CN105543171A - Method for amplifying regulatory T cells - Google Patents

Abstract

The present invention relates to a method for amplifying regulatory T cells. The method for expanding the regulatory T cells comprises the step of expanding CD4⁺CD25⁺Co-culturing Treg cells and hUC-MSCs by inoculating hUC-MSCs of P3 generation into upper chamber of Transwell chamber at certain density, and culturing CD4⁺CD25⁺Treg cells were seeded into the lower chamber of a Transwell chamber at a certain density, co-cultured for 14 days, and the cells were replenished every 2 days. The invention relates to a CD4⁺CD25⁺Treg cells are co-cultured with hUC-MSCs in vitro, and growth factors secreted by the hUC-MSCs can promote CD4⁺CD25⁺The proliferation of Treg cells enhances the amplification effect. CD4 amplified using the method of the invention⁺CD25⁺Treg cells are prepared into a preparation, and the effect of the reinfusion is good.

Description

The amplification method of regulatory T cells

Technical field

The present invention relates to cell field, particularly relate to a kind of amplification method of regulatory T cells.

Background technology

Regulatory T cells (Regulatorycell is called for short Treg) is the T cell subgroup that a group has immunosuppressive effect.Concerning the immunity system of human body, Treg cell is beyond doubt double-edged sword.It has very important effect to the tolerance of maintenance immunity of organism and immunne response stable state, can suppress strong immunne response by a series of mechanism, makes body effective removing antigen or pathogenic agent when self-inflicted injury is minimum.The quantity of control Treg cell can help us to control the process of disease with expression.Treg cell plays immune suppression function mainly through two kinds of modes.First kind of way is that Treg cell can play immunoregulation effect by the heterogeneous cytokine of secretion etc.; The second way is that Treg cell plays a role with direct contact of cell mainly through cell.

CD4 ⁺cD25 ⁺treg cell, as a class regulatory T cells, in the propagation of autoreactive T cell, maintains immunological tolerance aspect and plays an important role.Immunomagnetic beads two-step approach separation of C D4 is adopted in prior art ⁺cD25 ⁺treg cell, and use CD4 ⁺cD25 ⁺the perfect medium that Treg cell is special and certain density IL-2 cultivate amplification CD4 ⁺cD25 ⁺treg cell.The culture medium cost used in the above prior art is higher, immunological magnetic bead sorting CD4 ⁺cD25 ⁺treg cell needed post, complex operation.And, be separated the CD4 obtained ⁺cD25 ⁺the amplification efficiency of Treg cell is low.Therefore, how simple separation improve CD4 ⁺cD25 ⁺the amplification efficiency of Treg cell is the focus instantly studied.

Summary of the invention

In view of this, be necessary, for above-mentioned problem, to provide a kind of amplification method of regulatory T cells.

To achieve these goals, the present invention adopts following technical scheme:

The amplification method of regulatory T cells, by CD4 ⁺cD25 ⁺treg cell and hUC-MSCs co-cultivation.

Preferably, described CD4 ⁺cD25 ⁺the method of Treg cell and hUC-MSCs co-cultivation is:

HUC-MSCs is inoculated into the upper room of Transwell cell, by CD4 ⁺cD25 ⁺treg cell is inoculated into the lower room of Transwell cell, and co-cultivation 14 days, carried out fluid infusion every 2 days to cell.

Preferably, the density of described hUC-MSCs is 1 × 10 ⁵cells/ml, CD4 ⁺cD25 ⁺the density of Treg cell is 1 × 10 ⁶cells/ml.

Preferably, the substratum used during co-cultivation is X-VIVO15 substratum, wherein adds the Flt3 (Tyrosylprotein kinase 3 of Fms-liketyrosinekinase, FMS sample) of IL-2 and 10-50ng/ml of 200-1000U/ml.

Preferably, CD4 ⁺cD25 ⁺treg cell is prepared by the following method:

Mononuclearcell (PBMC) is separated in S11, employing density gradient centrifugation autoblood;

S12, in PBMC, sub-elect CD4 ⁺cD25 ⁺treg cell.

More preferably, described blood is the blood of Patients with SLE.Patients with SLE, because of Autoantigen Immune tolerance disappearance, causes lymphocyte to activate in a large number producing in a large number with hyperplasia, autoantibody and a large amount of releases of inflammatory mediator.CD4 in Patients with SLE catabasis body ⁺cD25 ⁺to being crossed strong by immunity, autoimmune disorder that is that cause has good result for the treatment of to Treg cell.

More preferably, described step S12 uses EasySep ^tMpeople CD4 ⁺cD25 ⁺t cell sorting test kit sub-elects CD4 in PBMC ⁺cD25 ⁺treg cell.

Preferably, hUC-MSCs is prepared by the following method:

Arteriovenous in S21, removal umbilical cord and amnion; Umbilical cord is cut into fragment; With the II Collagenase Type Digestive system umbilical cord fragment 2-4h of 0.2m/v%, 2000rpm is centrifugal obtains primary hUC-MSCs;

S22, after cultivating primary hUC-MSCs, 24h containing the X-VIVO15 of 5-20ng/mlEGF β, change liquid, remove non-attached cell; Continue to be cultured to hUC-MSCs degree of converging and reach 90%, with the centrifugal P1 that obtains of 0.25m/v% tryptic digestion 2min, 1000rpm for hUC-MSCs; Continue to cultivate until obtain P3 for hUC-MSCs.

Compared with prior art, the amplification method of regulatory T cells of the present invention has following beneficial effect:

1, the present invention is by CD4 ⁺cD25 ⁺treg cell carries out co-cultivation with human umbilical cord mesenchymal stem cells (hUC-MSCs) in vitro, and the somatomedin secreted by hUC-MSCs can promote CD4 ⁺cD25 ⁺the propagation of Treg cell, enhances expanding effect.CD4 after the inventive method amplification will be used ⁺cD25 ⁺treg cell makes preparation, feeds back curative effect good.

2, the present invention adopts sorting test kit immunomagnetic beads to carry out sorting, without the need to crossing post, easy and simple to handle, saves time.

3, the present invention adopts basic medium to cultivate, and reduces cost.

Accompanying drawing explanation

Fig. 1 is CD4 in effect example 1 ⁺cD25 ⁺the growth curve of Treg cell.

Fig. 2 is the CD4 that in embodiment 1, sorting obtains ⁺cD25 ⁺the flow cytometer detection result of Treg cell.

Fig. 3 is the CD4 obtained after embodiment 1 co-cultivation ⁺cD25 ⁺the flow cytometer detection result of Treg cell.

Fig. 4 is the CD4 that in comparative example 1, sorting obtains ⁺cD25 ⁺the flow cytometer detection result of Treg cell.

Fig. 5 is the CD4 obtained after comparative example 1 co-cultivation ⁺cD25 ⁺the flow cytometer detection result of Treg cell.

Embodiment

In order to better the present invention is described, be described further below in conjunction with the drawings and specific embodiments.In the present invention, agents useful for same or instrument all can be buied by market, and the detection method of use etc. are all known in the art, do not repeat them here.

Embodiment 1

The amplification method of regulatory T cells, comprises the following steps:

S1, CD4 ⁺cD25 ⁺the separation of Treg cell and sorting; Specifically comprise the following steps:

Mononuclearcell (PBMC) is separated in S11, employing density gradient centrifugation autoblood; Described blood is the blood of Patients with SLE;

S12, use EasySep ^tMpeople CD4 ⁺cD25 ⁺t cell sorting test kit, sub-elects CD4 according to the step operation described in its specification sheets in PBMC ⁺cD25 ⁺treg cell.

The separation of S2, hUC-MSCs and going down to posterity; Specifically comprise the following steps:

Arteriovenous in S21, removal umbilical cord and amnion; Umbilical cord scissors is broken to about 1mm ³the fragment of size; With the II Collagenase Type digestion umbilical cord fragment 2-4h of 0.2m/v% (mass body volume concentrations), stop digestion with the substratum containing volume fraction 10%FBS, the centrifugal 5min of 2000rpm obtains primary hUC-MSCs;

S22, after cultivating primary hUC-MSCs, 24h containing the X-VIVO15 of 5-20ng/mlEGF β, change liquid, remove non-attached cell; Continue to be cultured to hUC-MSCs degree of converging and reach 90%, with 0.25m/v% tryptic digestion 2min; Stop digestion with the substratum containing volume fraction 10%FBS, the centrifugal 5min of 1000rpm obtains P1 for hUC-MSCs; Continue to be cultured to and obtain P3 for hUC-MSCs.

S3, by CD4 ⁺cD25 ⁺treg cell and hUC-MSCs co-cultivation; Specifically comprise the following steps: S31, be 1 × 10 by density ⁵the P3 of cells/ml is inoculated into the upper room in Transwell cell (CORNING, the aperture of polycarbonate membrane is 3 μm), every hole 2ml for hUC-MSCs;

S32, be 1 × 10 by density ⁶the CD4 of cells/ml ⁺cD25 ⁺treg cell is inoculated into the lower room in Transwell cell, every hole 2ml;

S33, co-cultivation 14 days, carried out fluid infusion every 2 days to cell, and the substratum used is X-VIVO15 substratum, wherein adds the Flt3 of IL-2 and 10-50ng/ml of 200-1000U/ml.

Comparative example 1

(1) mononuclearcell in the blood sample of Ficoll-Paque Midst density gradient centrifugation separation 20ml erythema wolf storehouse patient is used;

(2) 1 × 10 is got ⁶individual mononuclearcell MidiMACS method separation of C D4 ⁺t cell; Use FAC ^saria High speed cell sorts instrument (BDBiosciences) sorting CD4 ⁺cD4 in T cell ⁺cD25 ⁺treg cell; Use flow cytometry analysis CD4 ⁺cD25 ⁺the expression of Treg cell also identifies purity;

(3) external use X-VIVO15 culture medium culturing CD4 ⁺cD25 ⁺treg cell, for stimulating expansion of regulatory T, adding heavy dose of recombinant human il-2 (500-1000U/ml), stimulating cultivation 14 days in substratum.

Effect example 1, proliferative conditions detect

In embodiment 1 and comparative example 1 cell cultures, randomly drawed 20 μ l cell suspensions every 2 days and carry out Trypan Blue, carry out cell counting; Continuous counter 7 times, draw the growth curve of cell, result as shown in Figure 1.

As shown in Figure 1, when comparative example 1 method cultivates regulatory T cells, cell proliferation is slow, and after cultivating 3 weeks, cell quantity is also only 1 × 10 ⁶individual left and right.Use method described in embodiment 1 to cultivate regulatory T cells, cell proliferation is rapid, just reaches 1 × 10 at the content of cultivation about the 13rd day cell ⁶individual left and right, in cultivation after 3 weeks, the content of cell reaches 4.5 × 10 ⁶more than individual, be 4.5 times of comparative example 1 method.

Effect example 2, flow cytometer detection

CD4 after Example 1, comparative example 1 sorting and after co-cultivation ⁺cD25 ⁺treg cell carries out the flow cytometer detection of CD4 and CD25.Flow cytometer detection method is as follows: get 1 × 10 ⁶individual CD4 ⁺cD25 ⁺treg cell; 250g is centrifugal, and 5min removes supernatant; 2 times are cleaned by the PBS solution containing 10%FBS; Lucifuge adds CD4 and CD25 antibody 2.5 μ L incubated at room 30min; 2 times are cleaned by the PBS solution containing 10%FBS; Filter with 500mLRPMI1640 substratum re-suspended cell, flow cytometer detects.Flow cytometer detection result is as shown in Fig. 2-5 and table 1.

Table 1, CD4 ⁺cD25 ⁺treg cell streaming detected result

Group	After sorting	After co-cultivation
			Embodiment 1	95.7％	97.9％
Comparative example 1	82.5	83.5％

From Fig. 2-5 and table 1, the CD4 of the regulatory T cells obtained after the sorting of comparative example 1 method ⁺cD25 ⁺expression amount is 82.5%, with CD4 after hUC-MSCs co-cultivation ⁺cD25 ⁺expression amount is 83.5%; And the CD4 of the regulatory T cells obtained after the sorting of embodiment 1 method ⁺cD25 ⁺expression amount is 95.7%, with CD4 after hUC-MSCs co-cultivation ⁺cD25 ⁺expression amount is 97.9%, is significantly higher than the CD4 of the regulatory T cells that comparative example 1 method obtains ⁺cD25 ⁺expression amount, and difference has statistical significance.So the inventive method can significantly improve the CD4 of regulatory T cells ⁺cD25 ⁺expression amount.

Effect example 3, CD4 ⁺cD25 ⁺the detection of the cytokine content of Treg emiocytosis

The cell culture fluid that Example 1, comparative example 1 are collected concentrates rear employing ELASA standard measure and detects TGF-β secreted by it and IL-10 content.

Concentration and the absorbance of TGF-β standard substance are linear, and its linearity curve is: Y=0.047X+0.29.In embodiment 1 cell culture fluid survey TGF-β OD450 value be 1.12, in comparative example 1 cell culture fluid survey TGF-β OD450 value be 0.94.Calculate according to linearity curve, in embodiment 1, the content of the TGF-β of cell culture fluid is 17.66 μ g/ml, and in comparative example 1, the content of the TGF-β of cell culture fluid is 13.83 μ g/ml.

Concentration and the absorbance of IL-10 standard substance are linear, and its linearity curve is: Y=0.11X+0.8.In embodiment 1 cell culture fluid survey IL-10 OD450 value be 1.203, in comparative example 1 cell culture fluid survey IL-10 OD450 value be 1.04.Calculate according to linearity curve, in embodiment 1, the content of the IL-10 of cell culture fluid is 3.66 μ g/ml, and in comparative example 1, the content of the IL-10 of cell culture fluid is 2.19 μ g/ml.

The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (8)

1. the amplification method of regulatory T cells, is characterized in that, by CD4 ⁺cD25 ⁺treg cell and hUC-MSCs co-cultivation.

2. the amplification method of regulatory T cells according to claim 1, is characterized in that, described CD4 ⁺cD25 ⁺the method of Treg cell and hUC-MSCs co-cultivation is:

HUC-MSCs is inoculated into the upper room of Transwell cell, by CD4 ⁺cD25 ⁺treg cell is inoculated into the lower room of Transwell cell, and co-cultivation 14 days, carried out fluid infusion every 2 days to cell.

3. the amplification method of regulatory T cells according to claim 2, is characterized in that, the density of described hUC-MSCs is 1 × 10 ⁵cells/ml, CD4 ⁺cD25 ⁺the density of Treg cell is 1 × 10 ⁶cells/ml.

4. the amplification method of regulatory T cells according to claim 2, is characterized in that, the substratum used during co-cultivation is X-VIVO15 substratum, wherein adds the Flt3 of IL-2 and 10-50ng/ml of 200-1000U/ml.

5. the amplification method of regulatory T cells according to claim 1, is characterized in that, CD4 ⁺cD25 ⁺treg cell is prepared by the following method:

PBMC is separated in S11, employing density gradient centrifugation autoblood;

S12, in PBMC, sub-elect CD4 ⁺cD25 ⁺treg cell.

6. the amplification method of regulatory T cells according to claim 5, is characterized in that, described step S12 uses EasySep ^tMpeople CD4 ⁺cD25 ⁺t cell sorting test kit sub-elects CD4 in PBMC ⁺cD25 ⁺treg cell.

7. the amplification method of regulatory T cells according to claim 5, is characterized in that, described blood is the blood of Patients with SLE.

8. the amplification method of regulatory T cells according to claim 1, is characterized in that, hUC-MSCs is prepared by the following method:

Arteriovenous in S21, removal umbilical cord and amnion; Umbilical cord is cut into fragment; With the II Collagenase Type Digestive system umbilical cord fragment 2-4h of 0.2m/v%, 2000rpm is centrifugal obtains primary hUC-MSCs;

S22, after cultivating primary hUC-MSCs, 24h containing the X-VIVO15 of 5-20ng/mlEGF β, change liquid, remove non-attached cell; Continue to be cultured to hUC-MSCs degree of converging and reach 90%, with the centrifugal P1 that obtains of 0.25m/v% tryptic digestion 2min, 1000rpm for hUC-MSCs; Continue to cultivate until obtain P3 for hUC-MSCs.