A method for semi-quantitative analysis of the expression level of secreted proteins from human articular chondrocytes using immunoblotting

Abstract

The application discloses a method for semi-quantitative analysis of expression level of secreted protein of human joint cartilage cells by using immunoblotting technology. The method comprises the following steps: 1) preparing a secreted protein culture solution; 2) collecting a sample of the secreted protein culture solution; 3) performing dialysis desalting treatment on the sample; 4) concentrating the secreted protein culture solution to prepare a powder; 5) re-dissolving the secreted protein culture solution powder and quantifying the protein; 6) separating the protein components in the re-dissolved culture solution; and 7) detecting the expression level of the secreted protein in the re-dissolved culture solution sample by using immunoblotting method. The low-temperature dialysis and vacuum concentration technology can significantly improve the concentration of the secreted protein in the human joint cartilage tissue culture solution sample under the condition of maintaining the integrity of the protein structure, and is suitable for detecting the chemical fluorescence signal of the secreted protein in the sample by using the immunoblotting technology. The application provides an important cell reaction detection tool for studying the pathogenesis of bone and joint diseases caused by degenerative changes of cartilage tissue and researching and developing biological small-molecule drugs for treating the diseases.

Description

Technical Field

[0001] This invention relates to the field of biomolecular analysis technology, and in particular to a method for semi-quantitative analysis of the expression level of secreted proteins in human articular chondrocytes using immunoblotting technology. Background Technology

[0002] Articular cartilage, the bone endplate lining the synovial joint cavity, is a crucial and unique tissue type that protects bone from severe mechanical stress during movement. This tissue is composed of an extremely rich extracellular matrix and chondrocytes, with a matrix-to-cell volume ratio far exceeding that of other tissue types in the human body. This structural characteristic endows articular cartilage with the physiological functions of energy absorption and pressure resistance. Chondrocytes, as the only cell type within articular cartilage, are responsible not only for synthesizing extracellular matrix framework proteins, primarily proteoglycans and collagen fibers, and for tissue renewal under physiological conditions, but also for expressing a range of secreted proteins, including matrix metalloproteinases, cytokines, chemokines, and growth factors, in response to changes in their microenvironment.

[0003] Articular cartilage undergoes degenerative changes due to mechanical or inflammatory stimuli, and is a major cause of a range of diseases affecting joint function, including osteoarthritis. Because articular cartilage is a unique tissue type lacking blood vessels and nerve connections, chondrocytes obtain their daily metabolic nutrients solely from synovial fluid. Therefore, once tissue damage occurs, chondrocytes cannot obtain sufficient nutrients for repair, leading to progressively expanding lesions and eventual complete tissue degradation, ultimately resulting in the complete loss of physiological function of the affected joint. However, the molecular mechanisms by which articular cartilage maintains homeostasis under physiological conditions and undergoes degenerative changes under pathological conditions remain unclear. Total joint replacement surgery remains the ultimate treatment for this type of disease, placing a heavy burden on patients' postoperative quality of life, families, and national finances. Therefore, clarifying the pathogenesis of diseases characterized by articular cartilage damage and developing targeted small molecule biological drugs to replace total joint replacement surgery are urgent research goals.

[0004] In vitro culture systems for articular cartilage tissue or cells serve as crucial platforms for researchers to study the mechanisms of physiological renewal and the molecular pathological mechanisms of degenerative changes in this tissue. To verify whether a specific stimulating factor participates in these mechanisms and its specific role, researchers need to analyze the levels of proteins produced and secreted extracellularly by chondrocytes in response to stimulating factors in the culture medium of articular cartilage tissue or cells at specific time points. Human articular cartilage tissue specimens primarily come from patients who have undergone total hip or knee replacement surgery due to femoral neck fractures or end-stage osteoarthritis. These patients are all over 55 years old, and their articular cartilage cells are in a mature, terminally differentiated state. Due to the long-term hypoxic environment within the joint cavity, their metabolic activity is low, resulting in low levels of secreted proteins that are difficult to detect using commonly used protein analysis techniques.

[0005] Immunoblotting and enzyme-linked immunosorbent assay (ELISA) are currently the mainstream techniques for quantitatively analyzing the levels of secreted proteins in tissue or cell culture systems. Both rely on the principle of specific antigen-antibody binding and use fluorescence colorimetry to quantify proteins. However, ELISA does not pre-treat the culture medium sample. The accumulation of salt ions in the culture medium and acid radicals produced by cell metabolism can significantly interfere with antibody recognition of secreted proteins, leading to a significantly higher false-negative rate. Furthermore, since ELISA does not involve protein denaturation, antigen-specific amino acid sequences may be embedded in the peptide chain structure, increasing steric hindrance for antibody recognition and binding, thus reducing detection sensitivity. This is another important reason for false-negative results.

[0006] Furthermore, enzyme-linked immunosorbent assay (ELISA) not only requires more expensive plate-laying antibodies but also costly standard proteins for standard curve plotting. Moreover, the 96-well plate setup for ELISA inevitably results in wasted wells due to the significantly lower sample size compared to 96 wells per assay. Therefore, due to inherent technical limitations and the low levels of secreted proteins in human articular cartilage tissue or cell culture media, ELISA is insufficient for achieving ideal protein quantification. On the other hand, Western blotting has minimum protein concentration requirements for the sample, which is difficult to meet in cartilage culture media. Additionally, the high salt ion concentration in the culture medium severely interferes with the electrophoretic separation of secreted proteins on SDS-polyacrylamide gels. These two factors make it impossible to directly use this technique to detect the expression levels of secreted proteins in cartilage culture media samples. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for semi-quantitative analysis of the expression levels of secreted proteins in human articular chondrocytes using immunoblotting technology. This detection method, centered on immunoblotting technology, effectively solves the antigen-antibody recognition barrier by dialyzing out salt ions from the culture medium, concentrating the secreted proteins in the culture medium at low temperatures, and reconstituted the secreted proteins with ultrapure water in a low volume. It also significantly improves the signal intensity of secreted proteins in the culture medium sample, enhancing detection sensitivity and quantification accuracy. This provides an important tool for analyzing cell response results, enabling research into the pathogenesis of osteoarthritis caused by degenerative changes in cartilage tissue and the development of small molecule biological drugs for treating such diseases.

[0008] The technical solution of the present invention is as follows:

[0009] A method for semi-quantitative analysis of the expression level of proteins secreted by human articular chondrocytes using Western blotting, the method comprising the following steps:

[0010] (1) Preparation of culture medium containing secretory proteins:

[0011] Adding pro-inflammatory factors to the culture medium containing human articular cartilage tissue slices to stimulate the culture medium yielded a culture medium containing secretory proteins.

[0012] (2) Collect samples of culture medium containing secreted proteins:

[0013] The culture medium containing secretory protein obtained in step (1) was transferred into a centrifuge tube, and the cap and hinge of the centrifuge tube were cut off to obtain a sample of culture medium containing secretory protein.

[0014] (3) Dialysis desalination of the sample:

[0015] Cover the centrifuge tube opening of the sample obtained in step (2) with the dialysis membrane tube, tie it tightly, place it in ultrapure water and stir to dialyze, and obtain a sample of culture medium containing secretory protein after desalting;

[0016] (4) Concentrate and prepare culture medium powder containing secretory proteins:

[0017] After removing the dialysis membrane of the sample obtained in step (3), vacuum concentration was performed to obtain a culture medium powder sample containing secretory proteins.

[0018] (5) Reconstituted culture medium containing secretory protein powder and protein quantification:

[0019] After reconstituted culture medium powder samples obtained in step (4) with ultrapure water, the samples were transferred to new centrifuge tubes to obtain reconstituted culture medium samples. The absorbance value was measured to obtain the total secreted protein concentration in each sample.

[0020] (6) Separation of protein components from the reconstituted culture medium:

[0021] Protein components were separated in a separating gel according to their molecular weight by polyacrylamide gel electrophoresis, resulting in a separating gel containing the secreted protein to be tested.

[0022] (7) Detection of the expression level of secreted proteins in reconstituted culture medium samples by Western blotting:

[0023] The separating gel containing the secreted protein to be tested obtained in step (6) was transferred to a membrane, blocked, and incubated with antibodies to detect the relative expression level of the secreted protein to be tested.

[0024] Further, in step (1), the diameter of the tissue slice is 0.5-1.0 cm, and the amount added is 80-100 mg wet weight / culture well; the tissue slice is added to stimulate with pro-inflammatory factors after equilibration; the equilibration specifically involves: placing the tissue slice in a culture well containing culture medium, the culture medium including DMEM / F12 and penicillin-streptomycin, the amount of which is 1.0-1.5 mL / culture well; the volume fraction of penicillin-streptomycin in the culture medium is 0.5-1.0%; the equilibration time is 2-3 days, and the culture medium is changed every 24 hours during the equilibration period.

[0025] Further, the pro-inflammatory factor includes one of the following: N-telopeptide of type II collagen (NT), C-telopeptide of type II collagen (CT), helical peptide of type II collagen (HP), and IL-1β; the amino acid sequence of the N-telopeptide of type II collagen is shown in SEQ ID NO.1, the amino acid sequence of the C-telopeptide of type II collagen is shown in SEQ ID NO.2, and the amino acid sequence of the helical peptide of type II collagen is shown in SEQ ID NO.2. As shown in NO.3; the concentration of the type II collagen N-terminal telopeptide in the culture medium is 30 μmol / L, the concentration of the type II collagen C-terminal telopeptide in the culture medium is 30 μmol / L, the concentration of the type II collagen triple helix polypeptide in the culture medium is 30 μmol / L, and the concentration of IL-1β is 10 ng / mL; the stimulation time is 4-7 days; and the volume of the culture medium is kept consistent in each well.

[0026] SEQ ID NO.1

[0027] QMAGG FDEKA GGAGL GVMQG PMGPM GPRGP P

[0028] SEQ ID NO.2

[0029] IDMSA FAGLG PREKG PDPLQ YMRA

[0030] SEQ ID NO.3

[0031] GPEGA QGPRG EPGTP GSPGP AGAS

[0032] Furthermore, the stimulation period is 4 days, with pro-inflammatory factors added on days 0 and 2 of stimulation, and the culture medium being changed each time they are added.

[0033] Furthermore, the stimulation period is 7 days, and pro-inflammatory factors are added on days 0, 2, and 4 of stimulation, with the culture medium being changed each time they are added.

[0034] Further, in step (3), the length of the dialysis membrane tube is 2-3 cm; the stirring speed is 70-100 rpm; the dialysis temperature is 0-4℃; dialysis is stopped when the sample solution is clear, colorless and transparent; and the ultrapure water is replaced every 12 hours during the dialysis process.

[0035] Furthermore, in step (4), the vacuum concentration temperature is 4-20℃, the vacuum degree is 0.01-0.02atm, and the time is 5-7h.

[0036] Furthermore, in step (5), the volume of the ultrapure water is 40-60 μL / sample tube, and the absorbance value is measured at a wavelength of 280 nm.

[0037] Further, in step (6), the separation specifically includes adding sample buffer, denaturation, and sample loading; the sample buffer contains sodium dodecyl sulfate at a concentration of 2%; the denaturation temperature is 95-100℃ and the time is 3-5 min.

[0038] Further, in step (7), the transfer is the transfer of the secreted protein to be tested from the separating gel to a nitrocellulose membrane, and the antibody is labeled with a fluorescent molecule; the antibody includes a fluorescently labeled primary antibody or a combination of an unlabeled primary antibody and a horseradish peroxidase-modified secondary antibody; the primary antibody is a rabbit-derived antibody against human chondrocyte secretory protein, and the human chondrocyte secretory protein includes matrix metalloproteinase-3 and matrix metalloproteinase-13; the secondary antibody is a sheep-derived antibody against rabbit IgG; the incubation temperature of the primary antibody is 2-6℃ for 10-15h, and the incubation temperature of the secondary antibody is 15-30℃ for 1-2h.

[0039] The beneficial technical effects of this invention are as follows:

[0040] 1. This invention addresses the problem that complex salt ion components in human articular cartilage tissue or cell culture medium interfere with the separation of protein components in polyacrylamide gel electrophoresis samples, and that interference with the binding of secretory proteins to their antibodies leads to detection difficulties. The invention involves dialysis of the culture medium samples to remove salt ions. A dialysis membrane with a pore size at least 10 kDa smaller than the molecular weight of the secretory protein to be detected is used. Under low temperature conditions, continuous agitation is employed to replace salt ions in the culture medium sample with ultrapure water. Simultaneously, this process also removes protein molecules with molecular weights smaller than the pore size of the dialysis membrane, further reducing interference with the detection of secretory proteins and significantly improving the signal detection rate of secretory proteins in the sample.

[0041] 2. This invention addresses the problem of low yield of secreted proteins from in vitro cultured human articular chondrocytes, which makes detection difficult. It utilizes low-temperature vacuum concentration technology to remove water from the desalted culture medium sample, followed by reconstitution and concentration of the concentrated culture medium sample containing secreted proteins using a small volume of ultrapure water. This significantly increases the concentration of secreted proteins in the sample, enabling the sample to be loaded onto a small volume of sodium dodecyl sulfate (SDS)-polypropylene gel. Electrophoresis then separates the secreted proteins to be detected from other protein components in the sample, preparing for subsequent specific antibody recognition, color development, and quantification.

[0042] 3. The immunoblotting protein detection technology used in this invention utilizes specific antibodies that bind to fluorescent molecules to make the secretory proteins of human articular cartilage visible to the naked eye and exhibiting measurable grayscale fluorescent bands. Compared to enzyme-linked immunosorbent assay (ELISA), which only displays the spectrophotometric value of the sample, the immunoblotting technology used in this invention provides more intuitive experimental results. Furthermore, the expression level of human articular cartilage secretory proteins calculated by ELISA based on spectrophotometric values ​​is subject to significant errors compared to the true value due to the influence of background spectrophotometric values, standard protein purity, and spectrophotometer measurement accuracy. The immunoblotting technology used in this invention can more accurately determine the expression level of human articular cartilage secretory proteins.

[0043] 4. The cost of antibodies, reagents, and consumables required by this invention is much lower than that required for detecting secretory proteins in human articular cartilage using enzyme-linked immunosorbent assay (ELISA). Furthermore, samples can be flexibly combined according to the number of samples to be tested and the number of electrophoresis loading wells per gel, avoiding the waste of detection wells that occurs when using ELISA.

[0044] In summary, this invention provides a method for semi-quantitatively analyzing the expression levels of secreted proteins in cultured human articular chondrocytes using immunoblotting technology. By employing low-temperature dialysis and vacuum concentration techniques, the ionic strength of the sample can be significantly reduced and the protein concentration increased while maintaining the structural integrity of the articular chondrocyte secreted proteins. This method is suitable for separating secreted proteins in the sample according to their molecular weight using SDS-polyacrylamide gel electrophoresis. Then, immunoblotting technology and image analysis software are used to semi-quantitatively analyze the expression level of articular chondrocyte secreted proteins in the sample. This method solves the problem of detection difficulties caused by the low amount of secreted proteins produced in vitro by these low-metabolic cells. It provides an important cellular result analysis tool for studying the pathogenesis of osteoarthritis caused by degenerative changes in cartilage tissue and for the development of small molecule drugs for treating such diseases. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the culture medium containing secretory proteins prepared according to the present invention.

[0046] Figure 2 This is a schematic diagram illustrating the collection and transfer of culture medium samples containing secretory proteins according to the present invention.

[0047] In the figure: A) Collection of the culture medium sample containing secretory protein according to the present invention; B) Transfer of the culture medium sample containing secretory protein according to the present invention.

[0048] Figure 3 This is a schematic diagram illustrating the dialysis desalination process of the sample according to the present invention.

[0049] In the diagram: A) Dialysis membrane wetting; B) Dialysis membrane stretching; C) Culture medium sample centrifugation and falling back; D) Removing the cap from the microcentrifuge tube; E) Covering and fixing the dialysis membrane; F) Culture medium sample inverted in ultrapure water after membrane covering; G) Dialysis sample stirred at low temperature.

[0050] Figure 4 This is a schematic diagram of the concentration of the culture medium sample after desalting according to the present invention.

[0051] Figure 5 The images show the secretory protein band diagram and grayscale relative value diagram obtained in Example 1 of this invention.

[0052] In the figure: A, Matrix metalloproteinase-3 protein band; B, Matrix metalloproteinase-13 protein band; C, Relative gray values ​​of the Matrix metalloproteinase-3 and -13 protein bands.

[0053] Figure 6 This is a band diagram of secretory protein obtained in Example 2 of the present invention.

[0054] Figure 7 This is a band diagram of secretory protein obtained in Example 3 of the present invention. Detailed Implementation

[0055] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0056] In the following examples, the human articular cartilage tissue slices were all obtained from middle-aged or elderly patients who underwent total hip or knee replacement surgery in clinical practice;

[0057] The amino acid sequences of the pro-inflammatory factor type II collagen (SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3) from the literature report (“Telopeptides of type II collagen upregulate proteinases and damage cartilage but are less effective than highly active fibronectin fragments”, Guo D et al., Inflammation Research, Vol. 58, No. 3, pp. 161-169, published on 20090204) were sent to China Jier Biochemical (Shanghai) Co., Ltd., and customized peptides were synthesized using a peptide synthesizer.

[0058] The following example, using the analysis of matrix metalloproteinases secreted by human articular chondrocytes, further illustrates the specific implementation of the present invention.

[0059] Example 1

[0060] A method for semi-quantitatively analyzing the expression level of proteins secreted by human articular chondrocytes using Western blotting, the method specifically comprising the following steps:

[0061] (1) Preparation of culture medium containing secretory proteins:

[0062] In a sterile fume hood in a BSL-2 laboratory, 1.0 mL of DMEM / F12 serum-free culture medium containing 1% penicillin-streptomycin was added to the wells of a sterile 24-well tissue culture plate. Then, using a sterile 22-24 gauge scalpel, full-thickness articular cartilage tissue was cut from the femoral head or tibial plateau into tissue slices of similar size and shape (each slice approximately 1 cm in diameter and approximately 100 mg wet weight). One slice per well was added to the culture medium. The slices were equilibrated for 48 hours at 37°C, 5% CO2, and 100% humidity. During the equilibration period, the culture medium was replaced with fresh medium every 24 hours to remove matrix metalloproteinase-3 (MMP-3) or matrix metalloproteinase-13 (MMP-13) secreted by chondrocytes induced by mechanical cutting stimulation of the scalpel.

[0063] After equilibration, each well was replaced with 1.0 mL of fresh DMEM / F12 serum-free culture medium containing 1% penicillin-streptomycin. According to the experimental design, the culture medium was divided into four experimental groups: 1) Untreated control (UC); 2) PBS treated (PBS); 3) Negative control; and 4) Pro-inflammatory factor treated. The Pro-inflammatory factor treated group received NT at a concentration of 30 μmol / L; the Negative control group received a biologically inactive stimulating factor, out-of-order type II collagen N-terminal telopeptide, at a concentration of 30 μmol / L; the PBS treated group received 1×PBS as a peptide solvent; and the Untreated control group received no treatment. Three replicate wells were set up for both the Negative control and Pro-inflammatory factor treated groups. The corresponding cell stimulant was added to each well, marked "Day 0". The culture medium was replaced and the cell stimulant replenished in each well on Day 2 and Day 4, respectively. The experiment ended on Day 7.

[0064] (2) Collect samples of culture medium containing secreted proteins:

[0065] Culture medium samples were collected at the following time points in step (1): Day 2, Day 4, and Day 7, and transferred to clean microcentrifuge tubes. The caps and hinges of the centrifuge tubes were then removed to create a dialysis membrane covering surface, resulting in a culture medium sample containing secretory proteins.

[0066] (3) Dialysis desalting treatment of culture medium samples containing secretory proteins:

[0067] Cut a 3cm section of dialysis membrane (10K MWCO, 22mm, Thermo Scientific). TM SnakeSkin TM (Cat#68100, Lot#UG281775) immerse the dialysis membrane completely in ultrapure water for 10 minutes to one hour. Perform short-term centrifugation to allow the culture medium samples in each tube to settle. Cut one edge of the immersed dialysis membrane to extend it into a single layer. Then, cover the centrifuge tube opening with the single-layer dialysis membrane and secure it with a rubber band. Insert the microcentrifuge tube covered with the dialysis membrane into a float, invert it into a beaker containing 1.0L of ultrapure water, and perform continuous dialysis at 100rpm and 4℃ until the phenol red color in the culture medium sample completely disappears and the sample becomes clear, colorless, and transparent. Stop dialysis at this point. The total dialysis time is approximately 48 hours, with the ultrapure water replaced every 12 hours.

[0068] (4) Concentrate and prepare culture medium powder containing secretory proteins:

[0069] After the dialysis desalting process is completed, the dialysis membrane is removed, and the microcentrifuge tube containing the desalted culture medium sample is placed in a vacuum concentrator. The sample is dehydrated and concentrated at 4℃ and 0.02 atm for 7 hours to obtain a culture medium powder sample containing secretory proteins.

[0070] (5) Reconstitution of concentrated culture medium samples and protein quantification:

[0071] Add 50 μL of ultrapure water to the culture medium powder obtained in step (4), repeatedly pipette and transfer to another clean microcentrifuge tube, mix thoroughly using a vortex mixer to complete the culture medium sample reconstitution step, take 1 μL of culture medium sample, and use NanoDrop... TM The A280 value and corresponding protein concentration were read using a One / OneC micro-UV-Vis spectrophotometer, and the results are shown in Table 1.

[0072] Table 1 Sample A of each group 280 Value and total protein concentration

[0073]

[0074] (6) Separation of protein components in the reconstituted culture medium by polypropylene gel electrophoresis:

[0075] Add loading buffer containing 2% SDS to the culture medium sample after reconstitution in step (5), heat at 100℃ for 5 min, load the culture medium sample with a total protein amount of 2-10 μg, load the sample onto a 4% polyacrylamide concentrator gel, electrophores at a constant current of 20 mA / gel, and complete the separation of secretory proteins on a 10% polyacrylamide separation gel.

[0076] (7) Detection of the expression level of the secreted protein to be tested in culture medium samples by immunoblotting:

[0077] The proteins separated in the gel were imprinted onto a nitrocellulose membrane and then incubated overnight at 4°C in a buffer containing a specific antibody (primary antibody) for matrix metalloproteinase-3 or matrix metalloproteinase-13. After incubation at room temperature for 1 hour in a secondary antibody labeled with HRP, HRP substrate was added to the membrane, and an imager captured and presented the chemiluminescent signals emitted by the protein bands. The grayscale values ​​of the matrix metalloproteinase-3 or matrix metalloproteinase-13 bands were analyzed and bar graphs were plotted using ImageJ image analysis software.

[0078] Test results are as follows Figure 5As shown in the figure, the matrix metalloproteinase-3 (MMP-3) and MMP-13 (MMP-13) protein bands (Lane No. 7, 8, 9) of the pro-inflammatory factor group are clearly visible, and the average gray value of the bands is significantly higher than that of the untreated group (Lane No. 2; UC), the pro-inflammatory factor solvent group (Lane No. 3; PBS), or the negative control group (Lane No. 4, 5, 6; Negative Control).

[0079] Example 2

[0080] A method for semi-quantitatively analyzing the expression level of proteins secreted by human articular chondrocytes using Western blotting, the method specifically comprising the following steps:

[0081] (1) Preparation of culture medium containing secretory proteins:

[0082] In a sterile fume hood in a BSL-2 laboratory, 1.5 mL of DMEM / F12 serum-free culture medium containing 0.5% penicillin-streptomycin was added to the wells of a sterile 24-well tissue culture plate. Then, using a sterile 22-24 gauge scalpel, full-thickness articular cartilage tissue was cut from the femoral head or tibial plateau into tissue slices of similar size and shape (each slice approximately 0.5 cm in diameter and approximately 80 mg wet weight). One slice per well was added to each culture well containing culture medium. The culture was equilibrated for 72 hours at 37°C, 5% CO2, and 100% humidity. During the equilibration period, the culture medium was replaced with fresh medium every 24 hours to remove matrix metalloproteinase-3 (MMP-3) or matrix metalloproteinase-13 (MMP-13) secreted by chondrocytes induced by mechanical cutting stimulation of the scalpel.

[0083] After equilibration, each well was replaced with 1.5 mL of fresh DMEM / F12 serum-free culture medium containing 0.5% penicillin-streptomycin antibiotics. Simultaneously, according to the experimental design, the culture medium was divided into eight experimental groups: 1) Nontreated control (NC); 2) PBS treated; 3) Negative control of NT (scrambled NT; SN); 4) Negative control of CT (scrambled CT; SC); 5) HP treated; 6) NT treated; 7) CT treated; 8) IL-1β treated. The following groups were added to the culture medium: HP treated group (containing type II collagen triple helix peptides at a concentration of 30 μmol / L); NT treated group (containing type II collagen N-terminal telopeptides at a concentration of 30 μmol / L); CT treated group (containing type II collagen C-terminal telopeptides at a concentration of 30 μmol / L); IL-1β treated group (containing recombinant human IL-1β (rhIL-1β) at a final concentration of 10 ng / mL); Nontreated control (NC) group (containing no treatment); PBS treated group (containing peptides or rhIL-1β solvent 1×PBS); Negative control of NT group (containing disordered type II collagen N-terminal telopeptides (SN) at a concentration of 30 μmol / L); and Negative control of CT group (containing disordered type II collagen C-terminal telopeptides (SC) at a final concentration of 30 μmol / L. Add the corresponding cell stimulant to each group of culture wells, and mark it as "Day 0". On Day 2, change the culture medium and replenish the cell stimulant in each well. The experiment ends on the 4th day.

[0084] (2) Collect samples of culture medium containing secreted proteins:

[0085] Collect culture medium samples at the following time points in step (1): Day 2 and Day 4, and transfer them to clean microcentrifuge tubes. Cut off the caps and hinges of the centrifuge tubes to create a dialysis membrane covering surface, and obtain a culture medium sample containing secretory proteins.

[0086] (3) Dialysis desalting treatment of culture medium samples containing secretory proteins:

[0087] Cut a 2cm section of dialysis membrane (10K MWCO, 22mm, Thermo Scientific). TM SnakeSkin TM(Cat#68100, Lot#UG281775) immerse the dialysis membrane completely in ultrapure water for 10 minutes to one hour. Perform short-term centrifugation to allow the culture medium samples in each tube to settle. Cut one edge of the immersed dialysis membrane to extend it into a single layer. Then, cover the centrifuge tube opening with the single-layer dialysis membrane and secure it with a rubber band. Insert the microcentrifuge tube covered with the dialysis membrane into a float, invert it into a beaker containing 1.0L of ultrapure water, and perform continuous dialysis at 70rpm and 0℃ until the phenol red color in the culture medium sample completely disappears and the sample becomes clear, colorless, and transparent. Replace the ultrapure water every 12 hours during this process.

[0088] (4) Concentrate and prepare culture medium powder containing secretory proteins:

[0089] After the dialysis desalting process is completed, the dialysis membrane is removed, and the microcentrifuge tube containing the desalted culture medium sample is placed in a vacuum concentrator. The sample is dehydrated and concentrated for 5 hours at 20°C and 0.01 atm to obtain a culture medium powder sample containing secretory proteins.

[0090] (5) Reconstitution of concentrated culture medium samples and protein quantification:

[0091] Add 40 μL of ultrapure water to the culture medium powder obtained in step (4), repeatedly pipette and transfer it into another clean microcentrifuge tube, mix it using a vortex mixer to complete the culture medium sample reconstitution step, take 1 μL of the culture medium sample, and use NanoDrop... TM One / OneC micro UV-Vis spectrophotometer, read A280 value and corresponding protein concentration.

[0092] (6) Separation of protein components in the reconstituted culture medium by polypropylene gel electrophoresis:

[0093] Add loading buffer containing 2% SDS to the culture medium sample after reconstitution in step (5), heat at 95°C for 3 min, load the culture medium sample with a total protein amount of 2-10 μg, load the sample onto a 4% polyacrylamide concentrator gel, electrophores at a constant current of 20 mA / gel, and complete the separation of secretory proteins on a 10% polyacrylamide separation gel.

[0094] (7) Detection of the expression level of the secreted protein to be tested in culture medium samples by immunoblotting:

[0095] The proteins separated in the gel were imprinted onto a nitrocellulose membrane and then incubated overnight at 4°C in a buffer containing a specific antibody (primary antibody) for matrix metalloproteinase-3 or matrix metalloproteinase-13. After incubation at room temperature for 2 hours in a secondary antibody labeled with HRP, HRP substrate was added to the membrane, and an imager captured and presented the chemiluminescent signals emitted by the protein bands. The grayscale values ​​of the matrix metalloproteinase-3 or matrix metalloproteinase-13 bands were analyzed and bar graphs were plotted using ImageJ image analysis software.

[0096] Test results are as follows Figure 6 As shown in the figure, on the fourth day of culture, the protein bands of matrix metalloproteinase-3 (MMP-3) and MMP-13 (MMP-13) in the groups of inflammatory factors recombinant human interleukin-1β (IL-1β), type II collagen telopeptide (NT or CT), and helical peptide (HP) were clearly visible to the naked eye, while the protein band signals in the control groups (NC, PBS, SN, SC) were basically undetectable.

[0097] Example 3

[0098] A method for semi-quantitatively analyzing the expression level of proteins secreted by human articular chondrocytes using Western blotting, the method specifically comprising the following steps:

[0099] (1) Preparation of culture medium containing secretory proteins:

[0100] In a sterile fume hood in a BSL-2 laboratory, 1.0 mL of DMEM / F12 serum-free culture medium containing 1% penicillin-streptomycin was added to the wells of a sterile 24-well tissue culture plate. Then, using a sterile 22-24 gauge scalpel, full-thickness articular cartilage tissue was cut from the femoral head or tibial plateau into tissue slices of similar size and shape (each slice approximately 1 cm in diameter and approximately 100 mg wet weight). One slice per well was added to the culture medium. The slices were equilibrated for 48 hours at 37°C, 5% CO2, and 100% humidity. During the equilibration period, the culture medium was replaced with fresh medium every 24 hours to remove matrix metalloproteinase-3 (MMP-3) or matrix metalloproteinase-13 (MMP-13) secreted by chondrocytes induced by mechanical cutting stimulation of the scalpel.

[0101] After equilibration, each well was replaced with 1.0 mL of fresh DMEM / F12 serum-free culture medium containing 1% penicillin-streptomycin. At the same time, according to the experimental design, the culture medium was divided into eight experimental groups: 1) Non-treated control (NC); 2) PBS treated; 3) Negative control of NT (scrambled NT; SN); 4) Negative control of CT (scrambled CT; SC); 5) HP treated; 6) NT treated; 7) CT treated; 8) IL-1β treated. The following groups were added to the culture medium: HP treated group (containing type II collagen triple helix peptides at a concentration of 30 μmol / L); NT treated group (containing type II collagen N-terminal telopeptide at a concentration of 30 μmol / L); CT treated group (containing type II collagen C-terminal telopeptide at a concentration of 30 μmol / L); IL-1β treated group (containing recombinant human IL-1β (rhIL-1β) at a concentration of 10 ng / mL); Nontreated control group (containing no treatment); PBS treated group (containing peptide solvent 1×PBS); Negative control of NT group (containing disordered type II collagen N-terminal telopeptide (SN) at a concentration of 30 μmol / L); Negative control of CT group (containing disordered type II collagen C-terminal telopeptide (SC) at a concentration of 30 μmol / L. Add the corresponding cell stimulant to each group of culture wells, and mark it as "Day 0". Change the culture medium and replenish the cell stimulant in each well on Day 2 and Day 4 respectively. End the experiment on the 7th day.

[0102] (2) Collect samples of culture medium containing secreted proteins:

[0103] Culture medium samples were collected at the following time points in step (1): Day 2, Day 4, and Day 7, and transferred to clean microcentrifuge tubes. The caps and hinges of the centrifuge tubes were then removed to create a dialysis membrane covering surface, resulting in a culture medium sample containing secretory proteins.

[0104] (3) Dialysis desalting treatment of culture medium samples containing secretory proteins:

[0105] Cut a 3cm section of dialysis membrane (10K MWCO, 22mm, Thermo Scientific). TM SnakeSkin TM(Cat#68100, Lot#UG281775) immerse the dialysis membrane completely in ultrapure water for 10 minutes to one hour. Perform short-term centrifugation to allow the culture medium samples in each tube to settle. Cut one edge of the immersed dialysis membrane to extend it into a single layer. Then, cover the centrifuge tube opening with the single-layer dialysis membrane and secure it with a rubber band. Insert the microcentrifuge tube covered with the dialysis membrane into a float, invert it into a beaker containing 1.0L of ultrapure water, and perform continuous dialysis at 100rpm and 4℃ until the phenol red color in the culture medium sample completely disappears and the sample becomes clear, colorless, and transparent. Replace the ultrapure water every 12 hours during this process.

[0106] (4) Concentrate and prepare culture medium powder containing secretory proteins:

[0107] After the dialysis desalting process is completed, the dialysis membrane is removed, and the microcentrifuge tube containing the desalted culture medium sample is placed in a vacuum concentrator. The sample is dehydrated and concentrated at 10℃ and 0.02 atm for 6 hours to obtain a culture medium powder sample containing secretory proteins.

[0108] (5) Reconstitution of concentrated culture medium samples and protein quantification:

[0109] Add 60 μL of ultrapure water to the culture medium powder obtained in step (4), repeatedly pipette and transfer it into another clean microcentrifuge tube, mix it using a vortex mixer to complete the culture medium sample reconstitution step, take 1 μL of the culture medium sample, and use NanoDrop... TM One / OneC micro UV-Vis spectrophotometer, read A280 value and corresponding protein concentration.

[0110] (6) Separation of protein components in the reconstituted culture medium by polypropylene gel electrophoresis:

[0111] Add loading buffer containing 2% SDS to the culture medium sample after reconstitution in step (5), heat at 100℃ for 5 min, load the culture medium sample with a total protein amount of 2-10 μg, load the sample onto a 4% polyacrylamide concentrator gel, electrophores at a constant current of 20 mA / gel, and then separate the secreted protein onto a 10% polyacrylamide separation gel to complete the separation of secreted proteins.

[0112] (7) Detection of the expression level of the secreted protein to be tested in culture medium samples by immunoblotting.

[0113] The proteins separated in the gel were imprinted onto a nitrocellulose membrane and then incubated overnight at 4°C in a buffer containing a specific antibody (primary antibody) for matrix metalloproteinase-3 or matrix metalloproteinase-13. After incubation at room temperature for 1 hour in a secondary antibody labeled with HRP, HRP substrate was added to the membrane, and an imager captured and presented the chemiluminescent signals emitted by the protein bands. The grayscale values ​​of the matrix metalloproteinase-3 or matrix metalloproteinase-13 bands were analyzed and bar graphs were plotted using ImageJ image analysis software.

[0114] Test results are as follows Figure 7 As shown in the figure, on the seventh day of culture, the matrix metalloproteinase-3 (MMP-3) and MMP-13 (MMP-13) protein bands of the inflammatory factors recombinant human interleukin-1β (IL-1β), type II collagen telopeptide (NT or CT), and helical peptide (HP) groups showed extremely strong signals, while the protein band signals of the control groups (NC, PBS, SN, SC) were extremely weak.

[0115] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for semi-quantitatively analyzing the expression level of proteins secreted by human articular chondrocytes using Western blotting technology, characterized in that, The method includes the following steps: (1) Preparation of culture medium containing secretory proteins: Adding pro-inflammatory factors to the culture medium containing human articular cartilage tissue slices to stimulate the culture medium yielded a culture medium containing secretory proteins. (2) Collect samples of culture medium containing secretory proteins: The culture medium containing secretory protein obtained in step (1) was transferred into a centrifuge tube, and the cap and hinge of the centrifuge tube were cut off to obtain a sample of culture medium containing secretory protein. (3) Dialysis desalination of the sample: Cover the centrifuge tube opening of the sample obtained in step (2) with the dialysis membrane tube, tie it tightly, place it in ultrapure water and stir to dialyze, and obtain a sample of culture medium containing secretory protein after desalting; (4) Concentrate and prepare culture medium powder containing secretory proteins: After removing the dialysis membrane of the sample obtained in step (3), vacuum concentration was performed to obtain a culture medium powder sample containing secretory proteins. (5) Reconstituted culture medium containing secretory protein powder and protein quantification: After reconstituted culture medium powder samples obtained in step (4) with ultrapure water, the samples were transferred to new centrifuge tubes to obtain reconstituted culture medium samples. The absorbance value was measured to obtain the total secreted protein concentration in each sample. (6) Separation of protein components from the reconstituted culture medium: Protein components were separated in a separating gel by polyacrylamide gel electrophoresis to obtain a separating gel containing the secreted protein to be tested. (7) Detection of the expression level of secretory proteins in reconstituted culture medium samples by Western blotting: The separating gel containing the secreted protein to be tested obtained in step (6) was transferred to a membrane, blocked, and incubated with antibodies to detect the relative expression level of the secreted protein to be tested. The pro-inflammatory factor is a polypeptide from the triple helix region of type II collagen; the amino acid sequence of the polypeptide from the triple helix region of type II collagen is shown in SEQ ID NO. 3; The dialysis temperature is 0-4℃; The vacuum concentration temperature is 4-20℃.

2. The method according to claim 1, characterized in that, In step (1), the diameter of the tissue slice is 0.5-1.0 cm, and the amount added is 80-100 mg wet weight / culture well; the tissue slice is added to stimulate with pro-inflammatory factors after equilibration; the equilibration is specifically: the tissue slice is placed in a culture well containing culture medium, the culture medium including DMEM / F12 and penicillin-streptomycin, the amount is 1.0-1.5 mL / culture well; the volume fraction of penicillin-streptomycin in the culture medium is 0.5-1.0%; the equilibration time is 2-3 days, and the culture medium is changed every 24 h during the equilibration period.

3. The method according to claim 1, characterized in that, In step (1), the concentration of the type II collagen triple helix peptide in the culture medium is 30 µmol / L; the stimulation time is 4-7 days; and the volume of the culture medium is kept consistent in each well.

4. The method according to claim 3, characterized in that, The stimulation period was 4 days, with pro-inflammatory factors added on days 0 and 2 of stimulation, and the culture medium was changed each time they were added.

5. The method according to claim 3, characterized in that, The stimulation period was 7 days, with pro-inflammatory factors added on days 0, 2, and 4 of stimulation, and the culture medium was changed each time they were added.

6. The method according to claim 1, characterized in that, In step (3), the length of the dialysis membrane tube is 2-3 cm; the stirring speed is 70-100 rpm; dialysis is stopped when the sample solution is clear, colorless and transparent, and the ultrapure water is replaced every 12 hours during the dialysis process.

7. The method according to claim 1, characterized in that, In step (4), the vacuum degree is 0.01-0.02 atm and the time is 5-7 h.

8. The method according to claim 1, characterized in that, In step (5), the volume of ultrapure water is 40-60 μL / sample tube, and the absorbance value is measured at a wavelength of 280 nm.

9. The method according to claim 1, characterized in that, In step (6), the separation specifically includes adding a loading buffer, denaturation, and loading; the loading buffer contains sodium dodecyl sulfate at a concentration of 2%; the denaturation temperature is 95-100℃ and the time is 3-5 min.

10. The method according to claim 1, characterized in that, In step (7), the transfer is the process of transferring the secreted protein to be tested from the separating gel to a nitrocellulose membrane, and the antibody is labeled with a fluorescent molecule; the antibody includes a fluorescently labeled primary antibody or a combination of an unlabeled primary antibody and a secondary antibody modified with horseradish peroxidase; the primary antibody is a rabbit-derived antibody against human chondrocyte secretory protein, and the human chondrocyte secretory protein includes matrix metalloproteinase-3 and matrix metalloproteinase-13; the secondary antibody is a sheep-derived antibody against rabbit IgG; the incubation temperature of the primary antibody is 2-6℃ for 10-15h, and the incubation temperature of the secondary antibody is 15-30℃ for 1-2h.

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