A method for promoting growth of hematopoietic stem cells

Abstract

The application discloses a method for promoting growth of hematopoietic stem cells, comprising the following steps: S101, constructing Nov gene recombinant Nov-MSC, and expressing Nov protein on the surface of MSC; S102, preparing Nov-MSC exosome Nov-MSC-EXO by using the Nov-MSC; and S103, stimulating HSC by using the Nov-MSC or the Nov-MSC-EXO, so as to promote growth of the HSC. The Nov gene recombinant Nov-MSC is prepared, so that the Nov protein does not need to be prepared separately, the preparation process of reagents is greatly simplified, and a better effect is obtained than that of using MSC or the Nov protein alone or using the MSC and the Nov protein in combination; and the Nov-MSC-EXO has similar biological activity to the Nov-MSC.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for promoting the growth of hematopoietic stem cells (HSCs). Background Technology

[0002] Hematopoietic stem cell (HSC) transplantation (HSCT) is one of the most effective treatments for various hematological diseases. HSCs used for transplantation are typically derived from bone marrow, peripheral blood, or umbilical cord blood. Compared to bone marrow and peripheral blood-derived HSCs, umbilical cord blood HSCs have advantages such as abundant and readily available sources, relatively lower HLA matching requirements, and a lower risk of graft-versus-host disease (GVHD). However, a problem is that umbilical cord blood contains a lower number of total nucleated cells (TNCs), leading to lower CD34 counts. + Insufficient HSC numbers typically lead to delayed chimerism of neutrophils and platelets after transplantation, making it unsuitable for older children and adults, severely impacting the application and efficacy of HSCT. Therefore, scientists in this field have been searching for methods to effectively increase umbilical cord blood hematopoietic stem cells (CB-HSCs); these include co-culture with bone marrow mesenchymal stem cells (BM-MSCs) and combined cytokine culture.

[0003] BM-MSCs are mesodermal-derived adult stem cells and an important component of the bone marrow hematopoietic microenvironment. Studies have shown that BM-MSCs can secrete or express a large number of hematopoietic regulatory factors, thereby regulating the functional characteristics of HSCs. Furthermore, researchers successfully used BM-MSCs as a feeder layer to co-culture CB-HSCs, expanding CB-HSCs in vitro. Compared with composite growth factor culture systems, co-culturing with CB-MSCs significantly improved the expansion efficiency of CB-HSCs, increasing their number while maintaining their "stemness."

[0004] The therapeutic effects of MSCs are primarily attributed to their paracrine functions. It is now recognized that, in addition to soluble factors, MSCs can secrete large amounts of microvesicles, or exosomes (EXOs). EXOs are important mediators of intercellular communication, consisting of a mixture of vesicular organelle-like structures released from different cell types. Proteins, lipids, mRNA, and miRNA derived from parental cells are selectively packaged within EXOs and transferred between cells via EXOs, mediating the programming of target cells. Increasing evidence suggests that EXOs play a crucial role in a wide range of physiological and pathological processes. Ratajczak et al. found that Exo derived from embryonic stem cells significantly promoted the in vitro expansion of HSCs and upregulated the expression of early pluripotency and early HSC markers, suggesting that Exo may be an important factor regulating HSC function (J. Ratajczak, K. Miekus, M. Kucia et al. Embryonic stem cell derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia, vol. 20, no. 5, pp. 847–856, 2006). Furthermore, data from both in vitro and in vivo experiments indicate that MSC-derived MSC-EXO is a potential key mediator of MSC biological function (TNLamichhane, S. Sokic, J.S. Schhardt, R.S. Raiker, J.W. Lin, and S.M. Jay. Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine. Tissue Engineering - Part B: Reviews, vol. 21, no. 1, pp. 45–54, 2015). The therapeutic effects of MSC-EXO have been demonstrated in several animal models of tissue injury (Y.-G. Zhu, X.-M. Feng, J. Abbott et al. Human mesenchymal stem cell microvesicles for treatment of Escherichia coliendotoxin-induced acute lung injury in mice. Stem Cells, vol. 32, no. 1, pp. 116–125, 2014).These studies found that the therapeutic effects of MSC-EXO are comparable to those of their parent MSCs. Therefore, MSC-EXO can be used to expand CB-HSCs in vitro, with the effectiveness depending on the quality of the MSCs.

[0005] Among numerous cytokines, the Nov / CCN3 protein can rapidly increase the number of hematopoietic stem cells in a single cord blood unit. Studies using cell cultures and mouse models have found that cord blood units exposed to the Nov protein exhibit more transplantable properties than conventional samples, increasing the number of hematopoietic stem cells several times over (Rajeev Gupta, Virginia Turati, Duncan Brian, et al. Nov / CCN3 Enhances Cord Blood Engraftment by Rapidly Recruiting Latent Human Stem Cell Activity, Cell StemCell (2020)).

[0006] DOI:10.1016 / j.stem.2020.02.012).

[0007] In summary, MSCs and compound cytokines are two different means of promoting HSC growth, each with its own role, especially the role of Nov / CCN3 protein, which deserves further investigation. However, in actual experiments using MSCs and Nov / CCN3 protein, the inventors found that the process for preparing high-quality Nov / CCN3 protein is quite complicated, difficult, and costly, which greatly limited the experimental progress. Summary of the Invention

[0008] The main objective of this invention is to provide a method for promoting the growth of hematopoietic stem cells (HSCs). The inventors prepared recombinant MSCs (Nov-MSCs) using the Nov gene themselves. Therefore, not only is it not necessary to prepare Nov protein separately, which greatly simplifies the reagent preparation process, but it also produces better results than using MSCs or Nov protein alone, or using MSCs and Nov protein in combination.

[0009] To achieve the above objectives, the present invention proposes a method for promoting HSC growth, comprising the following steps:

[0010] S101: Construct Nov gene recombinant MSCs (Nov-MSCs) and express Nov proteins on the surface of MSCs;

[0011] S102: Preparation of Nov-MSC exosomes (Nov-MSC-EXO) using Nov-MSCs;

[0012] S103: Stimulation of HSCs with Nov-MSCs and Nov-MSC-EXO can promote HSC growth.

[0013] Preferably, in step S101, the process for preparing recombinant Nov-MSCs includes:

[0014] S1011: Preparing MSC: Taking each well of a 6-well plate as an example, add an appropriate amount (5×10) 5 MSCs are suspended in 2 ml of culture medium and seeded into one well of a 6-well plate. Transfection is performed when the cell density reaches 50-80% confluency.

[0015] S1012: Prepare transfection reagents: Add 4 μgp D-Nov plasmid DNA to 600 μl Opti-MEM low serum culture medium and mix well; gently mix Lipofectamine LTX reagent, take 5 μl and add it to the DNA tube; gently mix and incubate at room temperature for 30 min.

[0016] S1013: Add 100 μL of DNA-LipofectamineLTX complex to each well of cells and gently shake the culture plate back and forth and side to side to mix.

[0017] S1014: Incubate at 37°C in a 5% CO2 incubator for 24 hours. Take the transfected Nov-MSCs and add mouse anti-human Nov (Abcam) monoclonal antibody. Incubate at 37°C for 1 hour. After washing twice, add FITC-labeled rabbit anti-mouse secondary antibody (Abcam). Incubate at 30°C for 30 minutes. After washing twice, suspend in 50 μL of physiological saline.

[0018] S1015: Fluorescence microscopy (Olympus) observation results show that the positive rate of Nov protein is approximately 55%.

[0019] S1016: Monoclonal Nov-MSCs were prepared using the limiting dilution method and stored in liquid nitrogen for later use.

[0020] Preferably, in S101, the Nov protein is expressed on the MSC surface by linking the 3' end of the Nov gene to the PDGFR transmembrane polypeptide gene on the pDisplay expression vector.

[0021] Preferably, in S101, the Nov protein gene clone is included, specifically including:

[0022] 1.1. Primer Design

[0023] 1) Primer 1: Nov-upSacII:gacgtcCCGCGGatgcagagtgtgcagagcacg;

[0024] 2) Primer 2: Nov-DnSalI:gacgtcGTCGACcattttccctctggtagtctt;

[0025] 1.2. RNA preparation: 50 ml of peripheral blood was collected from volunteers, lymphocytes (PBMCs) were separated using lymphocyte separation medium, and total RNA was prepared using TRIzol reagent;

[0026] 1.3.RT-PCR: Using total RNA as a template and primers 1 and 2 as primers, the Nov gene fragment was prepared using the Invitrogen SuperScript IV one-step RT-PCR kit and purified using the ESSpin column PCR product purification kit.

[0027] 1.4. Restriction endonuclease digestion of pDisplay vector and PCR products

[0028] 1) The pDisplay vector was digested with SacII / SalI restriction endonuclease, followed by agarose gel electrophoresis. The gel containing the digested pDisplay vector DNA fragment was excised and purified with EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain the pDisplayBglII / SalI digested vector product.

[0029] 2) The Nov gene fragment was digested with SacII / SalI restriction endonuclease, followed by agarose gel electrophoresis. The gel containing the digested Nov gene DNA fragment was excised and purified using the EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain the Nov gene BglII / SalI fragment digested gene product.

[0030] 3) Ligation: The pDisplaySacII / SalI and Nov gene SacII / SalI fragments were ligated using DNA ligase to obtain the pD-Nov product;

[0031] 4) Transfection of competent cells: Transfect DH5α competent cells with pD-Nov product;

[0032] 5) Colony selection and verification: Select positive colonies, culture them in small quantities, extract plasmid DNA, perform initial selection by digestion with the corresponding restriction endonuclease, confirm by sequencing, and preserve the pD-Nov recombinant bacteria;

[0033] 6) Cultivate pD-Nov recombinant bacteria and prepare pD-Nov recombinant plasmids.

[0034] Preferably, S102 includes:

[0035] S1021. Culture of MSCs and Nov-MSCs

[0036] S1022. Exosomes in cell culture supernatant were enriched using LifeTechnology Total Exosome Isolation (TEI) kit;

[0037] 1) Collect approximately 1000 ml of MSC and Nov-MSC stem cell culture supernatant respectively, aliquot them into 50 ml conical centrifuge tubes, and centrifuge at 2000Xg for 30 minutes;

[0038] 2) Transfer the supernatant to a new centrifuge tube, add 0.5 times the volume of TEI reagent, mix thoroughly, incubate at 4°C overnight, and centrifuge at 10,000Xg for 1 hour the next day;

[0039] 3) Aspirate the supernatant and suspend the precipitate in 5 ml of PBS;

[0040] 4) Centrifuge at 10,000Xg for 1 hour, aspirate the supernatant, suspend the precipitate in PBS, and adjust the concentration to 1mg / ml, which is an exosome with an 80-150nm wavelength.

[0041] S1023. Exosomes are aliquoted and stored at -20°C for later use.

[0042] Preferably, step S103 includes the preparation of HSCs and the amplification of HSCs.

[0043] This invention also discloses a method for promoting the growth of hematopoietic stem cells (HSCs). The inventors prepared MSCs with recombinant Nov genes (Nov-MSCs), which not only eliminates the need to prepare Nov protein separately, but also greatly simplifies the reagent preparation process. Moreover, it produces better results than using MSCs or Nov protein alone, or using MSCs and Nov protein in combination. Nov-MSC-EXO has similar biological activity to Nov-MSCs. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0045] Figure 1 A technical roadmap for a method to promote the growth of hematopoietic stem cells (HSCs) provided in an embodiment of the present invention;

[0046] Figure 2This is a schematic diagram of the structure of the pDisplay expression vector provided in an embodiment of the present invention.

[0047] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0049] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0050] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0051] In this embodiment of the invention, reference is made to Figure 1 The method for promoting the growth of hematopoietic stem cells (HSCs) includes:

[0052] 1. Construction of recombinant Nov-MSC cells

[0053] 1.1 This invention discloses a method for improving the quantity and quality of HSCs, one of which is recombinant Nov-MSCs. The source of the MSCs is not limited, and can be bone marrow MSCs (BM-MSCs), umbilical cord MSCs (UC-MSCs), umbilical cord blood MSCs (UCB-MSCs), adipose tissue MSCs (Ad-MSCs), dental pulp MSCs (DentalPulpMSCs, Dp-MSCs), etc.

[0054] 1.2. The Nov protein consists of 357 amino acids, and its sequence is shown in Sequence 1.

[0055] Sequence 1: Nov protein amino acid sequence

[0056] MQSVQSTSFCLRKQCLLCLTFLLLHLLGQVAATQRCPPQCPGRCPATPPTCAPGVRAVLDGCSCCLVCARQRGESCSDLEPCDESSGLYCDRSADPSNQTGICTAVEGDNCVFDGVIYRSGEKFQPSCKFQCTCRDGQIGCVPRCQLDVLLPEPNCPAPRKVEVPGECCEKWICGPDEED SLGGLTLAAYRPEATLGVEVSDSSVNCIEQTTEWTACSKSCGMGFSTRVTNRNRQCEMLKQTRLCMVRPCEQEPEQPTDKKGKKCLRTKKSLKAIHLQFKNCTSLHTYKPRFCGVCSDGRCCTPHNTKTIQAEFQCSPGQIVKKPVMVIGTCTCHTNCPKNNEAFLQELELKTTRGKM*

[0057] 1.3. The gene for the Nov protein may have one of the following DNA sequences:

[0058] 1) As shown in sequence 2, DNA consists of 1071 bases, and its coding sequence is from the 1st to the 1059th base from the 5' end;

[0059] 2) A DNA sequence that has more than 90% homology with the DNA sequence defined by sequence 2 and encodes the amino acid sequence of sequence 1;

[0060] 3) A nucleotide sequence that can hybridize with the DNA defined by sequence 2 under highly stringent conditions (washing the membrane at 65°C after hybridization with a solution containing 0.1X SSPE or 0.1X SSC, 0.1% SDS).

[0061] Sequence 2: DNA sequence of the Nov protein

[0062] atgcagagtgtgcagagcacgagcttttgtctccgaaagcagtgcctttgcctga

[0063] ccttcctgcttctccatctcctgggacaggtcgctgcgactcagcgctgccctcc

[0064] ccagtgcccgggccggtgccctgcgacgccgccgacctgcgcccccggggtgcgc

[0065] gcggtgctggacggctgctcatgctgtctggtgtgtgcccgccagcgtggcgaga

[0066] gctgctcagatctggagccatgcgacgagagcagtggcctctactgtgatcgcag

[0067] cgcggaccccagcaaccagactggcatctgcacggcggtagagggagataactgt

[0068] gtgttcgatggggtcatctaccgcagtggagagaaatttcagccaagctgcaaat

[0069] tccagtgcacctgcagagatgggcagattggctgtgtgccccgctgtcagctgga

[0070] tgtgctactgcctgagcctaactgcccagctccaagaaaagttgaggtgcctgga

[0071] gagtgctgtgaaaagtggatctgtggcccagatgaggaggattcactgggaggcc

[0072] ttacccttgcagcttacaggccagaagccaccctaggagtagaagtctctgactc

[0073] aagtgtcaactgcattgaacagaccacagagtggacagcatgctccaagagctgt

[0074] ggtatggggttctccacccgggtcaccaataggaaccgtcaatgtgagatgctga

[0075] aacagactcggctctgcatggtgcggccctgtgaacaagagccagagcagccaac

[0076] agataagaaaggaaaaaagtgtctccgcaccaagaagtcactcaaagccatccac

[0077] ctgcagttcaagaactgcaccagcctgcacacctacaagcccaggttctgtgggg

[0078] tctgcagtgatggccgctgctgcactccccacaataccaaaaccatccaggcaga

[0079] gtttcagtgctccccagggcaaatagtcaagaagccagtgatggtcattgggacc

[0080] tgcacctgtcacaccaactgtcctaagaacaatgaggccttcctccaggagctgg

[0081] agctgaagactaccagagggaaaatgtaa

[0082] 1.4. Cell membrane surface expression vectors

[0083] Expression of Nov proteins on the cell membrane surface is one of the core methods of this invention. This special structure, on the one hand, adds new functions to Nov proteins in MSCs, and on the other hand, MSCs provide a solidified surface for Nov proteins to interact, which is beneficial for Nov proteins to better exert their role in improving the quantity and quality of HSCs. Figure 2 The diagram shows the structure of the pDisplay expression vector. This invention expresses the Nov protein on the surface of MSCs by linking the 3' end of the Nov gene to the PDGFR transmembrane polypeptide gene on the pDisplay expression vector. However, this invention is not limited to this transmembrane region and this vector; any other expression vector with a transmembrane region structure is within the scope of this invention.

[0084] 2. Preparation of umbilical cord MSCs (UC-MSCs)

[0085] The umbilical cord was taken from a healthy pregnant woman who underwent a full-term cesarean section. The pregnant woman tested negative for HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-syphilis antibody, mycoplasma, and anti-cytomegalovirus antibody. All of the pregnant woman's prenatal examinations, including TORCH and Down syndrome screening, were normal.

[0086] Under aseptic conditions, collect the umbilical cord, cut it into small segments, each about 1 cm long, remove the arteries and veins (two arteries and one vein), cut the segments into small pieces in a glass bottle, and place them in 15 ml centrifuge tubes (each tube contains 2 segments, about 2 cm long). Add 8 ml of collagenase (type IV) for digestion, and after digestion for 3 hours, filter through a 100-mesh filter (or centrifuge at 500 rpm for 3-5 minutes). Discard the undigested tissue pieces, transfer the supernatant to 15 ml centrifuge tubes, about 5 ml per tube, add PBS to 10 ml, centrifuge at high speed (3000-4000 rpm), discard the supernatant, and transfer the precipitate to a culture dish for incubation. Incubate at 37°C in a 5% CO2 saturated humidity incubator in STEMPROhMSCSFM—human mesenchymal stem cell culture medium. Change half of the medium after 48 hours, and change the entire medium after 4 days, discarding a large number of suspended cells.

[0087] Change the medium every 3-4 days according to the color of the medium. Once the primary cells reach 60-80% coverage of the culture dish, passage them at a 1:2 ratio.

[0088] Fourth-generation UC-MSCs were cryopreserved and subjected to testing and identification.

[0089] Identification and usage criteria of UC-MSCs: Use cells from passages 3-10, when the cell number reaches 102 8 After rinsing twice with PBS, 0.05% Ttypsin-EDTA solution was added for digestion, followed by the addition of culture medium to terminate the digestion, yielding a product of 1-3 × 10⁻⁶. 6 The cell suspension was analyzed by flow cytometry and the results showed that: CD14 or CD11b, CD79a or CD19, CD34, CD45, CD109, and HLA-DR were negative <2%; the positive rates of CD29, CD44, CD73, CD90, and CD105 were all >95%; and EB virus, cytomegalovirus, HIV, hepatitis B virus, mycoplasma, bacterial culture, and fungal culture were all negative.

[0090] If all the above test indicators are normal, the sample can be used directly as a UC-MSC or stored in liquid nitrogen for future use.

[0091] 3. Cloning of Nov protein genes

[0092] 3.1. Primer Design

[0093] 1) Primer 1: Nov-upSacII:gacgtcCCGCGGatgcagagtgtgcagagcacg;

[0094] 2) Primer 2: Nov-DnSalI:gacgtcGTCGACcattttccctctggtagtctt;

[0095] 3.2. RNA preparation: 50 ml of peripheral blood was collected from volunteers, lymphocytes (PBMCs) were separated using lymphocyte separation medium, and total RNA was prepared using TRIzol reagent;

[0096] 3.3.RT-PCR: Using total RNA as a template and primers 1 and 2 as primers, the Nov gene fragment was prepared using the Invitrogen SuperScript IV one-step RT-PCR kit and purified using the ESSpin column PCR product purification kit.

[0097] 3.4. Restriction endonuclease digestion of pDisplay vector and PCR products

[0098] 1) The pDisplay vector was digested with SacII / SalI restriction endonuclease, followed by agarose gel electrophoresis. The gel containing the digested pDisplay vector DNA fragment was excised and purified with EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain the pDisplayBglII / SalI digested vector product.

[0099] 2) The Nov gene fragment was digested with SacII / SalI restriction endonuclease, followed by agarose gel electrophoresis. The gel containing the digested Nov gene DNA fragment was excised and purified using the EZ-10 Spin Column DNA PAGE Gel Extraction Kit to obtain the Nov gene BglII / SalI fragment digested gene product.

[0100] 3) Ligation: The pDisplaySacII / SalI and Nov gene SacII / SalI fragments were ligated using DNA ligase to obtain the pD-Nov product;

[0101] 4) Transfection of competent cells: Transfect DH5α competent cells with pD-Nov product;

[0102] 5) Colony selection and verification: Select positive colonies, culture them in small quantities, extract plasmid DNA, perform initial selection by digestion with the corresponding restriction endonuclease, confirm by sequencing, and preserve the pD-Nov recombinant bacteria;

[0103] 6) Cultivate pD-Nov recombinant bacteria and prepare pD-Nov recombinant plasmids.

[0104] 4. Preparation of recombinant Nov-MSCs

[0105] 4.1. Preparing MSC: Taking each well of a 6-well plate as an example, add an appropriate amount (5×10) 5 MSCs are suspended in 2 ml of culture medium and seeded into one well of a 6-well plate. Transfection is performed when the cell density reaches 50-80% confluency.

[0106] 4.2. Preparation of transfection reagents: Add 4 μg pD-Nov plasmid DNA to 600 μL Opti-MEM low serum culture medium and mix well; gently mix Lipofectamine LTX reagent, take 5 μL and add it to the DNA tube; gently mix and incubate at room temperature for 30 min;

[0107] 4.3. Add approximately 100 μL of DNA-Lipofectamine LTX complex to each well of cells, and gently shake the culture plate back and forth and side to side to mix.

[0108] 4.4.37℃, 5% CO2 incubator culture, after 24 hours take the transfected Nov-MSCs, add mouse anti-human Nov (Abcam) monoclonal antibody, incubate at 37℃ for 1 hour, wash twice, add FITC fluorescently labeled rabbit anti-mouse second antibody (Abcam), incubate at 30℃ for 30 minutes, wash twice, and then suspend in 50ul physiological saline.

[0109] 4.5. Fluorescence microscopy (Olympus) observation results showed that the positive rate of Nov protein was approximately 55%.

[0110] 4.6. Nov-MSC cells were cultured in STEMPRO hMSC SFM—human mesenchymal stem cell culture medium, and monoclonal Nov-MSCs were prepared by limiting dilution method and stored in liquid nitrogen for later use.

[0111] 5. Preparation of MSC-EXO and Nov-MSC-EXO

[0112] 5.1. Culture of MSCs and Nov-MSCs

[0113] 5.2. Exosomes in cell culture supernatant were enriched using Life Technology's Total Exosome Isolation (TEI) kit;

[0114] 1) Collect approximately 1000 ml of MSC and Nov-MSC stem cell culture supernatant respectively, aliquot them into 50 ml conical centrifuge tubes, and centrifuge at 2000Xg for 30 minutes;

[0115] 2) Transfer the supernatant to a new centrifuge tube, add 0.5 times the volume of TEI reagent, mix thoroughly, incubate at 4°C overnight, and centrifuge at 10,000Xg for 1 hour the next day;

[0116] 3) Aspirate the supernatant and suspend the precipitate in 5 ml of PBS;

[0117] 4) Centrifuge at 10,000Xg for 1 hour, aspirate the supernatant, suspend the precipitate in PBS, and adjust the concentration to 1mg / ml, which is an exosome with an 80-150nm wavelength.

[0118] 5.3. After aliquoting, exosomes are stored at -20℃ for later use.

[0119] 6. Functional determination of Nov-MSC and Nov-MSC-EXO

[0120] 6.1. Preparation of HSC

[0121] 1) Collection of umbilical cord blood for mononuclear cell (MNC) isolation: Umbilical cord blood is collected from the umbilical cord of a healthy full-term fetus delivered vaginally; after centrifugation, the white membrane layer is collected, and mononuclear cells are isolated using human lymphocyte separation solution and counted for later use;

[0122] 2) CD34+ cells were isolated and counted using the miniMACS isolation system;

[0123] 3) Purity analysis: After the immunomagnetic bead-labeled cells were purified twice by magnetic field, they were incubated with anti-CD34-PE, and the purity of CD34+ cells was detected by flow cytometry to be 98.25%.

[0124] 5.2. Amplification of HSCs

[0125] 1) Materials: (1) Cells: MSC, Nov-MSC, and HSC; (2) Exosomes: MSC-EXO, Nov-MSC-EXO; (3) Nov protein (Peprotech); (4) Complete culture medium: IMDM (Life Technology) with 0.1% HSA (Sigma), 10 ng / ml rhCSF, 20 ng / ml rhFlt3, and 100 ng / ml rhTPO (all from Peprotech);

[0126] 2) Cell Culture

[0127] (1) The 24-well culture plates were divided into 7 groups: A (HSC control), B (HSC+MSC), C (HSC+NOV protein), D (HSC+NOV-MSC), E (HSC+MSC-EXO), and F (HSC+Nov-MSC-EXO), with 3 replicates in each group (Table 1).

[0128] Table 1. Experimental Groups

[0129] HSC HSC HSC HSC HSC HSC HSC MSC - MSC - - - - NOV protein - - NOV protein - - - NOV-MSC - - - NOV-MSC - - MSC-EXO - - - - MSC-EXO - Nov-MSC-EXO - - - - - Nov-MSC-EXO

[0130] (2) Seed MSC and NOV-MSC cells into wells of groups B and D respectively. When the culture reaches 85% confluence, gently aspirate the culture medium from the wells; add 0.5 ml of medium containing 1X10 4 Complete culture medium for HSCs;

[0131] (3) Add 0.5 ml of complete culture medium to the wells of group A; add Nov protein, MSC-EXO and Nov-MSC-EXO diluted with complete culture medium to groups C, E and F respectively.

[0132] (4) Add 0.5 ml of 1X10 to each well of all groups. 4 Complete culture medium for HSCs;

[0133] (5) Place the culture plate in a 37℃, 5% CO2 incubator for 3 weeks, and change half of the medium every week;

[0134] (6) After digestion with trypsin-EDTA, all cells in the culture wells were collected, mouse anti-human CD34 (Abcam) monoclonal antibody was added, and the cells were incubated at 37 degrees for 1 hour. After washing twice, FITC-labeled rabbit anti-mouse secondary antibody (Abcam) was added, and the cells were incubated at 30 degrees for 30 minutes. After washing twice, the cells were suspended in 500 μL of physiological saline. Fluorescent positive cells were counted by flow cytometry (BD).

[0135] (7) Results: See Table 2.

[0136] Table 2. HSC Count

[0137]

[0138] Group A represents the results of HSC cultured alone for 3 weeks, serving as a baseline;

[0139] Group B was the result of co-culturing HSCs and MSCs, which stimulated HSC proliferation and increased the number of HSCs by 4 times.

[0140] Group C was the result of co-culturing HSCs with Nov proteins, which stimulated HSC proliferation, resulting in a 6-fold increase in the number of HSCs.

[0141] Group D represents the results of co-culturing HSCs with Nov-MSCs, which stimulated HSC proliferation. The number of HSCs was 120 times higher than that of HSCs alone, 30 times higher than that of MSCs alone, and 20 times higher than that of Nov protein alone. There were very significant differences between these groups.

[0142] Group E represents the result of HSC culture with the addition of MSC-EXO to stimulate HSC proliferation, which increased the number of HSCs by 3.6 times, and there was no significant difference from the effect of MSCs.

[0143] Group F was the result of adding Nov-MSC-EXO to HSC culture to stimulate HSC proliferation, increasing the number of HSCs by 92 times, which was not much different from the effect of Nov-MSC.

[0144] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made under the concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for promoting the proliferation of hematopoietic stem cells, characterized in that, Includes the following steps: S101: Construct Nov / CCN3 gene recombinant Nov-MSCs and express Nov / CCN3 protein on the surface of MSCs; The Nov / CCN3 protein is the protein with the amino acid sequence shown in SEQ ID NO.1; S102: Preparation of Nov-MSC exosomes (Nov-MSC-EXO) using Nov-MSCs; S103: Stimulate HSCs with Nov-MSCs or Nov-MSC-EXO to promote HSC amplification; In S101, the Nov / CCN3 protein is expressed on the surface of MSCs by linking the 3' end of the NoV / CCN3 gene to the PDGFR transmembrane polypeptide gene on the pDisplay expression vector. S103 includes the preparation of HSCs and the in vitro HSC amplification experiment; The MSC is a UC-MSC.

2. The method for promoting hematopoietic stem cell proliferation according to claim 1, characterized in that, In step S101, the preparation of recombinant Nov-MSCs includes: S1011: Preparing MSC: Taking each well of a 6-well plate as an example, prepare 5×10 5 One MSC was suspended in 2 ml of culture medium and seeded into one well of a 6-well plate. Transfection was performed when the cell density reached 50-80% confluency. S1012: Prepare transfection reagents: Add 4ug of pD-Nov / CCN3 plasmid DNA to 600ul of Opti-MEM low serum culture medium and mix well; gently mix Lipofectamine LTX reagent, take 5ul and add it to the DNA tube; gently mix and incubate at room temperature for 30min; S1013: Add 100 μL of DNA-Lipofectamine LTX complex to each well of cells, and gently shake the culture plate back and forth and side to side to mix. S1014: Incubate at 37℃ in a 5% CO2 incubator for 24 hours. Take the transfected Nov-MSCs and add mouse anti-human Nov / CCN3 monoclonal antibody. Incubate at 37℃ for 1 hour. After washing twice, add FITC-labeled rabbit anti-mouse secondary antibody. Incubate at 30℃ for 30 minutes. After washing twice, suspend in 50 μL of physiological saline. S1015: Fluorescence microscopy observation results show that the positive rate of Nov / CCN3 protein is 55%; S1016: Monoclonal Nov-MSCs were prepared using the limiting dilution method and stored in liquid nitrogen for later use.

3. The method for promoting hematopoietic stem cell proliferation according to claim 1, characterized in that, S101 includes the Nov / CCN3 protein gene clone, specifically including: Primer design: 1) Primer 1: Nov-upSacII:gacgtcCCGCGGatgcagagtgtgcagagcacg; 2) Primer 2: Nov-DnSalI:gacgtcGTCGACcattttccctctggtagtctt; RNA preparation: 50 ml of peripheral blood was collected from volunteers, and PBMCs were separated using lymphocyte separation medium, followed by the preparation of total RNA using TRIzol reagent; RT-PCR: Using total RNA as a template and primers 1 and 2 as primers, the Nov / CCN3 gene fragment was prepared using the Invitrogen SuperScript IV one-step RT-PCR kit and purified using the ES Spin column PCR product purification kit. Restriction endonuclease digestion of pDisplay vector and PCR products: 1) The pDisplay vector was digested with SacII / SalI restriction endonuclease, followed by agarose gel electrophoresis. The gel containing the digested pDisplay vector DNA fragment was excised and purified using the EZ-10 Spin Column DNAPAGE Gel Extraction Kit to obtain the pDisplay SacII / SalI digested vector product. 2) Digest the Nov / CCN3 gene fragment with SacII / SalI restriction endonuclease, perform agarose gel electrophoresis, cut the gel containing the digested Nov / CCN3 gene DNA fragment, and purify it separately with EZ-10 Spin Column DNAPAGE Gel Extraction Kit to obtain the Nov / CCN3 gene SacII / SalI digestion product. 3) Ligation: The pDisplay SacII / SalI and Nov / CCN3 gene SacII / SalI fragments were ligated using DNA ligase to obtain the pD-Nov / CCN3 product; 4) Transfection of competent cells: Transfect DH5α competent cells with pD-Nov / CCN3 product; 5) Colony selection and verification: Select positive colonies, culture them in small quantities, extract plasmid DNA, perform initial selection by digestion with the corresponding restriction endonuclease, confirm by sequencing, and preserve the pD-Nov / CCN3 recombinant bacteria. 6) Cultivate pD-Nov / CCN3 recombinant bacteria and prepare pD-Nov / CCN3 recombinant plasmid.

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