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

Abstract

The present application provides an activity analysis detection method for a stress-induced mesenchymal stem cell extract, comprising step one, culturing human primary articular chondrocytes; step two, aspirating the culture fluid, adding an equal volume of a composition to be tested, and incubating, wherein the composition to be tested includes a mesenchymal stem cell extract; step three, then adding an equal volume of a lipopolysaccharide solution with a concentration of 80ng / mL-120ng / mL, continuing to incubate, and collecting a cell lysate, which is used to detect inflammatory cytokines. The present application determines the optimal use concentration and cell incubation time of a mesenchymal stem cell extract (denoted as Alituo) and a lyophilized composition (denoted as Alip) and a lipopolysaccharide solution by measuring cell viability and the levels of inflammatory factors IL-6, IL-1β, and TNF-α. By detecting cell viability and inflammatory factors, the quality of the mesenchymal stem cell extract can be efficiently evaluated, and the activity analysis detection method has high repeatability and good 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] When 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 the activation of synovial macrophages; 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 generally cannot penetrate cell membranes and can only act on the cell surface or through extracellular pathways, mesenchymal stem cell extracts may have problems with reproducibility and stability in efficacy testing for the treatment of joint diseases. Consequently, there is no good evaluation mechanism to assess and identify the quality of mesenchymal stem cell extracts. This problem is even more complex when evaluating the efficacy of mesenchymal stem cell extracts in combination with other macromolecular drugs. Therefore, it is urgent to develop a test method that can effectively evaluate the performance of mesenchymal stem cell extracts and / or other macromolecular drugs. It is also of great significance to explore the optimal concentration of mesenchymal stem cell extracts under this test method. Summary of the Invention

[0004] Based on this, this application intends to use lipopolysaccharide (LPS) to induce inflammation in human articular chondrocytes, introduce mesenchymal stem cell extracts, and explore the changes in 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] In one aspect, 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: aspirate the culture medium, add an equal volume of a test composition, and incubate, wherein the test composition includes a mesenchymal stem cell extract;

[0009] Step 3: Then, an equal volume of lipopolysaccharide solution containing 80 ng / mL-120 ng / mL is added, the incubation is continued, and the cell lysate is collected. The cell lysate 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 trypsin working solution to 2×10 4 cells / mL-5×10 4 cells / mL, and seeded 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, for example 24h, and the cell culture plate is a 96-well plate.

[0011] The mass-to-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 primary human articular chondrocytes are cells in good growth state before being digested with pancreatic enzyme working solution.

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

[0014] In some embodiments, in step 2, the composition to be tested further includes a lyophilized composition, wherein the lyophilized composition 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 according to 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 a human primary articular chondrocyte suspension, culture for 12 h-30 h, aspirate the culture medium in the experimental group, add 100 μL of the test composition, continue incubation for 24 h-96 h, then add 10 μL of CCK-8 solution, incubate for 1 h-2 h, and detect the cell viability of the human primary articular chondrocytes.

[0017] The culture conditions are 37° C. and 5% CO 2 in a 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 heat 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, for example 100 ng / mL.

[0020] Wherein, when the test composition 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] 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] 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 to 96 h, preferably 72 h to 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 stock 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 25 mg / bottle of LPS in 5 mL of sterile PBS to prepare a stock solution with a concentration of 5.0 mg / mL, packaging 100 μL / vial, and diluting 1:50,000 with complete cell culture medium to obtain a 100 ng / mL LPS solution.

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

[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 lyophilized composition has a concentration of 10 μg / mL-1 mg / mL and an incubation time of 48 hours, which can reduce the content of IL-6 in a human primary articular chondrocyte inflammation model.

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

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

[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 to 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 a human primary articular chondrocyte inflammation model can be reduced.

[0036] The beneficial effects of this application are:

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

[0038] The inventors have discovered that both Alito and Alip have a significant inhibitory effect on inflammation in human primary articular chondrocytes. In a human articular chondrocyte inflammation assay, by controlling the LPS concentration range between 80ng / mL and 120ng / mL, particularly at 100ng / mL, the addition of Alito and / or Alip significantly reduced LPS-induced levels of IL-1β, TNF-α, and IL-6 in human primary articular chondrocytes, effectively inhibiting LPS-induced inflammation in human articular chondrocytes. 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 Alip and 10mg / mL Alip combined with 100ng / mL Alito showed an inhibitory effect on primary human articular chondrocytes, and the inhibitory effect was positively correlated with the incubation time. 1mg / mL Alip significantly enhanced the cell viability of human articular chondrocytes, while the inhibitory effect of Alito on the cell viability of human articular chondrocytes decreased from D3 to D4 at 1ng / mL-10μg / mL. These test results also verified that the inhibitory effects of Alip and Alito on chondrocytes are highly reproducible and stable, providing a reliable reference for the concentration screening of test compositions 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 trends of cell viability in the blank control group and the different doses of alituo groups in Example 1;

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

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

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

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

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

[0048] Figure 9 The IL-1β content of each group of cells in the exploratory experiment of LPS-induced inflammation in human articular chondrocytes in Example 4;

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

[0050] Figure 11 The TNF-α content of each group of cells in the LPS-induced human articular chondrocyte inflammation test results of Example 5;

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

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

[0053] For the sake of convenience, mesenchymal stem cells are used as examples in the examples. 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] Mesenchymal stem cells can theoretically induce the production of corresponding stress proteins after being subjected to stress conditions. 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, hyperoxia, 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 CO2, low CO2. Various stress treatments 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 Alipur and Alipre on the proliferation of human articular chondrocytes, and conduct exploratory tests on the effects of different doses of test substances on cell viability, and based on the trial results, set up different concentrations of test substances and test substance combinations to detect the effects on cell viability; use MSD to study the effects of Alipur and Alipre on the content of inflammatory factors in LPS-induced articular chondrocyte inflammation, and based on the cell viability exploration results, set up different concentrations of Alipur and Alipre combined with different concentrations of LPS to explore the anti-inflammatory activity.

[0056] Through the cell survival rate exploration test, it was found that when the dose of Alip was 1 mg / mL, it had no obvious inhibitory effect on the growth of human articular chondrocytes. The inhibitory effect of 10 mg / mL of Alip on cell survival was obvious, 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 80 ng / mL-120 ng / mL, especially 100 ng / mL of LPS, the gene expression levels of inflammatory factors IL-6 and IL-1β in articular chondrocytes were significantly increased, and Alip and Alito could inhibit the activation of inflammatory response to varying degrees. The inflammatory treatment effect of Alip 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 stress-induced mesenchymal stem cell extracts, the method comprising:

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

[0059] Step 2: aspirate the culture medium, add an equal volume of a test composition, and incubate, wherein the test composition includes a mesenchymal stem cell extract;

[0060] Step 3: Then, an equal volume of lipopolysaccharide solution containing 80 ng / mL-120 ng / mL is added, the incubation is continued, and the cell lysate is collected. The cell lysate 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 A suspension of human primary articular chondrocytes (100 μL / well) was cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours. In the experimental group, the culture medium was aspirated and 100 μL of the test composition was added. 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 and incubated for 1-2 hours. The cell viability of the human primary articular chondrocytes was measured.

[0062] In some embodiments, the activity analysis method comprises the following steps: according to the test results of the cell viability, a blank control group, a model control group, and experimental groups of preset concentrations of Alip and Alituo are set up respectively, and 2 mL of a 5×10 4 cells / mL of human primary articular chondrocyte suspension was cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours. In the experimental group, the cell culture medium was aspirated, and a preset concentration of Alip or Alito 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. Cell lysates were collected. The content of inflammatory factors was detected by MSD.

[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 experimental group of preset concentrations of Alipin and Alituo, an Alipin heating experimental group, and an Alipin and Alituo combined experimental group, and adding 2 mL of a 5×10 4cells / mL of human primary articular chondrocyte suspension was cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours. In the experimental groups, the cell culture medium was aspirated, and each well was added with 1 mg / mL of Alipros; 1 mg / mL of Alipros heated at 72°C for 20s-120s; 100 ng / mL of Alituo; and 1 mg / mL of Alipros combined with 100 ng / mL of Alituo. 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 lipopolysaccharide was added to each well in the experimental group and incubated for another 24 hours. An equal volume of complete culture medium and the corresponding lipopolysaccharide was added to the blank and model control groups, respectively. The negative control group was incubated for 24 hours with 1 mg / mL of Aliporin, 100 ng / mL of Alitox, and 1 mg / mL of Aliporin combined with 100 ng / mL of Alitox. Cell lysates were collected from each well. Inflammatory cytokine levels were measured using MSD.

[0064] In some embodiments, in step one, the culturing of primary human articular chondrocytes includes adjusting the concentration of the primary human articular chondrocytes in good growth condition after digestion with pancreatic enzyme working solution using human chondrocyte culture medium, and inoculating the cells into 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 hours to 30 hours, preferably 24 hours to 30 hours, and more preferably 24 hours.

[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. and 5% CO 2 in a cell culture incubator.

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

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

[0072] In some embodiments, the properties of the Alip and the Alito are both lyophilized powders.

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

[0074] In the following examples, experimental methods without specific experimental conditions were performed according to conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0075] 1. Experimental materials:

[0076] 1. Reagents:

[0077] Reagent name Batch number / article number Brand manufacturers Lipopolysaccharide (LPS) 0000115516 Sigma Corporation of America 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 type:

[0081] Human primary articular chondrocytes were purchased from Beina Chuanglian Biotechnology Co., Ltd. and stored in the cell room of Guokesaifu Hebei Pharmaceutical Technology Co., Ltd., growing adherently under culture conditions of 37°C and 5% CO2 and frozen under liquid nitrogen.

[0082] 2. Experimental operation steps:

[0083] 1. Preparation of test samples

[0084] Vehicle: complete cell culture medium;

[0085] Preparation method: Use complete cell culture medium to dissolve Alipe (dissolve in 6 mL of complete cell culture medium per bottle, concentration is 10 mg / mL) and Alito (dissolve in 1.3 mL of complete cell culture medium per bottle, concentration is 130 μg / mL) as stock solutions for later use, and then continue to use complete cell culture medium to dilute Alipe and Alito 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 Alip 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 25 mg / bottle of LPS in 5 mL of sterile PBS to prepare a stock solution with a concentration of 5.0 mg / mL. Aliquot 100 μL / vial. When used, dilute with complete cell culture medium to obtain LPS solutions of different concentrations. For example, dilute 1:50,000 with complete cell culture medium to obtain a 100 ng / mL LPS solution.

[0090] 3. Examples

[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 a blank control group and test substance groups with different concentration gradients (Alipin at 10 mg / mL, 1 mg / mL, 100 μg / mL, 10 μg / mL, and 1 μg / mL; Alito at 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL), see Table 1 for details:

[0095] Table 1

[0096]

[0097]

[0098] S2. Take well-grown primary human articular chondrocytes, digest them with 0.25% trypsin 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 culture in a 37°C, 5% CO2 cell culture incubator;

[0099] S3. After culturing for 24 h to allow all cells to adhere, 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), respectively.

[0100] (2) CCK-8 assay for 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 a microplate reader 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: Detection of Articular Chondrocyte Survival Rate

[0104] (1) Experimental grouping and treatment

[0105] S1. Set up a blank control group, a group receiving 10 mg / mL of Aliprin, and a group receiving a combination of 10 mg / mL of Aliprin and 100 ng / mL of Alitro. 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 assay for 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 well-grown primary human articular chondrocytes, digest them with 0.25% trypsin working solution, and use human chondrocyte complete culture medium to prepare 5×10 4 cells / mL of cell suspension, use a pipette to accurately inoculate the cell suspension into 96-well plates, adding 2 mL of cell suspension to each well; culture in a 37°C, 5% CO2 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, aspirate the blank control group in the 96-well plate, add 2 mL of complete culture medium to each of the blank control group and the model control group, and add 2 mL of LPS solution with concentrations of 100 ng / mL, 300 ng / mL, and 500 ng / mL to the model control group and the remaining experimental groups, respectively. After incubation for 24 h, collect the cell lysate.

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

[0120] Using MSD electrochemiluminescence (ECLA) detection technology, the SULFO-TAG™ marker is applied to the electrodes of the MULTI-ARRAY and MULTI-SPOT microplates. When electricity is applied to the electrodes, the electrochemical reaction excites the SULFO-TAG™ marker to emit strong light, which is then used to detect cytokine levels.

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

[0122] (1) Experimental grouping and treatment

[0123] S1. Based on the results of the "Exploratory Study on the Effect of Different Doses of Test Substances on the Survival Rate of Human Articular Chondrocytes," 1 mg / mL, 100 μg / mL, and 10 μg / mL were selected as the high, medium, and low concentrations of Alipin, respectively; and 10 μg / mL, 1 μg / mL, and 100 ng / mL were selected as the high, medium, and low concentrations of Alito, respectively. See Table 4 for the specific group design:

[0124] Table 4

[0125]

[0126] S2. Take well-grown primary human articular chondrocytes, digest them with 0.25% trypsin working solution, and use human chondrocyte complete culture medium to prepare 5×10 4 cells / mL of cell suspension, use a pipette to accurately inoculate the cell suspension into 96-well plates, adding 2 mL of cell suspension to each well; culture in a 37°C, 5% CO2 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, aspirate the blank control group in the 96-well plate, add 2 mL of complete culture medium to each of the blank control group and the model control group, and add 2 mL of 100 ng / mL LPS solution to each of the model control group and the remaining experimental groups. After incubation for 24 h, collect the cell lysate.

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

[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 Alip negative control group, an Alitol negative control group, an Alip + Alitol combined negative control group, an Alip heating group 1, an Alip heating group 2, an Alip heating group 3, an Alip administration group, an Alitol administration group, and an Alip + Alitol combined administration group were designed. 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 lysates

[0137] 4. Experimental Results

[0138] Cell viability test

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

[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** Alipin dose group 1 94.16±3.07* 72.94±7** 56.35±3.55 57.36±2.35 Alipin dose group 2 104.96±4.49* 110.68±7.21** 100.77±5.55 105.98±3.74 Alipin dose group 3 99.29±5.51 92.44±4.87* 92.52±5.59 95.55±2.89 Alipin dose group 4 101.24±3 90.21±5.83* 92.48±7.07 94.71±3.06 Alipin 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 Alipin dose group 2 showed a significant increase on D1 and D2, and a slight increase on D3 and D4; the Alipin dose group 1 showed a significant decrease during D1-D4, and continued to decrease until D3 and then remained stable. Figure 1 The cell viability change trend diagram of the blank control group and the Alip dose group in Table 6 is shown in the horizontal axis, the vertical axis is the cell viability, and the Figure 1 It can also be seen that during the experiment, the cell viability of Alipin dose groups 3, 4, and 5 remained stable, and was slightly lower than that of the blank control group overall; the cell viability of Alipin concentration group 2 showed a trend of first increasing and then decreasing, and was slightly higher than that of the blank control group overall; the cell viability of Alipin concentration group 1 showed a downward trend, and was lower than that of the blank control group overall.

[0145] The statistical data in Table 6 show that during the experiment, the cell viability of Alito dose groups 1, 2, 3, 4, and 5 showed a trend of first decreasing and then increasing. Except for Alito dose group 5, which had a slightly higher cell viability than the blank control group on D4, the cell viability of the other groups was lower than that of the blank control group during the experiment. 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 diagram of the blank control group and the aritol dosage group in Table 6 is shown in FIG. 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 is shown in the horizontal axis, the number of incubation days, and the vertical axis, the cell viability. Figure 3 It can be seen that as time goes by, the cell viability in the Alipin-treated group and the Alipin + Alituo combined-treated group showed a downward trend compared with the blank control group.

[0153] These results demonstrate that, from Days 1 to 4, Alip (10 mg / mL) and the combination of Alip (10 mg / mL) and Alito (100 ng / mL) exhibited inhibitory effects on primary human articular chondrocytes, with the inhibitory effect positively correlated with incubation time. These results also demonstrate the highly reproducible and stable inhibitory effects of Alip and Alito on chondrocytes.

[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 on the content of inflammatory factors 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 This is a trend chart showing the effects 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 IL-6, TNF-α, and IL-1β factor content. Figure 4-6 The results showed that compared with the blank control group, the IL-6, TNF-α, and IL-1β levels in the model control group increased at different incubation times, and Figure 4-6 The test results showed that during the experiment, compared with the model control group, the degree of reduction in the content 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 degree of reduction in the content 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 on the content of inflammatory factors 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 This is a trend chart showing the effects 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 IL-6, TNF-α, and IL-1β factor content. 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 level in the model control group showed a decreasing trend with incubation time, the IL-1β level showed an increasing trend with incubation time, and the TNF-α level showed a trend of first increasing and then decreasing with time.

[0167] Figure 7 The test results of the changes in IL-6 content with incubation time showed that during the experiment, compared with the model control group, the IL-6 content in the high-dose Alip group decreased more than that in the medium- and low-dose Alip groups; the IL-6 content in the low-dose Alipre 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, according to the experimental data of the model control group, the content of TNF-α was very low at this concentration of LPS.

[0169] Figure 9 The results of the test on the changes of IL-1β content with incubation time showed that during the experiment, the IL-1β content of each Alip and Alito dose group was reduced to a greater extent on D4 compared with the model control group.

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

[0171] Table 10 is a statistical table of inflammatory factor levels in each group of Example 5, with the unit being pg / mL. 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 Aliprin-treated group.

[0175] Figure 10-12This is a graphical representation of the corresponding IL-6, TNF-α, and IL-1β factor contents 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, incubation with 100 ng / mL LPS increased the levels of IL-6 and IL-1β in the cells. Compared with the blank control group, IL-6 levels were elevated in the negative control group treated with alipin (P < 0.05). In the negative control group treated with alipin plus alituo, IL-6 levels were significantly increased (P < 0.01), while IL-1β levels were significantly decreased (P < 0.01). Compared with the model control group, IL-6 levels were significantly decreased in all treatment groups (P < 0.01).

[0177] Compared with the model control group, IL-6 inflammatory factors in cells were reduced to some extent in Alip heating group 1, Alip heating group 2, Alip heating group 3, Alip administration group, Alital administration group, and Alip + Alital combination administration group, with significant differences (P < 0.01). Compared with the Alip administration group, Alip heating group 3 decreased by 20.67% (P < 0.01).

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

[0179] The TNF-α detection value 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 and should not be considered 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 deductions or substitutions that do not deviate from the concept of the present application are within the scope of protection 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: aspirate the culture medium, add an equal volume of the test composition, and incubate; Step 3: Then, an equal volume of lipopolysaccharide solution with a concentration of 80 ng / mL to 120 ng / mL is added, the incubation is continued, and the cell lysate is collected. The cell lysate is used to detect inflammatory cytokines; The inflammatory cytokines include one or more of IL-6, IL-1β and TNF-α; Satisfy any one of the given conditions, including: a. The test composition is a mesenchymal stem cell extract, the mesenchymal stem cell extract is alituo, and the concentration of the mesenchymal stem cell extract is 100 ng / mL; b. The composition to be tested is a mesenchymal stem cell extract and a lyophilized composition, the mesenchymal stem cell extract is Aletolide, the lyophilized composition is Aletolide, and the concentration of the mesenchymal stem cell extract is 100 ng / mL, and the concentration of the lyophilized composition is 1 mg / mL.

2. The activity analysis and 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 seeded in a cell culture plate at a density of 2000 cells-5000 cells per well, and cultured for 12 h-30 h. The cell culture plate is a 96-well plate; b. The culture conditions are 37°C, 5% CO2 in a cell culture incubator.

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

4. The activity analysis and detection method according to claim 2, wherein The step 2 also includes cell viability detection, and the cell viability detection 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 a human primary articular chondrocyte suspension, culture for 12 h-30 h, aspirate the culture medium in the experimental group, add 100 μL of the test composition, continue incubation for 24 h-96 h, then add 10 μL of CCK-8 solution, incubate for 1 h-2 h, and detect the cell viability of the human primary articular chondrocytes.

5. The activity analysis and detection method according to claim 1, wherein The lyophilized composition in the given condition b is heated at a temperature of 72° C. for a heating time of 15 s to 150 s.

6. The activity analysis and detection method according to claim 1, wherein In the step 2, the incubation time is 48 h-96 h.

7. The activity analysis and detection method according to claim 1, characterized in that: In the step 3, the preparation method of the lipopolysaccharide solution includes soaking the lipopolysaccharide in phosphate buffer to prepare a mother solution, and then diluting it with complete cell culture medium.

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

9. The activity analysis and detection method according to claim 7, wherein The concentration of the mother solution is 2 mg / mL-8 mg / mL.

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