CN113512528B - Use and method of miR-29a-3p inhibitor to induce human mBreg cells in vitro - Google Patents

Abstract

The present invention proposes a use and method for miR-29a-3p inhibitor to induce human mBreg cells in vitro, using miR-29a-3p inhibitor to induce human mBreg cells in vitro, and further, miR-29a-3p inhibitor to induce CD19 ⁺ B cells Transform into mBreg cells, and mBreg cells are CD19 ⁺ CD24 ^hi CD27 ⁺ Breg cell subsets; the method of using miR‑29a‑3p inhibitor to induce human mBreg cells in vitro has high purity, little damage to cell activity, high transformation efficiency, and cell viability It can make up for the shortage of the current mBreg basic and clinical research.

Description

Use and method of miR-29a-3p inhibitor to induce human mBreg cells in vitro

technical field

The present invention relates to the field of separation, purification and induction of cells, in particular to the use of human miR-29a-3p inhibitor to induce human mBreg cells in vitro and a method for separation and induction of mBreg cells.

Background technique

At present, it has been confirmed in animal models and clinical patients that Breg plays an important role in the occurrence of acute and chronic transplant rejection. Detection of Breg can not only predict the risk of acute and chronic rejection in postoperative patients, but also found in animal experiments that adoptive transplantation Injection of Breg can alleviate rejection. mBreg cells can inhibit the proliferation and suppressive effects of T cells by secreting IL-10 and cell-to-cell contact involving CTLA-4, thereby providing protection and maintaining the body's immune balance. However, due to the very small number of Breg in the human body, the survival time in vitro is short, and it cannot be effectively induced by ordinary B cells. The current experiments are mostly limited to animal models, and the current Breg adoptive infusion experiments also have great limitations. Therefore, to further study Breg cells, we must first establish an efficient cell isolation and induction method.

The main feature of Breg is the production and secretion of cytokines such as interleukin IL-10 and/or transforming growth factor TGF-β, which play a negative regulatory role in inhibiting inflammatory responses, but there is no recognized expression of Breg cells. Among them, CD19 ⁺ CD24 ^hi CD27 ⁺ is the most widely studied Breg, and this group of cells is significantly reduced in the chronic rejection of liver transplant patients, which can be used to predict the occurrence of acute rejection after liver transplantation. At present, the way to induce Breg in the world mainly induces ordinary B cells through BAFF (B-cell activating factor), and the induction purity of this technology is only about 10-30%, and the amount obtained is small, which cannot meet the needs of further scientific research and clinical research. need.

MicroRNAs are a class of small non-coding RNAs that down-regulate the expression levels of target proteins by inhibiting their translation or degrading their mRNAs by complementary binding to the 3'-untranslated region of target genes. MicroRNAs regulate many physiological and disease processes by affecting the expression of target genes. At present, studies have confirmed that MicroRNA plays a key role in the growth and differentiation of immune cells, and is further involved in the occurrence of acute rejection after liver transplantation. We hope to promote the differentiation of B cells into CD19 ⁺ CD24 ^hi CD27 ⁺ Breg cells by regulating the expression of miR-29a-3p in B cells to meet the needs of clinical research and treatment.

Cell therapy is currently a promising treatment method in the fields of autoimmune diseases and organ rejection. Recently, the adoptive infusion of Breg has become a new branch of cell therapy, but the main problem that has always limited the clinical application of Breg cells is the source of Breg cells. Relatively few, the current in vitro induction efficiency is low and its activity is also low.

SUMMARY OF THE INVENTION

In order to solve the technical problems of few sources of Breg cells, low in vitro induction efficiency and low activity, the present invention proposes the use of a miR-29a-3p inhibitor to induce human Breg cells in vitro. miR-29a-3p promotes the degradation of NFAT5 protein by binding to the 3'UTR region of NFAT5 target genes. The NFAT5 signaling pathway is an important pathway of immune cells in vivo and plays an important role in immune homeostasis. The inventors accidentally discovered in the experiment The miR-29a-3 pinhibitor can be used to induce the transformation of ordinary B cells into mBreg cells, thereby providing a new method for basic and clinical human mBreg related research.

Therefore, the applicant proposes a use of miR-29a-3p inhibitor to induce human mBreg cells in vitro, and a method for using miR-29a-3p inhibitor to induce human mBreg cells in vitro.

Further, the present invention selects induced CD19 ⁺ CD24 ^hi CD27 ⁺ B cells as mBreg.

The purpose of the present invention is to propose the use of miR-29a-3 pinhibitor to induce human Breg cells in vitro, aiming at the deficiencies of the existing Breg induction and isolation technology. And a new method for inducing Breg cells, which meets the experimental conditions, is simple to operate, takes a short time, and has high transformation efficiency, which can make up for the current shortage of Breg basic and clinical research problems.

Further, the present invention also proposes the use of miR-29a-3p inhibitor to induce the transformation of CD19 ⁺ B cells into mBreg cells.

Further, common B cells were induced to be CD19 ⁺ CD24 ^hi CD27 ⁺ Breg cell subsets.

In the effect of miR-29a-3p inhibitor in inducing human mBreg cells in vitro, and in the effect of miR-29a-3p inhibitor in inducing the transformation of CD19 ⁺ B cells into Breg cells, using miR-29a-3p inhibitor to induce Breg cells to induce high purity, Breg cells after induction The secretion of pro-inflammatory cytokines was decreased, and the inhibitory function of Breg was enhanced after induction.

Further, the CD19 ⁺ B cells are sorted by the following steps:

Step 1: Obtain peripheral blood mononuclear lymphocytes, use a hemocytometer to obtain 50~80ml of concentrated human peripheral blood mononuclear cells, mix with normal saline or PBS in an equal ratio of 1:1, and slowly add each 30ml portion along the wall of the test tube. 15ml mononuclear lymphocyte separation solution (Ficoll), do not mix the two; 20°C, 2300rpm, decelerate 1, centrifuge for 23 minutes, take the cell cloud layer between the plasma and Ficoll solution to obtain a mononuclear lymphocyte suspension;

The second step: centrifuge the mononuclear lymphocyte suspension obtained in the first step for 5 minutes at 20°C and 1500rpm, add and wash twice, discard the supernatant, count the number of cells using a hemocytometer, and add PBS at 25ul/10 ⁷ Resuspend the cells; add 5ul/10 ⁷ CD19+ Microbeads, incubate at room temperature for 30 minutes, wash once with PBS, resuspend with 4ml AUTOMACS BUFFER, start the AUTOMACS machine, set the positive selection program, and sort to obtain CD19 positive cells.

The present invention also provides a method for separating and inducing Breg cells using miR-29a-3p inhibitor, comprising the following steps:

Step 1: Obtain mononuclear lymphocytes, use a hemocytometer to obtain 50~80ml of concentrated human peripheral blood mononuclear lymphocytes, mix with PBS in an equal ratio of 1:1, and slowly add 15ml of mononuclear cells along the wall of the test tube for every 30ml Lymphocyte separation solution (Ficoll); 20°C, 2300rpm, deceleration 1, centrifugation for 23 minutes, take the middle cloud layer of the cell suspension to obtain a mononuclear lymphocyte suspension;

The second step: to obtain CD19 ⁺ B cells, the mononuclear lymphocyte suspension obtained in the first step was centrifuged at 20°C and 1500rpm for 5 minutes, added and washed twice, the supernatant was discarded, and the cells were resuspended by adding 25ul/10 ⁷ of normal saline; Add 5ul/10 ⁷ CD19 Microbeads, incubate at room temperature for 30 minutes, wash once with normal saline and resuspend with 4 ml AUTOMACS BUFFER, start the AUTOMACS machine, set the positive selection program, and sort to obtain CD19 positive cells;

The third step: Induction of regulatory B cells (Breg); the CD19 ⁺ cells obtained in the third step were centrifuged at 20°C and 1500 rpm for 5 minutes, added and washed twice, the supernatant was discarded, and the cells were cultured at a cell concentration of 0.5*10 ⁶ /ml. Base resuspended, add B cell activating factor (BAFF) and/or miR-29a-3p inhibitor for culture;

Step 4: Purity and function test, collect 10 ⁶ cells after 3 days, resuspend in 100 μl PBS, add CD19FITC, CD24 PERPCY5.5, CD27 APC flow antibody, incubate at 4°C for 30-45 minutes in the dark, PBS After washing twice, the cell purity was checked by flow cytometry.

Further preferably, the step of obtaining the mononuclear lymphocytes comprises:

Screening healthy volunteers and obtaining blood from healthy volunteers;

The steps of concentrating the obtained blood cell blood sample with a hemocytometer;

The steps of using Ficoll lymphocyte separation medium to obtain mononuclear lymphocytes;

Procedure for the isolation of CD19 ⁺ normal B cells by magnetic bead sorting.

Further preferably, using Ficoll lymphocyte separation solution, the step of obtaining mononuclear lymphocytes includes: taking blood, PBS and Ficoll lymphocyte separation solution (1:1:1), fully mixing the blood and PBS, and slowly along the test tube wall. Add to the top of the Ficoll liquid, avoid mixing the two. The cells were centrifuged at 37°C and 2300 rpm for 23 minutes, and the middle cloud cell layer was taken to obtain mononuclear lymphocytes.

The CD19 ⁺ CD24 ^hi CD27 ⁺ Breg cell subsets sorted and induced by the method of the present invention are more in line with the functional state of Breg cells when the body is in immune steady state, which cannot be accomplished by traditional BAFF induction methods.

In order to obtain a better effect of inducing human Breg cells, the applicant of the present invention proposes a method to separate CD19 ⁺ B cells using a magnetic bead sorter (AUTOMACS), and add human miR-29a-3p inhibitor to induce mBreg cells in vitro. This method can make up for the shortage of the current Breg basic and clinical research. The CD19 ⁺ CD24 ^hi CD27 ⁺ mBreg cell subsets sorted and induced according to the method of the present invention are more in line with the functional state of Breg cells when the body is in immune steady state, which cannot be accomplished by traditional BAFF induction methods. Using the method of the present invention to separate and induce Breg cells, its advantages are as follows:

1. Wide source. The use of magnetic bead sorting to separate and induce common B cells has a large base, and the absolute number of Breg cells obtained has obvious advantages compared with the direct sorting of CD19 ⁺ CD24 ^hi CD27 ⁺ mBreg cell subsets from the human body;

2. High cell activity. The immunomagnetic bead sorting process has high efficiency and can maintain cell viability to the greatest extent, so that it can survive longer in vitro;

3. The possibility of pollution is small;

4. It is obviously superior to the current BAFF induction method in terms of purity and function. The successful establishment of this cell isolation and induction technology provides a new experimental method for the further functional study of Breg cells.

Description of drawings

Figure 1 Schematic diagram of the acquisition of ordinary CD19 ⁺ B cells;

Figure 2 Schematic diagram of the proportion of CD19 ⁺ CD24 ^hi CD27 ⁺ cells before induction;

Figure 3 Induction purity chart of common B cells;

Figure 4 shows the reduction of Breg pro-inflammatory cytokine secretion after induction;

Figure 5 shows the enhanced inhibitory function of Breg after induction;

Figure 6 is a schematic flow chart of the present invention.

Detailed ways

Example 1:

Use of miR-29a-3p inhibitor to induce human mBreg cells in vitro.

Further, the use of miR-29a-3p inhibitor to induce the transformation of CD19 ⁺ B cells into mBreg cells.

Further, common B cells were induced to be CD19 ⁺ CD24 ^hi CD27 ⁺ Breg cell subsets.

In the effect of miR-29a-3p inhibitor in inducing human mBreg cells in vitro, and in the effect of miR-29a-3p inhibitor in inducing the transformation of ordinary B cells into mBreg cells, using miR-29a-3p inhibitor to induce Breg cells to induce high purity is shown in Figure 3. After induction Figure 4 shows the decrease in the secretion of Breg inflammatory cytokines, and Figure 5 shows the enhanced inhibitory function of Breg after induction.

As shown in Figure 6, a method for isolating and inducing Breg cells, comprising the following steps:

Step 1: Obtain mononuclear lymphocytes, use a hemocytometer to obtain 50~80ml of concentrated human peripheral blood mononuclear lymphocytes, mix with normal saline in a ratio of 1:1, and slowly add 15ml mononuclear lymphocytes per 30ml portion along the test tube wall. Put on nuclear lymphocyte separation liquid (Ficoll); centrifuge at 20°C and 2300rpm for 23 minutes, and take the middle cloud layer of the cell suspension to obtain a mononuclear lymphocyte suspension;

The second step: to obtain CD19+ B cells, the mononuclear lymphocyte suspension obtained in the first step was centrifuged at 20°C and 1500rpm for 5 minutes, added and washed twice, the supernatant was discarded, and 25ul/10 ⁷ was added to the normal saline to resuspend the cells; 5ul/107 ^{CD19Microbeads} , incubated at room temperature for 30 minutes, washed once with normal saline, resuspended with 4ml AUTOMACS BUFFER, started the AUTOMACS machine, set the positive selection program, and sorted to obtain CD19 ⁺ cells;

The third step: induction of regulatory B cells (Breg); the CD19 positive cells obtained in the third step were centrifuged at 20°C and 1500 rpm for 5 minutes, added and washed twice, discarded the supernatant, and used it at a cell concentration of 0.5*10 ⁶ /ml. The medium was resuspended, and B cell activating factor (BAFF) and/or miR-29a-3p inhibitor were added for culture;

Step 4: Purity and function test, collect 10 ⁶ cells after 3 days of culture, resuspend in 100 μl PBS, add CD19 FITC, CD24 PERPCY5.5, CD27 APC flow antibody, and incubate at 4°C for 30-45 minutes in the dark , washed twice with PBS, and detected cell purity by flow cytometry.

Further preferably, the CD19 ⁺ CD24 ^hi CD27 ⁺ Breg cell subsets are sorted by immunomagnetic bead sorting technology.

Figure 1 is a schematic diagram of the acquisition of CD19+ B cells: as shown in Figure 1, volunteers (5) were recruited, and after signing the informed consent form, the peripheral blood mononuclear cell samples of the volunteers were collected by a hematology professional physician using a hemocytometer. After the samples were obtained, PBMCs were isolated by density gradient centrifugation with lymphocyte separation medium. The cell surface was incubated with CD19 magnetic beads, and then the CD19-positive B cells were sorted and purified by the autoMACS immunomagnetic bead cell sorter, and detected by flow cytometry.

Further preferably, the step of obtaining the mononuclear lymphocytes comprises:

Screening healthy volunteers and obtaining blood from healthy volunteers;

The steps of concentrating the obtained blood cell blood sample with a hemocytometer;

The steps of using Ficoll lymphocyte separation medium to obtain mononuclear lymphocytes;

Procedure for the isolation of CD19 ⁺ normal B cells by magnetic bead sorting.

Further preferably, using Ficoll lymphocyte separation solution, the step of obtaining mononuclear lymphocytes includes: taking blood, PBS and Ficoll lymphocyte separation solution (1:1:1), fully mixing the blood and PBS, and slowly along the test tube wall. Add to the top of the Ficoll liquid. The cells were centrifuged at 37°C and 2300 rpm for 23 minutes, and the middle cloud cell layer was taken to obtain mononuclear lymphocytes.

Figure 2 is a schematic diagram of the proportion of CD24+CD27+ cells before induction. The CD19+ B cells sorted by the autoMACS magnetic bead cell sorter were detected by flow cytometry. Flow-through antibodies to CD19, CD24, and CD27 were stained on the cell surface. Through flow cytometry analysis and detection, CD19+ B cells were circled, and the proportion of CD24 and CD27 double positive cells in this group of cells was further analyzed. It can be seen from Figure 2 that the proportion of CD24hiCD27+ cells before induction. As shown in Figure 2, it accounts for about 5.58%.

Example 2:

1 Experimental material

1.1 Sample acquisition

Obtain blood samples from healthy volunteers.

1.2 Required reagents and experimental equipment

CD19 FITC, CD24 PERPCY5.5, CD27 APC flow antibody (Biolegend);

Medium (RPMI 1640, 10% FBS, 100mg/ml streptomycin, 10000U/mlpenicilin, Gibco);

AUTOMACS(MACS);

Flow cytometer flow cytometer (BD);

Cell incubator (Thermo);

Microscope (Zeiss Axiovert).

2 methods

2.1 Acquisition of peripheral blood mononuclear lymphocytes

As shown in Figure 1, about 2*10 ⁹ mononuclear cells (about 80ml of blood) were isolated from the human body (healthy volunteers) using a hemocytometer (apheresis machine). Take blood, PBS and Ficoll lymphocyte separation solution (1:1:1), mix blood and PBS well and slowly add them to the surface of Ficoll along the wall of the test tube to prevent the two from mixing. 37° C., 2300 rpm, deceleration 1, centrifugation for 23 minutes, and take the middle cloud cell layer, that is, mononuclear lymphocytes.

2.2 Separation of CD19 ⁺ common B cells by magnetic bead sorting.

According to the results of cell counting, add the corresponding dose of CD19 Microbeads for 30 minutes at room temperature. CD19 ⁺ normal B cells were obtained by positive selection by AUTOMACS magnetic bead sorter, and washed once with PBS.

2.3 Induction of regulatory B cells.

The cells were resuspended in culture medium at a concentration of 0.5*10 ⁶ /ml, and cultured for 3 days by adding B cell activating factor (BAFF) or miR-29a-3p inhibitor.

2.4 Purity testing.

After 3 days, 10 ⁶ cells were resuspended in 100 microliters of PBS, CD19 FITC, CD24 PERPCY5.5, CD27APC flow antibodies were added, incubated at 4°C for 30-45 minutes in the dark, washed twice with PBS, and detected by flow cytometry. The purity of the miR-29a-3p inhibitor-induced group was significantly higher (see Figure 3).

Figure 3 is a graph of the induced purity of mBreg cells: CD19+ B cells sorted by autoMACS were artificially induced in vitro to induce differentiation into CD24+CD27+ cells. The untreated group, B cell activating factor (BAFF) group and miR-29a-3p inhibitor group were set respectively. Three days after adding the corresponding reagents, the proportion of CD24+CD27+ cells in the CD19+ B cells of each group of samples was detected by flow cytometry. The results showed that miR-29a-3p inhibitor could significantly increase the proportion of CD24+CD27+ cells (control group; BAFF group; inhibitor group: 4.43; 13.7%; 23.1%, respectively).

2.5 Detection of cytokines and inhibitory ability

The cells isolated by the above method were tested for inflammatory cytokines (IFN-α1, IFN-β1) after 3 days, and their secretion levels were significantly decreased (see Figure 4).

Figure 4 is a graph showing the decrease in the secretion of Breg pro-inflammatory cytokines after induction, h is the number of hours of culture, none is the control group, BAFF is the B cell activator group, and inhibitor is the miR-29a-3p inhibitor group. The expression of inflammatory factors was detected in Breg cells 24 hours and 72 hours after treatment, respectively. Both IFN-α1 and IFN-β1 are two types of the interferon (interferon, IFN) family, and both are involved in the occurrence and development of the inflammatory environment. It was found by flow cytometry that in the early period (24h) after treatment, there was no difference in the secretion of IFN among the three groups. But at the late stage (72h), the secretion of IFN in the inhibitor group was significantly lower than that in the control group and BAFF group.

Co-culture with effector CD8+ T cells at a certain ratio (1:2, 1:4, 1:8, B cell:T cell), and after 4 days, the T cell proliferation level was detected, and it was found that by magnetic bead sorting and adding miR-29a The Breg cells obtained by the -3p inhibitor method have a strong function of inhibiting the proliferation of T cells (see Figure 5).

Figure 5 shows the enhanced inhibitory function of Breg after induction: the isolated and purified CD19+ B cells were artificially induced in vitro and treated with B cell activating factor (BAFF) and miR-29a-3p inhibitor, respectively. After 3 days of treatment, B cells and effector T cells in each group were mixed and co-cultured at the ratio of Breg:T cell=1:2, 1:4, and 1:8, respectively. After 4 days of co-culture, CFSE was used to detect the proliferation of effector T cells by flow cytometry. It was found that the inhibitory ability of Breg treated with miR-29a-3pinhibitor was enhanced. At 1:2, 1:4, and 1:8 ratios, the inhibitory efficiency of the inhibitor group was 65%, 58%, and 52%, respectively. While the control group and BAFF group were 56%, 45%, 37% and 50%, 35%, 24%.

In addition to the above-mentioned implementations, the present invention may also have other implementations. All technical solutions formed by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims (3)

1. Application of miR-29a-3p inhibitor to in-vitro induction of human mBreg cells, wherein miR-29a-3p inhibitor induces CD19⁺Transformation of B cells into mBreg cells, which are CD19⁺CD24^hiCD27⁺Breg cell subpopulation.
2. 2. Use of miR-29a-3p inhibitor according to claim 1 for inducing human mBreg cells in vitro, wherein the CD19 is characterized in that⁺B cells were sorted using the following steps:

   the first step is as follows: obtaining peripheral blood mononuclear lymphocytes, obtaining 50-80 ml of concentrated human peripheral blood mononuclear cells by using a blood cell apheresis instrument, mixing with physiological saline or PBS 1: 1, mixing in equal proportion, slowly adding 15ml of mononuclear lymphocyte separation solution Ficoll along the wall of a test tube by every 30ml, and not mixing the two solutions uniformly; centrifuging at 20 deg.C and 2300rpm for 23 min, and collecting cell cloud layer between blood plasma and mononuclear lymphocyte separation liquid Ficoll to obtain mononuclear lymphocyte suspension;

   the second step is that: the mononuclear cell suspension obtained in the first step was centrifuged at 20 ℃ and 1500rpm for 5 minutes, added and washed twice, the supernatant was discarded, and the number of cells was counted using a hemocytometer at 25. mu.l/10⁷Adding PBS to resuspend the cells; adding 5 mul/10⁷CD19 (1)⁺Microbeads, incubated at room temperature for 30 minutes, washed once with PBS, resuspended in 4ml AUTOMACS BUFFER, the AUTOMACS machine was started, the positive selection program was run, and CD19 positive cells were obtained by sorting.
3. 3. A method for separating and amplifying mBreg cells in vitro by using miR-29a-3p inhibitor is characterized by comprising the following steps: the method comprises the following steps:

   the first step is as follows: and a step of obtaining peripheral blood mononuclear lymphocytes, namely obtaining 50-80 ml of concentrated human peripheral blood mononuclear cells by using a blood cell apheresis instrument, mixing with normal saline or PBS 1: 1, mixing in equal proportion, slowly adding 15ml of mononuclear lymphocyte separation solution Ficoll along the wall of a test tube by every 30ml, and not mixing the two solutions uniformly; centrifuging at 20 deg.C and 2300rpm for 23 min, and collecting cell cloud layer between blood plasma and mononuclear lymphocyte separation solution Ficoll to obtain mononuclear lymphocyte suspension;

   the second step is that: sorting to obtain CD19⁺B cell step, the mononuclear cell suspension obtained in the first step was centrifuged at 20 ℃ and 1500rpm for 5 minutes, added and washed twice, the supernatant was discarded, and the number of cells was counted using a hemocytometer at 25. mu.l/10⁷Adding PBS to resuspend the cells; adding 5 mul/10⁷CD19 (1)⁺Microbeads, incubation for 30 minutes at room temperature, washing with PBS once, then resuspending with 4ml AUTOMACS BUFFER, starting the AUTOMACS machine, setting a positive selection program, and sorting to obtain CD19 positive cells;

   the third step: a step of inducing regulatory B cells; the second step obtained CD19 positive cells, 20 ℃, 1500rpm centrifugal 5 minutes add washing two times, abandon the supernatant, at 0.5 x 10⁶Cell concentration per ml resuspended in culture Medium and mi was addedCulturing R-29a-3p inhibitor;

   the fourth step: procedure for purity and functional testing, 10 days after 3⁶Resuspending the cells in 100 microliters of PBS, adding CD19 FITC, CD24 PERPCY5.5 and CD27 APC flow antibodies, incubating for 30-45 minutes at 4 ℃ in the dark, washing twice with PBS, and detecting the proportion of mBreg cells by using a flow cytometer;

   the mBreg cell is CD19⁺CD24^hiCD27⁺Breg cell subpopulation.

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