Detection method for inhibiting maturation of dendritic cells by using mesenchymal stem cells

Abstract

The invention discloses a detection method for inhibiting maturation of dendritic cells by using mesenchymal stem cells, and relates to the technical field of cytobiology and immunological detection.The detection method for inhibiting maturation of dendritic cells by using mesenchymal stem cells comprises the following steps: S1, preparing an experimental MSC: adopting a commercial MSC cell suspension; s2, CD14 + cell sorting: separating CD14 + cells from the PBMC, wherein the CD14 expression rate is more than 80% through flow detection; s3, inducing the CD14 + cells by using GM-CSF and IL-4, so as to obtain immature DC (iDC); s4, co-culturing the iDC and the LPS, inducing the iDC and the LPS to be mature, and setting an iDC + LPS + MSCs experimental group; s5, detecting indexes: detecting the expression rates of CD83, CD40, CD80, CD86 and HLA-DR, and calculating the inhibition rate of the MSCs on DC maturation; through CD14 + cell sorting, DC cell induced differentiation and flow cytometry of a DC maturation marker, quantitative evaluation of the MSCs immunomodulatory function is realized. The method is standard in operation and reliable in result, and can be used for curative effect evaluation and mechanism research of the MSCs in immune disease treatment.

Description

Technical Field

[0001] The present invention relates to the field of cell biology and immunological detection technology, and in particular to a method for detecting whether mesenchymal stem cells inhibit the maturation of dendritic cells. The method can be used to evaluate the immunoregulatory function of mesenchymal stem cells and develop related drugs. Background Art

[0002] Dendritic cells (DCs) are the strongest antigen-presenting cells, and their maturation state directly affects the immune response. Mesenchymal stem cells (MSCs) exert immunomodulatory effects by inhibiting DC maturation, but their detection methods have problems such as complex operation and insufficient stability. In the prior art, the efficiency of DC induction differentiation is low and the accuracy of marker detection is poor, making it difficult to accurately evaluate the inhibitory effect of MSCs. The present invention provides a method for detecting the inhibition of dendritic cell maturation by mesenchymal stem cells by optimizing cell sorting, induction conditions and detection procedures. The method has the characteristics of high efficiency and stability, and can significantly improve the detection accuracy and practicality. Summary of the Invention

[0003] In view of this, the present invention addresses the deficiencies in the existing technology, and its main purpose is to provide a detection method for mesenchymal stem cells to inhibit dendritic cell maturation. Through standardized processes and optimization of key parameters, it can achieve quantitative evaluation of the ability of MSCs to inhibit DC maturation, providing a reliable technical means for evaluating the efficacy of MSCs in immune diseases.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for detecting whether mesenchymal stem cells inhibit the maturation of dendritic cells comprises the following steps:

[0006] S1. Prepare MSCs for the experiment: Use commercial MSC cell suspension;

[0007] S2, CD14 + Cell sorting: CD14 was isolated from PBMC using CD14 magnetic bead sorting. + Cells, flow cytometry detection of CD14 expression rate> 80%;

[0008] S3. Immature DC (iDC) induction: CD14 induced by GM-CSF and IL-4 + cells, to obtain immature DCs (iDCs);

[0009] S4. Mature DC (mDC) induction: iDCs were co-cultured with LPS to induce maturation, and an iDC+LPS+MSCs experimental group was set up;

[0010] S5. Detection indicators: Flow cytometry was used to detect the expression rates of CD83, CD40, CD80, CD86, and HLA-DR, and the inhibition rate of MSCs on DC maturation was calculated.

[0011] As a preferred solution: in step S4, iDCs are co-cultured with LPS to induce maturation as a positive control group, and iDCs + culture medium (without LPS) are set as a negative control group; the co-culture conditions are: 37° C., 5% CO 2 incubator, and co-culture for 2-3 days.

[0012] As a preferred solution: the concentrations of GM-CSF and IL-4 in step S3 are both 10-100 ng / mL.

[0013] As a preferred solution: the concentrations of GM-CSF and IL-4 in step S3 are both 50 ng / mL, and GM-CSF and IL-4 are used to induce CD14 + Cells for 5-6 days.

[0014] As a preferred solution: the LPS concentration in step S4 is 10-100 ng / mL.

[0015] As a preferred solution: the LPS concentration in step S4 is 50 ng / mL.

[0016] As a preferred solution: in the iDC+LPS+MSCs experimental group in step S4, the cell ratio of MSCs to iDCs is 1:1.

[0017] As a preferred solution: the inhibitory rate of MSCs on DC maturation calculated in step S5 is specifically: inhibition rate = (positive group expression rate - negative group expression rate) / (positive group expression rate - experimental group expression rate) × 100%.

[0018] As a preferred solution: in step S5, MSCs are labeled with CD73 for flow cytometry clustering to eliminate the interference of MSCs in the detection of DC markers.

[0019] As a preferred solution: the MSCs in step S1 are fourth-generation MSCs, and the PBMCs in step S2 are mixed sources of commercially established lines.

[0020] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical solution that:

[0021] First, high accuracy: Through CD14 magnetic bead sorting and CD73 clustering technology, interference from miscells is eliminated to ensure the specificity of DC marker detection.

[0022] Second, good stability: the induction conditions (50 ng / mL GM-CSF / IL-4 induction for 5-6 days) made the iDC viability reach 91%, and the inhibition rate of maturation markers was stable.

[0023] Third, it is highly practical: the method of inducing DCs with frozen PBMCs reduces experimental costs, and the inhibition rate test results are comparable to those of fresh PBMCs, making it suitable for large-scale sample analysis.

[0024] Fourth, provide technical means: Through standardized processes and optimization of key parameters, quantitative evaluation of the ability of MSCs to inhibit DC maturation can be achieved, providing reliable technical means for evaluating the efficacy of MSCs in immune diseases (such as systemic lupus erythematosus).

[0025] To more clearly illustrate the structural features and effects of the present invention, it is described in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the cell morphology of the mDC-negative group of the present invention;

[0027] Figure 2 This is a schematic diagram of the cell morphology of the mDC-positive group of the present invention;

[0028] Figure 3 This is a schematic diagram of the cell morphology of the mDC experimental group of the present invention;

[0029] Figure 4 The flow cytometry results after CD14 magnetic bead separation of the present invention (CD14 + Schematic diagram of cell purity >90%);

[0030] Figure 5 Schematic diagram comparing the viability of iDCs induced by fresh PBMC and frozen PBMC of the present invention;

[0031] Figure 6 This is a bar graph showing the inhibition rate of MSC on DC maturation markers of the present invention;

[0032] Figure 7 Schematic diagram of the CD73 clustering flow cytometry results of the present invention. DETAILED DESCRIPTION

[0033] The present invention Figures 1 to 7 As shown, a method for detecting whether mesenchymal stem cells inhibit the maturation of dendritic cells comprises the following steps:

[0034] S1. Prepare MSCs for the experiment: Use commercial MSC cell suspension;

[0035] S2, CD14 +Cell sorting: CD14 was isolated from PBMC using CD14 magnetic bead sorting. + The CD14 expression rate of cells was >80% by flow cytometry to ensure the sorting purity;

[0036] S3. Immature DC (iDC) induction: CD14 induced by GM-CSF and IL-4 + cells, to obtain immature DCs (iDCs);

[0037] CD14 after sorting + The cells were induced and cultured with effective concentrations of GM-CSF and IL-4 for 5 days, with half of the medium replaced on the third day. After induction, the iDC morphology showed dendritic generation, and the CD14 expression rate decreased from 80%-100% to less than 20%;

[0038] S4. Mature DC (mDC) induction: iDCs were co-cultured with LPS to induce maturation, and an iDC+LPS+MSCs experimental group was set up;

[0039] iDCs were co-cultured with 50 ng / mL LPS for 2 days. A MSC co-culture experimental group (MSC to iDC ratio of 1:1) was also set up. After induction, mDCs showed suspended growth with dendrite-like protrusions on the surface.

[0040] S5. Detection indicators: Flow cytometry was used to detect the expression rates of CD83, CD40, CD80, CD86, and HLA-DR, and the inhibition rate of MSCs on DC maturation was calculated.

[0041] Result determination: If the marker expression rate of the experimental group is between the negative and positive groups or lower than that of the positive group, that is, the inhibition rate is greater than 0, it indicates that MSCs effectively inhibit DC maturation.

[0042] In step S4, iDCs were co-cultured with LPS to induce maturation as a positive control group, and iDCs + culture medium (without LPS) were set as a negative control group; the co-culture conditions were: 37° C., 5% CO 2 incubator, and co-cultured for 2-3 days.

[0043] In step S3, the concentrations of GM-CSF and IL-4 are both 10-100 ng / mL.

[0044] In step S3, the concentrations of GM-CSF and IL-4 were both 50 ng / mL. GM-CSF and IL-4 were used to induce CD14 + Cells for 5-6 days.

[0045] The LPS concentration in step S4 is 10-100 ng / mL.

[0046] The LPS concentration in step S4 is 50 ng / mL.

[0047] In step S4, the cell ratio of MSCs to iDCs in the iDC+LPS+MSCs experimental group was 1:1.

[0048] The specific calculation of the inhibition rate of MSCs on DC maturation in step S5 is: inhibition rate = (positive group expression rate - negative group expression rate) / (positive group expression rate - experimental group expression rate) × 100%.

[0049] In step S5, MSCs are labeled with CD73 for flow cytometry clustering to eliminate the interference of MSCs on DC marker detection; single-tube mixed detection scheme: a fluorescent antibody combination is used to label MSCs with CD73 to achieve cell clustering, thereby reducing the interference of MSCs on DC detection.

[0050] The MSCs in step S1 are fourth-generation MSCs, and the PBMCs in step S2 are mixed sources of commercially established lines.

[0051] Example 1: Detection of MSC inhibition of DC maturation from fresh PBMCs

[0052] Experimental materials: Fresh PBMC; human umbilical cord mesenchymal stem cells (passage 4, viability >85%);

[0053] Induction factors: GM-CSF 50 ng / mL, IL-4 50 ng / mL, LPS 50 ng / mL.

[0054] A method for detecting whether mesenchymal stem cells inhibit the maturation of dendritic cells is provided, comprising the following steps

[0055] S1. MSCs for experiments: Commercial MSC cell suspension was used.

[0056] S2, CD14 + Cell sorting: Human peripheral blood mononuclear cells (PBMCs) were magnetically separated using a CD14 magnetic bead sorting kit, and the CD14 expression rate was detected by flow cytometry.

[0057] S3. Induction of immature DCs (iDCs): 2×10 6 CD14 + Cells were seeded in 12-well plates and added with 1640 complete medium containing 50 ng / mL GM-CSF and 50 ng / mL IL-4. The cells were cultured at 37°C, 5% CO2 for 5 days. On the third day, half of the medium was changed (centrifugation at 400g for 3 minutes, supernatant discarded, and fresh medium containing the same concentration of cytokines was added).

[0058] S4. Mature DC (mDC) induction: iDC were co-cultured with 50 ng / mL LPS for 2 days. At the same time, a MSC co-culture experimental group was set up (MSC to iDC ratio was 1:1).

[0059] Co-culture experimental design

[0060] (1) Group settings:

[0061] Negative control group: iDC + culture medium (without LPS);

[0062] Positive control group: iDC+LPS;

[0063] Experimental group: iDC+LPS+MSCs.

[0064] (2) Culture conditions: 37°C, 5% CO2 incubator, total culture time 2 days.

[0065] S5. Detection indicators

[0066] (1) Flow cytometry:

[0067] Markers: CD83, CD40, CD80, CD86, HLA-DR (maturation marker, upregulated after LPS induction).

[0068] Single-tube mixed detection scheme: Using a combination of fluorescent antibodies, MSCs are labeled with CD73 to achieve cell clustering and reduce the interference of MSCs on DC detection.

[0069] (2) Calculation of inhibition rate:

[0070] Inhibition rate = (expression rate of positive group - expression rate of negative group) / (expression rate of positive group - expression rate of experimental group) × 100%

[0071] Test results

[0072] (1) mDC morphology results

[0073] The cells in the negative group grew semi-adherently with irregular morphology and a small number of short spiny processes;

[0074] The cells in the positive group grew in suspension, their volume increased, and a large number of dendrite-like protrusions of varying thickness appeared on the surface;

[0075] The cell morphology of the experimental group was between the negative and positive groups, and the number of processes was significantly reduced.

[0076] (2) Inhibition rate results

[0077] Table 1: MSC inhibition rate on DC maturation from fresh PBMC

[0078] markers Negative expression rate Positive expression rate Expression rate in the experimental group Inhibition rate CD86 2.34％ 25.40％ 9.66％ 68.26％ CD80 0.90％ 82.42％ 44.60％ 46.39％ CD40 33.27％ 82.31％ 27.68％ 100.00％ HLA-DR 79.84％ 94.23％ 56.39％ 100.00％ CD83 6.31％ 80.06％ 40.77％ 53.27％

[0079] (3) Conclusion: MSCs can effectively inhibit the maturation of DCs derived from fresh PBMCs.

[0080] Example 2: Detection of MSC-induced DC maturation inhibition by cryopreserved PBMC (activated 10 min after resuscitation)

[0081] Experimental Materials: PBMC sources: frozen PBMC (activated for 10 minutes after thawing); human umbilical cord mesenchymal stem cells (passage 4, viability >85%);

[0082] Induction factors: GM-CSF 50 ng / mL, IL-4 50 ng / mL, LPS 50 ng / mL.

[0083] Provided is a method for detecting whether mesenchymal stem cells inhibit dendritic cell maturation, comprising the following steps:

[0084] S1. MSCs for experiments: Commercial MSC cell suspension was used.

[0085] Cell sorting and differentiation induction

[0086] S2, CD14 + Cell sorting: Human peripheral blood mononuclear cells (PBMCs) were magnetically separated using a CD14 magnetic bead sorting kit, and the CD14 expression rate was detected by flow cytometry.

[0087] S3. Induction of immature DCs (iDCs): 2×10 6 CD14 + Cells were seeded in 12-well plates and cultured in 1640 complete medium containing 50 ng / mL GM-CSF and 50 ng / mL IL-4 at 37°C and 5% CO2 for 5 days. On the third day, half of the medium was changed (centrifuged at 400 g for 3 minutes, the supernatant was discarded, and fresh medium containing the same concentration of cytokines was added).

[0088] S4. Mature DC (mDC) induction: iDC were co-cultured with 50 ng / mL LPS for 2 days. At the same time, a MSC co-culture experimental group was set up (MSC to iDC ratio was 1:1).

[0089] Co-culture experimental design

[0090] (1) Group settings:

[0091] Negative control group: iDC + culture medium (without LPS);

[0092] Positive control group: iDC+LPS;

[0093] Experimental group: iDC+LPS+MSCs.

[0094] (2) Culture conditions: 37°C, 5% CO2 incubator, total culture time 2 days.

[0095] S5. Detection indicators

[0096] (1) Flow cytometry:

[0097] Markers: CD83, CD40, CD80, CD86, HLA-DR (maturation marker, upregulated after LPS induction).

[0098] Single-tube mixed detection scheme: Using a combination of fluorescent antibodies, MSCs are labeled with CD73 to achieve cell clustering and reduce the interference of MSCs on DC detection.

[0099] (2) Calculation of inhibition rate:

[0100] Inhibition rate = (expression rate of positive group - expression rate of negative group) / (expression rate of positive group - expression rate of experimental group) × 100%

[0101] Test results

[0102] (1) mDC morphology results

[0103] The cells in the negative group grew semi-adherently with irregular morphology and a small number of short spiny processes;

[0104] The cells in the positive group were partially differentiated, but there were still monocytes remaining;

[0105] The cell morphology of the experimental group was between the negative and positive groups, and the number of processes was reduced.

[0106] (2) Inhibition rate results

[0107] Table 2: MSC inhibition rate of DC maturation from frozen PBMC (activated 10 min after thawing)

[0108]

[0109]

[0110] (3) Conclusion: MSCs can effectively inhibit the maturation of DCs derived from cryopreserved PBMCs (activated for 10 min after resuscitation) Example 3: Detection of the inhibitory effect of MSCs on the maturation of DCs derived from cryopreserved PBMCs (activated for 4 h after resuscitation)

[0111] Experimental Materials: PBMC sources: cryopreserved PBMC (activated 4 hours after thawing); human umbilical cord mesenchymal stem cells (passage 4, viability > 85%);

[0112] Induction factors: GM-CSF 50 ng / mL, IL-4 50 ng / mL, LPS 50 ng / mL.

[0113] Provided is a method for detecting whether mesenchymal stem cells inhibit dendritic cell maturation, comprising the following steps:

[0114] S1. MSCs for experiments: Commercial MSC cell suspension was used.

[0115] Cell sorting and differentiation induction

[0116] S2, CD14 + Cell sorting: Human peripheral blood mononuclear cells (PBMCs) were magnetically separated using a CD14 magnetic bead sorting kit, and the CD14 expression rate was detected by flow cytometry.

[0117] S3. Induction of immature DC (iDC): 2×10 6 CD14 + Cells were seeded in 12-well plates and cultured in 1640 complete medium containing 50 ng / mL GM-CSF and 50 ng / mL IL-4 at 37°C and 5% CO2 for 5 days. On the third day, half of the medium was changed (centrifuged at 400 g for 3 minutes, the supernatant was discarded, and fresh medium containing the same concentration of cytokines was added).

[0118] S4. Mature DC (mDC) induction: iDC were co-cultured with 50 ng / mL LPS for 2 days. At the same time, a MSC co-culture experimental group was set up (MSC to iDC ratio was 1:1).

[0119] Co-culture experimental design

[0120] (1) Group settings:

[0121] Negative control group: iDC + culture medium (without LPS);

[0122] Positive control group: iDC+LPS;

[0123] Experimental group: iDC+LPS+MSCs.

[0124] (2) Culture conditions: 37°C, 5% CO2 incubator, total culture time 2 days.

[0125] S5. Detection indicators

[0126] (1) Flow cytometry:

[0127] Markers: CD83, CD40, CD80, CD86, HLA-DR (maturation marker, upregulated after LPS induction).

[0128] Single-tube mixed detection scheme: Using a combination of fluorescent antibodies, MSCs are labeled with CD73 to achieve cell clustering and reduce the interference of MSCs on DC detection.

[0129] (2) Calculation of inhibition rate:

[0130] Inhibition rate = (expression rate of positive group - expression rate of negative group) / (expression rate of positive group - expression rate of experimental group) × 100%

[0131] Test results

[0132] (1) mDC morphology results

[0133] The cells in the negative group grew semi-adherently with irregular morphology and a small number of short spiny processes;

[0134] The cells in the positive group were partially differentiated, but there were still monocytes remaining;

[0135] The cell morphology of the experimental group was between the negative and positive groups, and the number of processes was reduced.

[0136] (4) Inhibition rate results

[0137] Table 3: MSC inhibition of DC maturation from frozen PBMC (activated 4 hours after resuscitation)

[0138]

[0139]

[0140] (5) Conclusion: MSCs can effectively inhibit the maturation of DCs derived from cryopreserved PBMCs (activated 4 hours after resuscitation).

[0141] Table 4: Flow cytometry CD14 expression rate

[0142]

[0143] According to the analysis in Table 4, the CD14 + The cells showed the highest purity, with a CD14 expression rate of up to 99.70%, and the best differentiation efficacy. After induction into iDCs, CD14 expression dropped to 4.99%, indicating that most monocytes had differentiated into DCs. Due to the freezing process, the sorting purity and differentiation efficiency of cryopreserved cells are lower than those of fresh PBMCs. However, by extending the activation time to 4 hours, the sorting purity and differentiation efficiency can be relatively improved.

[0144] Table 5: Cell viability

[0145]

[0146] According to the analysis in Table 5, the CD14 +Cell viability was the highest, reaching 94.60%, and after induction into iDCs, the cell viability reached 91.00%, indicating that freshly derived PBMCs were in better condition. Cryopreserved cells had lower cell viability than freshly derived PBMCs due to freezing, but extending the activation time to 4 hours can improve cell viability and maintain a better cell state.

[0147] Table 6: Inhibition rate of MSC on DC cell maturation markers

[0148] Example CD86 CD80 CD40 HLA-DR CD83 Example 1 68.26％ 46.39％ 100.00％ 100.00％ 53.27％ Example 2 24.97％ 38.86％ 43.76％ 100.00％ 100.00％ Example 3 80.19％ 50.90％ 100.00％ 100.00％ 100.00％

[0149] As analyzed in Table 6, MSCs showed an inhibitory rate greater than 0 for DC maturation markers induced by PBMCs from different sources and at different activation times, indicating that MSCs can effectively inhibit DC maturation induced by PBMCs from different sources and at different activation times. Mainly for CD83, fresh PBMCs showed a better state of induced DC differentiation, with an inhibition rate of only 53.27%. However, frozen PBMCs showed a relatively weaker state of induced DC differentiation, with an inhibition rate of up to 100%.

[0150] Table 7: Cost Control

[0151] Example Operational costs Features Example 1 high Fresh blood sample required Example 2 Low Can be frozen in batches Example 3 Low Can be frozen in batches

[0152] As shown in Table 7, freshly sourced PBMCs need to be sorted each time, which has high operating costs and requires a high level of fresh blood samples. Cryopreserved PBMCs can be used at any time, have low operating costs, and are suitable for large-scale testing in enterprises.

[0153] Table 8: CD73 expression rate

[0154]

[0155]

[0156] Table 8 shows that mDCs and MSCs are co-cultured in direct contact. Directly testing for mDC maturation markers may affect the expression of mDCs. Therefore, CD73, a positive marker for MSCs, was used for sorting. The CD73 expression rate of sorted mDCs was less than 2%, while that of the MSC group was greater than 80%, indicating positive expression. CD73 clustering technology allows this assay to eliminate interference from other cells and ensure the specificity of DC marker detection.

[0157] Comparative conclusions of Example 1 to Example 3:

[0158] (1) Expression rate and viability: CD14 cells were sorted from fresh PBMC (Example 1). +The cell flow cytometry CD14 purity, cell viability and differentiation efficiency were the highest, followed by the frozen PBMC group (Example 3). The cell expression rate and viability of the detection method were relatively ideal.

[0159] (2) Inhibitory effect: The CD83 inhibition rate in the frozen PBMC group (Example 2 and Example 3) was the highest (100.00%), which may be related to the change in cell activation state during the freezing process; all three groups of examples had an inhibitory effect on the expression of DC maturation markers, and the results of the detection method were highly stable.

[0160] (3) Cost and stability: The fresh PBMC group (Example 1) had the highest iDC viability (91%), which is suitable for scenarios with high requirements for cell status. From the perspective of cost control, the frozen group (Example 3) is more suitable for large-scale testing and has better results.

[0161] (4) Accuracy: CD14 magnetic bead sorting and CD73 clustering technology are used to eliminate interference from other cells, ensure the specificity of DC marker detection, and achieve high accuracy of the detection method.

[0162] The design emphasis of the present invention is:

[0163] First, high accuracy: Through CD14 magnetic bead sorting and CD73 clustering technology, interference from miscells is eliminated to ensure the specificity of DC marker detection.

[0164] Second, good stability: the induction conditions (50 ng / mL GM-CSF / IL-4 induction for 5-6 days) made the iDC viability reach 91%, and the inhibition rate of maturation markers was stable.

[0165] Third, it is highly practical: the method of inducing DCs with frozen PBMCs reduces experimental costs, and the inhibition rate test results are comparable to those of fresh PBMCs, making it suitable for large-scale sample analysis.

[0166] Fourth, provide technical means: Through standardized processes and optimization of key parameters, quantitative evaluation of the ability of MSCs to inhibit DC maturation can be achieved, providing reliable technical means for evaluating the efficacy of MSCs in immune diseases (such as systemic lupus erythematosus).

[0167] The above description is merely a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for detecting that mesenchymal stem cells inhibit the maturation of dendritic cells, characterized in that: The steps include: S1. Prepare MSCs for the experiment: Use commercial MSC cell suspension; S2, CD14 + Cell sorting: CD14 was isolated from PBMC using CD14 magnetic bead sorting. + Cells, flow cytometry detection of CD14 expression rate> 80%; S3. Immature DC (iDC) induction: CD14 induced by GM-CSF and IL-4 + cells, to obtain immature DCs (iDCs); S4. Mature DC (mDC) induction: iDCs were co-cultured with LPS to induce maturation, and an iDC+LPS+MSCs experimental group was set up; S5. Detection indicators: Flow cytometry was used to detect the expression rates of CD83, CD40, CD80, CD86, and HLA-DR, and the inhibition rate of MSCs on DC maturation was calculated.

2. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 1, wherein: In step S4, iDCs were co-cultured with LPS to induce maturation as a positive control group, and iDCs + culture medium (without LPS) was set as a negative control group; the co-culture conditions were: 37° C., 5% CO 2 incubator, and co-cultured for 2-3 days.

3. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 1, wherein: In step S3, the concentrations of GM-CSF and IL-4 are both 10-100 ng / mL.

4. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 3, wherein: The concentrations of GM-CSF and IL-4 in step S3 were both 50 ng / mL, and GM-CSF and IL-4 were used to induce CD14 + Cells for 5-6 days.

5. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 1, characterized in that: The LPS concentration in step S4 is 10-100 ng / mL.

6. The method for detecting the inhibition of dendritic cell maturation by mesenchymal stem cells according to claim 5, characterized in that: The LPS concentration in step S4 is 50 ng / mL.

7. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 1, characterized in that: In the iDC+LPS+MSCs experimental group in step S4, the cell ratio of MSCs to iDCs was 1:

1.

8. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 2, characterized in that: The specific calculation of the inhibition rate of MSCs on DC maturation in step S5 is: inhibition rate = (positive group expression rate - negative group expression rate) / (positive group expression rate - experimental group expression rate) × 100%.

9. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 1, characterized in that: In step S5, MSCs are labeled with CD73 for flow cytometry clustering to eliminate the interference of MSCs in the detection of DC markers.

10. The method for detecting that mesenchymal stem cells inhibit dendritic cell maturation according to claim 1, characterized in that: The MSCs in step S1 are fourth-generation MSCs, and the PBMCs in step S2 are mixed sources of commercially established lines.

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