

[0045] S5. Oil Red 0 staining, BODIPY, Nile Red staining, TG content determination, qPCR and WB were used to detect the regulatory effect of the interference fragment on bovine adipocyte differentiation.

[0046] Specifically, by transferring the interference fragment containing si-ZNF423 into bovine adipocytes, the ZNF423 gene is effectively silenced. The interference fragment directly acts on the key regulatory gene ZNF423, which is beneficial to accurately regulate the proliferation and differentiation of adipocytes at the genetic level. EdU cell proliferation detection, flow cytometry, qPCR analysis, Western Blot (WB) protein detection and triglyceride (TG) content determination and other technical means are used to systematically detect the effect of ZNF423 gene interference on the proliferation and differentiation of bovine adipocytes, revealing the effects of ZNF423 interference from both protein and gene aspects, and comprehensively presenting the mechanism of action of gene interference on adipocytes.

[0047] In this way, the expression of ZNF423 gene is interfered with by the interference fragment containing si-ZNF423, and the effect of inhibiting the proliferation and differentiation of bovine adipocytes is detected by various detection methods, which makes it easy to understand the role of the interference fragment in bovine adipocytes, verify the role of ZNF423 gene in adipocyte development, and interfere with the expression of ZNF423 gene by the interference fragment to achieve the regulation of bovine adipocyte proliferation and differentiation, which is beneficial to improving beef quality and optimizing beef cattle breeding.

[0048] In some feasible methods, the interference fragment of si-ZNF423 is forward: GCACCAACUCUCCCAUCUUTT; reverse: AAGAUGGAGAGUGUGCTT, and the transfection reagent is Lipofectamine TM 3000.

[0049] As a specific implementation of a verification method for gene interference regulation of bovine adipocyte proliferation and differentiation provided in the application, step S1 includes:

[0050] Adipose tissue pretreatment: The collected adipose tissue was washed several times with PBS solution containing 1% penicillin / streptomycin in the cell chamber, and the non-adipose tissue on the adipose tissue was removed using sterile instruments;

[0051] Tissue block preparation: After washing the adipose tissue three times, the adipose tissue was cut into small pieces of 3 × 3 mm and inoculated into collagen-treated culture dishes;

[0052] Initial culture: Place the culture dish in a cell culture incubator at 37°C and 5% CO2, invert and culture for 6 hours, then add growth medium containing 10% FBS and 90% DMEM / high glucose;

[0053] Cell expansion: After a large number of fat cells grow around the tissue block, remove the tissue block and continue culturing until the cell confluence reaches about 80%, then expand the culture and cryopreservation of the cells;

[0054] Cell differentiation induction: When the confluence of adipocytes reached 100%, the medium was replaced with differentiation medium when cell growth was inhibited;

[0055] Differentiation maintenance: After 2 days of differentiation induction, the culture medium was replaced with maintenance medium. Thereafter, the culture medium was replaced every two days to maintain the differentiation state.

[0056] In some feasible methods, non-adipose tissue includes visible blood vessels, muscle and connective tissue, etc., the differentiation medium composition includes 10% FBS, 10μg / mL insulin, 1μmol / L dexamethasone, 0.5mmmmol / L IBMX, 1μmol / L rosiglitazone and DMEM / highglucose, and the maintenance medium composition includes 5% FBS, 10μg / mL insulin, 1μmol / L rosiglitazone and DMEM / highglucose, please refer to Figure 1 , for the successful isolation and culture of bovine primary adipocytes.

[0057] As a specific implementation of a verification method for gene interference regulation of bovine adipocyte proliferation and differentiation provided in the application, step S2 includes:

[0058] Transfection pretreatment: Bovine adipocytes were seeded into 6-well culture dishes. During transfection, the cells reached a seeding density of 70-90% (3.0 x 10^5 cells / well), and 2 mL of growth medium without antibiotics was added.

[0059] Preparation of transfection complex: Dilute the control group and si-ZNF423 validation group solution with transfection reagent (Lipofectamine TM 3000) solution, and mix thoroughly using a vortex mixer, and incubate the mixture at room temperature for 10-15 minutes;

[0060] Add transfection complexes to cells: add 135 μL of the incubated complexes to a culture dish containing bovine adipocytes, and the base medium is 1.5 ml Opti-MEM culture medium without FBS for starvation treatment;

[0061] Culture and observation: Place the culture dish in a 37°C, 5% CO2 incubator. 24 hours after transfection, replace the culture medium with complete growth medium.

[0062] In some embodiments, the transfection complex is incubated at room temperature for 10-15 minutes with si-ZNF423 and Lipofectamine TM 3000 to form a complex, Table 1 shows the dosage of each reagent:

[0063] Table 1. Amounts of reagents used in preparing transfection complexes

[0064] Reagents Dose / well (μL) Opti-MEM 125 <![CDATA[Lipofectamine TM 3000]]> 4 Control group and si-ZNF423 validation group 6

[0065] When adding transfection complexes to cells, the culture dish should be gently shaken to ensure even distribution of the complexes. Replace with complete growth medium 24 hours after transfection to support cell growth and reduce the potential toxicity of transfection reagents. 48 hours after transfection, the knockdown effect of si-ZNF423 can be analyzed by WB, qPCR and other methods.

[0066] To facilitate the implementation of this method, the detection methods involved in S3-S5 are also disclosed in this embodiment:

[0067] In some feasible embodiments, step S3 includes real-time fluorescence quantitative qPCR detection, protein immunoblotting experiment WB

[0068] Real-time fluorescence quantitative qPCR detection includes:

[0069] RNA extraction: Total RNA was extracted from bovine adipocytes using the guanidine isothiocyanate-phenol (Trizol) method;

[0070] RNA quality detection: Use a multifunctional full-wavelength microplate reader to measure the concentration (ng / μL) and OD value of total RNA. The ratio of OD260 / OD280 should be between 1.8 and 2.0 to ensure the purity of RNA. At the same time, the integrity of total RNA is detected by 1% agarose gel electrophoresis. If the 28S and 18S bands can be clearly observed, the total RNA quality is qualified.

[0071] cDNA synthesis: Use a reverse transcription kit to reverse transcribe qualified total RNA into cDNA and store it at -20℃ for future use;

[0072] qPCR detection: Use 2xM5HiPer SYBR Premix EsTaq fluorescent quantitative kit to perform real-time quantitative detection of target genes. The qPCR reaction system is prepared as shown in Table 2:

[0073] Table 2 qPCR reaction system preparation table

[0074] Reagents Dose / well (μL) 2×M5HiPer SYBR Premix EsTaq (with Tli RNaseH) 7.5 cDNA 1.0 Upstream primer (10 μM) 0.3 Downstream primer (10μ M) 0.3 ddH20 5.9 Overall system 15

[0075] PCR reaction conditions: PCR reaction was performed on a CFX96 Real-Time PCR Detection System instrument using a two-step program: 95°C pre-denaturation for 30 seconds, 95°C denaturation for 5 seconds, 60°C annealing for 30 seconds, and 72°C extension for 30 seconds for 40 cycles, with GAPDH selected as the internal reference gene.

[0076] Data analysis: The 2-ΔΔCt method was used to perform relative quantitative analysis of the mRNA expression levels of target genes to evaluate changes in gene expression.

[0077] Table 3 Primer sequences for qPCR detection of ZNF423, proliferation-related genes, adipogenic differentiation-related genes, and internal reference genes

[0078]

[0079] Western blotting includes: extraction of total cell protein, protein quantification (BCA method), protein electrophoresis and membrane transfer, among which:

[0080] Extraction of total cell protein includes:

[0081] Wash cells: remove the culture medium and wash the cells three times with PBS solution;

[0082] Lyse cells: add 100 μL of lysis buffer to each well of a 6-well plate. The lysis buffer includes: 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor, and 5 μL of protease inhibitor (100 mM PMSF) per 1 mL of cell lysis buffer, and gently pipette the cells to promote cell lysis;

[0083] Transfer lysate: Transfer the lysate to a pre-cooled 1.5 mL centrifuge tube and vortex vigorously for 30 seconds to fully disperse the cell fragments. Place the centrifuge tube on ice for 4 minutes and repeat 5 times.

[0084] Centrifugal separation: Precool the low-temperature high-speed centrifuge to 4°C, place the centrifuge tube containing the sample in the centrifuge, and centrifuge at 12000rpm for 5 minutes to separate the total protein;

[0085] Collect the supernatant: Carefully transfer the supernatant (total protein) to a new centrifuge tube;

[0086] Preserve protein samples: Store the collected total protein in a -80°C ultra-low temperature freezer to maintain the stability and activity of the protein.

[0087] Protein quantification (BCA method) includes:

[0088] Protein sample pretreatment: Use the BCA protein content detection kit to detect the concentration of the extracted protein sample, mix 5× protein loading buffer and protein sample in a ratio of 4:1, boil the mixed sample at 100°C for 10 minutes to denature the protein, and store the denatured protein sample at -20°C;

[0089] Protein concentration measurement: according to the number of standard wells, prepare an appropriate amount of BCA working solution, the ratio of reagent A: reagent B = 50:1, add 200 μL of BCA working solution to each well of the ELISA plate;

[0090] Preparation of standard: Use 0.5 μg / μL BSA standard as the initial solution and add reagents according to Table 4:

[0091] Table 4 Standard preparation reagent addition table

[0092] Hole No. 0 1 2 3 4 5 6 7 Protein standard solution (μL) 0 1 2 4 8 12 16 20 Deionized water (μL) 20 19 18 16 12 8 4 0 Corresponding protein content (μg) 0 0.5 1.0 2.0 4.0 6.0 8.0 10.0

[0093] Sample treatment: Add the protein sample to be tested to the ELISA plate, and add appropriate amount of deionized water to make the total volume of the sample dilution liquid 20μL. Place the ELISA plate on an oscillator for 30s and place it in the dark at 37℃ for 30min to complete the color reaction.

[0094] Absorbance measurement: Measure the sample absorbance at 562nm;

[0095] Standard curve drawing and concentration calculation: Use protein content (μg) as the horizontal axis (0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0) and its corresponding absorbance value as the vertical axis to draw a standard curve. Substitute the absorbance value of the measured protein sample into the standard curve to calculate its concentration after dilution. Calculate its actual concentration based on the dilution multiple of the measured protein sample, and calculate the amount of protein loading required for electrophoresis.

[0096] Protein electrophoresis and membrane transfer include:

[0097] Gel preparation: align the glass plate, make sure the short plate faces the operator and clamp it on the plate rack to keep the bottom edge aligned to prevent gel leakage, prepare the separation gel (lower layer gel), inject it into the glass plate to a predetermined height after fully mixing, and cover it with an appropriate amount of anhydrous ethanol to level the gel surface, while removing bubbles. After the separation gel solidifies, remove the anhydrous ethanol and use filter paper to absorb the residual liquid. Then prepare the concentrated gel (upper layer gel), inject it until the glass plate is full after fully mixing, and quickly insert the comb and let it stand for 15 minutes to ensure that the gel is completely solidified;

[0098] Acrylamide gel electrophoresis: remove the glass plate from the plate rack and place it on the electrode plate, making sure the short plate faces inwards and snaps into the green slot of the electrode plate, placing it correctly according to the positive and negative polarities; pour in the electrophoresis buffer, first inject from the middle of the electrode plate, and then fill the entire electrophoresis tank; Note: For a single electrode plate, add the buffer to the 2GEL mark, and for a double electrode plate, add it to the 4GEL mark. If leakage is observed, remove it and add it again. If there is no leakage, pull out the comb vertically and add the protein sample. In the initial electrophoresis stage, run at 80V for 25 to 35 minutes to concentrate the sample in the separation gel area. In the second stage of electrophoresis, adjust the voltage to 120V until the sample migrates to the bottom of the gel;

[0099] Transfer (electrotransfer): prepare 1× transfer solution, the formula is 100mL of 10× transfer solution, 200mL of methanol and 700mL of ddH20; cut the PVDF membrane to match the size of the gel and pre-treat it with methanol, gently remove the glass plate, remove the small glass plate after it loosens, and remove the concentrated gel, place the transfer clip in the tray containing 1× transfer solution, make sure the black side is facing down (negative pole), place the special sponge, sponge, filter paper, gel, PVDF membrane, filter paper, sponge and sponge in sequence, remove bubbles and clamp the transfer clip (make sure it is properly tightened), then place the transfer clip in the transfer tank (pay attention to the positive and negative polarity, the clip faces up, black against black), pour in 1× transfer solution, and place an ice box in the tank to lower the temperature, and transfer the membrane at a constant current of 300mA for 1.5 hours;

[0100] Blocking: Add rapid blocking solution to the membrane and block on a shaker at the lowest speed for 15 minutes;

[0101] Primary antibody incubation: Incubate the membrane with primary antibody at 4°C overnight;

[0102] Secondary antibody incubation: After recovering the primary antibody, wash the membrane quickly on a shaker 3 times with TBST buffer for 10 minutes each time, incubate the secondary antibody on a shaker at a slow speed for 2 hours, and after recovering the secondary antibody, wash the membrane quickly on a shaker 3 times with TBST buffer for 10 minutes each time;

[0103] Development: Prepare the developer solution by mixing liquid A and liquid B in a ratio of 1:1, and add an appropriate amount of the developer solution to the film for development.

[0104] See also Figure 2 and Figure 3 , Figure 2 This is the result of ZNF423 mRNA expression in bovine adipocytes after ZNF423 interference. Figure 3 These are the protein expression results of ZNF423 in bovine adipocytes after interfering with ZNF423 and the gray value quantification results. It can be seen that the expression of ZNF423 in bovine adipocytes can be effectively reduced after transfection of si-ZNF423.

[0105] In some embodiments, step S4 includes EdU staining and flow cytometry to detect cell cycle:

[0106] EdU staining steps include:

[0107] EdU labeling: discard half of the culture medium in the bovine adipocyte culture plate, add EdU working solution preheated to 37°C, mix the EdU working solution and culture medium in equal volumes, EdU working solution: culture medium = 1:1, and then incubate the cells for 6 hours to label the proliferating cells;

[0108] Cell fixation: After incubation, discard the culture medium and add fixative to fix the cells for 15 minutes at room temperature;

[0109] Enhance permeability: After washing the cells three times with washing solution, add an appropriate amount of permeabilization solution and incubate at room temperature for 10 to 15 minutes;

[0110] Click chemical reaction: Wash the cells three times with washing solution, then add appropriate amount of Click reaction solution and incubate at room temperature in the dark for 30 minutes to allow the covalent binding of EdU and fluorophore;

[0111] Nucleus staining: discard the Click reaction solution and wash with washing solution, add appropriate amount of 1×Hoechst33342 solution, and continue to incubate at room temperature in the dark for 10 minutes to stain the cell nucleus;

[0112] Fluorescence detection and recording: Wash the cells three times with washing solution, perform fluorescence detection under an inverted microscope, and record and analyze cell proliferation.

[0113] Flow cytometry detection of cell cycle steps includes:

[0114] Sample preparation: Collect bovine adipocytes grown to an appropriate density, remove the culture medium and PBS, add trypsin to digest the adherent cells until the cells fall off the culture dish, then add serum-containing culture medium to terminate the digestion, transfer the cells to a centrifuge tube, centrifuge at room temperature at 300-400xg for 4-5 minutes, and discard the supernatant;

[0115] Cell fixation: resuspend the cells in pre-cooled PBS, transfer to a 1.5 mL centrifuge tube, centrifuge at 1000 g for 5 minutes at 4°C, discard the supernatant, retain 50 μL of the cell pellet, add the cell suspension to pre-cooled 70% ethanol, mix well, and fix at 4°C for at least 4 hours;

[0116] Cell resuspension and RNase treatment: Remove the fixed cells, centrifuge again at 1000g for 5 minutes at 4°C, discard the supernatant, add 1mL of pre-cooled PBS to resuspend the cells, then centrifuge again, discard the supernatant, retain 50μL of PBS buffer, add 100μL of RNaseA solution, fully suspend the cells, and incubate in a 37°C water bath for 30 minutes to digest RNA;

[0117] PI staining: Prepare PI staining solution in dark conditions, add 400 μL of PI staining solution, mix well, and incubate in dark at 4°C for 30 minutes;

[0118] Flow cytometer detection: Set the appropriate excitation wavelength (optionally 488 nm) on the flow cytometer and detect red fluorescence, acquire cells at a low speed, and use flow cytometric analysis software to analyze DNA content;

[0119] Data analysis: According to the distribution of DNA content, the cell cycle was divided into G1 / G0 phase, S phase and G2 / M phase, and the percentage of cells in each phase was calculated.

[0120] It should be understood that during the entire experiment, especially during the PI staining and RNase treatment steps, direct or indirect light exposure should be avoided to prevent photobleaching of fluorescent dyes. Cells should be kept on ice or at 4°C during fixation and staining to reduce cell degradation and DNA degradation. Before flow cytometry detection, ensure that cells are fully resuspended to avoid cell aggregation, which will affect the accuracy of the flow cytometer.

[0121] Flow cytometry detection of cell cycle steps includes:

[0122] Cell collection and processing: Bovine adipocytes were collected from the culture plate and gently washed with EDTA-free PBS buffer to remove the culture medium residue; the cells were centrifuged at 300 x g for 5 minutes at room temperature, the supernatant was discarded, the cells were resuspended with pre-cooled PBS, and centrifuged at 2000 rpm for 5 minutes;

[0123] Binding buffer preparation: dilute the Binding Buffer 1:4 with ddH20 to prepare the working concentration of the binding buffer;

[0124] Cell resuspension and staining: resuspend the cells with 250 μL binding buffer, take 100 μL of cell suspension into a new 1.5 mL centrifuge tube, and add 5 μL Annexin V-FITC. This fluorescently labeled ligand can specifically bind to the phosphatidylserine (PS) on the everted membrane of apoptotic cells.

[0125] Incubation: Incubate the mixed cell suspension at room temperature in the dark for 10 minutes to allow Annexin V-FITC to fully bind to PS;

[0126] PI staining: 5 minutes before flow cytometry, add 10 μL PI solution and mix well;

[0127] Cell resuspension and flow cytometry detection: discard the incubation solution, add 400 μL PBS to resuspend the cells, and then use flow cytometer for detection. Set the appropriate excitation wavelength and detect red and green fluorescence on the flow cytometer. Annexin V-FITC emits green fluorescence under 488 nm excitation, while PI emits red fluorescence under 635 nm excitation;

[0128] Data analysis: The collected data were analyzed using flow cytometric analysis software. According to the fluorescence intensity of Annexin V-FITC and PI, the cell cycle was divided into early apoptosis (Annexin V positive / PI negative), late apoptosis or necrosis (Annexin V positive / PI positive), and normal cells (Annexin V negative / PI negative). The cell ratio in each quadrant was calculated to evaluate the percentage of apoptotic cells.

[0129] It should be understood that the experimental operation should be carried out under light-proof conditions to prevent photobleaching of fluorescent dyes. Appropriate control groups should be set up in the experiment, including untreated cells and positive control groups. When analyzing the data, the threshold should be carefully set to distinguish apoptotic cells from other cells. To prevent fluorescence decay, flow cytometry should be performed within 1 hour. PI staining for too long may result in a higher apoptosis rate in the test. It is recommended to add PI staining 5 minutes before going on the machine.

[0130] See also Figure 4-Figure 7 , Figure 4 This is the EdU staining result and quantification result of bovine adipocytes after interfering with ZNF423. Figure 5 This is the cell cycle result diagram and quantification result diagram of bovine adipocytes after interfering with ZNF423. Figure 6 This is the result diagram of apoptosis of bovine adipocytes after interfering with ZNF423 and the quantitative result diagram. Figure 7 This is the result of mRNA expression of proliferation-related genes PCNA and CCNB1 in bovine adipocytes after interfering with ZNF423. It can be seen that interfering with ZNF423 will inhibit the proliferation of bovine adipocytes.

[0131] In some feasible embodiments, step S5 includes Oil Red O, BODIPY, Ni1e Red staining and TG determination.

[0132] The Oil Red 0 staining procedure includes:

[0133] Cell preparation: The culture medium of bovine adipocytes cultured for 4 days after transfection of the control group and si-ZNF423 validation group was discarded, and the cells were gently washed 3 times with EDTA-free PBS buffer to remove the residual culture medium;

[0134] Cell fixation: Use 4% paraformaldehyde preheated to 37°C to fix the cells for 5 minutes to maintain cell morphology and permeabilize the cell membrane. After discarding the formaldehyde solution, add an equal volume of 4% paraformaldehyde solution and incubate at room temperature for 1 hour.

[0135] Enhance cell permeability: Wash the cells with 60% isopropanol and let stand for about 5 minutes. After the culture dish is completely dry, proceed to the next step.

[0136] Oil Red 0 staining: Add Oil Red 0 working solution and incubate at room temperature for 30 minutes to allow the Oil Red 0 dye to bind to the lipid droplets in the cells, and the lipid droplets appear red;

[0137] Removal of staining solution and cell nucleus staining: After discarding the Oil Red 0 working solution, wash the cells 1 to 3 times with PBS, observe the images under a microscope and take photos to evaluate the formation of lipid droplets;

[0138] Data analysis: Images taken under a microscope were analyzed to quantify the number and size of lipid droplets to assess the degree of adipocyte differentiation and lipid synthesis capacity.

[0139] BODIOY dyeing steps include:

[0140] Cell preparation: Same as the steps for Oil Red 0 staining;

[0141] Cell fixation: Fix cells with 4% paraformaldehyde preheated to 37°C for 30-60 minutes to preserve cell morphology and permeabilize cell membranes.

[0142] Preparation of BODIPY working solution: Dissolve BODIPY powder in DMSO to prepare 1 mmmmol / L BODIPY stock solution, then dilute 1000 times with PBS to obtain BODIPY working solution;

[0143] B0DIPY staining: add 500 μL B0DIPY working solution to each well and incubate at room temperature in the dark for 30 minutes to allow B0DIPY dye to bind to lipid droplets in cells;

[0144] Post-staining washes: Wash cells 3 times with PBS for 5 minutes each to remove unbound dye;

[0145] Nuclear staining: Use DAPI (2000-fold dilution in PBS) to stain the nucleus for 10 min to clearly identify the nucleus under a fluorescence microscope;

[0146] Final washing and imaging observation: Wash the cells again 3 times with PBS, 5 minutes each time, remove the excess DAPI staining solution, perform imaging observation under a fluorescence microscope, and record the fluorescence signal of lipid droplets.

[0147] The Nile Red dyeing steps include:

[0148] Cell preparation: Same as the steps for Oil Red 0 staining;

[0149] Cell fixation: Fix cells with 4% paraformaldehyde preheated to 37°C for 20 min to preserve cell morphology and permeabilize cell membranes.

[0150] Preparation of Nile Red working solution: Dissolve Nile Red powder in DMSO to prepare 1 mmol / L Nile Red stock solution, then dilute 1000 times with PBS to obtain Nile Red working solution;

[0151] Nile Red staining: Add 500 μL of Nile Red working solution to each well and incubate at room temperature in the dark for 10 minutes to allow the Nile Red dye to bind to the lipid droplets in the cells;

[0152] Washing after staining: Same as B0DIPY staining washing steps.

[0153] Nuclear staining: Same as B0DIPY staining nuclear staining steps.

[0154] Final washing and imaging observation: the same as B0DIPY staining washing and imaging observation steps.

[0155] TG assay includes:

[0156] Sample preparation: Bovine adipocytes were collected after transfection with the control group and the si-ZNF423 validation group. After washing with PBS, the supernatant was discarded, 0.2 mL of PBS was added, and ultrasonic disruption was performed in an ice water bath (power 300 W, 3-5 s / time, interval 15 s, repeated 3-5 times);

[0157] Calculation of TG content: TG content in cells was detected using a TG assay kit.

[0158] See also Figure 8-Figure 12 , Figure 8 This is the Oil Red 0 staining result and quantification result of bovine adipocytes after interference with ZNF423; Fig. 9 This is the B0DIPY staining and Nile Red results of bovine adipocytes after interfering with ZNF423. Fig.10 This is the result of TG content in bovine adipocytes after interfering with ZNF423. Fig.11This is the result of mRNA expression of adipogenic differentiation-related genes PPARγ, C / EBPβ, SCD1 and FABP4 in bovine adipocytes after interference with ZNF423. Fig.12 These are the protein expression results of adipogenic differentiation-related proteins PPARγ and FABP4 in bovine adipocytes after interfering with ZNF423, as well as the grayscale value quantification results. It can be seen that interfering with ZNF423 will inhibit the differentiation and lipid metabolism of bovine adipocytes.

[0159] In some feasible ways, the sources of some cells and reagents used in the above embodiments include: bovine adipocytes are obtained by collecting newborn calf adipose tissue and isolating it using the tissue block method; siRNA genes and PCR amplification primers are synthesized by Shanghai Sangon Biotechnology Co., Ltd.; PBS, DMEM / High-Glucose, and 0.25% trypsin are purchased from Hyclone; LipofectamineTM3000 transfection reagent and Annexin V-FITC / PI cell apoptosis detection kit are purchased from Invigentech; BeyoClickTMEdU-555 cell proliferation detection kit, cell cycle and cell apoptosis detection kit, and 4% paraformaldehyde are purchased from Beyotech; IBMX, dexamethasone, insulin, and rosiglitazone are purchased from Sigma; fetal bovine serum (FBS) is purchased from BI; reverse transcription kits and RNAiso Plus are purchased from Takara; 2×M5Hiper SYBR PremixEsTaq is purchased from Polymer; Oil Red 0 is purchased from Solarbio; B0DIPY 493 / 503, Nile Red were purchased from Anjiekai; triglyceride (TG) determination kit (single reagent GPO-PAP method) (microplate reader and biochemical analyzer) were purchased from Nanjing Jiancheng Bioengineering Institute; anti-ZNF423 antibody (Bioss), anti-FABP4 antibody (Abways), anti-PPARγ antibody (Bioss), anti-GAPDH (Abways), Goat Anti-Rabbit IgG secondary antibody (Affinity), and other WB-related reagents were purchased from Yamei.

[0160] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. 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 verification method for gene interference regulation of bovine adipocyte proliferation and differentiation, characterized in that: The following steps are involved: S1. Cut the bovine fat cell tissue into small pieces and directly place them in a culture dish for culture, using the tissue's own nutrients and growth factors to promote cell growth and proliferation; S2. Construct an interference fragment containing si-ZNF423, use siRNA technology to target the ZNF423 gene, use a transfection reagent to transfect siRNA into bovine adipocytes to form a validation group, and set up a control group without interference fragments; S3, extract the total RNA and total protein of bovine adipocytes, and detect the ZNF423 gene interference efficiency by qPCR and WB; S4. EdU staining, flow cytometry, and qPCR techniques were used to detect the regulatory effects of the interference fragments on bovine adipocyte proliferation; S5. Oil Red 0 staining, BODIPY, Nile Red staining, TG content determination, qPCR and WB were used to detect the regulatory effect of the interference fragment on bovine adipocyte differentiation.

2. The verification method for gene interference regulation of bovine adipocyte proliferation and differentiation according to claim 1, characterized in that: The interference fragments are specifically: forward: GCACCAACUCUCCCAUCUUTT, reverse: AAGAUGGAGAGUGUGCTT.

3. The verification method for gene interference regulation of bovine adipocyte proliferation and differentiation according to claim 1, characterized in that: The step S1 comprises: Adipose tissue pretreatment: The collected adipose tissue was washed several times with PBS solution containing 1% penicillin / streptomycin in the cell chamber, and the non-adipose tissue on the adipose tissue was removed using sterile instruments; Tissue block preparation: After washing the adipose tissue three times, the adipose tissue was cut into small pieces of 3 × 3 mm and inoculated into collagen-treated culture dishes; Initial culture: Place the culture dish in a cell culture incubator at 37°C and 5% CO2, invert and culture for 6 hours, then add high-glucose DMEM medium containing 10% FBS; Cell expansion: After a large number of fat cells grow around the tissue block, remove the tissue block and continue culturing until the cell confluence reaches 80-90%, then expand the culture and cryopreservation of the cells; Cell differentiation induction: When the adipocyte confluence reached 100% and cell growth was inhibited, the medium was replaced with differentiation medium; Differentiation maintenance: After 2 days of differentiation induction, the culture medium was replaced with maintenance medium. Thereafter, the culture medium was replaced every two days to maintain the differentiation state.

4. The verification method for gene interference regulation of bovine adipocyte proliferation and differentiation according to claim 2, characterized in that: The step S2 comprises: Transfection pretreatment: Bovine adipocytes were seeded into 6-well culture dishes and 2 mL of growth medium without antibiotics was added. During transfection, the cells reached a seeding density of 70-80% of 3.0x10^5 cells / well; Preparation of transfection complex: dilute the control group and si-ZNF423 validation group solutions and transfection reagent solution with Opti-MEM medium, mix thoroughly using a vortex mixer, and incubate the mixture at room temperature for 10-15 minutes; Add transfection complexes to cells: Add 135 μL of the incubated complexes to a culture dish containing bovine adipocytes. At this time, replace the base solution with 1.5 ml of serum-free Opti-MEM culture medium to starve the cells. Culture and observation: The culture dish was placed in an incubator at 37°C and 5% CO2. Six hours after transfection, the culture medium was replaced with complete growth medium.

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