CN110982786A - Method for evaluating influence of human umbilical cord mesenchymal stem cells on secretion of TNF- α by T lymphocytes - Google Patents
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Abstract
The invention relates to a method for evaluating influence of human umbilical cord mesenchymal stem cells on T lymphocytes to secrete TNF- α, which belongs to the technical field of stem cells and regenerative medicine and comprises the following steps of co-culturing, adding T lymphocytes into a small pore plate coated with a CD3 antibody in advance, adding the umbilical cord mesenchymal stem cells, co-culturing the T lymphocytes and the umbilical cord mesenchymal stem cells, adding a CD28 antibody and interleukin 2 into a co-culture system, collecting culture supernatant after co-culturing for a preset time, detecting the content of TNF- α by an ELISA method, detecting the capacity of different subsets of T lymphocytes to secrete TNF- α by flow cytometry, detecting the expression quantity of TNF- α by qPCR, and comprehensively evaluating the immunoregulation capacity of the mesenchymal stem cells.
Description
Technical Field
The invention relates to the technical field of stem cells and regenerative medicine, in particular to a method for evaluating influence of human umbilical cord mesenchymal stem cells on secretion of TNF- α by T lymphocytes.
Background
The immune regulation is one of important indexes for evaluating the physiological functions of the umbilical cord mesenchymal stem cells, the T cells are important components of adaptive immunity, the activated T cells can secrete a large amount of TNF- α - α which is an important immune regulation factor and participate in various important physiological activities, such as regulating immune response, killing target cells, inducing tumor cell apoptosis and the like.
In the conventional method, a flow cytometer is used for detecting TNF- α secreted by T cells, an enzyme linked immunosorbent assay (ELISA) is used for detecting TNF- α secreted by the T cells, and a real-time fluorescence quantitative method (qPCR) is used for detecting the expression condition of TNF- α genes secreted by the T cells, however, qPCR, ELISA and flow cytometry have advantages and defects in detection of TNF- α secreted by the T cells, a single experimental method cannot accurately evaluate the capacity of the T lymphocytes to secrete the TNF- α, a patient suffering from rheumatoid arthritis has the defect that the content of TNF- α in joint synovial fluid in a joint cavity is increased and is one of main reasons for activating B cells and further damaging cartilage tissues, adalimumab (a neutralizing antibody of TNF- α) is mainly used for treating the autoimmune diseases caused by the content increase of TNF- α clinically at present, but the treatment effect of part of the patients is limited, and research shows that mesenchymal stem cells have the capacity of regulating immune response, so that the capacity of the mesenchymal stem cells to regulate the T- α secretion of other subgroups of the TNF- α is urgently needed for treating the autoimmune diseases caused by using the mesenchymal stem cells in the future.
Disclosure of Invention
Based on the above, there is a need to provide a method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes, which can accurately evaluate the capacity of the T lymphocytes to secrete TNF- α as a whole.
A method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes, comprising the following steps:
co-culturing: adding T lymphocytes into a small pore plate coated with a CD3 antibody in advance, adding umbilical cord mesenchymal stem cells, adding a culture medium, co-culturing the T lymphocytes and the umbilical cord mesenchymal stem cells, adding a CD28 antibody and interleukin 2 into a co-culture system, co-culturing for a preset time, collecting culture supernatant, and testing;
detecting the content of TNF- α by ELISA method, detecting the ability of different sub-group T lymphocyte to secrete TNF- α by flow cytometry, and detecting the expression quantity of TNF- α by qPCR;
evaluation: and (4) integrating the three detection results to evaluate the immunoregulatory capacity of the mesenchymal stem cells.
The inventor finds in practice that in conventional detection, T cells sorted out by magnetic beads are mixed with some NK cells and NKT cells, so when qPCR is used for detecting expression of TNF- α gene and ELISA is used for detecting content of TNF- α in solution, the capacity of synthesizing TNF- α by the T cells cannot be strictly shown, one part of data of the qPCR and ELISA is NKT cells or NK cells, flow cytometry can be used for detecting the capacity of secreting TNF- α by the T lymphocytes and even can be used for detecting the capacity of secreting TNF- α by T cell subsets, but due to the limitation of the principle of detecting secreting cytokines by flow cytometry, Golgi transport inhibitors are required to be added into a culture system to inhibit secretion of the cytokines to the outside of the cells, cytokine flow cytometry cannot be used for detecting the secreted before adding the inhibitors, and therefore, the detection of the qPCR, ELISA and the flow cytometry cannot be used for accurately evaluating the capacity of secreting the TNF- α by the T lymphocytes by a single experimental method, and the three comprehensive experimental methods are helpful for reading the overall obtained result.
In the method, the T lymphocyte and the human umbilical cord mesenchymal stem cell are co-cultured, and the CD3 antibody and the CD28 antibody are added in the culture process to simulate the process that the immune cell is activated by contacting pathogenic microorganisms, so that the method has the advantage of high reduction degree of in-vivo environment.
In one embodiment, in the co-culturing step, the T lymphocytes are obtained by: diluting blood with normal saline, adding lymphocyte separation liquid, keeping the blood layer on the upper layer, centrifuging, removing the upper layer transparent liquid, keeping the middle white layer, sucking the white layer, adding phosphate buffer solution, centrifuging, collecting cells, and sorting T lymphocytes with magnetic beads.
As can be understood, the umbilical cord mesenchymal stem cells are prepared by a conventional method, such as a tissue mass culture method or an enzyme digestion method.
In one example, the co-incubation step is performed by pre-coating the well plate with CD3 antibody, adding 4-6. mu.g/ml CD3 antibody to the well plate, and incubating at 36-38 ℃ for 1-3 hours. The CD3 antibody is pre-coated, and the antibody can be contacted with cells more fully and has better activation capability.
In one embodiment, in the co-culturing step, the medium is RPMI-1640 medium, the final concentration of the CD28 antibody is 4-6 μ g/ml, and the final concentration of interleukin 2 is 150-250U/ml. The condition is adopted for co-culture, so that the growth condition of the T cells is better.
In one embodiment, the co-culture step comprises collecting the culture supernatant after 2-4 days of co-culture, and collecting the culture supernatant for testing, wherein the T cells are fully activated and secrete more TNF- α, so that the evaluation is more accurate.
In one embodiment, in the co-culturing step, the ratio of the T lymphocytes to the umbilical cord mesenchymal stem cells is 100: 1-10: 1.
In one embodiment, the evaluation step calculates a comprehensive evaluation Index by the formula of Index (TNF- α) ═ value (ELISA) x 0.5/(1ng/ml) + value (TNF- α + CD3+ cells%). x 0.4/(1%) + value (qPCR) x 0.1
Wherein value (ELISA) represents the secretion of TNF- α detected by ELISA, value (TNF- α + CD3+ cells%) represents the percentage of T cells secreting TNF- α, and value (qPCR) represents the relative value of TNF- α gene expression.
In one embodiment, in the evaluating step, the T cell is judged to secrete TNF- α when Index (TNF- α) is not less than 10.6, and the T cell is judged to secrete TNF- α when Index (TNF- α) is not more than 10.6.
The invention also discloses application of the method for evaluating the influence of the human umbilical cord mesenchymal stem cells on the secretion of TNF- α by the T lymphocytes in establishing a detection model of the influence of the stem cells on the secretion of TNF- α by the T lymphocytes.
In one embodiment, the stem cell assay model for influencing TNF- α secretion from T lymphocytes is applied to scientific research.
Compared with the prior art, the invention has the following beneficial effects:
the method for evaluating the influence of the human umbilical cord mesenchymal stem cells on the T lymphocytes to secrete the TNF- α can comprehensively and comprehensively evaluate the inhibition degree of the umbilical cord mesenchymal stem cells on the T lymphocytes to secrete the TNF- α.
In addition, the method can accurately calculate the proportion of T lymphocytes secreting TNF- α, can calculate the total amount of TNF- α secreted by T cells in a culture environment, combines the result of the expression change of TNF- α genes, comprehensively and efficiently evaluates the function of the mesenchymal stem cells for regulating the T cells to secrete TNF- α according to an index formula, and can better guide the use of the mesenchymal stem cells for treating autoimmune diseases caused by the increase of the content of TNF- α or evaluate the prognosis.
Drawings
FIG. 1 is a flowchart of an evaluation method in an embodiment;
FIG. 2 shows the results of ELISA assay in examples;
FIG. 3 shows the results of flow cytometry in the examples;
FIG. 4 shows the qPCR detection results in the examples.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Raw materials:
CD3 antibody (Invitrogen, cat # 16-0037-85)
CD28 antibody (Invitrogen, cat # 16-0289-85)
Lymphocyte separation (Tianjin sea, cat number LTS1077)
Sorting T lymphocyte magnetic bead (Meitian whirlwind, merchandise number 130-
Umbilical cord mesenchymal stem cells (Guangdong Weitai biotechnology limited)
RPMI-1640 medium (Gibco, cat # C11995500BT)
Examples
A method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes, comprising the following steps:
first, co-culturing.
1. A small well plate pre-coated with CD3 antibody was obtained by adding 200. mu.l of CD3 antibody (CD3 neutralizing antibody, anti-CD3) at 5. mu.g/ml to a 96 well plate and incubating at 37 ℃ for 2 hours. It is understood that the above 96-well plate can be selected from other solid phase materials according to the requirement.
2. Obtaining 10ml of blood donated by healthy people, and diluting the blood with physiological saline to 25 ml; slowly adding 15mlm into a 50ml centrifuge tube, wherein no residue is left on the wall of the centrifuge tube; the diluted peripheral blood was then slowly added to the lymphocyte separation medium, taking care to prevent mixing of the two phases.
3. 50ml centrifuge tubes were placed in a centrifuge (manufacturer: Thermo model: ST16R) at 20 ℃ for 20 minutes at 400g with an acceleration of 7 and a deceleration of 0.
4. After the centrifugation, the tube was slowly removed, the upper layer of the transparent liquid was carefully removed by using a pipette, the white layer was retained, the white layer was carefully aspirated by using a pipette, and Phosphate Buffered Saline (PBS) was added to 50ml, and the mixture was centrifuged at 4 ℃ for 400g for 8 minutes at an ascending rate of 9 and a descending rate of 9.
5. Discarding the supernatant, adding Phosphate Buffer Solution (PBS) to 50ml, centrifuging at 4 deg.C for 8 min at an ascending rate of 9 and a descending rate of 9;
6. discarding the supernatant, dispersing the cells, removing PBS from the 96-well plate, sorting T lymphocytes by magnetic beads according to the conventional method, adding 1X10 to each well5T lymphocytes, as 100: 1. 10:1 and 1:0 adding umbilical cord mesenchymal stem cells, namely adding 1000, 10000 or no umbilical cord Mesenchymal Stem Cells (MSC), and co-culturing the T lymphocytes and the umbilical cord mesenchymal stem cells in a total volume of 200 mul of RPMI-1640 culture medium (containing 10% fetal calf serum). And adding CD28 antibody (neutralizing antibody of CD28, anti-CD28) to the co-culture system to make the final concentration 5 mug/ml, simultaneously adding interleukin 2(IL-2, final concentration 200U/ml), co-culturing for 3 days, collecting culture supernatant, and testing.
The above steps are shown in fig. 1.
And II, detecting.
And (3) taking the culture supernatant, respectively detecting the content of TNF- α by an ELISA method, detecting the capacity of different subgroups of T lymphocytes for secreting TNF- α by flow cytometry, and detecting the expression level of TNF- α by qPCR.
1. And (5) ELISA method results.
The kit (Invitrogen, cat # BMS223HS) was used to detect TNF- α in the culture supernatant, and the results are shown in FIG. 2, wherein, T cell: MSC represents the ratio of T lymphocytes to umbilical cord mesenchymal stem cells, 1:0 represents only T cells, 100:1 and 50:1 represent that the T cells and the mesenchymal stem cells are co-cultured in different ratios, and the results in FIG. 2 show that the umbilical cord mesenchymal stem cells inhibit the T lymphocytes from secreting TNF- α.
2. And detecting the result by flow cytometry.
The flow cytometry detection results are shown in FIG. 3, wherein MSC represents the ratio of T lymphocytes to umbilical cord mesenchymal stem cells, 1:0 represents only T cells, 100:1 and 50:1 represent that T cells and mesenchymal stem cells are co-cultured according to different ratios, and the graphical results show that about 23.7% of T cells secrete TNF- α, and after co-culture with mesenchymal stem cells, the ratio of T cells secreting TNF- α is reduced to 16.6% and 9.3%, respectively.
3. And (5) qPCR detection results.
The qPCR detection result is shown in figure 4, wherein the ratio of the T lymphocyte to the umbilical cord mesenchymal stem cell is shown by the T cell: MSC, only the T cell is shown by 1:0, the T cell and the mesenchymal stem cell are co-cultured according to different ratios by 100:1 and 50:1, and the graphical result shows that the expression of the T lymphocyte TNF- α gene in the co-cultured group is obviously weakened compared with the simple T cell group.
And thirdly, evaluating.
The results of ELISA, flow and qPCR were combined and calculated as follows:
the TNF- α indexes of different groups are calculated by combining index (1:0) with 12.17, index (100:1) with 8.56 and index (50:1) with 4.87, and the TNF- α indexes of different groups are compared together, so that the mesenchymal stem cells remarkably inhibit the T cells from secreting TNF- α.
In the above formula, considering that ELISA directly detects TNF- α in solution, despite interference of TNF- α secreted by NK cells or NKT cells, the proportion of T cells sorted by magnetic beads can reach more than 90%, so the weight is set to be 0.5 for ELISA, flow cytometry can accurately detect the proportion of T cells secreting TNF- α, but due to the limitation of the detection principle, only the proportion of T cells secreting TNF- α within a few hours of adding a Golgi transporter inhibitor can be detected, so the weight is set to be 0.4 for flow cytometry, and considering that the result of real-time fluorescence quantitative PCR is a reference value relative to an internal reference gene, the weight is set to be 0.1 for qPCR.
Through the Index (TNF- α), the function of the mesenchymal stem cells for regulating the secretion of TNF- α by T cells can be comprehensively evaluated, and the autoimmune diseases caused by the increase of the content of TNF- α can be better guided by using the mesenchymal stem cells for treating the mesenchymal stem cells, the invention has the advantages that through the accumulation of a large amount of experimental data and evaluation after analysis, when the Index (TNF- α) is more than or equal to 10.6, the T cells are indicated to highly secrete TNF- α, when the Index (TNF- α) is less than 10.6, the T cells are indicated to lowly secrete TNF- α, when the Index (TNF- α) value of the T cells in a disease model is higher than 10.6, the T cells are indicated to highly secrete TNF- α, and after the mesenchymal stem cell treatment, the Index (TNF- α) is lower than 10.6, the stem cells are indicated to inhibit the T cells from secreting TNF- α.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes, which comprises the following steps:
co-culturing: adding T lymphocytes into a small pore plate coated with a CD3 antibody in advance, adding umbilical cord mesenchymal stem cells, adding a culture medium, co-culturing the T lymphocytes and the umbilical cord mesenchymal stem cells, adding a CD28 antibody and interleukin 2 into a co-culture system, co-culturing for a preset time, collecting culture supernatant, and testing;
detecting the content of TNF- α by ELISA method, detecting the ability of different sub-group T lymphocyte to secrete TNF- α by flow cytometry, and detecting the expression quantity of TNF- α by qPCR;
evaluation: and (4) integrating the three detection results to evaluate the immunoregulatory capacity of the mesenchymal stem cells.
2. The method for evaluating the influence of human umbilical cord mesenchymal stem cells on TNF- α secretion by T lymphocytes according to claim 1, wherein in the co-culturing step, the T lymphocytes are obtained by taking blood, diluting the blood with physiological saline, adding a lymphocyte separation solution, keeping a blood layer on an upper layer, centrifuging, removing an upper transparent liquid, retaining a middle leucocyte layer, sucking a leucocyte layer, adding a phosphate buffer solution, centrifuging, collecting cells, and sorting the T lymphocytes with magnetic beads.
3. The method for evaluating the influence of human umbilical cord mesenchymal stem cells on T lymphocyte secretion of TNF- α according to claim 1, wherein in the co-culturing step, the small well plate is pre-coated with the CD3 antibody by adding 4-6 μ g/ml of the CD3 antibody to the small well plate and incubating at 36-38 ℃ for 1-3 hours.
4. The method for evaluating the influence of human umbilical cord mesenchymal stem cells on T lymphocyte secretion of TNF- α as claimed in claim 1, wherein in the co-culturing step, the culture medium is RPMI-1640 culture medium, the final concentration of the CD28 antibody is 4-6 μ g/ml, and the final concentration of interleukin 2 is 150-250U/ml.
5. The method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes according to claim 1, wherein in the co-culturing step, after co-culturing for 2-4 days, culture supernatant is collected.
6. The method for evaluating the influence of the human umbilical cord mesenchymal stem cells on the secretion of TNF- α by the T lymphocytes, according to claim 1, wherein in the co-culturing step, the ratio of the T lymphocytes to the umbilical cord mesenchymal stem cells is 100: 1-10: 1.
7. The method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes according to any one of claims 1 to 6, wherein in the evaluating step, the comprehensive evaluation Index is calculated by the following formula:
Index(TNF-α)＝value(ELISA)×0.5/(1ng/ml)+value(TNF-α+CD3+cells％)×0.4/(1％)+value(qPCR)×0.1
wherein value (ELISA) represents the secretion of TNF- α detected by ELISA, value (TNF- α + CD3+ cells%) represents the percentage of T cells secreting TNF- α, and value (qPCR) represents the relative value of TNF- α gene expression.
8. The method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes according to claim 7, wherein in the evaluating step, when Index (TNF- α) is more than or equal to 10.6, the T cells are judged to highly secrete TNF- α, and when Index (TNF- α) is less than 10.6, the T cells are judged to lowly secrete TNF- α.
9. Use of the method for evaluating the influence of human umbilical cord mesenchymal stem cells on the secretion of TNF- α by T lymphocytes, as claimed in any one of claims 1 to 8, in establishing a detection model of the influence of stem cells on the secretion of TNF- α by T lymphocytes.
10. The use of claim 9, wherein the stem cell assay model for affecting T lymphocyte secretion TNF- α is used in scientific research.
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| CN113584116A (en) * | 2021-08-19 | 2021-11-02 | 深圳科诺医学检验实验室 | Detection method for immune regulation and control function of mesenchymal stem cells |
| CN117607423A (en) * | 2023-10-14 | 2024-02-27 | 武汉中生毓晋生物医药有限责任公司 | Method for detecting concentration of anti-human lymphocyte immunoglobulin active antibody |
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| 吴美: "无血清培养在体内外对间充质干细胞的免疫调节活性的影响", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 陈世优: "免疫调节因子ABIN1抑制HIV-1复制的机制研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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| CN111979186A (en) * | 2020-08-21 | 2020-11-24 | 遵义医科大学附属医院 | A method and application of rapid and efficient expansion of human mesenchymal stem cells in vitro |
| CN111979187A (en) * | 2020-08-21 | 2020-11-24 | 遵义医科大学附属医院 | Method for resisting aging of human mesenchymal stem cells and enhancing dryness characteristics of human mesenchymal stem cells |
| CN111979187B (en) * | 2020-08-21 | 2022-04-08 | 遵义医科大学附属医院 | A method for anti-aging and enhancing stemness characteristics of human mesenchymal stem cells |
| CN111979186B (en) * | 2020-08-21 | 2022-04-08 | 遵义医科大学附属医院 | A method and application of rapid and efficient expansion of human mesenchymal stem cells in vitro |
| CN113584116A (en) * | 2021-08-19 | 2021-11-02 | 深圳科诺医学检验实验室 | Detection method for immune regulation and control function of mesenchymal stem cells |
| CN117607423A (en) * | 2023-10-14 | 2024-02-27 | 武汉中生毓晋生物医药有限责任公司 | Method for detecting concentration of anti-human lymphocyte immunoglobulin active antibody |
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