CN108753714B - GSK-3 beta inhibitor induces the purposes of human body Breg cell and the method for separation and induction Breg cell in vitro - Google Patents
GSK-3 beta inhibitor induces the purposes of human body Breg cell and the method for separation and induction Breg cell in vitro Download PDFInfo
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Abstract
A kind of GSK-3 beta inhibitor induces the purposes of human body Breg cell in vitro, and the method using the external evoked human body Breg cell of GSK-3 beta inhibitor, human body Breg cell is induced using GSK-3 beta inhibitor in vitro, and GSK-3 beta inhibitor induces common B cell into the effect of Breg cell transformation, using GSK-3 beta inhibitor induction Breg cell induction purity is high, the secretion of Breg inflammatory cytokine is reduced after induction, and Breg inhibits function enhancing after induction.The method that the external evoked human body Breg cell of GSK-3 beta inhibitor is utilized using the present invention, it is easy to operate, it is time-consuming short, purity is high, cell activity damage small, high conversion efficiency, and cell activity is high, source is wide, possibility of pollution is small, can make up for it lazy weight in the current basis Breg and clinical research, provides a kind of new experimental method for the further functional study of Breg cell.
Description
Technical field
The present invention relates to separation and inducing cell field, specifically GSK-3 beta inhibitor induces human body Breg in vitro
The purposes of cell and separation and the method for inducing Breg cell.
Background technique
Confirm that Breg and the acute and chronic repulsion of kidney transplant are closely related in animal model and clinical patients at present,
Breg can not only predict that the risk of acute and chronic repulsion occurs for postoperative patient, also found adoptive infusion Breg in zoopery
It can treat rejection.Meanwhile Breg can also adjust other immunocytes by secreting relevant cell factor, realization is exempted from vivo
The reciprocal effect of epidemic disease reaction maintains immunity of organism balance.But Breg is because quantity is few in human body and can not be effectively by general
Logical B cell induction, experiment at present is confined to animal model, is not possible to effectively carry out people's Breg cell correlative study.To Breg
The further in-depth study of cell it may first have to establish a kind of efficient cell separation and abductive approach.
Breg's is mainly characterized by the generation cell factors such as interleukins IL-10 and (or) transforming growth factor TGF-β,
And then play the role of negative regulation, and but its specific cell phenotype of shortage, wherein CD19+CD24++CD27+It is current study most
For extensive Breg, and this group of cells occur to have significant change when acute and chronic repels in renal transplant recipients, it might even be possible to pre-
Survey the generation of postoperative repulsion.The mode of Breg is induced mainly to pass through BAFF(B-cell activating in the world at present
Factor) common B cell is induced, and the technological guide purity only 20 ~ 30% or so, be unable to satisfy further scientific research and
The needs of clinical research.
Cell therapy is a kind of New Scheme for being expected to give treatment to a variety of difficult diseases at present, the adoptive infusion of nearest Breg
As the new branch of cell therapy, but Breg adopts treatment is that Breg cell origin is fewer for puzzlement always, current body
Outer induced efficiency is low and its is active also low.
Summary of the invention
In view of the above technical problems, the present invention proposes that a kind of GSK-3 beta inhibitor induces the use of human body Breg cell in vitro
On the way.GSK-3 signal beta access is the important access of vivo immunization cell, is played an important role in immune homeostasis, and inventor is trying
Chancing in testing can use GSK-3 beta inhibitor and induces common B cell to Breg cell transformation, thus based on and it is clinical
Human body Breg correlative study provides new method.
Applicant proposed the purposes that a kind of GSK-3 beta inhibitor induces human body Breg cell in vitro, Yi Jili as a result,
With the method for the external evoked human body Breg cell of GSK-3 beta inhibitor.
Further present invention selection induction CD19+CD24++CD27+B cell is as Breg.
The purpose of the present invention is the deficiencies for existing Breg induction and isolation technics, propose GSK-3 beta inhibitor in vitro
Induce the purposes of human body Breg cell.And a kind of method of new separation and induction Breg cell, meet experiment condition, operates
Simply, time-consuming short, high conversion efficiency, can make up for it the problem of lazy weight in the current basis Breg and clinical research.
Further, the present invention also proposes the purposes that GSK-3 beta inhibitor induces common B cell to Breg cell transformation.
Further, inducing common B cell is CD19+CD24++CD27+Breg cell subsets.
GSK-3 beta inhibitor induce in vitro human body Breg cell and GSK-3 beta inhibitor induce common B cell to
In the effect of Breg cell transformation, using GSK-3 beta inhibitor induction Breg cell induction purity is high, Breg inflammatory is thin after induction
Intracellular cytokine secretion is reduced, and Breg inhibits function enhancing after induction.
The method that the present invention also proposes a kind of separation and induction Breg cell, comprising the following steps:
Step 1: obtaining monokaryon lymphocyte, instrument is adopted using haemocyte list and obtains concentration human peripheral monokaryon lymphocyte
50 ~ 80ml is mixed with physiological saline with 1:1 ratio, and slowly 15ml Ficoll monokaryon lymph is added along test tube wall in every 30ml portion
In cell separating liquid；20 DEG C, 2300rpm centrifugation 20 minutes, take cloud and mist layer among cell suspension outstanding to get monokaryon lymphocyte
Liquid；
Step 2: obtaining CD19+B cell, the monokaryon lymphocyte suspension that the first step obtains, 20 DEG C, 1500rpm centrifugation 5
Minute is added and washes twice, and supernatant is abandoned, with 25ul/107Physiological saline is added, cell is resuspended；5ul/10 is added7CD19
Microbeads is incubated at room temperature 30 minutes, and brine is once resuspended with 4mlAUTOMACS BUFFER afterwards, starting
AUTOMACS machine, setting sun select program, and sorting obtains CD19 positive cell；
Step 3: the step of induction modulability B cell Breg；The CD19 positive cell Breg that second step obtains, 20 DEG C,
1500rpm is centrifuged to be added for 5 minutes and wash twice, and supernatant is abandoned, with 0.5*106The cell concentration of/ml is resuspended using culture medium, is added
Enter B cell activation factor BAFF or GSK-3 beta inhibitor SB216763 to be cultivated；
Step 4: purity and Function detection, 10 after 3 days6A cell is resuspended in 100 microlitres of PBS, addition CD19 FITC,
CD24 PE, CD27 APC streaming antibody, 4 DEG C are protected from light incubation 30-45 minutes, and PBS is washed twice, flow cytomery cell
Purity.
It is further preferred that sub-electing CD19 using immunological magnetic bead sorting technology+CD24++CD27+Breg cell subsets.
It is further preferred that the step of obtaining the monokaryon lymphocyte includes:
The step of screening healthy volunteer, blood obtained out of healthy volunteer body；
The step of pachyemia cellular blood sample that will acquire using haemocyte Dan Caiyi；
Using Ficolll lymphocyte separation medium, the step of obtaining monokaryon lymphocyte；
Magnetic bead sorting separates the step of CD19 positive common B cell.
It is further preferred that using Ficolll lymphocyte separation medium, the step of obtaining monokaryon lymphocyte includes taking
Blood, PBS and Ficoll lymphocyte separation medium, blood, PBS and Ficoll lymphocyte separation medium proportion are 1:1:1, by blood
Liquid and PBS are slowly added on Ficoll liquid level along test tube wall after being sufficiently mixed.37 DEG C, 2300rpm centrifugation 20 minutes, take centre
Cloud and mist cellular layer obtains monokaryon lymphocyte.
The CD19 that the method according to the invention is sorted and induced+CD24++CD27+ Breg cell subsets, more meets
The functional status of Breg cell when body is in immune homeostasis, and this is that traditional BAFF abductive approach is impossible.Utilize this
The method of invention separates and induction Breg cell, advantage are as follows:
1, source is wide.Common B cell is separated using magnetic bead sorting and external evoked, the radix that is added GSK-3 beta inhibitor
Greatly, the Breg cell of acquisition is compared and directly sorts CD19 out of human body+CD24++CD27+Breg cell subsets, absolute quantity
It has a clear superiority；
2, cell activity is high.Immunological magnetic bead sorting procedure segment, it is high-efficient, cell activity can be kept to greatest extent；
3, possibility of pollution is small；
4, current BAFF abductive approach is substantially better than in purity and function.The success of the cell separation and inductive technology
It establishes, provides a kind of new experimental method for the further functional study of Breg cell.
The present invention uses immunological magnetic bead sorting, and immunological magnetic bead sorting is a kind of novel cell sorting technology.Magnetic bead combines thin
Target cell is obtained by yin choosing or sun choosing after the specific marker of cellular surface antigen, the aim cell that this method obtains has behaviour
Make the advantages that simple, time-consuming short, with high purity, cell activity damage is small.In order to obtain preferably induction human body Breg cell effect,
Present invention applicant proposes a kind of method for separating using magnetic bead sorting instrument (AUTOMACS) and inducing human body Breg cell, energy
The shortcomings that enough making up lazy weight in the current basis Breg and clinical research.What the method according to the invention was sorted and was induced
CD19+CD24++CD27+Breg cell subsets more meets the functional status of Breg cell when body is in immune homeostasis, and
This is that traditional BAFF abductive approach is impossible.It is as follows using method separation of the invention and induction Breg cell, advantage:
1, source is wide.Common B cell is separated using magnetic bead sorting and is induced, and radix is big, and the Breg cell of acquisition is compared
CD19 is directly sorted out of human body+CD24++CD27+Breg cell subsets, absolute quantity have a clear superiority；
2, cell activity is high.Immunological magnetic bead sorting procedure segment, it is high-efficient, cell activity can be kept to greatest extent；
3, possibility of pollution is small；
4, current BAFF abductive approach is substantially better than in purity and function.The success of the cell separation and inductive technology
It establishes, provides a kind of new experimental method for the further functional study of Breg cell.
Detailed description of the invention
The common B cell of Fig. 1 obtains schematic diagram；
CD24 before Fig. 2 is induced+CD27+Cell proportion schematic diagram；
The common B cell of Fig. 3 induces purity figure；
The secretion of Breg inflammatory cytokine reduces display figure after Fig. 4 induction；
Breg inhibits function enhancing display figure after Fig. 5 induction；
Flow diagram Fig. 6 of the invention.
Specific embodiment
Embodiment 1:
GSK-3 beta inhibitor induces the purposes of human body Breg cell in vitro.
Further, GSK-3 beta inhibitor induces common B cell to the purposes of Breg cell transformation.
Further, inducing common B cell is CD19+CD24++CD27+Breg cell subsets.
GSK-3 beta inhibitor induce in vitro human body Breg cell and GSK-3 beta inhibitor induce common B cell to
In the effect of Breg cell transformation, purity brilliant idea Fig. 3 is induced using GSK-3 beta inhibitor induction Breg cell, Breg is scorching after induction
Property cytokine secretion reduction see Fig. 4, after induction Breg inhibit function enhancing sees Fig. 5.
As shown in fig. 6, a kind of method of separation and induction Breg cell, comprising the following steps:
Step 1: obtaining monokaryon lymphocyte, instrument is adopted using haemocyte list and obtains concentration human peripheral monokaryon lymphocyte
50 ~ 80ml is mixed with physiological saline with 1:1 ratio, and slowly 15ml monokaryon lymphocyte point is added along test tube wall in every 30ml portion
In chaotropic (Ficoll)；20 DEG C, 2300rpm centrifugation 20 minutes, take cell suspension centre cloud and mist layer to get monokaryon lymphocyte
Suspension；
Step 2: obtaining CD19+B cell, the monokaryon lymphocyte suspension that the first step obtains, 20 DEG C, 1500rpm centrifugation 5
Minute is added and washes twice, and supernatant is abandoned, with 25ul/l07Cell is added physiological saline and cell is resuspended；5ul/10 is added7CD19
Microbeads is incubated at room temperature 30 minutes, and brine is once resuspended with 4mlAUTOMACS BUFFER afterwards, starting
AUTOMACS machine, setting sun select program, and sorting obtains CD19 positive cell；
Step 3: the step of induction modulability B cell (Breg)；The CD19 positive cell Breg that second step obtains, 20 DEG C,
1500rpm is centrifuged to be added for 5 minutes and wash twice, and supernatant is abandoned, with 0.5*106The cell concentration of/ml is resuspended using culture medium, is added
Enter B cell activation factor (BAFF) or GSK-3 beta inhibitor SB216763 is cultivated；
Step 4: purity and Function detection, 10 after 3 days6A cell is resuspended in 100 microlitres of PBS, addition CD19 FITC,
CD24 PE, CD27 APC streaming antibody, 4 DEG C are protected from light incubation 30-45 minutes, and PBS is washed twice, flow cytomery cell
Purity.
It is further preferred that sub-electing CD19 using immunological magnetic bead sorting technology+CD24++CD27+ Breg cell subsets.
As shown in Figure 1, it is further preferred that the step of obtaining the monokaryon lymphocyte includes:
The step of screening healthy volunteer, blood obtained out of healthy volunteer body；
The step of pachyemia cellular blood sample that will acquire using haemocyte Dan Caiyi；
Using Ficolll lymphocyte separation medium, the step of obtaining monokaryon lymphocyte；
Magnetic bead sorting separates the step of CD19 positive common B cell.
It is further preferred that using Ficolll lymphocyte separation medium, the step of obtaining monokaryon lymphocyte includes taking
Blood, PBS and Ficoll lymphocyte separation medium (1:1:1), slowly add to along test tube wall after blood is sufficiently mixed with PBS
On Ficoll liquid level.37 DEG C, 2300rpm centrifugation 20 minutes, take intermediate cloud and mist cellular layer, obtain monokaryon lymphocyte.
CD19+CD24 in healthy volunteer's peripheral blood before being induced with flow cytomery++CD27+ It is thin that Breg cell accounts for B
The ratio of born of the same parents is that 3.8 ± 0.7%(Fig. 2 is wherein CD19 in an example volunteer peripheral blood+CD24++CD27+ Breg cell proportion).
Embodiment 2:
1 experimental material
1.1 sample acquisition
Blood sample is obtained from Nanjing Medical University volunteer.
Reagent and experiment equipment needed for 1.2
CD19 FITC, CD24 PE, CD27 APC streaming antibody (Miltenyi Biotec)；
Culture medium (RPMI 1640,10% FBS, 100mg/ml streptomycin, 10000U/ml
Penicilin, Gibco)；
AUTOMACS(MACS)；
Flow cytometer flow cytometer (BD)；
Cell incubator (Thermo)；
Microscope (Zeiss Axiovert).
2 methods
The acquisition of 2.1 peripheral blood mononuclear lymphocytes
As shown in Figure 1, firstly, recruitment volunteer (6) is utilized after signing informed consent form by hematology's specialist
Haemocyte list adopts the peripheral blood mononuclear cells sample of instrument acquisition volunteer.Obtain sample after, using lymphocyte separation medium into
Row centrifuge separation, extracts lymphocyte and monocyte therein.
Specifically, being drenched using the interior monokaryon of haemocyte Dan Caiyi (haemocyte list milling machine) separation human body (6 healthy volunteers)
Bar cell about 2*109A (about 80ml blood).Take blood, PBS and Ficoll lymphocyte separation medium (1:1:1), by blood with
PBS is slowly added on Ficoll liquid level along test tube wall after being sufficiently mixed.37 DEG C, 2300rpm centrifugation 20 minutes, take intermediate cloud and mist
Cellular layer, i.e. monokaryon lymphocyte.
Then, cell surface is caught into CD19 magnetic bead, then by autoMACS immunomagnetic beads cell sorter, by CD19 sun
Property common B cell sort purification, and detected using flow cytometer.
Specifically, being examined using the CD19+B cell that flow cytometer sub-elects autoMACS magnetic cell sorter
It surveys.The streaming antibody of CD19, CD24, CD27 are caught in cell surface.It is detected by flow cytometry analysis, it is thin to iris out CD19+B
Born of the same parents further analyze the cell proportion of the bis- sun of CD24, CD27 in this group of cells, account for about 3.26% or so as shown in Figure 2.
2.2 magnetic bead sortings separate the positive common B cell of CD19.
According to cell counts, it is added corresponding dosage CD19 Microbeads, room temperature 30 minutes.Through AUTOMACS magnetic
The choosing of pearl sorter sun obtains the positive common B cell of CD19, and PBS washed once.
2.3 induction modulability B cells.
With 0.5*106The cell concentration of/ml is resuspended using culture medium, and B cell activation factor (BAFF) or GSK- is added
3 beta inhibitor SB216763 carry out culture 3 days.
Specifically, the CD19+B cell that autoMACS is sub-elected carries out artificial induction in vitro, induce it to CD24+
CD27+ cell differentiation.Untreated fish group, B cell activity factor (BAFF) group, GSK-3 beta inhibitor (SB216763) is respectively set
Group.
2.4 purity detecting.
Corresponding reagent is added after 3 days, detects CD24+ in the CD19+B cell of every group of sample respectively with flow cytometer
The ratio of CD27+ cell.
Specifically, 10 after 3 days6A cell is resuspended in 100 microlitres of PBS, and CD19 FITC, CD24 PE, CD27 is added
APC streaming antibody, 4 DEG C are protected from light incubation 30-45 minutes, and PBS is washed twice, flow cytomery cell purity, the results showed that
GSK-3 beta inhibitor group can be obviously improved the ratio (control group of CD24+CD27+ cell;BAFF group;Inhibitor component is not
5.7%；24.1%；42.6%).GSK-3 beta inhibitor induction group purity obviously increases (see figure 3).
2.5 cell factors and rejection ability detection
The cell got according to the above method is detected inflammatory cytokine (IFN-α 1, IFN-β 1) after 3 days, secretes water
It is flat to be decreased obviously (see figure 4).Certain proportion co-cultures (1:2,1:4,1:8, Breg:T cell) with effector T cell, after 4 days
T cell proliferation level is detected, discovery obtains Breg cell with stronger suppression by the method that magnetic bead sorting and inhibitor induce
Function (see figure 5) processed.
Fig. 4 be induction after Breg inflammatory cytokine secretion reduce display figure (h be cultivate hourage, none is control group,
BAFF is B cell activity factor group, and SB is GSK-3 beta inhibitor group), the Breg cell of different disposal group is 8 small after treatment
When, 72 hours, carry out respectively detection inflammatory factor expression.IFN-α 1 and IFN-β 1 be all interferon (interferon,
IFN) the two types of family have been involved in the occurrence and development of inflammatory environment.It is found by flow cytometer detection, early stage after treatment
(8h), three groups of IFN secrete indifference.But the IFN secretory volume of phase (72h) after treatment, SB group be considerably less than control group and
BAFF group.
Fig. 5 is Breg inhibition function enhancing display figure after induction: by the CD19 of separating-purifying+B cell carries out people in vitro
Work induction, respectively using B cell activity factor (BAFF), GSK-3 beta inhibitor (SB216763) processing.It, will after processing 3 days
For every group of B cell and effector T cell respectively with Breg:T cell=1:2, the ratio of 1:4,1:8 carry out mixing co-cultivation.Training altogether
After supporting 4 days, CFSE detection is carried out using streaming, detects the proliferative conditions of effector T cell.As a result, it has been found that processed through SB
Breg rejection ability is enhanced.In 1:2,1:4, under 1:8 ratio, the inhibition efficiency of SB group is respectively 60%, 45%, 30%.And it is right
It is respectively 42%, 35%, 15% and 52%, 39%, 17% according to group and BAFF group.
In addition to above-mentioned implementation, the present invention can also have other embodiments.It is all to be formed using equivalent substitution or equivalent transformation
Technical solution, fall within the scope of protection required by the present invention.
Claims (2)
1.GSK-3 beta inhibitor induces common B cell to CD19 in vitro+CD24++CD27+The purposes of Breg cell subsets conversion.
2. GSK-3 beta inhibitor according to claim 1 induces common B cell to CD19 in vitro+CD24++CD27+Breg
The purposes of cell subsets conversion, it is characterised in that: CD19 is sub-elected using immunological magnetic bead sorting technology+CD24++CD27+ Breg
Cell subsets.
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