Activity detection method of stress-induced mesenchymal stem cell extract

Abstract

The invention belongs to the field of medicines, and discloses an activity detection method of a stress-induced mesenchymal stem cell extract, which comprises the following steps: pre-culturing BV2 cells, preparing more than three groups of mesenchymal stem cell extract culture mediums with different concentrations, and detecting the anti-inflammatory performance of the mesenchymal stem cell extract. Wherein the model group and the drug delivery group are established, lipopolysaccharide induced inflammation stimulation is performed on BV2 cells in the model group and the drug delivery group, then the BV2 cells are respectively transferred to a corresponding blank culture medium and a culture medium containing a mesenchymal stem cell extract for incubation, and finally BV2 cell culture supernate is taken for Elisa detection to obtain the concentration of inflammatory factors. According to the detection method disclosed by the embodiment of the invention, a safe experimental concentration and a data result with good repeatability and significance can be screened out, a performance monitoring model of the mesenchymal stem cell extract can be established, the product quality can be stabilized, the change rule of the product concentration and the anti-inflammatory activity can be explored, and a basis is established for subsequently using the mesenchymal stem cell extract as an inflammation inhibitor.

Description

Technical Field

[0001] The present application belongs to the field of medicine, and specifically relates to a method for detecting the activity of stress-induced mesenchymal stem cell extracts. Background Art

[0002] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely found in bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, urine and other tissues. They have the characteristics of wide sources, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and convenient collection. By stimulating MSCs and then lysing MSCs, a mesenchymal stem cell extract with biorepair activity can be separated and purified from the cell lysate. The mesenchymal stem cell extract has achieved significant results in completed animal and clinical experiments, has a clear drug potential, and has now entered the clinical trial stage.

[0003] Mesenchymal stem cells can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN), etc., and participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, immune maturation and other processes. They can be used for immune regulation, joint tissue repair and treatment of acute lung injury, severe pneumonia, acute respiratory distress syndrome and other diseases. However, at the current stage, mesenchymal stem cell extracts are obtained by stimulating the expression of mesenchymal stem cells and separating and purifying them, which leads to significant performance differences between batches. Therefore, it is necessary to clarify a detection method for evaluating the anti-inflammatory and pro-repair effects of mesenchymal stem cell extracts on cells induced with inflammation, monitor the anti-inflammatory properties of mesenchymal stem cell extracts, reduce the batch differences of mesenchymal stem cell extracts, and improve the quality stability of the product. At the same time, it is of great significance to explore the changing rules of the anti-inflammatory activity of mesenchymal stem cell extracts and the optimal drug concentration. Summary of the invention

[0004] The inventors of the present application discovered that after culturing mesenchymal stem cells under appropriate stress conditions, multiple proteins can be separated from the lysates of the lysed stress-cultured stem cells. The separated mesenchymal stem cell extracts containing a combination of multiple proteins have anti-inflammatory and repair activity and can be used to treat nervous system and joint tissue damage. An activity detection method is designed to accurately evaluate the anti-inflammatory activity of mesenchymal stem cell extracts.

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

[0006] A method for detecting the activity of a stress-induced mesenchymal stem cell extract, comprising the following steps: S1: using BV2 cells, with N×10 4 The cells are inoculated into the well plate at a density of 10 cells per well, and pre-cultured for 20h-28h, 4.5≤N≤5.5; S2: The mesenchymal stem cell extract is used to prepare at least 3 groups of activity detection medium with different concentrations, and the concentration of the mesenchymal stem cell extract in each group of the activity detection medium is within the preset concentration range; S3: A model group and a drug administration group are established according to the BV2 cells, and the BV2 cells in the model group and the drug administration group are transferred to a culture medium containing 450ng / mL-550ng / mL lipopolysaccharide, and cultured for 20h-28h; S4: The BV2 cells in the model group are transferred to a blank culture medium, and the BV2 cells in the drug administration group are transferred to a blank culture medium. The BV2 cells are transferred to the activity detection culture medium of each group respectively: S5: continue to incubate the BV2 cells for 12 hours or 24 hours, centrifuge, and take the supernatant; and S6: take the supernatant for Elisa detection to obtain the concentration of inflammatory factors, and evaluate the anti-inflammatory activity of the mesenchymal stem cell extract by comparing the concentration of inflammatory factors in the model group and the drug-treated group; wherein, the preparation of the mesenchymal stem cell extract includes the following steps: S001: culturing mesenchymal stem cells and creating stress conditions to stimulate the mesenchymal stem cells; S002: lysing, separating, and purifying the mesenchymal stem cells obtained in S001 to obtain the mesenchymal stem cell extract.

[0007] In some embodiments of the method, the S3 also includes: establishing an anti-inflammatory positive control group and an anti-inflammatory negative control group based on the BV2 cells, and transferring the anti-inflammatory positive control group and the anti-inflammatory negative control group to the culture medium containing 450ng / mL-550ng / mL lipopolysaccharide for 20h-28h; the S4 also includes transferring the BV2 cells in the anti-inflammatory positive control group to a culture medium supplemented with 5mM sodium butyrate, and transferring the BV2 cells in the anti-inflammatory negative control group to a culture medium supplemented with 1000ng / mL placebo, wherein the placebo is human serum albumin; the S5 also includes continuing to incubate the anti-inflammatory positive control group The BV2 cells in the anti-inflammatory control group and the anti-inflammatory negative control group are subjected to 12h or 24h, centrifuged, and the supernatant is taken; S6 also includes taking the supernatant in the anti-inflammatory positive control group and the anti-inflammatory negative control group for Elisa detection to obtain the concentration of the inflammatory factor, and evaluating the credibility of the data of the Elisa detection by comparing the concentrations of the inflammatory factors in the model group, the anti-inflammatory negative control group, and the anti-inflammatory positive control group, and evaluating the anti-inflammatory activity of the mesenchymal stem cell extracts of different concentrations by comparing the concentrations of the inflammatory factors in the drug administration group, the anti-inflammatory negative control group, and the anti-inflammatory positive control group.

[0008] In some embodiments of the method, in S6, the inflammatory factors include one or a combination of more than one of IL-1β, TNF-α and IL-6.

[0009] In some embodiments of the method, before S1, the activity detection method further comprises the following steps: S01: using the BV2 cells, 3 The cells are inoculated into the well plate at a density of 10 cells per well, and pre-cultured for 20h-28h, 7.5≤M≤8.5; S02: at least 3 groups of toxicity detection medium with different concentrations are prepared using the mesenchymal stem cell extract; S03: a blank control group, a toxicity negative control group, a toxicity positive control group and a test group are established according to the BV2 cells, the blank control group is incubated in a blank medium, the toxicity negative control group and the toxicity positive control group are incubated in a medium with a placebo and sodium butyrate, respectively, and the test group is incubated in the toxicity detection medium of each group, and the placebo is human serum albumin; S04: the BV2 cells are incubated for 12h or 24h; S05: 10 μL of CCK-8 solution, and continue incubating for 1h-2h; and S06: using an ELISA instrument to measure the cell viability of the BV2 cells, and by comparing the cell viabilities of the blank control group with those of the toxicity negative control group and the toxicity positive control group, respectively, to evaluate the credibility of the data of the ELISA instrument; comparing the test group with the toxicity negative control group and the toxicity positive control group, respectively, wherein the toxicity detection medium corresponding to the test group with a cell viability greater than or equal to that of the toxicity negative control group is determined to be non-toxic to the BV2 cells, and the toxicity detection medium corresponding to the test group with a cell viability less than that of the toxicity positive control group is determined to be toxic to the BV2 cells, and after excluding the concentration range consisting of the maximum and minimum values ​​of the concentration of the mesenchymal stem cell extract included in the toxicity detection medium determined to be non-toxic, and the concentration range consisting of the maximum and minimum values ​​of the concentration of the mesenchymal stem cell extract included in the toxicity detection medium determined to be toxic, the remaining concentration range is used as the preset concentration range.

[0010] In some method embodiments, the preset concentration range is 10 ng / mL-1000 ng / mL.

[0011] In some method embodiments, the activity detection medium comprises 6 groups, and the concentrations of the mesenchymal stem cell extracts in the 6 groups of the activity detection medium are 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL and 1000 ng / mL, respectively.

[0012] In some embodiments of the method, the culture medium used to culture the BV2 cells in steps S01 to S03 and S1 to S4 is a culture medium including serum, and the incubation conditions of the BV2 cells are 37° C. and 5% CO 2 .

[0013] In some embodiments of the methods, the culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0014] In some embodiments of the method, the S6 includes the following steps: S61: taking a standard substance including the inflammatory factor, gradient dilution, and preparing a standard curve of the OD value of the standard substance at 450nm-the concentration of the inflammatory factor; S62: adding a buffer to the supernatant according to a predetermined dilution ratio to form a sample to be tested, taking a predetermined volume of the sample to be tested into the well of the experimental plate, fully mixing the sample to be tested with the specific antibody capable of binding to the inflammatory factor pre-coated in the well of the experimental plate, covering with a sealing film, incubating at 37°C for 2h, and after the incubation is completed, washing the well of the experimental plate, and discarding the upper liquid; S6 3: Add an equal volume of biotinylated antibody working solution to each well as the sample to be tested, cover with a new sealing film, and continue incubating at 37°C for 1 hour. After the incubation is completed, wash the experimental plate wells again and discard the upper liquid; S64: Add an equal volume of HRP-labeled detection antibody to each well as the sample to be tested, cover with a new sealing film, and continue incubating at 37°C for 30min-60min. After the incubation is completed, wash the experimental plate wells again and discard the upper liquid; S65: Add an equal volume of TMB substrate to each well as the sample to be tested, cover with a new sealing film, and incubate at 37°C in the dark for 15min-20 min; S66: after the incubation is completed, add 0.5 times the volume of the sample to be tested to each well of the stop solution, and immediately put it into the microplate reader to measure the OD value of the contents of each well at 450 nm within 5 minutes; and S67: according to the standard curve, substitute the OD value of each well at 450 nm to obtain the concentration of inflammatory factors in the contents of each well.

[0015] In some embodiments of the method, in S62, the predetermined dilution ratio refers to a ratio of the volume of the supernatant to the volume of the buffer solution being equal to 1:2, and the predetermined volume is 100 μL.

[0016] The beneficial effects of this application are:

[0017] The inventors have found that the extract isolated from mesenchymal stem cells cultured under stress conditions (denoted as Alito) has good biological anti-inflammatory activity.

[0018] In the cytotoxicity experiment, 10ng / mL, 50ng / mL, and 100 ng / mL of alituo had no effect on the viability of BV2 cells, while 200ng / mL, 500ng / mL, and 1000 ng / mL of alituo significantly increased the cell viability of BV2 cells. On the one hand, it proves that alituo has no cytotoxicity to BV2 cells under the above experimental concentration conditions and will not cause cell damage, providing a test concentration reference for further anti-inflammatory activity research; on the other hand, a rule can also be drawn: high concentrations of alituo have a certain promoting effect on BV2 cell viability; this may be related to the anti-inflammatory, antioxidant, or cytoprotective properties of the drug, providing guidance for further research on the role of alituo in neuroinflammation and related diseases.

[0019] In the anti-inflammatory property test of cells, after treatment with alituo, high concentration of alituo significantly reduced the levels of IL-1β, TNF-α and IL-6 inflammatory factors in BV2 cells induced by lipopolysaccharide (LPS), which can be used to characterize the improvement effect of alituo on the inflammatory level of BV2 cells induced by lipopolysaccharide (LPS), and can effectively inhibit the cell inflammation caused by lipopolysaccharide (LPS); further, in repeatable experiments, the standard deviation of the inhibitory effect of high concentration of alituo on inflammatory factors is relatively smaller, which means that the variability of experimental data is low. Therefore, the anti-inflammatory effect of high concentration of alituo is more credible, and the anti-inflammatory efficacy of alituo can be evaluated more accurately, and to a certain extent, the mechanism of action of alituo in alleviating damage caused by nerves and other inflammations by regulating the expression of inflammatory factors is revealed, which provides reference value for the subsequent clinical application of alituo products in the treatment of diseases related to neuroinflammation.

[0020] In addition, among the three inflammatory factors IL-1β, TNF-α and IL-6, Alituo has the best inhibitory effect on IL-6 inflammatory factor, which to a certain extent indicates that the anti-inflammatory effect of Alituo is related to its regulation of the NF-κB signaling pathway, which plays a core role in the inflammatory response. By inhibiting the activation of the NF-κB signaling pathway, it reduces the production of inflammatory factors and exerts its neuroprotective effect. These results provide experimental basis for Alituo as a potential neuroprotective agent and point out the direction for future clinical applications.

[0021] In summary, the experimental method proposed in this application can be used to evaluate and measure the feasibility of aritol as a potential inflammatory inhibitor, and is expected to be used as a test model for monitoring the performance of aritol. On the one hand, it can reduce the batch-to-batch differences of aritol and improve the quality stability of the product. On the other hand, it can explore the changes in the concentration and anti-inflammatory activity of aritol and calculate the optimal drug concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1: A bar graph showing the effects of different concentrations of alituo on BV2 cell viability; (A) shows the effect of alituo on BV2 cell viability after 12 h of alituo, and (B) shows the effect of alituo on BV2 cell viability after 24 h of alituo.

[0023] Figure 2 The following are bar graphs showing the effects of different concentrations of alituo on the concentrations of inflammatory factors in BV2 cells induced by lipopolysaccharide (LPS); (A) is the regulatory effect of alituo on the IL-1β inflammatory factor; (B) is the regulatory effect of alituo on the TNF-α inflammatory factor; (C) is the regulatory effect of alituo on the IL-6 inflammatory factor.

[0024] Figure 3 The following are bar graphs showing the concentrations of different inflammatory factors in BV2 cells under different conditions in Comparative Example 1; wherein (A) is the concentration level of IL-6 inflammatory factor when BV2 cells are cultured in medium containing FBS and medium without FBS for 6 h; (B) is the concentration level of IL-6 inflammatory factor when BV2 cells are cultured in medium containing FBS and medium without FBS for 24 h; (C) is the concentration level of TNF-α inflammatory factor when BV2 cells are cultured in medium containing FBS and medium without FBS for 6 h; (D) is the concentration level of TNF-α inflammatory factor when BV2 cells are cultured in medium containing FBS and medium without FBS for 24 h.

[0025] Figure 4 : A bar graph of the IL-6 inflammatory factor concentration in the control experiment in which BV2 cells were cultured in a culture medium containing both lipopolysaccharide (LPS) and mesenchymal stem cell extracts and in a culture medium containing lipopolysaccharide (LPS) and mesenchymal stem cell extracts step by step for 6 hours and 24 hours in Comparative Example 2. DETAILED DESCRIPTION

[0026] The technical solution of the present application is further explained below in conjunction with embodiments.

[0027] 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 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. In addition, in practical applications, it is not limited to mesenchymal stem cells, and other stem cells can also produce similar stress proteins after stress induction. Other stem cells that can be used include but are not limited to at least one of embryonic stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and MUSE cells.

[0028] After undergoing stress conditions, mesenchymal stem cells can theoretically induce the production of corresponding stress proteins. For the convenience of explanation, ultraviolet irradiation is used as a stress condition in the embodiment to prepare mesenchymal stem cell extracts with anti-inflammatory properties. 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, and low CO2. Various stress treatments can be performed one after another, or simultaneously when they do not conflict.

[0029] Example 1: Cultivation of human umbilical cord mesenchymal stem cells

[0030] Resuscitate a small crystal of P8 HUC-MSC (human umbilical cord-derived mesenchymal stem cells) into a T25 culture flask and add 2.5 mL of Huakai mesenchymal stem cell serum-free culture medium.

[0031] Irradiate the cells with 300nm LED UV for 6h. Remove the supernatant in the stem cell culture medium, wash twice with 1mL of saline, add 660μl of pure water and repeatedly blow and lyse for 10min, filter through a 0.22μm filter membrane, and store at 4℃.

[0032] The UV irradiation conditions are as follows:

[0033] Intermediate medium T25 bottle <![CDATA[UVA(μW / cm 2 )]]> 11.71 <![CDATA[UVB(μW / cm 2 )]]> 26.81 Peak wavelength (nm) 320.4

[0034] The lysate of mesenchymal stem cells (also referred to as ALT stock solution) was harvested, and the protein concentration was measured to be 0.718 mg / mL; the lysate was further purified to obtain a mesenchymal stem cell extract, which was referred to as ALT.

[0035] It should be noted that the method for purifying the lysate into aritol includes:

[0036] Instrument: AKTA explorer

[0037] Chromatographic column: Nano-micron superdex150 molecular sieve 8×500, column volume is about 30mL

[0038] Reagents: 0.1M NaOH, 20% ethanol, PBS (phosphate buffered saline), purified water

[0039] UV absorption wavelength: 280nm, 260nm as reference

[0040] Equilibration column sequence:

[0041] First, wash the column with 2CV of pure water, then balance the column with PBS for 2CV and return the 280 nm UV absorption value to zero.

[0042] Sample preparation: Prepare 20 mL of the lysate in Example 1. Use an ultrafiltration concentration tube with a molecular weight cutoff of 3 KD to reduce 20 mL of the lysate to about 600 μl to obtain a crude sample.

[0043] Experimental process: After balancing the chromatography column, select a 500μl sample loop for sample loading, the sample loading flow rate is 0.4mL / min, the PBS elution flow rate is 0.2mL / min, and the elution is eluted to the peak. The separated elution liquid is collected from the UV absorption value of 4mAU, and the samples with elution volume segments of 17.0 to 20.0mL are collected in order according to the order of the elution liquid, which is recorded as Alito. The protein concentration of Alito is measured to be 10μg / mL.

[0044] Example 2: BV2 cell culture method for biological activity detection of mesenchymal stem cell extract (Aritol)

[0045] S2.1) BV-2 cells from BJDRW were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (PS) at 37°C and 5% CO2.

[0046] S2.2) Digest the cultured BV2 cells with trypsin and dilute them to a cell density of 50,000 cells per 100 μL. Inoculate each well of a 96-well plate at a density of 100 μL and continue culturing at 37°C, 5% CO2 for 24 h.

[0047] Example 3: Method for preparing culture medium for experimental group for biological activity detection of mesenchymal stem cell extract (Aritol)

[0048] S3.1) Add 120 μL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (PS) to each well of a new 3599 plate;

[0049] S3.2) adding lipopolysaccharide (LPS) to the culture medium of each well in the 3599 plate to prepare a culture medium with a corresponding concentration;

[0050] S3.3) Add aritol, placebo (human serum albumin) and sodium butyrate prepared in Example 1 to the culture medium of each well of the 3599 plate to prepare culture medium with corresponding concentrations, set up 2 wells for each concentration and repeat the experiment, and mix for 10 minutes.

[0051] It can be understood that the BV2 cells induced and cultured with the medium containing lipopolysaccharide (LPS) in S3.2) are transferred one by one to the medium prepared in S3.3) and continue to be incubated.

[0052] Example 4: Cytotoxicity assay of mesenchymal stem cell extract (Aritol)

[0053] Experimental method: S4.1) Using the BV2 cells cultured in Example 2, 8×10 3 The density of each well was inoculated into the plate wells in S3.1) of Example 3, and pre-cultured for 24 hours; S4.2) according to S3.3) of Example 3, 6 groups of alitori were prepared with the concentrations of 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL of toxicity detection medium, wherein the toxicity detection medium is obtained by diluting the alituoga medium prepared in Example 1; S4.3) dividing the BV2 cells into a blank control group, a toxicity negative control group, a toxicity positive control group and 6 test groups on a well basis, wherein the blank control group is incubated with a blank medium, and the BV2 cells in the toxicity negative control group, the toxicity positive control group and the drug administration group are respectively transferred to the medium containing placebo (human albumin) and sodium butyrate prepared in S3.3) of Example 3 and the above 6 groups of toxicity detection medium for incubation; S4.4) incubating the BV2 cells for 12h or 24h; S4.5) adding 10 μL of CCK-8 solution to the BV2 cells in each well and continuing to incubate for 1-2h; and S4.6) measuring the cell viability of the BV2 cells using an ELISA instrument.

[0054] Basic test principle: WST-8 in CCK-8 reagent is reduced to yellow formazan by dehydrogenase in cells under the action of 1-Methoxy PMS (1-methoxy-5-methylphenazine dimethyl sulfate). The amount of formazan generated is proportional to the cell viability. Therefore, the cell viability can be indirectly reflected by measuring the absorbance (OD value) of the contents of each well.

[0055] To further illustrate the role of each experimental group setting, the blank control group is compared with the toxicity negative control group and the toxicity positive control group for cell viability. If the data difference between the blank control group and the toxicity positive control group is significant enough compared with the difference between the blank control group and the toxicity negative control group, the cell viability data characterized by the microplate reader can be used to evaluate the toxicity of the test group; based on this, it can be determined that the test group whose cell viability is greater than or equal to the toxicity negative control group is non-toxic, and the test group whose cell viability is less than that of the toxicity positive control group is toxic; considering that the concentration data of toxic alitate included in the test group determined to be toxic may be included in the range constituted by the non-toxic alitate concentration data included in the test group determined to be non-toxic, it is necessary to eliminate the concentration range constituted by the maximum and minimum values ​​in the toxic alitate concentration data from the concentration range constituted by the maximum and minimum values ​​in the non-toxic alitate concentration data, and the retained concentration range is the preset concentration range.

[0056] Experimental results: From Figure 1 It can be seen that 10ng / mL, 50ng / mL, and 100 ng / mL of alituo had no effect on the viability of BV2 cells, and 200ng / mL, 500ng / mL, and 1000 ng / mL of alituo significantly increased the cell viability of BV2 cells. On the one hand, it proves that alituo has no cytotoxicity to BV2 cells under the above experimental concentration conditions and will not cause cell damage. Therefore, subsequent anti-inflammatory activity studies can choose to use the safe concentration range of 10ng / mL-1000 ng / mL verified in this experiment; on the other hand, a rule can also be drawn: high concentrations of alituo have a certain promoting effect on BV2 cell viability; this can reflect the anti-inflammatory, antioxidant or cell protection properties of alituo to a certain extent, and provide guidance for further research on the role of alituo in neuroinflammation and related diseases.

[0057] Example 5: Anti-inflammatory activity detection of mesenchymal stem cell extract (Aritol)

[0058] Experimental method: S5.1) Using the BV2 cells cultured in Example 2, 5×10 4The density of each well was inoculated into the plate wells in S3.1) of Example 3, and pre-cultured for 24 hours; S5.2) 6 concentrations were selected within the preset concentration range that was non-toxic to BV2 cells screened out in Example 4, and 6 groups of activity detection culture media with alitoru added concentrations of 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL were prepared according to S3.3) of Example 3, and the activity detection culture media was obtained by diluting the alitoru added culture medium prepared in Example 1; S5.3) The BV2 cells were divided into a blank control group, an anti-inflammatory positive control group, an anti-inflammatory negative control group, a model group, and six drug administration groups on a well basis, and the BV2 cells of the anti-inflammatory positive control group, the anti-inflammatory negative control group, the model group, and the drug administration group were transferred to the culture medium containing 500 ng / mL lipopolysaccharide (LPS) prepared in S3.2) of Example 3, and the BV2 cells in the blank control group were transferred to the culture medium containing 500 ng / mL lipopolysaccharide (LPS) in Example 3 The blank culture medium in S3.1) of Example 3 was used for culturing for 24 hours; S5.4) the BV2 cells of the blank control group and the model group were transferred again to the blank culture medium in S3.1) of Example 3, and correspondingly, the BV2 cells of the six drug-administered groups were transferred to 6 groups of active detection culture medium containing alituo, the BV2 cells in the anti-inflammatory positive control group were transferred to a culture medium supplemented with 5mM sodium butyrate, and the BV2 cells in the anti-inflammatory negative control group were transferred to a culture medium supplemented with 1000ng / mL placebo (human albumin); S5.5) incubating the BV2 cells for 12h or 24h, centrifuging, and taking the supernatant; and S5.6) taking the supernatant for Elisa detection: S5.7) taking the standard sample including the inflammatory factor to be tested, gradient dilution, and preparing the standard sample at 4 A standard curve is formed by adding a buffer solution to the supernatant at a volume ratio of 1:2 between the supernatant and the buffer solution to form a test sample, taking 100 μL of the test sample into the well of the experimental plate, fully mixing the test sample with the specific antibody that can bind to the inflammatory factor pre-coated in the well of the experimental plate, covering with a sealing film, incubating at 37°C for 2 hours, and after the incubation is completed, washing the well of the experimental plate with a buffer solution, and discarding the upper liquid; S5.9) Add 100 μL of biotinylated antibody working solution to each well, cover with a new sealing film, and continue incubating at 37°C for 1 hour. After the incubation is completed, wash the well of the experimental plate again, and discard the upper liquid; S5.10) Add 100 μL of HRP-labeled (Horseradish) to each well. Peroxidase, horseradish peroxidase) detection antibody, cover with a new sealing film, continue incubation at 37°C for 30 minutes, after the incubation is completed, wash the experimental plate wells again and discard the upper liquid; S5.11) Add 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) substrate to each well, cover with a new sealing film, and incubate at 37°C in the dark for 15-20 min; S5.12) After the incubation is completed, add 50 μL of stop solution to each well, and immediately place it in the microplate reader to measure the OD value of the contents of each well at 450 nm within 5 minutes; and S5.13) According to the standard curve, substitute the OD value of each well at 450 nm to obtain the concentration of inflammatory factors in the contents of each well. .

[0059] Basic test principle: Elisa test box usually includes a 96-well test plate, and each well is pre-coated with a layer of adsorbed protein with fixed specific antibodies. It can be understood that this experiment selects specific antibodies that can bind to IL-1β, TNF-α or IL-6 inflammatory factors, and solidifies them by binding to the inflammatory factors through specific antibodies, and then adds biotinylated antibody working solution to further capture and label the specific antibodies bound to the inflammatory factors to improve the stability of the binding; then add the detection antibody labeled with HRP, use the catalytic properties of HRP, add TMB substrate, and finally add the stop solution, which can produce a strong color reaction in a short time, thereby improving the sensitivity of the detection. The depth of the color of the reaction reflects the level of the inflammatory factor content, so the concentration of the inflammatory factor included can be indirectly inferred by measuring the absorbance (OD value) of the contents of each well.

[0060] It is understandable that the difference between the blank control group and the other experimental groups is that the BV2 cells in the blank control group were cultured in blank culture medium without stimulation conditions throughout the experiment, which can be used to verify the fact that lipopolysaccharide (LPS) induces the expression of inflammatory factors IL-1β, TNF-α and IL-6. Furthermore, the model group was compared with the anti-inflammatory negative control group and the anti-inflammatory positive control group for inflammatory factor concentrations. The data difference between the model group and the anti-inflammatory positive control group was significant enough compared to the difference between the model group and the anti-inflammatory negative control group. The data of inflammatory factors characterized by the Elisa test can be used to evaluate the anti-inflammatory properties of the drug-treated group. Based on this, at least the data between the inflammatory factor content data of the anti-inflammatory negative control group and the anti-inflammatory positive control group can reflect the anti-inflammatory properties through the numerical value, where the smaller the value, the stronger the anti-inflammatory property, and from Figure 2 It can be seen that the data of the drug-treated groups are between the data of the anti-inflammatory negative control group and the anti-inflammatory positive control group, and the strength of the anti-inflammatory activity of each drug-treated group can be evaluated by numerical comparison.

[0061] Experimental results: From Figure 2It can be seen that the sodium butyrate in the figure is the anti-inflammatory positive control group, and the placebo is human serum albumin, which is the anti-inflammatory negative control group. In the anti-inflammatory performance test of cells, compared with the blank control group, lipopolysaccharide (LPS) in the model group can significantly increase the levels of IL-1β, TNF-α and IL-6 inflammatory factors in BV2 cells. After treatment with alitor, high concentrations of alitor significantly reduced the levels of IL-1β, TNF-α and IL-6 inflammatory factors in BV2 cells induced by lipopolysaccharide (LPS), which can be used to characterize the improving effect of alitor on the inflammatory levels of BV2 cells induced by lipopolysaccharide (LPS); further, in repeatable experiments, the standard deviation of the inhibitory effect of high concentrations of alitor on inflammatory factors was relatively smaller, which means that the variability of experimental data is low. Therefore, the anti-inflammatory effect of high concentrations of alitor is more credible, and the anti-inflammatory efficacy of alitor can be evaluated more accurately. It also reveals to a certain extent the mechanism of action of alitor in alleviating damage caused by nerves and other inflammations by regulating the expression of inflammatory factors, which provides reference value for the subsequent clinical application of alitor products in the treatment of diseases related to neuroinflammation.

[0062] In addition, among the three inflammatory factors IL-1β, TNF-α and IL-6, Alitol has the best inhibitory effect on the IL-6 inflammatory factor. This indicates to a certain extent that the anti-inflammatory effect of Alitol is related to its regulation of the NF-κB signaling pathway, which plays a core role in the inflammatory response. Alitol reduces the production of inflammatory factors by inhibiting the activation of the NF-κB signaling pathway, thereby exerting its neuroprotective effect.

[0063] In summary, the experimental method proposed in this application can be used to evaluate and measure the feasibility of Alittor as a potential inflammatory inhibitor, and is expected to serve as a quality control model for monitoring the performance of Alittor. On the one hand, it can unify the batch-to-batch quality standards of Alittor and improve the quality stability of the product; on the other hand, it can explore the variation patterns of the concentration and anti-inflammatory activity of Alittor and calculate the optimal drug concentration.

[0064] Comparative Example 1: Bioactivity Detection of Mesenchymal Stem Cell Extract (Aritol) Using Culture Medium Without Fetal Bovine Serum

[0065] The activity test was performed with reference to Example 5, wherein no fetal bovine serum was added when preparing the culture medium used in the experimental group, that is:

[0066] S1) Add 220 μL of DMEM culture medium containing 1% penicillin / streptomycin (PS) to a new 3599 plate; S2) Add the alituo and lipopolysaccharide (LPS) prepared in Example 1 to each well of the 3599 plate, respectively, and configure them into culture medium with corresponding concentrations, set 2 wells for each concentration to repeat the experiment, and mix for 10 minutes; wherein the alituo concentration is set to two groups, namely 10 ng / mL and 100 ng / mL, respectively, and the lipopolysaccharide concentration is set to one group, with a concentration of 500 ng / mL; and S3) Transfer the BV2 cells induced by lipopolysaccharide (LPS) to the culture medium containing alituo one by one, continue incubation for 6h or 24h, centrifuge, take the supernatant, and perform the Elisa detection steps S5.6)-S5.13) in Example 5 to obtain the content of IL-6 and TNF-α inflammatory factors.

[0067] The experimental results can be found in Figure 3 The inflammatory factor content of BV2 cells in the culture medium without fetal bovine serum is on the low side. The low data will make error analysis difficult, especially considering the inevitable system errors and operational errors. The low data will lead to poor statistical significance and it is difficult to draw a statistically reliable conclusion. Therefore, the experimental results using the culture medium containing fetal bovine serum are more reliable and more desirable for evaluating the anti-inflammatory properties of Alito.

[0068] Comparative Example 2: Bioactivity detection using a culture medium containing both lipopolysaccharide and mesenchymal stem cell extract (Alito)

[0069] The activity test was performed with reference to Example 5, wherein, when preparing the culture medium used in the experimental group, lipopolysaccharide (LPS) inflammatory inducer and the alituo prepared in Example 1 were added simultaneously, and the culture medium comprising 500 ng / mL lipopolysaccharide (LPS) and 200 ng / mL alituo, the culture medium containing only 500 ng / mL lipopolysaccharide (LPS), and the culture medium containing only 200 ng / mL mesenchymal stem cell extract were respectively configured, and a blank control group without stimulation conditions, an LPS group induced only by lipopolysaccharide (LPS), an ALT+LPS co-culture group cultured with a culture medium containing both lipopolysaccharide and alituo, and an ALT+LPS group cultured successively with culture medium containing lipopolysaccharide (LPS) and alituo, respectively, were set up, and the incubation was continued for 6 h or 24 h, centrifuged, the supernatant was taken, and the Elisa detection steps S5.6)-S5.13) in Example 5 were performed to obtain the content of IL-6 inflammatory factor.

[0070] Experimental results refer to Figure 4Among them, the inflammatory factor concentration of BV2 cells cultured in the ALT+LPS group increased much faster within 6 hours than that in the ALT+LPS co-culture group. After 24 hours, the inflammatory factor concentration of BV2 cells cultured in the former decreased significantly, reflecting the anti-inflammatory and repairing effect of alituo, while the inflammatory factor concentration of BV2 cells cultured in the latter increased significantly. It is difficult to clarify whether the mechanism of action of alituo is on BV2 cells or on LPS, and the uncertainty is strong. Although the final inflammatory factor concentration of the ALT+LPS co-culture group is lower, its data fluctuates greatly and the credibility of the data is low. Therefore, in this application, BV2 cells are cultured successively with culture media containing lipopolysaccharide (LPS) and alituo, and the data obtained have good repeatability and can more accurately reflect the anti-inflammatory performance of alituo.

[0071] In summary, the toxicity detection method and anti-inflammatory performance detection method of the mesenchymal stem cell extract provided in the present application can quickly screen out safe experimental concentrations and data results with good repeatability and significance, and can be used to establish a performance monitoring model for the mesenchymal stem cell extract (Aritol), stabilize the quality of the mesenchymal stem cell extract product (Aritol), explore the changes in product concentration and anti-inflammatory activity, and lay a foundation for subsequent use as an inflammatory inhibitor.

[0072] 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 detecting the activity of a stress-induced mesenchymal stem cell extract, characterized in that: The activity detection method The following steps are involved: S1: BV2 cells were used, with N×10 4 The cells are inoculated into the well plate at a density of 10 cells per well, and pre-cultured for 20h-28h, 4.5≤N≤5.5; S2: at least 3 groups of activity detection medium with different concentrations are prepared using the mesenchymal stem cell extract, and the concentration of the mesenchymal stem cell extract in each group of the activity detection medium is within the preset concentration range; S3: a model group and a drug administration group are established according to the BV2 cells, and the BV2 cells in the model group and the drug administration group are transferred to a culture medium containing 450ng / mL-550ng / mL lipopolysaccharide, and cultured for 20h-28h; S4: transferring the BV2 cells in the model group to a blank culture medium, and transferring the BV2 cells in the drug administration group to the activity detection culture medium of each group respectively; S5: continuing to incubate the BV2 cells for 12 hours or 24 hours, centrifuging, and taking the supernatant; and S6: taking the supernatant for Elisa detection to obtain the concentration of inflammatory factors, and evaluating the anti-inflammatory activity of the mesenchymal stem cell extract by comparing the concentration of inflammatory factors in the model group and the drug administration group; wherein, the preparation of the mesenchymal stem cell extract The method comprises the following steps: S001: culturing mesenchymal stem cells and creating stress conditions to stimulate the mesenchymal stem cells; S002: cracking, separating and purifying the mesenchymal stem cells obtained in S001 to obtain the mesenchymal stem cell extract.

2. The activity detection method according to claim 1, characterized in that: The S3 also includes: establishing an anti-inflammatory positive control group and an anti-inflammatory negative control group according to the BV2 cells, transferring the anti-inflammatory positive control group and the anti-inflammatory negative control group to the culture medium containing 450ng / mL-550ng / mL lipopolysaccharide for 20h-28h; the S4 also includes transferring the BV2 cells in the anti-inflammatory positive control group to a culture medium supplemented with 5mM sodium butyrate, and transferring the BV2 cells in the anti-inflammatory negative control group to a culture medium supplemented with 1000ng / mL placebo, wherein the placebo is human serum albumin; the S5 also includes continuing to incubate the anti-inflammatory positive control group and The BV2 cells in the anti-inflammatory negative control group are centrifuged for 12 hours or 24 hours to obtain the supernatant; S6 also includes taking the supernatant in the anti-inflammatory positive control group and the anti-inflammatory negative control group for Elisa detection to obtain the concentration of the inflammatory factor, and evaluating the credibility of the data of the Elisa detection by comparing the concentrations of the inflammatory factors in the model group, the anti-inflammatory negative control group and the anti-inflammatory positive control group, and evaluating the anti-inflammatory activity of the mesenchymal stem cell extracts of different concentrations by comparing the concentrations of the inflammatory factors in the drug administration group, the anti-inflammatory negative control group and the anti-inflammatory positive control group.

3. The activity detection method according to claim 1, characterized in that: In S6, the inflammatory factors include one or a combination of multiple of IL-1β, TNF-α and IL-6.

4. The activity detection method according to claim 1, characterized in that: Before S1, the activity detection method further includes the following steps: S01: using the BV2 cells, M×10 3 The cells are inoculated into the well plate at a density of 10 cells per well, and pre-cultured for 20h-28h, 7.5≤M≤8.5; S02: at least 3 groups of toxicity detection medium with different concentrations are prepared using the mesenchymal stem cell extract; S03: a blank control group, a toxicity negative control group, a toxicity positive control group and a test group are established according to the BV2 cells, the blank control group is incubated in a blank medium, the toxicity negative control group and the toxicity positive control group are incubated in a medium with a placebo and sodium butyrate, respectively, and the test group is incubated in the toxicity detection medium of each group, and the placebo is human serum albumin; S04: the BV2 cells are incubated for 12h or 24h; S05: 10 μL of CCK-8 solution, and continue incubating for 1h-2h; and S06: using an ELISA instrument to measure the cell viability of the BV2 cells, and by comparing the cell viabilities of the blank control group with those of the toxicity negative control group and the toxicity positive control group, respectively, to evaluate the credibility of the data of the ELISA instrument; comparing the test group with the toxicity negative control group and the toxicity positive control group, respectively, wherein the toxicity detection medium corresponding to the test group whose cell viability is greater than or equal to that of the toxicity negative control group is determined to be non-toxic to the BV2 cells, and the toxicity detection medium corresponding to the test group whose cell viability is less than that of the toxicity positive control group is determined to be toxic to the BV2 cells, and in the concentration range consisting of the maximum and minimum values ​​of the concentration of the mesenchymal stem cell extract included in the toxicity detection medium determined to be non-toxic, after excluding the concentration range consisting of the maximum and minimum values ​​of the concentration of the mesenchymal stem cell extract included in the toxicity detection medium determined to be toxic, the remaining concentration range is used as the preset concentration range.

5. The activity detection method according to claim 4, characterized in that: The preset concentration range is 10 ng / mL-1000 ng / mL.

6. The activity detection method according to claim 5, characterized in that: The activity detection medium includes 6 groups, and the concentrations of the mesenchymal stem cell extracts in the 6 groups of the activity detection medium are 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL and 1000 ng / mL, respectively.

7. The activity detection method according to claim 4, characterized in that: The culture medium used to culture the BV2 cells in steps S01 to S03 and S1 to S4 is a culture medium including serum, and the incubation conditions of the BV2 cells are 37° C. and 5% CO 2 .

8. The activity detection method according to claim 7, characterized in that: The culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

9. The activity detection method according to any one of claims 1 to 3, characterized in that: The S6 comprises the following steps: S61: taking a standard substance including the inflammatory factor, gradiently diluting, and preparing a standard curve of the OD value of the standard substance at 450nm-the concentration of the inflammatory factor; S62: adding a buffer to the supernatant according to a predetermined dilution ratio to form a sample to be tested, taking a predetermined volume of the sample to be tested into the well of the experimental plate, fully mixing the sample to be tested with the specific antibody that can bind to the inflammatory factor pre-coated in the well of the experimental plate, covering with a sealing film, incubating at 37°C for 2h, and after the incubation is completed, washing the wells of the experimental plate and discarding the upper liquid; S63: adding 1% HCl to each well; Add a biotinylated antibody working solution of the same volume as the sample to be tested, cover with a new sealing film, continue to incubate at 37°C for 1 hour, and after the incubation is completed, wash the experimental plate wells again, and discard the upper liquid; S64: add a detection antibody labeled with HRP of the same volume as the sample to be tested to each well, cover with a new sealing film, continue to incubate at 37°C for 30min-60min, after the incubation is completed, wash the experimental plate wells again, and discard the upper liquid; S65: add a TMB substrate of the same volume as the sample to be tested to each well, cover with a new sealing film, and incubate at 37°C in the dark for 15min-20 min; S66: after the incubation is completed, add 0.5 times the volume of the sample to be tested to each well of the stop solution, and immediately put it into the microplate reader to measure the OD value of the contents of each well at 450 nm within 5 minutes; and S67: according to the standard curve, substitute the OD value of each well at 450 nm to obtain the concentration of inflammatory factors in the contents of each well.

10. The activity detection method according to claim 9, characterized in that: In S62, the predetermined dilution ratio refers to that the ratio of the supernatant volume to the buffer volume is equal to 1:2, and the predetermined volume is 100 μL.

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