CN112080469B - Application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro - Google Patents

The invention discloses application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro. The invention cultures CD34+ hematopoietic stem cells in human umbilical cord blood under the serum-free condition, and tests the effect of T1(GPANVET) in the in-vitro amplification of human umbilical cord blood hematopoietic stem cells, and finds that the T1 peptide has better promotion effect on the self-renewal of the human umbilical cord blood hematopoietic stem cells. The invention also discovers that the combined culture medium of the Stem Cell Factor (SCF), the Thrombopoietin (TPO), the FMS-like tyrosine kinase 3 ligand (FLT3-L), the interleukin-6 (IL-6) and the T1 peptide has the proportion of promoting the expansion of hematopoietic stem cells and maintaining the stem cells, can improve the differentiation potential of the hematopoietic stem cells, and can provide a new method for clinical treatment in the future.

Application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro

Technical Field

The invention belongs to the technical field of stem cells, and particularly relates to application of T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro.

Background

Hematopoietic Stem Cells (HSCs) are stem cells that produce other blood cells in the blood system and have long-term self-renewal capacity and the potential to differentiate into various types of mature blood cells. Autologous or allogeneic transplantation has been successfully used to treat diseases that endanger human health, such as hematologic disorders, malignancies, primary immunodeficiency diseases and metabolic abnormalities in children and adult patients. However, hematopoietic stem cell transplantation is also limited by various factors, such as the need for highly similar HLA matching for bone marrow hematopoietic stem cells, and the possibility of mobilizing peripheral blood hematopoietic stem cells by G-CSF to cause donor spleen rupture.

Umbilical cord blood hematopoietic stem cells (UCB-HSCs) have the advantages of wide sources, long-term storage, low incidence rate of GVHD (graft versus host disease), low immunogenicity and the like, and become a stem cell product with important clinical application value. At present, the biggest bottleneck of clinical application of UCB-HSCs is insufficient cell number, so that HSCs implantation delay and immune reconstruction delay are caused, the risk of bacterial and viral infection of patients is increased, and the death rate of the patients is increased. Therefore, in vitro expansion of UCB-HSCs is the most direct solution to the current shortage of cell numbers.

Self-renewal and directed differentiation of HSCs are doubly regulated by factors internal to HSCs and external environmental factors. The self-renewal process is an integrated process involving the activation and maintenance of cell proliferation pathways as well as the inhibition of differentiation and cell death pathways. The internal factors are mainly transcription factor networks formed by HSCs specific transcription factors, the external microenvironment comprises stromal cells, cytokines, small molecular compounds, microRNA and the like, and in addition, the special culture system comprises hypoxia, perfusion culture, fed-batch culture and the like, and has important regulation and control effects on self-renewal and directed differentiation of HSCs.

According to different in vitro amplification modes, the method can be divided into two types: one is to simulate the growth environment and mechanism in bone marrow by using stromal cells, and perform perfusion culture under the support of stroma, so as to provide various components required by the growth of hematopoietic stem cells, adjust the growth and differentiation directions of the hematopoietic stem cells, and have good amplification effect. However, the method has the disadvantages of obvious cost, complex culture components and great difficulty in separating hematopoietic stem cells in the later period, which all limit the popularization of the amplification method. In another mode, the hematopoietic stem cells are amplified in vitro by adding cytokines or small molecular compounds and fetal calf serum, but the mode is easy to generate hypoxia, insufficient nutrition, complex serum components and increased influencing factors, and the expected amplification effect cannot be achieved.

Therefore, the research and development of the serum-free in-vitro culture medium which can promote the rapid expansion of the hematopoietic stem cells, is safe and convenient and can maintain the dryness of the hematopoietic stem cells have important value and application prospect.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the T1 peptide in promoting the proliferation of cord blood hematopoietic stem cells in vitro.

Another object of the present invention is to provide a method for the in vitro serum-free expansion of cord blood hematopoietic stem cells.

Still another object of the present invention is to provide a pharmaceutical composition for expanding umbilical cord blood hematopoietic stem cells.

Still another object of the present invention is to provide a serum-free medium for expanding cord blood hematopoietic stem cells.

The purpose of the invention is realized by the following technical scheme:

the application of a T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro, wherein the amino acid sequence of the T1 peptide is shown as follows: GPANVET.

The T1 peptide can increase the proportion and the number of cord blood hematopoietic stem cells and the differentiation potential of the hematopoietic stem cells (the differentiation potential of the expanded hematopoietic stem cells is the same as that of the cytokine FLT 3-L).

The cord blood hematopoietic stem cells are human cord blood hematopoietic stem cells; including human cord blood hematopoietic stem cell CD34⁺Cell, CD34⁺CD38^-A cell.

The application of the T1 peptide in preparing a product for promoting cord blood hematopoietic stem cell proliferation, wherein the amino acid sequence of the T1 peptide is shown as follows: GPANVET.

The product comprises a medicament, a kit, a culture medium and the like.

An in vitro serum-free expansion method of cord blood hematopoietic stem cells is characterized in that a culture medium containing T1 peptide is used for culturing the cord blood hematopoietic stem cells to improve the proportion and the number of the cord blood hematopoietic stem cells and the differentiation potential of the hematopoietic stem cells; wherein, the amino acid sequence of the T1 peptide is shown as follows: GPANVET.

The in vitro serum-free amplification method of the umbilical cord blood hematopoietic stem cells specifically comprises the following steps: the method comprises the steps of pre-enriching the mononuclear cells in the umbilical cord blood by adopting density gradient centrifugation, then sorting CD34+ hematopoietic stem cells in the mononuclear cells by using an immunomagnetic bead sorting method, and then culturing the umbilical cord blood hematopoietic stem cells by using a culture medium containing T1 peptide.

The concentration of the T1 peptide in the culture medium is 0-20 mu g/ml (excluding 0); preferably 5-20 mu g/ml; more preferably 10. mu.g/ml.

The culture medium is a StemBan SFEM culture medium; preferably StemBan SFEM medium containing Stem Cell Factor (SCF), Thrombopoietin (TPO), interleukin-6 (IL-6) and FMS-like tyrosine kinase 3 ligand (FLT 3-L); further preferably a StemBan SFEM culture medium containing 10-100 ng/ml stem cell factor, 10-100 ng/ml thrombopoietin, 10-100 ng/ml IL-6 and 10-100 ng/ml FMS-like tyrosine kinase 3 ligand; still further preferred is StemBan SFEM medium containing 100ng/ml stem cell factor, 100ng/ml thrombopoietin, 20ng/ml IL-6 and 50ng/ml FMS-like tyrosine kinase 3 ligand.

The cord blood hematopoietic stem cells are human cord blood CD34⁺Hematopoietic stem cells.

The density of the cord blood hematopoietic stem cells is 5 multiplied by 10⁴～10⁵One per ml.

The temperature of the culture was 37 ℃.

The culture time is 7-9 days; preferably for 7 days.

A pharmaceutical composition for expanding umbilical cord blood hematopoietic stem cells, comprising the following components: t1 peptide, Stem Cell Factor (SCF), Thrombopoietin (TPO), interleukin-6 (IL-6), and FMS-like tyrosine kinase 3 ligand (FLT 3-L); wherein the amino acid sequence of the T1 peptide is shown as follows: GPANVET.

The pharmaceutical composition preferably comprises the following components: 0-20 mug/ml T1 peptide (excluding 0), 10-100 ng/ml stem cell factor, 10-100 ng/ml thrombopoietin, 10-100 ng/ml IL-6 and 10-100 ng/ml FMS-like tyrosine kinase 3 ligand.

More preferably, the pharmaceutical composition comprises the following components: 5-20. mu.g/ml T1 peptide, 100ng/ml stem cell factor, 100ng/ml thrombopoietin, 20ng/ml IL-6 and 50ng/ml FMS-like tyrosine kinase 3 ligand.

The pharmaceutical composition for amplifying the cord blood hematopoietic stem cells is applied to promoting the cord blood hematopoietic stem cells to proliferate in vitro.

A serum-free culture medium for expanding cord blood hematopoietic stem cells comprises the following components: StemBan SFEM medium + 10-100 ng/ml stem cell factor + 10-100 ng/ml thrombopoietin + 10-100 ng/ml IL-6+ 10-100 ng/ml FMS-like tyrosine kinase 3 ligand + 0-20 μ g/ml T1 peptide (excluding 0); wherein, the amino acid sequence of the T1 peptide is shown as follows: GPANVET.

The serum-free culture medium preferably comprises the following components: StemBan SFEM medium +100ng/ml stem cell factor +100ng/ml thrombopoietin +20ng/ml IL-6+ 50-100 ng/ml FMS-like tyrosine kinase 3 ligand + 5-20 μ g/ml T1 peptide.

The serum-free culture medium further preferably comprises the following components: StemBan SFEM medium +100ng/ml Stem cell factor +100ng/ml thrombopoietin +20ng/ml IL-6+100ng/ml FMS-like tyrosine kinase 3 ligand +10 μ g/ml T1 peptide.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention cultures CD34+ hematopoietic stem cells in human umbilical cord blood under the serum-free condition, and tests the effect of T1 in the in-vitro amplification of human umbilical cord blood hematopoietic stem cells, finds that the T1 peptide has a better promotion effect on the self-renewal of the human umbilical cord blood hematopoietic stem cells, and can provide a new method for clinical treatment in the future.

(2) The invention finds that the combined culture medium containing the factors SCF + TPO + IL-6, SCF + TPO + IL-6+ FLT3-L, SCF + TPO + IL-6+ T1 has the advantages of promoting the expansion of hematopoietic stem cells and maintaining the proportion of stem cells and improving the differentiation potential of the hematopoietic stem cells by comparing the combined culture medium containing the factors SCF + TPO + IL-6+ FLT3-L, SCF + TPO + IL-6+ T1.

(3) The invention provides a serum-free culture medium for amplifying human cord blood hematopoietic stem cells, which is based on a Stemcell StemSpan culture medium and also comprises other components: stem Cell Factor (SCF), Thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), interleukin-6 (IL-6), and T1 peptide.

Drawings

FIG. 1 is a graph of the change in cell number 7 days after treatment with different concentrations of T1 peptide in combination with SCF, TPO, and IL-6.

FIG. 2 is a graph of CD34 after 7 days of treatment with different concentrations of T1 peptide in combination with SCF, TPO, and IL-6⁺Graph of cell ratio change.

FIG. 3 is CD34 after 7 days of treatment with different concentrations of T1 peptide in combination with SCF, TPO, IL-6⁺CD38^-Graph of cell ratio change.

FIG. 4 is CD34 of cord blood cultured with different concentrations of cytokine combinations⁺Colony formation results are shown.

FIG. 5 is a graph of the change in cell number 7 days after treatment with different concentrations of T1 peptide in combination with SCF, TPO, IL-6, FLT 3-L.

FIG. 6 is a graph of CD34 after 7 days of treatment with different concentrations of T1 peptide in combination with SCF, TPO, IL-6, FLT3-L⁺Graph of cell ratio change.

FIG. 7 is a graph of CD34 after 7 days of treatment with different concentrations of T1 peptide in combination with SCF, TPO, IL-6, FLT3-L⁺CD38^-Graph of cell ratio change.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

1. The culture medium and the components thereof are conventional commercial products:

the culture medium StemBan SFEM is purchased from squirrel Biotechnology Limited, Guangzhou, under the product number 09650, STEMCELL;

cytokines such as stem cell factor (Human SCF; SCF for short), thrombopoietin (Human TPO; TPO for short), interleukin-6 (Human IL-6; IL-6 for short), FMS-like tyrosine kinase 3 ligand (FLT 3-L; the like) are purchased from squirrel Biotech Limited, Guangzhou, and the product numbers are respectively: 300-07-10, 300-18-10, 200-06-5 and AF-300-19-10;

② sodium methylcellulose semisolid culture medium (MethoCult H4434 Classic) available from Guangzhou Poly research Biotech Co., Ltd., cat # 04414.

③ IMDM Medium, cat # C12440500BT, from Oncology, Inc., Guangzhou.

2. The T1 short peptide (GPANVET) of the present invention was synthesized by Shanghai Tanpu Biotech Co., Ltd.

3. The invention provides an in vitro culture medium for umbilical cord blood hematopoietic stem cells, which is a StemBan SFEM culture medium containing 10-100 ng/ml SCF, 10-100 ng/ml TPO, 10-100 ng/ml IL-6, 10-100 ng/ml FLT3-L and 0-20 mu g/ml T1 peptide.

Example 1 Effect of Stem cell factor, thrombopoietin, Interleukin-6 in combination with FLT3-L or varying concentrations of T1 peptide on the in vitro proliferation of hematopoietic Stem cells

1.1 reagent materials:

collecting Umbilical Cord Blood (UCB) from a first hospital affiliated with river-south university; lymphocyte isolates purchased from squirrel Biotech Limited, Guangzhou (cat # 07861, STEMCELL); the umbilical cord blood dilution buffer is purchased from Guangzhou Yuan Biotechnology instruments Co., Ltd (Cat. No. 130-.

1.2 Experimental methods:

1.2.1 cord blood CD34⁺Cell sorting and purity characterization

Diluting the collected umbilical cord blood with umbilical cord blood dilution buffer, performing monocyte separation with lymphocyte separation liquid to obtain umbilical cord blood mononuclear cells, and separating CD34 by immunomagnetic bead cell sorting⁺The sorted cells were subjected to flow cytometric purity characterization using anti-human-CD34-FITC (Oncon Biotechnology Co., Ltd., Guangzhou, 555821, BD) and anti-human-CD38-APC (Oncon Biotechnology Co., Ltd., Guangzhou, 555462, BD).

1.2.2 cord blood CD34⁺In vitro expansion of hematopoietic stem cells

(1) And centrifuging the cell suspension subjected to the sorting of the immunomagnetic beads, centrifuging for 8 minutes at 300g, and discarding the supernatant.

(2) The cell pellet was resuspended in StemBan SFEM +100ng/ml SCF +100ng/ml TPO +20ng/ml IL-6 medium (both final system concentrations) to adjust the cell concentration to 10⁵One per ml. mu.L of the culture medium was added to each well of 24-well plates, and then 500. mu.L of the same culture medium was added to each well to culture the cells in 6 rows, numbered A to F, and 3 wells in each row.

(3) Experiment setup control and experimental groups, where 5. mu.g/ml, 10. mu.g/ml, 15. mu.g/ml, 20. mu.g/ml of T1 peptide (SITH group) were added to the C-F column medium, and equal volumes of StemBan SFEM medium (SIT group) and FLT3-L (SITF group) were added to the two control groups A and B column medium, respectively) The cells Before culture (Beform culture) were used as a blank control. Gently shaking and mixing, adding 5% (v/v) CO at 37 deg.C₂An incubator.

(4) After 7 days, the cultured cells were counted, collected again, centrifuged at 300g for 5min, labeled antibodies anti-human-CD34-FITC and anti-human-CD38-APC, incubated at room temperature for 30 min, protected from light.

(5)300g centrifugation for 5 minutes, discarding unbound antibody, and 200 u L FACS buffer heavy suspension, flow cytometry analysis.

1.3 analysis of results

The results are shown in FIGS. 1 to 3: as is clear from FIG. 1, the number of cultured cells was increased by 10 times or more as compared with the number of cells initially cultured, and the number of cultured cells was 18.28 times in the SITF group, 11.03 times in the SIT group, and 12.16 times in the SITH group at a concentration of 10. mu.g/ml, indicating that the T1 peptide had the effect of promoting the proliferation of cord blood cells. Meanwhile, as can be seen from FIG. 2, the SITH group was able to increase CD34 in cord blood compared with the SITF group and the SIT group⁺The proportion of cells, and the concentration dependence, have significant differences. Finally, as can be seen from FIG. 3, the T1 peptide, at a concentration of 10. mu.g/ml, was CD34⁺CD38^-The proportion of cells reached a maximum, higher than the SITF and SIT groups. Thus, it was found that the T1 peptide can increase CD34⁺CD38^-The proportion and number of cord blood hematopoietic stem cells.

Example 2 colony formation assay of cord blood CD34+ cells and cultures thereof

2.1 reagent materials:

sodium methylcellulose semisolid medium (MethoCult H4434 Classic); IMDM medium.

2.2 Experimental methods:

sorted cord blood CD34⁺CD34 after culturing cells (sorting method as in example 1) and different combinations of factors⁺Cells were adjusted to a cell density of 1 × 10 using IMDM medium (containing 2% (v/v) Fetal Bovine Serum (FBS))⁴Mixing the cells per ml according to the proportion of 100ul cell suspension and 1ml sodium methylcellulose semisolid culture medium, fully and uniformly mixing the cells on a vortex apparatus, standing for 10-15 min, transferring the mixture to a 35mm culture dish, and flattening and culturing the mixtureThe culture is placed on a 100mm culture dish, 3 times of repetition are carried out on each experimental group, and 1 35mm culture dish filled with sterile water is added into the experimental group to maintain high humidity in the whole culture process and prevent the culture from drying up; place 100mm dish in incubator (37 ℃, 5% CO)₂). After 14 days of culture, cultures were removed and each colony was counted under an inverted microscope according to its morphological characteristics and representative colonies were photographed, stored and further analyzed. Wherein, the experiment is divided into 3 groups: and (6) SITH: CD34 in culture with 100ng/ml SCF +100ng/ml TPO +20ng/ml IL-6+ 10. mu.g/ml T1 peptide⁺(ii) a And (6) SITF: CD34 cultured with 100ng/ml SCF +100ng/ml TPO +20ng/ml IL-6+ FLT3-L⁺(ii) a fresh-uncultured CD34 after sorting⁺(ii) a (in the figure, Total: Total of colonies, CFU-B: erythroid colonies, CFU-GM: granulocyte-macrophage colony, CFU-GEMM: granulocyte-erythro-megakaryocyte-macrophage colony).

2.3 analysis of results:

the results are shown in FIG. 4: as can be seen from FIG. 4, cord blood CD34 cultured with a combination of SITF and SITH factor was compared with the sorted cells⁺The cells have higher capability of forming CFU-B, CFU-GM, CFU-GEMM and other colonies, and compared with the SITH group, the SITF group has no significant difference, namely the differentiation potential is similar.

Example 3 Effect of Stem cell factor, thrombopoietin, Interleukin-6 in combination with FLT3-L and varying concentrations of T1 peptide on the in vitro proliferation of hematopoietic Stem cells

3.1 reagent materials: the same as in example 1.

3.2 Experimental methods:

3.2.1 cord blood CD34+ cell sorting and purity identification: the same as in example 1.

3.2.2 in vitro expansion of cord blood CD34+ hematopoietic Stem cells

(1) And centrifuging the cell suspension subjected to the sorting of the immunomagnetic beads, centrifuging for 8 minutes at 300g, and discarding the supernatant.

(2) The cell pellet was resuspended in StemBan SFEM +100ng/ml SCF +100ng/ml TPO +20ng/ml IL-6 medium (both final system concentrations) to adjust the cell concentration to 10⁵500. mu.L/ml of each well was added to a 24-well plate, and 500. mu.L of the same medium was added to the wells to culture, 6 rows in totalNumbers A-F, 3 duplicate wells per column.

(3) The experiment sets up a control group and an experimental group, wherein 50ng/ml FLT3-L is added to each row of the culture medium of the C-F row, and then T1 peptide (SITFH group) of 5 mug/ml, 10 mug/ml, 15 mug/ml and 20 mug/ml is added to each row. The culture medium of two control groups A and B was supplemented with 50ng/ml and 100ng/ml FLT3-L (SITF group), respectively, as a blank control for the cells Before culture (Beform culture). Gently shaking and mixing, adding 5% (v/v) CO at 37 deg.C₂An incubator.

(4) After 7 days, the cultured cells were counted, collected again, centrifuged at 300g for 5min, labeled antibodies anti-human-CD34-FITC and anti-human-CD38-APC, incubated at room temperature for 30 min, protected from light.

(5)300g centrifugation for 5 minutes, discarding unbound antibody, and 200 u L FACS buffer heavy suspension, flow cytometry analysis.

3.3 analysis of results:

the results are shown in FIGS. 5 to 7: as shown in FIG. 5, in comparison with the SITF group, the addition of the T1 peptide to the SITFH group, in which the concentration of FLT3-L was halved, promoted cord blood CD34⁺Proliferation of cells; among them, SITFH (0.05,5) proliferated cells at the highest number, which was 15.96 times as large as the number of cells initially cultured. As can be seen from FIG. 6, cord blood CD34 was observed in all groups⁺The cells were cultured to reduce the cell ratio, wherein SITF (0.05,0), SITF (0.1,0), SITFH (0.05,5) and SITFH (0.05,15) group CD34⁺Cells were not significantly different, whereas SITFH (0.05,10) and SITFH (0.05,20) groups CD34⁺The proportion of cells decreased slightly. As is clear from FIG. 7, after the culture, cord blood hematopoietic stem cells CD34⁺CD38^-The cell proportion is increased by about 40%, and the increase of SITFH (0.05,10) group is the maximum and reaches 42.47%. Therefore, the T1 peptide can promote the proliferation of cord blood hematopoietic stem cells by cooperating with FLT3-L, and can play the function of FLT 3-L.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

Application of <120> T1 peptide in promoting cord blood hematopoietic stem cell proliferation in vitro

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> T1 peptide

<400> 1

Gly Pro Ala Asn Val Glu Thr

1 5