CN110592017A - Use of histone methyltransferase inhibitor in preparing product for promoting megakaryocyte proliferation or platelet production - Google Patents
Use of histone methyltransferase inhibitor in preparing product for promoting megakaryocyte proliferation or platelet production Download PDFInfo
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- CN110592017A CN110592017A CN201911029451.7A CN201911029451A CN110592017A CN 110592017 A CN110592017 A CN 110592017A CN 201911029451 A CN201911029451 A CN 201911029451A CN 110592017 A CN110592017 A CN 110592017A
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- histone methyltransferase
- inhibitor
- cells
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- megakaryocyte
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Classifications
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- C12N5/0634—Cells from the blood or the immune system
- C12N5/0644—Platelets; Megakaryocytes
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses application of a histone methyltransferase inhibitor in preparing a product for promoting megakaryocyte proliferation or platelet production, wherein the histone methyltransferase inhibitor is an inhibitor of histone methyltransferase G9 a. Histone methyltransferase G9a inhibitors of the present invention can promote single CD34+Differentiation of hematopoietic stem progenitor cells yields about 400 CD41a+CD42b+The platelet particles are improved by more than 10 times compared with a control group, the platelet generation efficiency is obviously higher than the general range reported in the literature, the processing time is short, the operability and the repeatability are strong, the production cost of the platelets is obviously reduced, and the foundation is laid for the large-scale production of functional platelets for clinical treatment in the future.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a histone methyltransferase inhibitor in preparing a product for promoting megakaryocyte proliferation or platelet production.
Background
Platelets are one of the visible components of blood, and play an important role in the hemostasis and thrombosis processes of the body through functions such as adhesion, aggregation, granule release and the like. Clinically, various causes such as hematopoietic system malignant tumor, aplastic anemia, malignant tumor, hematopoietic stem cell transplantation therapy and radiotherapy and chemotherapy can cause thrombocytopenia or dysfunction, so that the body is subjected to bleeding to different degrees, and even threatens life in severe cases. Donor-derived concentrated platelet transfusions are currently the only clinical treatment. According to statistics, the number of patients needing platelet infusion in China is nearly ten million each year, and the market scale of the platelets is estimated to be nearly 1000 million RMB. Currently, there is no effective substitute for clinically infused platelets, and only platelets can be collected from a non-compensated donor. In recent years, the shortage of clinical blood and platelet products due to the shortage of blood has been an important problem in clinical practice [ YINYH, Li CQ, Liu Z. blood administration in China: stabilizing effects and exchange accessing safety and availability. transfer. 2015; 55(10):2523-30], in vitro platelet availability is a potentially effective way to solve this problem.
Currently, differentiation into megakaryocytes and platelets can be induced starting from hematopoietic stem progenitor cells of different origins, such as bone marrow, peripheral blood, umbilical cord blood, human pluripotent stem cells, and the like. Choi et al isolated CD34 from human peripheral blood in 1995+Cells, co-cultured with re-compromised dog serum, produced platelet-like debris from the obtained megakaryocytes, which was the first report on the production of functional human platelets [ Choi ES, Nichol JL, Hokom MM, et al.proteins genetic protein from protein displaying human megakaryocytes arefection.blood 1995; 85(2):402-413]. With the discovery and widespread use of TPO, different groups have successively reported the in vitro culture of CD34 from different sources+Cells that, under different cytokine combinations including TPO, can obtain platelets with ultrastructure similar to platelets in vivo and partial physiological function [ Matsunaga T, Tanaka I, Kobune M, et. 24(12):2877-2887][Norol F,Vitrat N,Cramer E,et al.Effects of cytokines on platelet production from blood and marrow CD34+cells.Blood.1998；91(3):830–843][Lu SJ,Li F,Yin H,et al.Platelets generated from human embryonic stem cells are functional in vitro and in themicrocirculation of living mice.Cell Res.2011,21(3):530-545]. Although the research on the in vitro induction of the stem cells to generate the platelets has been greatly developed, the differentiation efficiency of the megakaryocytes in vitro is low, and the platelet generation efficiency of the megakaryocytes generated by differentiation is far lower than the in vivo generation efficiency, so that the clinical requirements are difficult to meet. Moreover, the high in vitro production costs limit the scale-up of platelets.
The small molecular compound has the characteristics of low price, high efficiency and controllability, can accurately adjust the time sequence of a target signal, and explores the small molecular compound which can effectively promote megakaryodifferentiation and thrombopoiesis so as to open up a new way for obtaining a large number of functional platelets which can be clinically needed. It is well known that the transcription factor arene receptor antagonist StemRegenin 1(SR1) specifically promotes human HS/PCs amplification [ Boitano AE, Wang J, Romeo R, et al, aryl hydrocarbon receptors promoter the expansion of human hematopic cells science 2010; 329(5997):1345-8], whereas during megakaryocyte differentiation, SR1 promotes megakaryocyte maturation and platemaking by maintaining a subpopulation of CD34+ CD41low megakaryocyte precursor cells that are more plateproducing competent [ Strassel C, Brouard N, Mallo L, et al, aryl hydrocarbon receptor-dependent expression of amegakara preventive with a high potential to product precursors.2016; 127(18):2231-40]. Small molecule iBET-151 selectively amplifies human pluripotent stem Cell-derived megakaryocyte precursor cells while inhibiting the amplification of other myeloid lineage cells by inhibiting the activity of c-MYC, thereby promoting late thrombopoiesis [ Feng Q, Shabrani N, Thon JN, et al.Scalable generation of unidentifiable pluripotent stem cells. Stem Cell Rep.2014.3(5): 817) 831 ]. NIP-004 is a non-peptidyl activator of the human TPO receptor c-MPL, and it has been reported that the addition of NIP-004 significantly promotes late megakaryocyte maturation and thrombopoiesis [ Nakamura T, Miyakawa Y, Miyamura A, et al.A novinonpeptidyl human c-MPL activators human antibodies human megakaryopeptides and thrombopoietins.blood.2006; 107(11):4300-7]. Similarly, the selective inhibitor of Src tyrosine kinase SU6656[ Kaminska J, Klimczak-Jajor E, Skierski J, Bany-Laszewicz U.S. effects of inhibitors of Src kinases, SU6656, on differentiation of megakaryocytic promoters and activity of alpha1, 6-fucosyltranferase. 55(3):499-506] and Rho kinase inhibitor Y-27632[ Avanzi MP, Goldberg F, Davila J, et al. Rho kinase inhibition drive megaparkaryocyte polymerization and proplatile formation through MYC and NFE2 downmodulation. Br J Haematol, 2014; 164(6) 867-876 can also be used as an inducer of megakaryocyte differentiation to promote the late polyploidization process of megakaryocytes and the release of functional platelet granules. However, the above-mentioned small molecule compounds act only at a specific stage of megakaryocyte differentiation, and hardly promote cell proliferation, megakaryocyte differentiation and platelet production at the same time, and have a limited effect of promoting the final yield of platelets.
Therefore, it is urgently required to find a compound to solve the above problems.
Disclosure of Invention
In one aspect, the present invention provides the use of a histone methyltransferase inhibitor in the preparation of a product for promoting megakaryocyte proliferation or platelet production, in response to the problems of the lack of a compound and a method for simultaneously promoting cell proliferation, megakaryocyte differentiation and platelet production in the prior art and the low level of platelet production promoted by the compound in the prior art.
The technical scheme provided by the invention is as follows:
use of a histone methyltransferase inhibitor which is an inhibitor of histone methyltransferase G9a in the manufacture of a product for promoting megakaryocyte proliferation or platelet production.
The inventor of the present invention found through creative work that early transient treatment of hematopoietic stem cells with histone methyltransferase G9a inhibitor (EHMTi) can continuously promote proliferation and maturation of megakaryocytes obtained after differentiation, and efficiently obtain activated platelets. Compared with the prior art, the invention has the advantages of more platelets, low application cost, stronger operability and repeatability, and capability of realizing efficient and continuous in-vitro generation of functional platelets.
Histone methyltransferase G9a, also known as euchromosonmlic histone lysine N-methyltransferase 2 (EHMT 2), catalyzes the methylation of lysine 9 (H3K9) and lysine 373(K373) of p53 of histone H3. The G9a mediated histone methylation is closely related to the occurrence and development of tumors, is considered as a novel promising anti-tumor target, and the development and the attention of inhibitors thereof are increased.
Histone methyltransferase G9a inhibitors are largely classified as natural product inhibitors and small molecule inhibitors. Such natural product inhibitors include, but are not limited to, for example, chaetocin and its derivatives (as shown in formula 1), and cinofungin (as shown in formula 2) and its analogs.
Preferably, in one embodiment of the invention, the inhibitor of histone methyltransferase G9a is a small molecule inhibitor of histone methyltransferase G9 a.
The small molecule inhibitors of histone methyltransferase G9a may include, but are not limited to, for example, BIX01294 (as shown in formula 3), UNC0224 (as shown in formula 4), UNC0321 (as shown in formula 5), UNC0638 (as shown in formula 6), UNC0631 (as shown in formula 7), UNC0646 (as shown in formula 8), UNC0737 (as shown in formula 9), UNC0642 (as shown in formula 10), UNC0965 (as shown in formula 11), E67 (as shown in formula 12), E70 (as shown in formula 13), E72 (as shown in formula 14), TM2-115 (as shown in formula 15), 867750 (as shown in formula 16), 867751 (as shown in formula 17), BIX01338 (as shown in formula 18), BRD9539 (as shown in formula 19), BRD4770 (as shown in formula 20), A-366 (as shown in formula 21), HKMTI-1-247 (as shown in formula 22), HKI-1-248 (as shown in formula 23), CPUY 0724), and CPUY 070 (as shown in formula 0725), DCG066 (shown as formula 26) and CBC-12 (shown as formula 27).
Preferably, in one embodiment of the invention, the small molecule inhibitor of histone methyltransferase G9a is one or more selected from a-366, UNC0631, or UNC 0638.
In the above use, the megakaryocytes or the platelets may be derived from CD34 in bone marrow, peripheral blood, umbilical cord blood or human pluripotent stem cells+Differentiation of cells, preferably, in one embodiment of the present invention, the megakaryocytes or the platelets are derived fromCD34 in cord blood+Differentiation of the cells.
The invention also provides a method of promoting megakaryocyte proliferation or platelet production, namely CD34+The histone methyltransferase G9a inhibitor was added at a final concentration of 100-500nM at the differentiation stage of the cells.
The methods described in the present invention are all non-therapeutic.
Preferably, in one embodiment of the invention, the histone methyltransferase G9a inhibitor is added to a final concentration of 200 nM.
The CD34+The differentiation stage of the cells may be any suitable differentiation stage. In one embodiment of the present invention, the inventors studied the effect of adding a histone methyltransferase G9a inhibitor at different stages on megakaryocyte proliferation or platelet production, and as a result, found that the histone methyltransferase G9a inhibitor acts not only at the pre-differentiation stage but also at each stage of differentiation.
Preferably, the histone methyltransferase G9a inhibitor is added at an early stage of megakaryocyte differentiation, i.e., at a proliferation stage of megakaryocytes on days 0 to 6.
Preferably, the above method comprises the steps of:
step 1) isolation of CD34+A cell;
step 2) resuspending the isolated CD34 obtained in step 1) in serum-free medium containing megakaryocyte growth-inducing factor+Cells were treated with the histone methyltransferase G9a inhibitor at a final concentration of 100-500nM, 5% CO at 37 ℃2Culturing for 3 days under the condition;
step 3) replacing the cell culture solution with the serum-free medium containing the megakaryocyte proliferation-inducing factor in step 2), adding the histone methyltransferase G9a inhibitor at a final concentration of 100-500nM, adjusting the cell density to be the same as the initial cell density in step 2), and adjusting the cell density to 5% CO at 37 deg.C2Culturing under the condition until day 6;
step 4) replacing the cell culture solution with serum-free medium containing megakaryocyte maturation inducing factorsMedium, adjusting the cell density to 5 times (2X 10) the initial cell density in step 2) or step 3)5/mL～5×105mL), culture is continued until the desired number of platelets is produced.
Step 1) in the above method is CD34+The cell separation step may be any suitable method for achieving the object of the present invention. Preferably, in one embodiment of the present invention, step 1) may specifically be:
firstly, separating umbilical cord blood mononuclear cells by using Ficoll separating medium, and then carrying out positive sorting twice by using CD34 immunomagnetic beads to obtain high-purity (more than or equal to 90 percent) CD34+Cells, and cell counting.
More preferably, in one embodiment of the present invention, step 1) may specifically be:
A. separating mononuclear cells from cord blood by using Ficoll separating medium, and counting the cells at a rate of 1X 108Adding 300. mu.l of sorting buffer into each cell for resuspension, adding 100. mu.l of FcR blocker and 100. mu.l of CD34 magnetic beads in a dark place, and standing in a refrigerator at 4 ℃ for 30 min;
B. taking out the sample, adding 10ml of sorting buffer to wash out the unbound cells, and rotating at 1200rpm for 5 min; completely removing supernatant, and adding 1ml of sorting buffer for heavy suspension;
C. placing the MS sorting column in a magnetic field corresponding to a MACS sorter, fully wetting the sorting column by using 500 mul sorting buffer, adding the cell suspension into the sorting column through a filter membrane (200 meshes), washing for 3 times by using the buffer, and collecting all cells as much as possible;
D. moving the MS sorting column out of the magnetic field, placing the MS sorting column on a 15ml centrifuge tube, adding a sorting buffer into the sorting column, and pressurizing by using a piston equipped with the MS sorting column to quickly push out the magnetic labeled cells;
E. repeating the above sorting step, and sorting the eluted CD34 with a new MS sorting column+Secondary sorting of the cells to obtain high-purity (more than or equal to 90 percent) CD34+Cells, and cell counting.
Steps 2) to 4) in the above method are cell differentiation steps.
Preferably, in one embodiment of the present invention, the megakaryocyte inducing factor in step 2) or step 3) is human thrombopoietin at a final concentration of 50ng/mL, stem cell factor at a final concentration of 20ng/mL, and interleukin-3 at a final concentration of 20 ng/mL;
the megakaryocyte maturation inducing factors in the step 4) are hTPO with a final concentration of 50ng/mL and IL-11 with a final concentration of 20 ng/mL.
Preferably, in one embodiment of the present invention, the initial cell density in step 1), step 2) or step 3) is 1 × 105one/mL.
Preferably, in an embodiment of the present invention, the step 2) to the step 4) may be specifically:
a. resuspension of CD34 with serum-free medium StemBan SFEM supplemented with human thrombopoietin (hTPO), Stem Cell Factor (SCF) and interleukin-3 (IL-3)+Cells were cultured in a medium supplemented with a histone methyltransferase inhibitor, seeded at an initial density of 1X 105/mL in 12-well plates, and incubated at 37 ℃ with 5% CO2Standing and culturing for 3 days in an incubator;
b. changing the culture solution and cytokine at 3 days, adding histone methyltransferase inhibitor into the original culture medium, and adjusting cell culture density to 1 × 105The solution is subjected to standing culture for 3 days;
c. on day 6, the culture medium and cytokines (hTPO, IL-11) were replaced in total, and the cell culture density was adjusted to 5X 105mL, culturing to 18 days, and changing the solution every 3 days; detection of CD41a every 3 days from day 12 to day 18+CD42b+Platelet particles.
The invention also provides application of the product for promoting the proliferation of megakaryocytes or the production of platelets in preparing products for treating severe blood loss, anemia, immune thrombocytopenia, chronic liver disease combined thrombocytopenia or thrombocytopenia caused by other reasons.
In another aspect of the present invention, a pharmaceutical composition comprising the megakaryocytes or platelets prepared by the above method is provided.
The invention has the beneficial effects that:
in the present inventionHistone methyltransferase G9a inhibitors may promote a single CD34+Differentiation of hematopoietic stem progenitor cells yields about 400 CD41a+CD42b+The platelet particles are improved by more than 10 times compared with a control group, the platelet generation efficiency is obviously higher than the general range reported in the literature, the processing time is short, the operability and the repeatability are strong, the production cost of the platelets is obviously reduced, and the foundation is laid for the large-scale production of functional platelets for clinical treatment in the future.
Drawings
FIG. 1 is a graph of single CD34 after treatment with histone methyltransferase inhibitor added in an example of the present invention+Cell proliferation profiles of the inoculated cells;
FIG. 2 is a graph of single CD34 after treatment with histone methyltransferase inhibitor+Generating a kinetic map of megakaryocyte numbers of the seeded cells;
FIG. 3 is a graph of single CD34 after treatment with histone methyltransferase inhibitor+A kinetic map of the number of platelet particles produced from the seeded cells;
FIG. 4 is a graph of the ratio of platelet counts after treatment with histone methyltransferase inhibitors at different windows in an example of the invention.
Detailed Description
The invention discloses application of a histone methyltransferase inhibitor in preparing a product for promoting megakaryocyte proliferation or platelet production, and the method can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. The main terms appearing in the present invention are explained below.
The term "megakaryocyte" is a cell in the bone marrow differentiated from hematopoietic stem cells, and is a mature cell in normal bone marrow capable of producing platelets, the precursor of which is a granular megakaryocyte. The cells are large in size, and platelets are formed after the edge parts of mature megakaryocytes break and fall off. On average, about 2000 platelets are produced per megakaryocyte.
The term "platelet" is a small cytoplasm that is shed from the cytoplasm of a mature megakaryocyte of the bone marrow. When blood is lost due to vascular trauma, the functional activity of platelets in the physiological hemostasis process can be roughly divided into two stages, namely, the first stage is mainly that after trauma occurs, the platelets are rapidly adhered to the trauma and are aggregated into clusters to form softer hemostatic emboli; the second segment is primarily intended to promote blood clotting and form a firm hemostatic plug.
The term "histone methyltransferase G9a," also known as euchromosonic histone lysine N-methyltransferase 2 (EHMT 2), catalyzes the methylation of lysine 9 (H3K9) and lysine 373(K373) of p53 of histone H3. The G9a mediated histone methylation is closely related to the occurrence and development of tumors, is considered as a novel promising anti-tumor target, and the development and the attention of inhibitors thereof are increased.
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1:
1) cord blood CD34+Isolation of cells
a. Pouring the cord blood into a plasma bottle or a 50ml centrifugal tube according to VUmbilical cord blood:VSorting bufferAdding sorting buffer (formula shown below) at a ratio of 1:1, adding the hydroxyethyl starch with the mixed volume of 1/4, mixing, and standing at room temperature for 45 min;
sorting buffer formula: phosphate Buffered Saline (PBS), 0.5% Bovine Serum Albumin (BSA), 0.4% 500mM ethylenediaminetetraacetic acid (EDTA), 1% streptomycin mixture (P/S)
b. The supernatant was transferred to 50Centrifuging the solution in a ml centrifuge tube at room temperature and 1500rpm for 10 min; discarding the supernatant, and adding a sorting buffer to resuspend the cells; taking 15ml centrifuge tubes, adding 4.5ml Ficoll separating medium (V) into each tubeFicoll:VSorting buffer1:1), slowly adding the heavy suspension to the Ficoll separating medium to avoid breaking the liquid level stratification, 1800rpm, 15min (setting the centrifuge to 0 liter and 0 fall);
c. discarding the upper water sample, carefully absorbing the leucocyte layer (mononuclear cell layer), adding a sorting buffer for dilution at 1200rpm for 5 min; discarding the supernatant, adding sorting buffer to resuspend the cells, taking 10 μ l (diluted by 10-100 times) for counting the cells, and centrifuging the rest suspension at 1200rpm for 5 min;
d. discard the supernatant, every 1X 108Adding 300. mu.l of sorting buffer into each cell for resuspension, adding 100. mu.l of FcR blocker and 100. mu.l of CD34 magnetic beads in a dark place, and standing in a refrigerator at 4 ℃ for 30 min;
e. taking out the sample, adding 10ml of sorting buffer to wash out the unbound cells, and rotating at 1200rpm for 5 min; completely removing supernatant, and adding 1ml of sorting buffer for heavy suspension;
f. placing the MS sorting column in a magnetic field corresponding to a MACS sorter, fully wetting the sorting column by using 500 mul sorting buffer, adding the cell suspension into the sorting column through a filter membrane (200 meshes), washing for 3 times by using the sorting buffer, and collecting all cells as much as possible;
g. moving the MS sorting column out of the magnetic field, placing the MS sorting column on a 15ml centrifuge tube, adding a sorting buffer into the sorting column, and rapidly pushing out the magnetic labeled cells by using a piston matched with the MS sorting column under pressure;
h. repeating the above sorting step, and sorting the eluted CD34 with a new MS sorting column+Secondary sorting of the cells to obtain high-purity (more than or equal to 90 percent) CD34+Cells, and cell counting.
2) Cell culture
a. Resuspension of CD34 with serum-free medium StemBan SFEM supplemented with human thrombopoietin (hTPO, final concentration 50ng/mL), stem cell factor (SCF, final concentration 20ng/mL) and interleukin-3 (IL-3, final concentration 20ng/mL)+Adding histone methyltransferase inhibitor (one or more selected from A-366, UNC0631 or UNC0638, concentration is 200nM) at 1X 10 to the original culture medium5Initial density of one/mL was inoculated in 12-well plates at 37 ℃ with 5% CO2Standing and culturing for 3 days in an incubator;
b. on day 3, the culture medium and cytokines were replaced, and histone methyltransferase inhibitor (one or more selected from A-366, UNC0631 and UNC0638 at 200nM) was added to the original culture medium to adjust the cell culture density to 1X 105mL, and the static culture was continued for 3 days.
c. On day 6, the culture medium and cytokines (hTPO, final concentration 50 ng/mL; IL-11, final concentration 20ng/mL) were all replaced, and the cell culture density was adjusted to 5X 105mL, culturing to 18 days, and changing the solution every 3 days; detection of CD41a every 3 days from day 12 to day 18+CD42b+Platelet particles.
Experimental example 1: cell proliferation assay
The cells at each stage in example 1 were counted, and different control groups were set.
a. The control group (DMSO) was cultured under the same conditions as the experimental group (EHMTi), and the effect of the small molecule compound on cell proliferation was examined by cell counting every three days.
b. As shown in fig. 1, compared to the DMSO control group, the treatment with histone methyltransferase inhibitor (a366) added on days 0 to 3 or days 3 to 6 of the culture significantly promoted cell proliferation, and the fold of cell expansion was increased by 15 times or more.
In FIG. 1, the total number of cells in the control group (DMSO group) was decreased at days 9-18 of megakaryocyte differentiation; after the histone methyltransferase inhibitor is added for treatment, the total number of cells still shows an ascending trend on the 9 th to 12 th days of megakaryocyte differentiation, the total number of cells of an experimental group is increased by about 15 times compared with that of a control group on the 12 th day of megakaryocyte differentiation, and the total number of cells does not begin to decline until the 15 th to 18 th days of megakaryocyte differentiation, but is still higher than that of the control group.
Experimental example 2: megakaryocyte flow assay
The megakaryocytes in example 1 were examined.
a. Approximately 1X 10 at days 3, 6, 9 and 12 of megakaryocyte-induced differentiation5The cell suspension was centrifuged at 300g for 5min and the cell pellet resuspended with 100. mu.L of 0.2% BSA.
b. Adding 1 μ L anti-CD41a-APC (BD) and 1 μ L anti-CD42b-PE (BD) flow antibody to each group, incubating in dark for 30min, and detecting CD41a with flow cytometer (FACS Canto II; BD Biosciences)+CD42b+The proportion of megakaryocytes.
Experimental example 3: flow assay for platelet particles
Platelet particles from example 1 were tested.
a. Cell suspensions were collected on days 12, 15, and 18 of culture, centrifuged at 300g for 5min, and 200. mu.L of supernatant was collected.
b. mu.L of anti-CD41a-APC (BD) and 1. mu.L of anti-CD42b-PE (BD) flow antibody were added to each group, incubated for 30min in the dark, and then tested on a computer (FACS Canto II; BD Biosciences).
c. And (4) performing flow type result analysis by taking platelets from normal peripheral blood as a positive control gate.
Experimental example 4: megakaryocyte and platelet quantification
a. Calculating the number of megakaryocytes and platelets produced by the single human pluripotent stem cells according to the flow analysis result, wherein the calculation formula is as follows: megakaryocyte production (day (t) cell number/day (0) cell number × CD41a+CD42b+Percentage of megakaryocytes; platelet production by day (t) number of particles × CD41a+CD42b+Percent platelets/day (t) megakaryocyte count CD41a+CD42b+Megakaryocyte yield.
b. As shown in FIG. 2, the addition of histone methyltransferase inhibitor (A366) significantly promoted CD41a compared to the DMSO-added group+CD42b+Megakaryocytopoiesis.
Dynamic monitoring shows that the total number of megakaryocytes reaches a peak value at the 12 th day of megakaryocyte differentiation; the treatment with the addition of histone methyltransferase inhibitor resulted in an increase of the total number of megakaryocytes by about 6-fold compared with the control group (DMSO group).
c. As shown in FIG. 3, the addition of histone methyltransferase inhibitor (A366) significantly increased CD41a during the platelet production phase+CD42b+Yield of active platelets.
Dynamic monitoring shows that the platelet yield of the histone methyltransferase inhibitor treated group is maintained at a higher level on the 12 th to 18 th days of megakaryocyte differentiation; platelet production increased nearly 10-fold by day 15 of megakaryocyte differentiation compared to the control group (DMSO group).
Experimental example 5: action window experiment
Platelet particles from example 1 were tested.
a. The platelet yield of each group was measured by adding histone methyltransferase inhibitor (A366) to each of the culture days 0-3, 3-6, 6-9, 9-12, 12-15 and 0-15, and setting the control group (DMSO) and megakaryocyte differentiation day 18.
b. The results are shown in fig. 4, compared with the DMSO control group, the addition of histone methyltransferase inhibitor (a366) at days 0-3 or days 3-6 of culture increased platelet production by nearly 30-fold, which is substantially the same as the full-course (days 0-15) addition effect; the platelet yield is improved by more than 10 times by adding histone methyltransferase inhibitor (A366) in the culture days 6-9; the treatment of adding histone methyltransferase inhibitor (A366) on the 9 th-12 th and 12 th-15 th days of culture has no obvious promotion effect on the yield of the platelet.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. Use of a histone methyltransferase inhibitor for the manufacture of a product for promoting megakaryocyte proliferation or platelet production, wherein the histone methyltransferase inhibitor is an inhibitor of histone methyltransferase G9 a.
2. The use according to claim 1, wherein said megakaryocytes or said platelets are derived from CD34 in bone marrow, peripheral blood, cord blood, or human pluripotent stem cells+Differentiation of the cells, preferably CD34 in cord blood+Differentiation of the cells.
3. Use according to claim 1 or 2, wherein the inhibitor of histone methyltransferase G9a is a small molecule inhibitor of histone methyltransferase G9 a.
4. Use according to claim 3, wherein the small molecule inhibitor of histone methyltransferase G9a is one or more selected from A-366, UNC0631 or UNC 0638.
5. The use according to claim 1, 2 or 4, wherein the method of promoting megakaryocyte proliferation or platelet production is CD34+Adding the histone methyltransferase G9a inhibitor with the final concentration of 100-500nM in the differentiation stage of the cells, wherein the method is used for non-treatment purposes;
the final concentration is preferably 200 nM.
6. Use according to claim 5, characterized in that the method comprises the following steps:
step 1) isolation of CD34+A cell;
step 2) resuspending the isolated CD34 obtained in step 1) in serum-free medium containing megakaryocyte growth-inducing factor+Cells were treated with the histone methyltransferase G9a inhibitor at a final concentration of 100-500nM, 5% CO at 37 ℃2Culturing for 3 days under the condition;
step 3) replacing the cell culture solution with the serum-free medium containing the megakaryocyte proliferation-inducing factor in step 2), adding the histone methyltransferase G9a inhibitor at a final concentration of 100-500nM, adjusting the cell density to be the same as the initial cell density in step 2), and adjusting the cell density to 5% CO at 37 deg.C2Culturing under the condition until day 6;
step 4) replacing the cell culture solution obtained in the step 3) with a serum-free culture medium containing megakaryocyte maturation inducing factors, and adjusting the cell density to 5 times (2X 10) of the initial cell density in the step 2) or the step 3)5/mL～5×105mL), culture is continued until the desired number of platelets is produced.
7. The use according to claim 6, wherein the megakaryocyte proliferation-inducing factor of step 2) or step 3) is human thrombopoietin at a final concentration of 50ng/mL, stem cell factor at a final concentration of 20ng/mL, and interleukin-3 at a final concentration of 20 ng/mL;
the megakaryocyte maturation inducing factors in the step 4) are hTPO with a final concentration of 50ng/mL and IL-11 with a final concentration of 20 ng/mL.
8. The use according to claim 6, wherein the initial cell density in step 1), step 2) or step 3) is 1 x 105one/mL.
9. The use according to claim 1, 2 or 4, wherein the product for promoting megakaryocyte proliferation or platelet production is used for treating severe blood loss, anemia, immune thrombocytopenia, chronic liver disease with thrombocytopenia or thrombocytopenia due to other causes.
10. A pharmaceutical composition comprising megakaryocytes or platelets produced by the method of any one of claims 5-8.
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