CN113512528B

CN113512528B - Application and method for inducing human mBreg cells in vitro by miR-29a-3p inhibitor

Info

Publication number: CN113512528B
Application number: CN202110444251.9A
Authority: CN
Inventors: 夏永祥; 李近阳
Original assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Current assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2022-04-22
Anticipated expiration: 2041-04-23
Also published as: CN113512528A

Abstract

The invention provides application and a method of miR-29a-3p inhibitor for inducing human mBreg cells in vitro, wherein miR-29a-3p inhibitor is adopted to induce human mBreg cells in vitro, and further miR-29a-3p inhibitortor induced CD19⁺B cells were transformed into mBreg cells, which were CD19⁺CD24^hiCD27⁺Breg cell subpopulation; the method for inducing the mBreg cells of the human body in vitro by using the miR-29a-3p inhibitor has the advantages of high purity, small damage to cell activity, high transformation efficiency, high cell activity, wide source and low pollution possibility, and can make up for the defects of insufficient quantity in the conventional mBreg basis and clinical research.

Description

Application and method for inducing human mBreg cells in vitro by miR-29a-3p inhibitor

Technical Field

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

Background

At present, Breg is proved to play an important role in the occurrence of acute and chronic rejection of transplantation in both animal models and clinical patients, the detection of Breg can predict the risk of acute and chronic rejection of postoperative patients, and adoptive Breg infusion is also found to relieve rejection reaction in animal experiments. mBreg cells can inhibit the proliferation and suppression of T cells by secreting IL-10 and by cell-cell contact involving CTLA-4, thereby providing protection and maintaining the immune balance of the body. However, Breg has a very small amount in human body, the survival time in vitro is short, and the Breg cannot be effectively induced by common B cells, so the current experiment is mostly limited to an animal model, and the current Breg adoptive feedback experiment also has great limitation. Therefore, further intensive research on Breg cells is required to establish a highly efficient cell isolation and induction method.

Breg is mainly characterized by the production and secretion of cytokines such as interleukin IL-10 and/or transforming growth factor TGF-beta, and thus plays a role in inhibiting the negative regulation of inflammatory response, but there is no recognized phenotype of Breg cells, among which CD19⁺CD24^hiCD27⁺Is currently the most widely studied Breg, and this population of cells is significantly reduced in the case of chronic rejection in liver transplant patients, and can be used to predict the occurrence of acute rejection after liver transplantation. At present, the Breg is induced mainly by BAFF (B-cell activating factor) in the world, but the induction purity of the technology is only about 10-30%, the obtained amount is small, and the requirements of further scientific research and clinical research cannot be met.

MicroRNAs are small, non-coding RNAs that base pair with the 3' -untranslated region of a target geneInhibits its translation or degrades its mRNA thereby down-regulating the expression level of the target protein. Micrornas regulate many physiological and disease processes by affecting the expression of target genes. Research has now confirmed that MicroRNA plays a key role in the growth and differentiation of immune cells and is further involved in the development of acute rejection after liver transplantation. We hope to promote B cell transfer to CD19 by regulating miR-29a-3p expression in B cells⁺CD24^hiCD27⁺Breg cells differentiate to meet the needs of clinical research and therapy.

Cell therapy is a treatment method which is expected to achieve certain curative effect in the fields of autoimmune diseases, organ rejection and the like at present, adoptive infusion of Breg is a new branch of cell therapy recently, but the main problems which always limit the clinical application of Breg cells are that the sources of the Breg cells are few, the current in vitro induction efficiency is low and the activity of the Breg cells is low.

Disclosure of Invention

In order to solve the technical problems of low sources of Breg cells, low in-vitro induction efficiency and low activity of the Breg cells, the invention provides application of miR-29a-3p inhibitor in-vitro induction of human Breg cells. miR-29a-3p promotes the degradation of NFAT5 protein by binding to the 3' UTR region of NFAT5 target gene, the NFAT5 signal pathway is an important pathway of immune cells in vivo and plays an important role in immune homeostasis, and the inventor accidentally finds that miR-29a-3p inhibitor can be used for inducing the transformation of common B cells to mBreg cells in experiments, thereby providing a new method for basic and clinical mBreg related research of human bodies.

Therefore, the applicant provides the application of the miR-29a-3p inhibitor in-vitro induction of the human mBreg cells and a method for in-vitro induction of the human mBreg cells by using the miR-29a-3p inhibitor.

Further selection of the invention induces CD19⁺CD24^hiCD27⁺B cells were treated as mwreg.

The invention aims to provide application of miR-29a-3p inhibitor in-vitro induction of human Breg cells, aiming at the defects of the existing Breg induction and separation technology. And a novel method for inducing Breg cells, which meets experimental conditions, has the advantages of simple operation, short time consumption and high transformation efficiency, and can make up for the problem of insufficient quantity in the current Breg basis and clinical research.

Further, the invention also provides miR-29a-3p inhibitor induced CD19⁺Use of a B cell transformed into a mBreg cell.

Further, the common B cell was induced to be CD19⁺CD24^hiCD27⁺Breg cell subpopulation.

miR-29a-3p inhibitor induces human mBreg cells in vitro, and miR-29a-3p inhibitor induces CD19⁺In the effect of converting B cells into Breg cells, miR-29a-3p inhibitor is adopted to induce the Breg cells to have high induction purity, secretion of Breg proinflammatory inflammatory cytokines is reduced after induction, and the Breg inhibition function is enhanced after induction.

Further, the CD19⁺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) into each 30ml of sample tube wall, and not mixing the two solutions uniformly; decelerating at 20 ℃ and 2300rpm for 1, centrifuging for 23 minutes, and taking a cell cloud layer between the plasma and the Ficoll liquid to obtain a mononuclear lymphocyte suspension;

the second step is that: centrifuging the mononuclear lymphocyte suspension obtained in the first step at 20 ℃ and 1500rpm for 5 minutes, adding and washing twice, discarding the supernatant, counting the number of cells by using a hemocytometer at 25ul/10⁷Adding PBS to resuspend the cells; adding 5ul/10⁷The CD19+ Microbeads was incubated at room temperature for 30 minutes, washed once with PBS, resuspended in 4ml of AUTOMACS BUFFER, the AUTOMACS machine was started, the positive selection program was set, and CD19 positive cells were obtained by sorting.

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

the first step is as follows: obtaining mononuclear lymphocytes, obtaining 50-80 ml of concentrated human peripheral blood mononuclear lymphocytes by using a blood cell apheresis instrument, mixing with PBS 1: 1, mixing in equal proportion, and slowly adding 15ml of mononuclear lymphocyte separation solution (Ficoll) into each 30ml portion along the wall of a test tube; decelerating at 20 ℃ and 2300rpm for 1 minute, centrifuging for 23 minutes, and taking the middle cloud layer of the cell suspension to obtain the mononuclear lymphocyte suspension;

the second step is that: acquiring CD19⁺B cells, the monocyte suspension obtained in the first step, were centrifuged at 1500rpm at 20 ℃ for 5 minutes, washed twice, and the supernatant was discarded to 25ul/10⁷Adding physiological saline to resuspend the cells; adding 5ul/10⁷The CD19 Microbeads are incubated for 30 minutes at room temperature, washed once by physiological saline, resuspended by 4ml of AUTOMACS BUFFER, an AUTOMACS machine is started, a positive selection program is set, and CD19 positive cells are obtained by sorting;

the third step: induction of regulatory B cells (Breg); CD19 obtained in the third step⁺Cells were centrifuged at 20 ℃ and 1500rpm for 5 minutes, washed twice with additions, the supernatant discarded and the cell pellet washed at 0.5 x 10⁶The cell concentration of/ml is resuspended by using a culture medium, and B cell activating factor (BAFF) and/or miR-29a-3p inhibitor are added for culture;

the fourth step: purity and function test, collecting 10 days after 3 days⁶The cells are resuspended in 100 microliters of PBS, CD19 FITC, CD24 PERPCY5.5 and CD27 APC flow antibody are added, the cells are incubated for 30-45 minutes at 4 ℃ in the dark, the cells are washed twice by PBS, and the purity of the cells is detected by a flow cytometer.

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

screening healthy volunteers and obtaining blood from the healthy volunteers;

concentrating the obtained blood into a cell blood sample by using a blood cell apheresis instrument;

adopting Ficoll lymphocyte separation liquid to obtain mononuclear lymphocytes;

magnetic bead sorting separation of CD19⁺And (4) carrying out a step of common B cells.

Preferably, the step of obtaining mononuclear lymphocytes by using the Ficoll lymphocyte separation solution comprises the steps of taking blood, PBS and the Ficoll lymphocyte separation solution (1: 1: 1), fully mixing the blood and the PBS, and slowly adding the mixture to the Ficoll liquid level along the wall of the test tube to avoid uniformly mixing the blood, the PBS and the Ficoll lymphocyte separation solution. Centrifuging at 37 deg.C and 2300rpm for 23 min, and collecting the intermediate nebula cell layer to obtain mononuclear lymphocytes.

CD19 sorted and induced according to the method of the invention⁺CD24^hiCD27⁺The Breg cell subset is more consistent with the functional state of Breg cells when the body is in immune homeostasis, which cannot be achieved by the traditional BAFF induction method.

In order to obtain better effect of inducing human Breg cells, the applicant of the present invention proposed a method for separating CD19 by using magnetic bead sorter (AUTOMACS)⁺B cells and a method for inducing the cells into mBreg cells by adding human miR-29a-3p inhibitor in vitro can make up for the defect of insufficient quantity in the current Breg basis and clinical research. CD19 sorted and induced according to the method of the invention⁺CD24^hiCD27⁺The mBreg cell subset is more consistent with the functional state of Breg cells when the body is in immune homeostasis, which cannot be achieved by the traditional BAFF induction method. Breg cells are isolated and induced using the method of the invention, with the following advantages:

1. the source is wide. The magnetic bead is used for sorting and separating common B cells and inducing the common B cells, the base number of the common B cells is large, and the obtained Breg cells are directly compared with CD19 sorted from human bodies⁺CD24^hiCD27⁺The absolute number of mBreg cell subsets has obvious advantages;

2. the cell activity is high. The immune magnetic bead sorting process has high efficiency, can maintain the cell activity to the maximum extent, thereby having longer survival time in vitro;

3. the pollution possibility is low;

4. is obviously superior to the prior BAFF induction method in purity and function. The successful establishment of the cell separation and induction technology provides a new experimental method for further functional research of Breg cells.

Drawings

FIG. 1 common CD19⁺B cell acquisition schematic;

FIG. 2 Pre-Induction CD19⁺CD24^hiCD27⁺A schematic cell ratio diagram;

FIG. 3 general B cell induction purity profile;

FIG. 4 is a graph showing the reduction in Breg pro-inflammatory cytokine secretion following induction;

FIG. 5 is a graph showing enhanced Breg inhibitory function after induction;

FIG. 6 is a schematic flow diagram of the present invention.

Detailed Description

Example 1:

application of miR-29a-3p inhibitor in-vitro induction of mBreg cells of a human body.

Further, miR-29a-3p inhibitor induces CD19⁺Use of a B cell transformed into a mBreg cell.

In the effect that miR-29a-3p inhibitor induces mBreg cells of a human body in vitro, and miR-29a-3p inhibitor induces common B cells to be transformed into mBreg cells, the induction purity of the Breg cells induced by miR-29a-3p inhibitor is shown in figure 3, the secretion of Breg inflammatory cytokines after induction is reduced in figure 4, and the inhibition function of Breg after induction is enhanced in figure 5.

As shown in fig. 6, a method of isolating and inducing Breg cells comprising the steps of:

the first step is as follows: obtaining mononuclear lymphocytes, and obtaining 50-80 ml of concentrated human peripheral blood mononuclear lymphocytes by using a blood cell apheresis instrument, wherein the volume of the concentrated human peripheral blood mononuclear lymphocytes is 1: 1, mixing according to a proportion, slowly adding 15ml of mononuclear lymphocyte separation liquid (Ficoll) into each 30ml of sample tube wall; centrifuging at 20 ℃ and 2300rpm for 23 minutes, and taking the intermediate cloud layer of the cell suspension to obtain the mononuclear lymphocyte suspension;

the second step is that: obtaining CD19+ B cells, centrifuging the mononuclear lymphocyte suspension obtained in the first step at 20 ℃ and 1500rpm for 5 minutes, adding and washing twice, discarding the supernatant, and adding 25ul/10⁷Adding physiological saline to resuspend the cells; adding 5ul/10⁷The CD19 Microbeads was incubated at room temperature for 30 minutes, washed once with physiological saline, resuspended in 4ml of AUTOMACS BUFFER, the AUTOMACS machine was started, the positive selection program was set, and CD19 was obtained by sorting⁺A cell;

the third step: induction of regulatory BCells (Breg); centrifuging the CD19 positive cells obtained in the third step at 20 deg.C and 1500rpm for 5 min, adding the mixture to wash twice, discarding the supernatant, and adding 0.5 × 10⁶The cell concentration of/ml is resuspended by using a culture medium, and B cell activating factor (BAFF) and/or miR-29a-3p inhibitor are added for culture;

the fourth step: purity and function detection, collecting 10 after culturing for 3 days⁶The cells are resuspended in 100 microliters of PBS, CD19 FITC, CD24 PERPCY5.5 and CD27 APC flow antibody are added, the cells are incubated for 30-45 minutes at 4 ℃ in the dark, the cells are washed twice by PBS, and the purity of the cells is detected by a flow cytometer.

Further preferably, the CD19 is sorted out by immunomagnetic bead sorting⁺CD24^hiCD27⁺Breg cell subpopulation.

FIG. 1 is a schematic of CD19+ B cell acquisition: as shown in fig. 1, volunteers (5) were recruited, and after signing an informed consent, peripheral blood mononuclear cell specimens of the volunteers were collected by a hematology specialist using a blood cell apheresis instrument. After obtaining the sample, PBMC is separated by density gradient centrifugation using lymphocyte separation liquid. The cell surface is incubated with CD19 magnetic beads, and then the CD19 positive B cells are sorted and purified by an automatic MACS immunomagnetic bead cell sorter and detected by a flow cytometer.

screening healthy volunteers and obtaining blood from the healthy volunteers;

adopting Ficoll lymphocyte separation liquid to obtain mononuclear lymphocytes;

Preferably, the step of obtaining mononuclear lymphocytes by using the Ficoll lymphocyte separation solution comprises the steps of taking blood, PBS and the Ficoll lymphocyte separation solution (1: 1: 1), fully mixing the blood and the PBS, and slowly adding the mixture to the surface of the Ficoll liquid along the wall of the test tube. Centrifuging at 37 deg.C and 2300rpm for 23 min, and collecting the intermediate nebula cell layer to obtain mononuclear lymphocytes.

FIG. 2 is a schematic diagram showing the proportion of CD24+ CD27+ cells before induction, and CD19+ B cells sorted out by an automatic MACS magnetic bead cell sorter were detected by a flow cytometer. The cell surface is stained with flow antibodies of CD19, CD24 and CD 27. The CD19+ B cells were circled by flow cytometry analysis and the population was further analyzed for the proportion of cells that were double positive for CD24 and CD27, as can be seen in FIG. 2, the proportion of CD24hi CD27+ cells before induction. As shown in fig. 2, is about 5.58%.

Example 2:

1 materials of the experiment

1.1 sample acquisition

Blood samples were obtained from healthy volunteers.

1.2 reagents and laboratory Equipment

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

culture medium (RPMI 1640, 10% FBS, 100mg/ml streptomycin, 10000U/ml penicilin, Gibco);

AUTOMACS（MACS）；

flow cytometer Flow cytometry (BD);

cell culture incubator (Thermo);

microscope (Zeiss Axiovert).

2 method

2.1 acquisition of peripheral blood mononuclear lymphocytes

As shown in FIG. 1, mononuclear cells were isolated from human (healthy volunteers) by a blood cell sampling apparatus (blood cell sampling machine) at 2X 10⁹One (about 80ml blood). Taking blood, PBS and Ficoll lymphocyte separation fluid (1: 1: 1), fully mixing the blood and the PBS, slowly adding the mixture to the surface of the Ficoll liquid along the wall of the test tube, and preventing the mixture from being mixed uniformly. Decelerating at 37 deg.C and 2300rpm for 1 min, centrifuging for 23 min, and collecting the intermediate cloud cell layer, i.e. mononuclear lymphocytes.

2.2 magnetic bead sorting separation of CD19⁺Normal B cells.

According to the cell count results, CD19 Microbeads were added at the corresponding dose for 30 minutes at room temperature.Obtaining CD19 by positive selection of an AUTOMACS magnetic bead sorter⁺Normal B cells, PBS washed once.

2.3 Induction of regulatory B cells.

At 0.5 x 10⁶The cell concentration per ml was resuspended in culture medium and cultured for 3 days by adding B cell activating factor (BAFF) or miR-29a-3p inhibitor.

2.4 purity detection.

After 3 days 10⁶The cells are resuspended in 100 microliters of PBS, CD19 FITC, CD24 PERPCY5.5 and CD27 APC flow antibody are added, the cells are incubated for 30-45 minutes at 4 ℃ in the dark, the cells are washed twice by PBS, the purity of the cells is detected by a flow cytometer, and the miR-29a-3p inhibitor induced group purity is obviously increased (see figure 3).

FIG. 3 is a graph of mBreg cell induction purity: the CD19+ B cells sorted by autoMACS were artificially induced in vitro to differentiate into CD24+ CD27+ cells. An untreated group, a B cell activating factor (BAFF) group and a miR-29a-3p inhibitor group are respectively arranged. 3 days after the addition of the corresponding reagents, the proportion of CD24+ CD27+ cells in the CD19+ B cells of each group of samples was measured by flow cytometry, respectively. The results show that the miR-29a-3p inhibitor can remarkably improve the proportion of CD24+ CD27+ cells (a control group, a BAFF group, an inhibitor group and a cell growth inhibitor group are respectively 4.43, 13.7 and 23.1 percent).

2.5 cytokine and inhibitory Capacity assays

The cells obtained by the above method were examined for inflammatory cytokines (IFN-. alpha.1, IFN-. beta.1) after 3 days, and their secretion levels were significantly decreased (see FIG. 4).

FIG. 4 is a graph showing the reduction in Breg pro-inflammatory cytokine secretion after induction, h is the number of hours in culture, none is the control group, BAFF is the B cell activator group, inhibitor is the miR-29a-3p inhibitor group: breg cells from different treatment groups were tested for inflammatory factor expression at 24 hours and 72 hours post-treatment, respectively. IFN-alpha 1 and IFN-beta 1 are both types of Interferon (IFN) families, and are involved in the development of an inflammatory environment. Three groups were found to have no differences in IFN secretion early after treatment (24 h) by flow assay. However, in the latter treatment period (72 h), the IFN secretion amount of the inhibitor group was significantly smaller than that of the control group and BAFF group.

The Breg cells are co-cultured with effector CD8+ T cells in a certain proportion (1: 2, 1:4, 1:8, B cells: T cells), the proliferation level of the T cells is detected after 4 days, and the Breg cells obtained by magnetic bead sorting and a method of adding miR-29a-3p inhibitor are found to have a strong function of inhibiting the proliferation of the T cells (see figure 5).

FIG. 5 is a graph showing enhanced Breg inhibitory function following induction: the separated and purified CD19+ B cells are artificially induced in vitro and are respectively treated by B cell activating factor (BAFF) and miR-29a-3p inhibitor. After 3 days of treatment, B cells and effector T cells from each group were treated with Breg: t cell = 1:2, 1:4 and 1:8 in proportion. After 4 days of co-culture, the proliferation of effector T cells was examined by CFSE assay using flow cytometry. The results show that the Breg inhibition capability treated by the miR-29a-3p inhibitor is enhanced. The inhibition efficiency of the inhibitor group is 65%, 58% and 52% respectively at the ratio of 1:2, 1:4 and 1: 8. While the control and BAFF groups were 56%, 45%, 37% and 50%, 35%, 24%, respectively.

In addition to the above, other embodiments of the present invention are possible. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Claims

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. 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. 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.