CN101402941A

CN101402941A - Method for amplifying Valpha24NKT cell from umbilical stalk blood

Info

Publication number: CN101402941A
Application number: CNA2008102025173A
Authority: CN
Inventors: 高峰
Original assignee: Shanghai Stem Cell Technology Co Ltd
Current assignee: Shanghai Stem Cell Technology Co Ltd
Priority date: 2008-11-11
Filing date: 2008-11-11
Publication date: 2009-04-08

Abstract

The invention relates to a blood product of umbilical blood, and discloses a method for amplifying VAlpha24 NKT cell from umbilical blood, aiming at overcoming the defect that little content of VAlpha24 NKT cell in umbilical blood and the difficulty in clinical research and treatment of umbilical blood. The method comprises the following steps: A. the collection and storage of umbilical blood; B. purification of a single karyocyte in umbilical blood; and C. amplication in vitro of VAlpha24 NKT cell of a single karyocyte in umbilical blood. The method amplifying VAlpha24 NKT cell from umbilical blood is characterized by high amplification efficiency and strong specificity, and in that the acquired VAlpha24 NKT cell has a two-way immunoregulation capability of secreting INF-Gamma and IL-4, and has extensive application in clinical research and treatment.

Description

The method of amplification V α 24NKT cell from Cord blood

Technical field

The present invention relates to the Cord blood blood products, be specifically related to a kind of method of the V α 24NKT cell that from Cord blood, increases.

Background technology

Natural killer T cells (NKT) is the lymphocyte that a class has NK acceptor and TXi Baoshouti and demonstration NK cell and T cell two aspect character.Because its phenotypic function and immunocytes such as T cell, B cell and NK cell are different, are quite paid attention in recent years.The NKT cell has the TCR phenotype (TCR V α 14/V β 8---mouse V α 14 NKT cells, TCR V α 24/V β 11---people V α 24 NKT cells) of high conservative, simultaneously the peculiar sign of coexpression NK cell CD161.The special glycolipid molecule that V α 24 NKT cell recognition are offered by the CD1d molecule also has a large amount of cytokines of rapid secretion, participates in the innate immunity of body and the ability of the acquired immune response.Discover that V α 24 NKT cells are in control infection, and are antitumor, overcome transplant rejection, and bringing into play crucial effects in the generation of inhibition autoimmune disorder (type i diabetes, systemic lupus erythematous, scleroderma etc.).

Glycolipid class antigen, (α-Galcer) can be used for inducing the NKT cell expansion ex vivo as alpha-galactosylceramide.Because α-GalCer is limited by the CD1d molecule to the activation of NKT, general at present employing dendritic cell is offered α-GalCer, by immunomagnetic beads purifying CD4 ^-CD8 ^-NKT sets up NKT clone.α-the GalCer of the CD1d submission of the TCR identification dendritic cell of V α 24 NKT cells, simultaneously its surperficial CD28 and CD154 interact with the CD80/CD86 and the CD40 on dendritic cell surface respectively, activate dendritic cell and produce IL-12 and IL-18, act on the IL-12R and the IL-18R on V α 24NKT surface, make the IL-4 and the IFN-γ of V α 24NKT secreting high levels.V α 24NKT cell produces IL-4 only need costimulatory signal of CD28-CD80/CD86, then needs CD28-CD80/CD86 and two costimulatory signals of CD154-CD40 but produce IFN-γ, and needs the participation of the endogenous IL-12 of dendritic cell generation.Monokaryon is an also weak expression CD1d molecule and have the restricted immunoreactive ability of very strong activation CD1d of cell surface, so V α 24NKT cell can increase by adding α-GalCer in peripheral blood mononuclear cell (PBMNC) the vitro culture process.

V α 24 NKT cells have the panimmunity regulatory function:

1.NKT the anti-infectious function of cell

Studies show that the NKT cell has participated in replying of multiple infection, for example participate in the immunoreaction process to simplexvirus, mycobacterium, listeria bacteria, plasmodium etc., this activates with the NKT cell-specific and can cause a large amount of propagation and the cellulotoxic effect of NK cell relevant.The NKT cell also may be regulated the generation of Th1 cell by secretion IL-4 negativity, as replying the infection of bacterium or bacterium composition such as lipopolysaccharides.

2.NKT the antitumor action of cell

Cell can directly play a role as the anti-tumour effect cell although NKT is supported in vitro tests, and the anti-tumor function that can not get rid of the NKT cell mainly is by activating other immune effector cell, as the NK cell realize may.α-GalCer produces the transfer that IFN-γ suppresses tumour by activating the NKT cell, also may produce anti-tumour effect by NKT cell-stimulating bone-marrow-derived lymphocyte.The NKT cell kills and wounds various tumour cells by the cell-mediated mode of NK that pore-forming protein relies on after being subjected to α-GalCer and IL-12 and activating, and does not rely on the tumour target cell and whether express CD1d.

3.NKT cell is to the restraining effect of autoimmune disorder

The quantity that has confirmed the NKT cell descends, or changing function is relevant with the generation of autoimmune disorder.Between the identical twin, the ill individuality of diabetes is compared with the non-diabetic individuality, and the quantity of NKT cell obviously reduces at the former.There is defective by the V α 24NKT cell clone that obtains through separation in these diabetic subject's bodies aspect the generation IL-4.The NKT cell plays a part in steady very important at immunity of organism, contain a high proportion of self killer T cell in the NKT cell, but this kill mechanism is different with its antitumor mechanism, realizes by the Fas/FasL approach.

4.NKT cell is to the restraining effect of GVHD

CD4 in the marrow ^-CD8 ^-The NKT cell is found in behind the allogeneic bone marrow transplantation and relies on mode with a kind of IL-4, plays an important role in preventing GVHD, and the NKT cell can generate a large amount of IL-4 abilities prompting NKT cells and Th2 is replied have the driving effect.Research also finds, in v α 14 NKT Cell Deficient Mice Infected, adopting property infusion v α 14 NKT cells can cause graft to plant work for a long time in that the receptor animal is intravital.

Cord blood is a kind of new resources of wide material sources, and reduced immunogenicity cell wherein is subjected to people's attention just day by day.Because the content of V α 24 NKT cells in bleeding of the umbilicus is few, is difficult to clinical study and treatment, the method that therefore searches out a kind of bleeding of the umbilicus of amplification in vitro efficiently NKT cell is significant.

Summary of the invention

It is few to the objective of the invention is to overcome the content of V α 24 NKT cells in bleeding of the umbilicus, is difficult to the defective of clinical study and treatment, and a kind of method of the V α 24 NKT cells that increase from Cord blood is provided.

The invention discloses a kind of method of the V α 24 NKT cells that from Cord blood, increase, comprise the following steps:

A. Cord blood collection and preservation;

B. Cord blood mononuclearcell purifying;

C. V α 24 NKT cell expansion ex vivo in the Cord blood mononuclearcell;

Among the above-mentioned steps A, Cord blood picks up from neonatal umbilical cord and placenta, adopts ACD-B anticoagulation bag to collect, and continues to add in the HES liquid, after the piping and druming evenly, places 0-6 hour in 4 ℃ of refrigerators.

Among the above-mentioned steps B, Cord blood adopts lymphocyte separation medium to isolate mononuclearcell, is suspended in the complete culture solution.

Among the above-mentioned steps C,, add IL-2 and alpha-galactosylceramide, in 37 ℃, 5%CO with the Cord blood mononuclearcell of fresh separated ₂And cultivate under the condition of saturated humidity.Wherein, IL-2 content is 5-20U/mL, and alpha-galactosylceramide content is 100-200ng/mL.

Among the above-mentioned steps C, concentration was 2 * 10 when the Cord blood mononuclearcell was cultivated ⁵-1 * 10 ⁶/ mL, optimum concn is 5 * 10 ⁵/ mL.

Among the above-mentioned steps C, Cord blood mononuclearcell proliferation time is 7-14 days, and Best Times is 7 days.

The invention discloses from Cord blood the method for amplification V α 24 NKT cells, more particularly adopt IL-2 and alpha-galactosylceramide (the V α 24 NKT cells in the amplification bleeding of the umbilicus of α-Galcer).The method that increases V α 24 NKT cells from Cord blood of the present invention has the amplification efficiency height, and high specificity, the V α 24 NKT cells of acquisition have the two-way immunoregulation ability of secretion INF-γ and IL-4.

Description of drawings

Fig. 1 adopts IL-2 and alpha-galactosylceramide (α-Galcer) amplification Cord blood (CB) and peripheral blood (PB) V α 24 NKT cell amplification design sketchs.

Fig. 2 (a) is the cell content flow cytometer detected result figure before the amplification.

Fig. 2 (b) is the 7th day UCB-NKT cell content flow cytometer detected result figure of amplification.

Fig. 2 (c) is the 14th day PB-NKT cell content cell content flow cytometer detected result figure of amplification.

Embodiment

Below enumerate specific examples and further set forth the present invention, should be understood that example is not to be used to limit protection scope of the present invention.

The embodiment 1 V α 24 NKT cells that from Cord blood, increase

1, the neonatal umbilical cord of the fresh collection of preparation of Cord blood mononuclearcell and the Cord blood in the placenta are collected with ACD-B anticoagulation bag, continue and add in the 6%HES liquid at 4: 1 with volume ratio, blow and beat under even back 4 ℃ and leave standstill 1h, the tunica albuginea layer is used the 0.6%ACD-PBS two-fold dilution in the middle of drawing, and the ratio with 2: 1 behind the mixing adds Ficoll upper strata, centrifugal (2000rpm, 20min), get the intermediate cell ring, PBS washes 3 times, is suspended in the complete culture solution that contains 10%FCS.

2, the amplification of V α 24 NKT cells is inoculated in the Cord blood mononuclearcell of fresh separated in the 12 porocyte culture plates in the Cord blood, and every experimental port inoculum density is 5 * 10 ⁵/ ml adds IL-2 (5U/mL) and α-Galcer (100ng/mL), 37 ℃, 5%CO ₂And cultivate under the condition of saturated humidity, cultivate and detect V α 24 NKT cell contents after 7 days.

The embodiment 2 V α 24 NKT cells that from Cord blood, increase

1, the neonatal umbilical cord of the preparation of Cord blood mononuclearcell collection and the Cord blood in the placenta, collect with ACD-B anticoagulation bag, place 6h in 4 ℃, continue adds in the 6%HES liquid with volume ratio at 4: 1, blow and beat under even back 4 ℃ and leave standstill 1h, the tunica albuginea layer is used the 0.6%ACD-PBS two-fold dilution in the middle of drawing, and the ratio with 2: 1 behind the mixing adds Ficoll upper strata, centrifugal (2000rpm, 20min), get the intermediate cell ring, PBS washes 3 times, is suspended in the complete culture solution that contains 10%FCS.

2, the amplification Cord blood mononuclearcell of V α 24 NKT cells is inoculated in the 12 porocyte culture plates in the Cord blood, and every experimental port inoculum density is 1 * 10 ⁶/ ml adds IL-2 (20U/mL) and α-Galcer (200ng/mL), 37 ℃, 5%CO ₂And cultivate under the condition of saturated humidity, cultivate and detect V α 24 NKT cell contents after 14 days.

The content of embodiment 3 V α 24 NKT cells and phenotype detect

Testing method: draw 100 μ l cultured cells respectively, each adds CD3, TCR V β 11 monoclonal antibodies of TCRV α 24, CD56, CD161 and the FITC mark of 20 μ l PE marks, lucifuge is hatched 15min, add 0.5ml PBS, do the two fluoroscopic examinations of flow cytometer, measure the 7th day and the 14th day V α 24 NKT cell content and other phenotypic characteristics.

Test result: adopt IL-2 and alpha-galactosylceramide (the V α 24 NKT cells in the amplification bleeding of the umbilicus of α-Galcer), the amplification efficiency of V α 24 NKT is significantly improved, account for lymphocytic ratio and be (24.48 ± 4.19) % in the time of the 7th day, be (12.15 ± 2.02) % in the time of the 14th day, the V α 24 NKT cells when especially cultivating the 7th day are original (8.74 ± 4.37) * 10 ²Doubly (see Fig. 1, Fig. 2 a b c).Amplification back Cord blood V α 24 NKT cell TCR V β 11 expression amounts are much larger than TCR V α 24, and the expression of CD161 is lower than the V α 24 NKT cells that the adult peripheral mononuclear cell amplification obtains, illustrate that Cord blood V α 24 NKT cells are in crudity, cell phenotype with become human peripheral V α 24 NKT cells incomplete same (seeing Table 1).

The phenotype of table 1 Cord blood (CB) and peripheral blood (PB) V α 24 NKT cells

Claims

1. the method for an amplification Va24NKT cell from Cord blood comprises the following steps:

A. Cord blood collection and preservation;

B. Cord blood mononuclearcell purifying;

C. Va24NKT cell expansion ex vivo in the Cord blood mononuclearcell.

2. amplification Va24NKT cell method from Cord blood according to claim 1 is characterized in that, in the described steps A, Cord blood picks up from neonatal umbilical cord and placenta, adopts ACD-B anticoagulation bag to collect, and continues to add in the HES liquid, after the piping and druming evenly, in 4 ℃ of refrigerators, placed 0-6 hour.

3. amplification Va24NKT cell method from Cord blood according to claim 1 is characterized in that among the described step B, Cord blood adopts lymphocyte separation medium to isolate mononuclearcell, is suspended in the complete culture solution.

4. the method for amplification Va24NKT cell from Cord blood according to claim 1 is characterized in that, among the described step C, with the Cord blood mononuclearcell of fresh separated, adds IL-2 and a-galactosyl ceramide, in 37 ℃, 5%CO ₂And cultivate under the condition of saturated humidity.

As described in the claim 4 from Cord blood the method for amplification Va24NKT cell, it is characterized in that IL-2 content is 5-20U/mL, a-galactosyl ceramide content is 100-200ng/mL.

6. amplification Va24NKT cell method from Cord blood according to claim 1 is characterized in that among the described step C, concentration was 2 * 10 when the Cord blood mononuclearcell was cultivated ⁵-1 * 10 ⁶/ mL.

7. amplification Va24NKT cell method from Cord blood according to claim 1 is characterized in that among the described step C, concentration was 5 * 10 when the Cord blood mononuclearcell was cultivated ⁵/ mL.

8. amplification Va24NKT cell method from Cord blood according to claim 1 is characterized in that among the described step C, Cord blood mononuclearcell proliferation time is 7-14 days.

9. amplification Va24NKT cell method from Cord blood according to claim 1 is characterized in that among the described step C, Cord blood mononuclearcell proliferation time is 7 days.