Activity analysis and detection method of stress-induced mesenchymal stem cell extract

Abstract

The invention provides an activity analysis and detection method of a stress-induced mesenchymal stem cell extract. The activity analysis and detection method comprises the following steps: step 1, culturing human primary articular cartilage cells; 2, the culture solution is sucked out, a to-be-detected composition with the same volume is added, incubation is conducted, and the to-be-detected composition comprises a mesenchymal stem cell extract; and step 3, then adding a lipopolysaccharide solution with the isovolumetric concentration of 80 ng / mL-120 ng / mL, continuing incubation, and collecting a cell lysis solution which is used for detecting inflammatory cytokines. According to the present invention, the optimal use concentration and the optimal cell incubation time of the mesenchymal stem cell extract (marked as ALITO), the freeze-drying agent composition (marked as ALIP) and the lipopolysaccharide solution are determined by determining the cell viability and the levels of the cell inflammatory factors IL-6, IL-1beta and TNF-alpha, and the cell viability and the inflammatory factors are detected so as to determine the optimal use concentration and the optimal cell incubation time of the mesenchymal stem cell extract (marked as ALITO), the freeze-drying agent composition (marked as ALIP) and the lipopolysaccharide solution; the quality of the mesenchymal stem cell extract can be efficiently evaluated, and the activity analysis and detection method is high in repeatability and good in stability.

Description

Technical Field

[0001] The present application relates to the technical field of biopharmaceuticals, and in particular to a method for analyzing and detecting the activity of stress-induced mesenchymal stem cell extracts. Background Art

[0002] After mesenchymal stem cells are exposed to an inflammatory environment, they produce very high levels of immunosuppressive factors such as TNF-α, IL-1β, and interferon (IFN)γ. Intra-articular injection of mesenchymal stem cells is considered a promising option for the treatment of osteoarthritis. For example, in mice, a single intra-articular injection of purified mesenchymal stem cells reduced synovitis and cartilage damage by inhibiting synovial macrophage activation; the potential therapeutic effects of mesenchymal stem cell-based osteoarthritis therapies have also been observed in various animal models, including sheep, rabbits, miniature pigs, and even clinical studies. Many related technologies have conducted various mesenchymal stem cell clinical studies, most of which focus on the evaluation of therapeutic effects.

[0003] Mesenchymal stem cell extracts are macromolecules obtained by stimulating the expression of mesenchymal stem cells and isolating and purifying them. Since macromolecules usually cannot penetrate the cell membrane and can only act on the cell surface or through extracellular pathways, mesenchymal stem cell extracts may have problems with poor repeatability and stability in the efficacy testing of joint disease treatment, and there is no good evaluation mechanism to evaluate the quality of mesenchymal stem cell extracts. This problem is even more complicated when evaluating the efficacy of mesenchymal stem cell extracts and other macromolecular drugs. Therefore, it is urgent to develop a detection method that can effectively evaluate the performance of mesenchymal stem cell extracts and / or other macromolecular drugs, and it is also of great significance to explore the optimal drug concentration of mesenchymal stem cell extracts under this detection method. Summary of the invention

[0004] Based on this, the present application intends to use lipopolysaccharide (LPS) to induce inflammation in human articular chondrocytes, introduce mesenchymal stem cell extracts, and explore the changing patterns of the anti-inflammatory activity of mesenchymal stem cell extracts and the optimal drug concentration by monitoring the relevant indicators of inflammatory cytokines.

[0005] The technical solution adopted in this application is:

[0006] On the one hand, the present application provides a method for analyzing the activity of a stress-induced mesenchymal stem cell extract, the method comprising:

[0007] Step 1: Cultivating primary human articular chondrocytes;

[0008] Step 2, aspirating the culture medium, adding an equal volume of a test composition, and incubating, wherein the test composition includes a mesenchymal stem cell extract;

[0009] Step 3: Then add an equal volume of 80 ng / mL-120 ng / mL lipopolysaccharide solution, continue incubation, and collect cell lysate, which is used to detect inflammatory cytokines.

[0010] In some embodiments, in step 1, the culturing of human primary articular chondrocytes comprises adjusting the concentration of the human primary articular chondrocytes digested with pancreatic enzyme working solution to 2×10 4 cells / mL-5×10 4 cells / mL, and inoculated in a cell culture plate at a density of 2000 cells-5000 cells per well, and cultured until all cells adhere to the wall. Preferably, the culture time is 12h-30h, more preferably 24h-30h, such as 24h, and the cell culture plate is a 96-well plate.

[0011] The mass volume ratio of the pancreatic enzyme working solution is 0.25%, which means that 0.25 g of pancreatic enzyme is dissolved in 100 mL of phosphate buffered saline (PBS).

[0012] The human primary articular chondrocytes before being digested with pancreatic enzyme working solution are cells in good growth state.

[0013] In some embodiments, in step 1, the culture conditions are 37°C, 5% CO 2 Culture in a cell culture incubator.

[0014] In some embodiments, in step 2, the composition to be tested also includes a lyophilized composition, which includes collagen, chitosan and a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is selected from one or more of a buffer salt, an osmotic pressure regulator, a lyoprotectant, and a pH regulator.

[0015] In the present application, the lyophilized composition is prepared using Example 1 in CN202311344207.6.

[0016] In some embodiments, the composition to be tested in step 2 further comprises a cell viability test, wherein the cell viability test comprises setting up a blank control group and an experimental group, wherein the blank control group and the experimental group are both added with a density of 2×10 3 cells / well-5×10 3cells / well of human primary articular chondrocyte suspension, culture for 12h-30h, aspirate the culture medium in the experimental group, add 100μL of the test composition, continue incubation for 24h-96h, then add 10μL of CCK-8 solution, incubate for 1h-2h, and detect the cell viability of the human primary articular chondrocytes.

[0017] The culture conditions are 37°C, 5% CO 2 Cell culture incubator.

[0018] The concentration of the lyophilized composition is generally 10 μg / mL-1 mg / mL, or the lyophilized composition of 1 mg / mL is subjected to a heating treatment, preferably, the heating temperature is 72° C., the heating time is 15s-150s, preferably 20s-120s.

[0019] The concentration of the mesenchymal stem cell extract is generally 100 ng / mL-10 μg / mL, preferably 100 ng / mL-1 μg / mL, such as 100 ng / mL.

[0020] Wherein, when the composition to be tested is the mesenchymal stem cell extract and the lyophilized composition, the concentration of the mesenchymal stem cell extract is 100 ng / mL, and the concentration of the lyophilized composition is 1 mg / mL.

[0021] The preparation method of the lyophilized composition and the mesenchymal stem cell extract comprises using complete cell culture medium to dissolve the lyophilized composition and the mesenchymal stem cell extract respectively as mother solutions, and then diluting them with complete cell culture medium.

[0022] Among them, in some possible embodiments, the preparation method of the lyophilized composition includes dissolving the lyophilized composition in 6 mL of complete cell culture medium to prepare a mother solution with a concentration of 10 mg / mL, and then diluting it with complete cell culture medium before use.

[0023] Among them, in some possible embodiments, the preparation method of the mesenchymal stem cell extract includes dissolving the mesenchymal stem cell extract in 1.3 mL of complete cell culture medium to prepare a mother solution with a concentration of 130 μg / mL, and then diluting it with complete cell culture medium before use.

[0024] In some embodiments, in step 2, the incubation time is 48 h-96 h, preferably 72 h-96 h, and more preferably 96 h.

[0025] In some embodiments, in step three, the method for preparing the lipopolysaccharide solution comprises soaking the lipopolysaccharide in sterile PBS to prepare a mother solution, which is then diluted with complete cell culture medium before use.

[0026] The concentration of the mother solution is 2 mg / mL-8 mg / mL, preferably 5.0 mg / mL-8 mg / mL, and more preferably 5.0 mg / mL.

[0027] In some possible embodiments, in step three, the preparation method of the 100 ng / mL lipopolysaccharide solution includes dissolving LPS with a specification of 25 mg / bottle in 5 mL of sterile PBS to prepare a mother solution with a concentration of 5.0 mg / mL, packaging it in 100 μL / tube, and diluting it 1:50000 with complete cell culture medium to obtain a 100 ng / mL LPS solution when used.

[0028] In some embodiments, in step three, the continued incubation time is 12h-30h, preferably 18h-24h, and more preferably 24h.

[0029] In the present application, the inflammatory cytokines include one or more of IL-6, IL-1β and TNF-α.

[0030] In some embodiments, the concentration of the lyophilized composition is 10 μg / mL-1 mg / mL, the incubation time is 48 hours, and the content of IL-6 in the human primary articular chondrocyte inflammation model can be reduced.

[0031] In some embodiments, the concentration of the lyophilized composition is 10 μg / mL-1 mg / mL, the incubation time is 96 hours, and the content of IL-1β in the human primary articular chondrocyte inflammation model can be reduced.

[0032] In some embodiments, the concentration of the mesenchymal stem cell extract is 100 ng / mL-10 μg / mL, the incubation time is 48h-96h, and the content of IL-6 and IL-1β in the human primary articular chondrocyte inflammation model can be reduced.

[0033] In some embodiments, the lyophilized composition has a concentration of 1 mg / mL and is heated at 72° C. for 20 s to 120 s, which can reduce the content of IL-6 in a human primary articular chondrocyte inflammation model.

[0034] In some embodiments, the lyophilized composition has a concentration of 1 mg / mL and is heated at 72° C. for 60 s-120 s, which can reduce the content of IL-1β in a human primary articular chondrocyte inflammation model.

[0035] In some embodiments, when the concentration of the lyophilized composition is 1 mg / mL and the concentration of the mesenchymal stem cell extract is 100 ng / mL, the content of IL-6 in the human primary articular chondrocyte inflammation model can be reduced.

[0036] The beneficial effects of this application are:

[0037] The present application provides an activity analysis detection method for stress-induced mesenchymal stem cell extracts. The optimal use concentrations and cell incubation times of the mesenchymal stem cell extract (denoted as Aleto), the lyophilized agent composition (denoted as Aleto), and the lipopolysaccharide solution are determined by measuring the cell viability and the levels of the cell inflammatory factors IL-6, IL-1β, and TNF-α. The quality of the mesenchymal stem cell extract can be efficiently evaluated by detecting the cell viability and inflammatory factors. The activity analysis detection method has high repeatability and good stability.

[0038] The inventors have found that both Alituo and Alip have good inhibitory effects on inflammation of human primary articular chondrocytes. In the detection of human articular chondrocyte inflammation, by controlling the concentration range of LPS in 80ng / mL-120ng / mL, especially in the in vitro human primary articular chondrocyte inflammation detection system induced by 100ng / mL LPS, when Alituo and / or Alip are added for treatment, Alituo and Alip have significant effects on reducing the levels of IL-1β, TNF-α and IL-6 inflammatory factors in human primary articular chondrocytes induced by LPS, and can effectively inhibit the inflammation of human articular chondrocytes caused by LPS. Furthermore, 100 ng / mL Alital, 100 ng / mL Alital combined with 1 mg / mL Aliprex, 1 mg / mL Aliprex, and 1 mg / mL Aliprex after heating at 72°C for 20s-120s can all reduce the content of inflammatory IL-6 in human primary articular chondrocytes; 100 ng / mL Alital, 1 mg / mL Aliprex, 1 mg / mL Aliprex, and 1 mg / mL Aliprex after heating at 72°C for 60s-120s can reduce the content of inflammatory IL-1β in human primary articular chondrocytes.

[0039] In the cell viability test, 10mg / mL Alipe and 10mg / mL Alipe combined with 100ng / mL Alito showed inhibitory effects on human primary articular chondrocytes, and the inhibitory effect was positively correlated with the incubation time. 1mg / mL Alipe significantly improved the cell viability of human articular chondrocytes, and the inhibitory effect of Alito 1ng / mL-10μg / mL on the cell viability of human articular chondrocytes decreased from D3 to D4. The test results also verified that the inhibitory effects of Alipe and Alito on chondrocytes have strong repeatability and stability, providing a reliable reference for the concentration screening of the test composition in further inflammation detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a graph showing the changing trend of cell viability in the blank control group and the groups with different doses of Alipin in Example 1;

[0041] Figure 2 This is a graph showing the changing trend of cell viability in the blank control group and different doses of alituo groups in Example 1;

[0042] Figure 3 This is a graph showing the changing trend of cell viability in each group in Example 2;

[0043] Figure 4 The IL-6 content of each group of cells in the exploratory test of different doses of lipopolysaccharide combined with the test substance for human articular chondrocytes in Example 3;

[0044] Figure 5 The TNF-α content of each group of cells in the exploratory test of different doses of lipopolysaccharide combined with the test substance on human articular chondrocytes in Example 3;

[0045] Figure 6 The IL-1β content of each group of cells in the exploratory test of different doses of lipopolysaccharide combined with the test substance for human articular chondrocytes in Example 3;

[0046] Figure 7 The IL-6 content of each group of cells in the exploratory test of LPS-induced inflammation in human articular chondrocytes in Example 4;

[0047] Figure 8 is the TNF-α content of each group of cells in the exploratory test of LPS-induced inflammation in human articular chondrocytes in Example 4;

[0048] Fig. 9 is the IL-1β content of each group of cells in the exploratory test of LPS-induced inflammation in human articular chondrocytes in Example 4;

[0049] Fig.10 The IL-6 content of each group of cells in the detection results of LPS-induced human articular chondrocyte inflammation in Example 5;

[0050] Fig.11 is the TNF-α content of each group of cells in the detection results of LPS-induced human articular chondrocyte inflammation in Example 5;

[0051] Fig.12 This is the IL-1β content of each group of cells in the LPS-induced human articular chondrocyte inflammation detection result in Example 5. DETAILED DESCRIPTION

[0052] The technical solution of the present application is further explained below in conjunction with embodiments.

[0053] For the convenience of explanation, mesenchymal stem cells are used as examples in the embodiments, and the sources of the mesenchymal stem cells include but are not limited to at least one of human umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

[0054] After undergoing stress conditions, mesenchymal stem cells can theoretically induce the production of corresponding stress proteins. For the convenience of explanation, the mesenchymal stem cell extract in this application is a frozen protein polymer prepared by the method shown in the culture test 1 in CN202410944325.9. In practical applications, other stress conditions can achieve similar effects. Other stress conditions include, but are not limited to, at least one of the following stress methods: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, hypoxia, high oxygen, oxidation (under conditions containing oxidants such as hydrogen peroxide or hypochlorous acid), high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, rays, rays, rays, ion beams, high CO 2 , low CO 2 . Various stress management methods can be carried out one after another or simultaneously when they do not conflict.

[0055] The research idea of ​​this application is to culture human primary articular chondrocytes, first use the CCK-8 method to detect the inhibitory effect of alituo and alipin on the proliferation of human articular chondrocytes, and then conduct exploratory tests on the effects of different doses of test substances on cell viability and detection tests on the effects of different concentrations of test substances and combinations of test substances on cell viability based on the trial results; use MSD to study the effects of alipin and alipin on the content of inflammatory factors in LPS-induced articular chondrocyte inflammation, and based on the results of cell viability exploration, set different concentrations of alipin and alipin combined with different concentrations of LPS to explore the anti-inflammatory activity.

[0056] Through the cell survival rate exploration experiment, it was found that when the dose of Alipe was 1 mg / mL, it had no obvious inhibitory effect on the growth of human articular chondrocytes, and 10 mg / mL of Alipe had a significant inhibitory effect on cell survival, which was also verified by the cell survival rate test results; the results of the cell inflammation exploration test showed that under the stimulation of 80ng / mL-120ng / mL, especially 100ng / mL of LPS, the gene expression levels of inflammatory factors IL-6 and IL-1β in articular chondrocytes were significantly increased, while Alipe and Alito could inhibit the activation of inflammatory response to varying degrees. The inflammatory treatment effect of Alipe and Alito was also verified by the cell inflammation test results, indicating that the model has high repeatability and good stability.

[0057] The present application provides a method for analyzing and detecting the activity of a stress-induced mesenchymal stem cell extract, the method comprising:

[0058] Step 1: Cultivating primary human articular chondrocytes;

[0059] Step 2, aspirating the culture medium, adding an equal volume of a test composition, and incubating, wherein the test composition includes a mesenchymal stem cell extract;

[0060] Step 3: Then add an equal volume of 80 ng / mL-120 ng / mL lipopolysaccharide solution, continue incubation, and collect cell lysate, which is used to detect inflammatory cytokines.

[0061] In some embodiments, the activity analysis detection method includes: setting a blank control group and experimental groups with different concentration gradients; adding a density of 2×10 3 cells / well of human primary articular chondrocyte suspension and incubate at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator for 24 hours. In the experimental group, the culture medium was aspirated, 100 μL of the composition to be tested was added, and the blank control group and the experimental group were incubated for 24 hours, 48 ​​hours, 72 hours, and 96 hours, respectively. Then, 10 μL of CCK-8 solution was added, incubated for 1 hour to 2 hours, and the cell viability of the human primary articular chondrocytes was detected.

[0062] In some embodiments, the activity analysis detection method comprises the following steps: according to the detection results of the cell viability, a blank control group, a model control group, and an experimental group of Alip and Alituo with a preset concentration are set up respectively, and 2 mL of a 5×10 4 cells / mL of human primary articular chondrocyte suspension and placed in a 37°C, 5% CO 2 Cultured in a cell culture incubator for 24 hours. In the experimental group, the cell culture medium was aspirated, and a preset concentration of Alip or Alituo was added to each well. The blank control group, model control group, and experimental group were incubated for 48 hours, 72 hours, and 96 hours, respectively. After incubation, an equal volume of 100 ng / mL lipopolysaccharide was added to each well of the experimental group and incubated for 24 hours. An equal volume of complete culture medium and corresponding lipopolysaccharide were added to the blank control group and the model control group, respectively, and incubated for 24 hours, and cell lysates were collected. MSD was used to detect the content of inflammatory factors.

[0063] In some embodiments, the activity analysis detection method comprises the following steps: setting up a cell control group, a model control group, a negative control group, an experiment group with preset concentrations of Alip and Alituo, an Alip heating experiment group, and an Alip and Alituo combined experiment group, and adding 2 mL of 5×10 4cells / mL of human primary articular chondrocyte suspension and placed in a 37°C, 5% CO 2 Cultured in a cell culture incubator for 24 hours. In the experimental group, the cell culture medium was aspirated, and each well was added with 1 mg / mL of Alip; 1 mg / mL of Alip heated at 72°C for 20s-120s; 100 ng / mL of Alituo; 1 mg / mL of Alip combined with 100 ng / mL of Alituo, and the blank control group, model control group, negative control group and experimental group were incubated for 48 hours, 72 hours and 96 hours respectively. After incubation, an equal volume of 100 ng / mL of lipopolysaccharide was added to each well of the experimental group, and the incubation continued for 24 hours; the blank control group and the model control group were added with an equal volume of complete culture medium and the corresponding lipopolysaccharide; the negative control group was added with 1 mg / mL of Alip, 100 ng / mL of Alituo, and 1 mg / mL of Alip combined with 100 ng / mL of Alituo, and incubated for 24 hours, and the cell lysate was collected. MSD was used to detect the levels of inflammatory factors.

[0064] In some embodiments, in step one, the human primary articular chondrocyte culture includes adjusting the concentration of the human primary articular chondrocytes in good growth state after digestion with pancreatic enzyme working solution with human chondrocyte culture solution, and inoculating them in a cell culture plate at a density of 2000 cells-5000 cells per well, and culturing until all cells are attached to the wall.

[0065] The concentration of human primary articular chondrocytes is generally 2×10 4 cells / mL-5×10 4 cells / mL.

[0066] The culture time is not particularly limited as long as all cells are attached to the wall, for example, 12 h to 30 h, preferably 24 h to 30 h, and more preferably 24 h.

[0067] The mass-to-volume ratio of the pancreatic enzyme working solution is a conventional choice in the art, which is 0.25%, meaning that 0.25 g of pancreatic enzyme is dissolved in 100 mL of PBS.

[0068] The cell culture plate is generally conventional in the art, such as a 96-well plate.

[0069] The culture conditions are 37°C, 5% CO 2 Cell culture incubator.

[0070] In some embodiments, in step 2, the incubation time is 48 h-96 h, preferably 72 h-96 h, and more preferably 96 h.

[0071] In some embodiments, in step three, the continued incubation time is 12h-30h, preferably 18h-24h, and more preferably 24h.

[0072] In some embodiments, the Aliprex and Alito are both in the form of lyophilized powder.

[0073] The present application is further described in detail below with reference to examples and drawings, but the implementation methods of the present application are not limited thereto.

[0074] The experimental methods in the following examples without specific experimental conditions were carried out according to conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used were obtained from commercial sources unless otherwise specified.

[0075] 1. Experimental materials:

[0076] 1. Reagents:

[0077] Reagent name Batch number / article number Brand manufacturers Lipopolysaccharide (LPS) 0000115516 Sigma Sterile PBS Batch number: 20230901-2277 Item number: PB2004Y Tianjin Haoyang 0.25% pancreatin working solution Batch number: 2537170 Item number: 25200-072 Gibco

[0078] 2. Consumables and instruments and equipment:

[0079] Consumables Name Batch number / article number Brand manufacturers Cell culture incubator 3311 ThermoFisher 96-well plate Batch number: 1101228L01 Item number: 1101A NEST

[0080] 3. Cell types:

[0081] Human primary articular chondrocytes were purchased from Beina Chuanglian Biotechnology Co., Ltd. and stored in the cell room of Guoke Saifu Hebei Pharmaceutical Technology Co., Ltd. at 37°C and 5% CO 2 Grow adherently under culture conditions and freeze under liquid nitrogen conditions.

[0082] 2. Experimental operation steps:

[0083] 1. Preparation of test samples

[0084] Medium: complete cell culture medium;

[0085] Preparation method: Use complete cell culture medium to dissolve Alipe (dissolved in 6 mL of complete cell culture medium per bottle, with a concentration of 10 mg / mL) and Alitol (dissolved in 1.3 mL of complete cell culture medium per bottle, with a concentration of 130 μg / mL) as stock solutions for later use, and then continue to use complete cell culture medium to dilute Alipe and Alitol to appropriate concentrations.

[0086] Among them, the Alito is a frozen protein polymer prepared by the method shown in the culture experiment 1 in CN202410944325.9, and the Alipul is a freeze-dried powder prepared by the example 1 in CN202311344207.6.

[0087] 2. Preparation of modeling drugs

[0088] Solvent: PBS, complete cell culture medium;

[0089] Preparation method: Dissolve LPS with a specification of 25 mg / bottle in 5 mL of sterile PBS to prepare a mother solution with a concentration of 5.0 mg / mL, and package it in 100 μL / tube. When used, dilute it with complete cell culture medium to obtain LPS solutions of different concentrations. For example, dilute it with complete cell culture medium at 1:50000 to obtain 100 ng / mL LPS solution.

[0090] 3. Embodiment

[0091] Cell viability assay

[0092] Example 1 Exploratory test on the survival rate of human articular chondrocytes treated with different doses of test substances

[0093] (1) Experimental grouping and treatment

[0094] S1. Set up blank control group and test substance groups with different concentration gradients (Alipin 10mg / mL, 1mg / mL, 100μg / mL, 10μg / mL, 1μg / mL, Alito 10μg / mL, 1μg / mL, 100ng / mL, 10ng / mL, 1ng / mL), see Table 1 for details:

[0095] Table 1

[0096]

[0097]

[0098] S2. Take primary human articular chondrocytes in good growth state, digest them with 0.25% pancreatic enzyme working solution, and use corresponding culture medium to prepare 2×10 4 cells / mL of cell suspension, use a pipette to accurately inoculate the cell suspension into a 96-well plate, add 100 μL of cell suspension to each well (i.e., 2000 cells / well), and incubate at 37°C and 5% CO 2 Culture in a cell culture incubator;

[0099] S3. After culturing for 24 h to allow all cells to adhere to the wall, the culture medium in the 96-well plate was aspirated, and 100 μL of different preparations were added according to the groups in Table 1, and incubated for 24 h (first day D1), 48 h (second day D2), 72 h (third day D3) and 96 h (fourth day D4).

[0100] (2) CCK-8 detection of cell viability

[0101] Add 10 μL of CCK-8 detection solution to each well and incubate in a 37°C incubator for 2 h. Use an enzyme-labeled instrument to measure the absorbance of each well at 450 nm, and calculate the cell survival rate according to the following formula.

[0102] Cell proliferation rate (%) = (OD value of experimental group - bottom value of plate) / (OD value of blank control group - bottom value of plate) × 100%.

[0103] Example 2: Test for the Survival Rate of Articular Chondrocytes

[0104] (1) Experimental grouping and treatment

[0105] S1. Set up a blank control group, an Aliprin 10 mg / mL administration group, and an Aliprin 10 mg / mL and Alituo 100 ng / mL combined administration group. See Table 2 for details:

[0106] Table 2

[0107]

[0108] Steps S2 and S3 are the same as the above exploratory test.

[0109] (2) CCK-8 detection of cell viability

[0110] Cellular inflammation assay

[0111] Example 3: Exploratory test on the effect of different doses of lipopolysaccharide combined with test substances on inflammation of human articular chondrocytes

[0112] (1) Experimental grouping and treatment

[0113] S1. Design a blank control group, a model control group, and different concentration gradient lipopolysaccharide groups (100 ng / mL, 300 ng / mL, 500 ng / mL). The concentrations and treatment methods corresponding to the specific groups are shown in Table 3:

[0114] Table 3

[0115]

[0116] S2. Take primary human articular chondrocytes in good growth state, digest them with 0.25% pancreatic enzyme working solution, and use human chondrocyte complete culture medium to prepare 5×10 4 cells / mL of cell suspension, and use a pipette to accurately inoculate the cell suspension into a 96-well plate, adding 2 mL of cell suspension to each well; incubate at 37°C and 5% CO 2 Culture in a cell culture incubator;

[0117] S3. After culturing for 24 h to allow all cells to adhere, the culture medium in the 96-well plate was aspirated, and different test substances were added according to the groups in Table 3, and incubated for 48 h, 72 h, and 96 h;

[0118] S4. After incubation, the blank control group in the 96-well plate was aspirated, 2 mL of complete culture medium was added to the blank control group and the model control group, and 2 mL of LPS solution with concentrations of 100 ng / mL, 300 ng / mL and 500 ng / mL was added to the model control group and the remaining experimental groups, respectively. After incubation for 24 h, the cell lysate was collected.

[0119] (2) MSD was used to detect the contents of IL-6, IL-1β, and TNF-α in cell lysate.

[0120] MSD electrochemiluminescence (ECLA) detection technology is used to detect cytokine content by using SULFO-TAGTM markers. After the surface of the MULTI-ARRAY and MULTI-SPOT microplate electrodes is energized, the electrochemical action excites the SULFO-TAGTM markers to emit strong light.

[0121] Example 4: Exploratory test on LPS-induced inflammation of human articular chondrocytes treated with different doses of test substances

[0122] (1) Experimental grouping and treatment

[0123] S1. According to the results of the "exploratory study on the effect of different doses of test substances on the survival rate of human articular chondrocytes", 1mg / mL, 100μg / mL, and 10μg / mL were selected as the high, medium, and low concentrations of Alip, and 10μg / mL, 1μg / mL, and 100ng / mL were selected as the high, medium, and low concentrations of Alito. See Table 4 for the specific group design:

[0124] Table 4

[0125]

[0126] S2. Take primary human articular chondrocytes in good growth state, digest them with 0.25% pancreatic enzyme working solution, and use human chondrocyte complete culture medium to prepare 5×10 4 cells / mL of cell suspension, and use a pipette to accurately inoculate the cell suspension into a 96-well plate, adding 2 mL of cell suspension to each well; incubate at 37°C and 5% CO 2 Culture in a cell culture incubator;

[0127] S3. After culturing for 24 h to allow all cells to adhere, the culture medium in the 96-well plate was aspirated, and different test substances were added according to the groups in Table 4, and incubated for 48 h, 72 h and 96 h;

[0128] S4. After incubation, the blank control group in the 96-well plate was aspirated, 2 mL of complete culture medium was added to the blank control group and the model control group, and 2 mL of LPS solution with a concentration of 100 ng / mL was added to the model control group and the remaining experimental groups, and the cell lysate was collected after incubation for 24 h.

[0129] (2) MSD detection of IL-6, IL-1β, and TNF-α in cell lysate

[0130] Example 5: Detection of LPS-induced inflammation in human articular chondrocytes

[0131] (1) Experimental grouping and treatment

[0132] A blank control group, a model control group, an alipin negative control group, an alituo negative control group, an alipin + alituo combined administration negative control group, an alipin heating group 1, an alipin heating group 2, an alipin heating group 3, an alipin administration group, an alituo administration group, and an alipin + alituo combined administration group were designed respectively; the concentrations and treatment methods corresponding to the specific groups are shown in Table 5:

[0133] Table 5

[0134]

[0135] Steps S2-S4 are the same as the above exploratory test of LPS-induced inflammation in human articular chondrocytes, except that the incubation time in step S3 is 96 hours.

[0136] (2) MSD detection of IL-6, IL-1β, and TNF-α in cell lysate

[0137] IV. Experimental Results

[0138] Cell viability test

[0139] 1. Exploratory test results of different doses of test substances on the survival rate of human articular chondrocytes

[0140] Table 6 is a statistical table of cell viability data corresponding to each group in Example 1, and all data are expressed as mean ± standard deviation.

[0141] Table 6

[0142] Group D1 D2 D3 D4 Blank control group 100±1.19 100±4.45 100±5.37** 100±3.89** Aliprin dose group 1 94.16±3.07* 72.94±7** 56.35±3.55 57.36±2.35 Aliprin dose group 2 104.96±4.49* 110.68±7.21** 100.77±5.55 105.98±3.74 Aliprin dose group 3 99.29±5.51 92.44±4.87* 92.52±5.59 95.55±2.89 Aliprin dose group 4 101.24±3 90.21±5.83* 92.48±7.07 94.71±3.06 Aliprin dose group 5 98.41±3.73 93.4±3.61** 93.94±7.98 95.36±4.81 Alitol dose group 1 99.76±4.1 91.25±3.62** 88.87±6.07** 92.4±3.59* Alitol dose group 2 99.03±5.84 91.38±3.67** 88.94±4.6** 92.71±4.72* Alitol dose group 3 99.91±4.29 91.25±6.37* 90.02±5.79** 92.89±4.19* Alitol dose group 4 99.76±2.67 92.98±5.2 92.14±4.13* 94.52±1.85 Alitol dose group 5 100.21±5.05 95.64±5.01 93.12±3.12* 104.13±6.48

[0143] In Table 6, “*” indicates P<0.05 compared with the blank control group, and “**” indicates P<0.01 compared with the blank control group.

[0144] From the statistical data in Table 6, it can be seen that compared with the blank control group, the levels of Alipin dose group 2 increased significantly at D1 and D2, and increased slightly at D3 and D4; the levels of Alipin dose group 1 decreased significantly during D1-D4, and continued to decrease until D3, after which they remained stable. Figure 1 The cell viability change trend chart of the blank control group and the Alipin dosage group in Table 6, the horizontal axis is the incubation days, the vertical axis is the cell viability, combined with Figure 1 It can also be seen that during the experiment, the cell viability of Alip dose groups 3, 4, and 5 remained stable, and was slightly lower than that of the blank control group as a whole; the cell viability of Alip concentration group 2 showed a trend of first increasing and then decreasing, and was slightly higher than that of the blank control group as a whole; the cell viability of Alip concentration group 1 showed a downward trend, and was lower than that of the blank control group as a whole.

[0145] From the statistical data in Table 6, it can be seen that during the test, the cell viability of Alito dose groups 1, 2, 3, 4, and 5 showed a trend of first decreasing and then increasing. Except that the cell viability of Alito dose group 5 was slightly higher than that of the blank control group at D4, the cell viability of the other groups was lower than that of the blank control group during the test. Overall, the degree of decrease in Alito dose group 5 was lower than that of Alito dose groups 1, 2, 3, and 4. Figure 2 The cell viability change trend chart of the blank control group and the alituo dosage group in Table 6, combined with Figure 2 It can also be seen that the cell viability trends of the alituo dosage groups 1-5 are the same as the statistical data analysis in Table 1 above.

[0146] Figure 1 and 2 “*” indicates P < 0.05 compared with the blank control group, and “**” indicates P < 0.01 compared with the blank control group. The colors represent the respective groups.

[0147] 2. Test results of human articular chondrocyte survival rate

[0148] Table 7 is a statistical table of cell viability data corresponding to each group in Example 2, and all data are expressed as mean ± standard deviation.

[0149] Table 7

[0150]

[0151] In the table, “*” indicates P < 0.05 compared with the blank control group, and “**” indicates P < 0.01 compared with the blank control group.

[0152] Figure 3 The cell viability change trend diagram of the blank control group and the experimental group in Table 7, the horizontal axis is the incubation days, the vertical axis is the cell viability, Figure 3 It can be seen that with the passage of time, the cell viability in the Alipin-treated group and the Alipin + Alituo combined treatment group showed a downward trend compared with the blank control group.

[0153] The above results show that during D1-D4, Alipin 10mg / mL and Alipin 10mg / mL + Alito 100ng / mL combined had an inhibitory effect on human primary articular chondrocytes, and the inhibitory effect was positively correlated with the incubation time. The test results also verified that the inhibitory effects of Alipin and Alito on chondrocytes were highly repeatable and stable.

[0154] Cellular inflammation test

[0155] 3. Exploratory study on the effect of different doses of lipopolysaccharide combined with test substances on inflammation of human articular chondrocytes

[0156] Table 8 is a table of individual data of inflammatory factor contents in each group of Example 3, with the unit being pg / mL. “-” in the table indicates that the content is below the detection limit.

[0157] Table 8

[0158]

[0159]

[0160] Figure 4-6 It is a trend chart of the influence of each group of cells corresponding to the individual data table in Table 10 on the content of IL-6, TNF-α, and IL-1β factors. Figure 4-6 The horizontal axis is the incubation time, and the vertical axis is the content of IL-6, TNF-α, and IL-1β factors. Figure 4-6 The results showed that compared with the blank control group, the levels of IL-6, TNF-α, and IL-1β in the model control group at different incubation times were increased. Figure 4-6 The test results showed that during the experiment, compared with the model control group, the reduction in the levels of cellular inflammatory factors IL-6 and IL-1β in the 1mg / mL alipe combined with 100ng / mL lipopolysaccharide group was greater than the inhibitory effect of alipe combined with other concentrations of lipopolysaccharide; the reduction in the levels of cellular inflammatory factors IL-6 and IL-1β in the 100ng / mL alipe combined with 100ng / mL lipopolysaccharide group was greater than the inhibitory effect of alipe combined with other concentrations of lipopolysaccharide.

[0161] 4. Exploratory test results of different doses of test substances on LPS-induced inflammation in human articular chondrocytes

[0162] Table 9 is a table of individual data of inflammatory factor contents in each group of Example 4, with the unit being pg / mL. “-” in the table indicates that the content is below the detection limit.

[0163] Table 9

[0164]

[0165]

[0166] Figure 7-9 It is a trend chart of the influence of each group of cells corresponding to the individual data table in Table 9 on the content of IL-6, TNF-α, and IL-1β factors. Figure 7-9 The horizontal axis is the incubation time, and the vertical axis is the content of IL-6, TNF-α, and IL-1β factors. Figure 7-9 The results showed that compared with the blank control group, the levels of IL-6, TNF-α, and IL-1β in the model control group increased at different incubation times. During the experiment, the IL-6 content in the model control group decreased with the incubation time, the IL-1β content increased with the incubation time, and the TNF-α content increased first and then decreased with time.

[0167] Figure 7 The test results of IL-6 content changing with incubation time showed that during the experiment, compared with the model control group, the IL-6 content in the high-dose alipin group decreased more than that in the medium- and low-dose alipin groups; the IL-6 content in the low-dose alipin group decreased more significantly on D2 and D3.

[0168] Figure 8 The test results of the change of TNF-α content with incubation time showed that during the experiment, the experimental data of the model control group showed that the TNF-α content was very low at this concentration of LPS.

[0169] Fig. 9 The results of the test on the changes of IL-1β content with incubation time showed that during the experiment, the reduction of IL-1β content at D4 in each dose group of Alip and Alituo was greater than that in the model control group.

[0170] 5. Results of LPS-induced inflammation in human articular chondrocytes

[0171] Table 10 is a statistical table of the inflammatory factor content in each group of Example 5, the unit is pg / mL, and all data are expressed as mean ± standard deviation.

[0172] Table 10

[0173]

[0174] In the table, “*” indicates P < 0.05 compared with the blank control group, “**” indicates P < 0.01 compared with the blank control group; “#” indicates P < 0.05 compared with the model control group, “##” indicates P < 0.01 compared with the model control group; “^^” indicates P < 0.01 compared with the group treated with Aliprin.

[0175] Figure 10-12It is a graphical representation of the corresponding contents of IL-6, TNF-α, and IL-1β factors in the cells of each group in Table 10. Figure 10-12 The horizontal axis represents the corresponding group, and the vertical axis represents the content of IL-6, TNF-α, and IL-1β factors.

[0176] Combined with Table 10 and Figure 10-12 The results showed that compared with the blank control group, the levels of IL-6 and IL-1β in the cells were increased after incubation with the modeling agent LPS 100 ng / mL. Compared with the blank control group, the IL-6 content in the negative control group of Alipu increased (P<0.05); the IL-6 content in the negative control group of Alipu + Alituo combined administration increased significantly (P<0.01), and the IL-1β content decreased significantly (P<0.01). Compared with the model control group, the IL-6 content in each drug administration group decreased significantly (P<0.01).

[0177] Compared with the model control group, the IL-6 inflammatory factor in the cells of Alipin heating group 1, Alipin heating group 2, Alipin heating group 3, Alipin administration group, Alito administration group, and Alipin + Alito combined administration group all decreased to a certain extent, and there were significant differences (P<0.01). Compared with the Alipin administration group, Alipin heating group 3 decreased by 20.67% (P<0.01).

[0178] Compared with the model control group, the IL-1β inflammatory factor in the cells was reduced by 20.00% in the alituo-treated group (P>0.05).

[0179] The detection value of TNF-α was near the detection limit.

[0180] The above results show that an in vitro human primary articular chondrocyte inflammation model was established by LPS 80ng / mL-120ng / mL, especially 100ng / mL. Alital 100ng / mL, Alital 100ng / mL + Aliprex 1mg / mL, Aliprex 1mg / mL and Aliprex 1mg / mL after heating at 72℃ for 20s-120s can all reduce the IL-6 content in the human primary articular chondrocyte inflammation model; Alital 100ng / mL, Aliprex 1mg / mL, Aliprex 1mg / mL and Aliprex 1mg / mL after heating at 72℃ for 60s-120s can reduce the IL-1β content in the human primary articular chondrocyte inflammation model.

[0181] The above is a further detailed description of the present application, which shall not be regarded as a limitation on the specific implementation of the present application. For ordinary technicians in the technical field to which the present application belongs, simple deduction or replacement without departing from the concept of the present application shall fall within the protection scope of the present application.

Claims

1. A method for analyzing the activity of stress-induced mesenchymal stem cell extracts, characterized in that: include: Step 1: Cultivating primary human articular chondrocytes; Step 2, aspirating the culture medium, adding an equal volume of a test composition, and incubating, wherein the test composition includes a mesenchymal stem cell extract; Step 3: Then add an equal volume of lipopolysaccharide solution with a concentration of 80 ng / mL-120 ng / mL, continue incubation, and collect cell lysate, which is used to detect inflammatory cytokines.

2. The activity analysis detection method according to claim 1, characterized in that: The step 1 satisfies the following conditions at the same time: a. The human primary articular chondrocyte culture comprises adjusting the concentration of the human primary articular chondrocytes to 2×10 4 cells / mL-5×10 4 cells / mL, and inoculated in a cell culture plate at a density of 2000 cells-5000 cells per well, and cultured for 12h-30h, wherein the cell culture plate is a 96-well plate; b. The culture conditions are 37° C. and 5% CO 2 in a cell culture incubator.

3. The activity analysis detection method according to claim 2, characterized in that: The mass volume ratio of the pancreatic enzyme working solution is 0.25%.

4. The analysis and detection method according to any one of claims 1 to 3, characterized in that: In step 2, the composition to be tested also includes a lyophilized composition, which includes collagen, chitosan and a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is selected from one or more of a buffer salt, an osmotic pressure regulator, a lyoprotectant, and a pH regulator.

5. The activity analysis detection method according to claim 4, characterized in that: The composition to be tested in step 2 also includes a cell viability test, and the cell viability test includes setting up a blank control group and an experimental group, wherein the blank control group and the experimental group are both added with a density of 2×10 3 cells / well-5×10 3 cells / well of human primary articular chondrocyte suspension, culture for 12h-30h, aspirate the culture medium in the experimental group, add 100μL of the test composition, continue incubation for 24h-96h, then add 10μL of CCK-8 solution, incubate for 1h-2h, and detect the cell viability of the human primary articular chondrocytes.

6. The activity analysis detection method according to claim 5, characterized in that: Meet any of the following conditions: a. The composition to be tested is the mesenchymal stem cell extract, and the concentration of the mesenchymal stem cell extract is 100 ng / mL-10 μg / mL; b. The composition to be tested is the lyophilized composition, and the concentration of the lyophilized composition is 10 μg / mL-1 mg / mL; c. The composition to be tested is the lyophilized composition, and the concentration of the lyophilized composition is 1 mg / mL, the heating temperature is 72° C., and the heating time is 15 s-150 s; d. The composition to be tested is the mesenchymal stem cell extract and the lyophilized composition, and the concentration of the mesenchymal stem cell extract is 100 ng / mL, and the concentration of the lyophilized composition is 1 mg / mL.

7. The activity analysis detection method according to claim 1, characterized in that: In the step 2, the incubation time is 48h-96h.

8. The activity analysis detection method according to claim 1, characterized in that: In the step three, the method for preparing the lipopolysaccharide solution includes soaking the lipopolysaccharide in a phosphate buffer to prepare a mother solution, and then diluting it with a complete cell culture medium.

9. The activity analysis detection method according to claim 1, characterized in that: In the step 3, the incubation time is 12h-30h.

10. The activity analysis detection method according to claim 8, characterized in that: The concentration of the mother solution is 2 mg / mL-8 mg / mL.

11. The activity analysis detection method according to claim 1, characterized in that: The inflammatory cytokines include one or more of IL-6, IL-1β and TNF-α.

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