CD47 mesenchymal stem cell as well as preparation method and application thereof

Abstract

The invention relates to the technical field of stem cell biological medicine, and discloses an allogenic mesenchymal stem cell preparation for treating liver injury as well as a preparation method and application thereof, and an exogenous CD47 gene is introduced into mesenchymal stem cells. The cell line stably carries the CD47 gene, the immunological rejection resistance of the mesenchymal stem cells can be remarkably improved, the residence capacity of the mesenchymal stem cells can be remarkably enhanced, and a new solution is provided for a host to eliminate the immunological rejection problem of the mesenchymal stem cells. The mesenchymal stem cells are derived from healthy donors, the source is wide, the multiplication capacity is high, the mesenchymal stem cells still have the capacity of self-renewal and multidirectional differentiation after multiple passages, the technology is expected to solve the problem that stem cell transplantation is limited by the number of autologous cells and immunological rejection, and the mesenchymal stem cells have great application prospects.

Description

Technical Field

[0001] The present invention relates to the field of stem cell biomedicine, and in particular to mesenchymal stem cells and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are multipotent adult stem cells originating from the mesoderm, and are widely found in umbilical cord blood, bone marrow, fat and other parts. They have a wide range of sources and are not ethically controversial, have low tumorigenicity, low immunogenicity, and have the paracrine function of secreting multiple cytokines. Currently, many studies have demonstrated the therapeutic potential of mesenchymal stem cells in various diseases such as diabetes, cartilage injury, spinal cord injury, and liver injury. However, due to the fact that their cells are easily cleared by the body's immune system, their survival time in the body is limited, which seriously affects the effective use of mesenchymal stem cells in clinical treatment.

[0003] Studies have shown that canine adipose tissue-derived mesenchymal stem cells can be effectively used to prevent and treat major clinical diseases such as diabetes, liver damage and renal failure. However, canine adipose tissue-derived mesenchymal stem cells are easily affected by damage such as ROS, and their passage and vitality are limited.

[0004] CD47, also known as integrin-associated protein (IAP), is a transmembrane protein widely expressed on the surface of various cell types and has important biological functions. CD47 inhibits the phagocytosis of macrophages by binding to signal regulatory protein α (SIRPα). This mechanism plays a protective role in the immune escape of normal cells and prevents autologous cells from being mistakenly killed by the immune system. However, there have been no reports on the use of the CD47 gene to modify mesenchymal stem cells to enhance their survival efficiency in vivo, and it has broad application prospects. Summary of the invention

[0005] The purpose of the present invention is to overcome the above problems existing in the prior art and to provide a mesenchymal stem cell and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention provides a mesenchymal stem cell in a first aspect, into which an exogenous gene encoding CD47 is introduced.

[0007] The second aspect of the present invention provides a method for preparing a mesenchymal stem cell line, the method comprising: introducing a CD47 gene into the mesenchymal stem cells.

[0008] The third aspect of the present invention provides mesenchymal stem cells prepared by the above preparation method.

[0009] The fourth aspect of the present invention provides that the above-mentioned mesenchymal stem cells enhance their ability to escape from macrophage phagocytosis or enhance their survival efficiency in vivo after transplantation.

[0010] Through the above technical scheme, the cell line stably carries CD47, which can significantly improve the anti-macrophage phagocytic ability of mesenchymal stem cells and significantly improve their survival efficiency in the body after transplantation, and exert their therapeutic effect in the body for a longer time, providing a new solution to the dilemma of host clearance of mesenchymal stem cells after transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the map of the newly constructed CD47 lentiviral expression vector.

[0013] Figure 2 is the CD47 mRNA expression level of ADMSC / CD47 cells.

[0014] Figure 3 is the expression level of CD47 protein in ADMSC / CD47 cells.

[0015] Figure 4 This is the result of co-culture of ADMSCs and macrophages for 96 h.

[0016] Figure 5 It is the survival efficiency of ADMSC transplanted into the body. DETAILED DESCRIPTION

[0018] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0019] The first aspect of the present invention provides a mesenchymal stem cell, wherein the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein. The mesenchymal stem cells have an exogenous gene encoding CD47 introduced into them (that is, the mesenchymal stem cells are mesenchymal stem cells overexpressing CD47).

[0020] In the present invention, the CD47 protein can be derived from humans or dogs. Preferably, the amino acid sequence of CD47 is shown in SEQ ID NO: 2. SEQ ID NO: 2 is: MWPLAALLLLGSASCGSAQLIFNITKSVEYTACNESAIIPCFVNNVEATNINEMYVKWKFRGKDIFTFDGAVQKTTHGDKFKSTKIVPQKLLNGIASLEMSKEEAVVGNYTCEVTELSREGETIIELKYRIVSWFSPNENILIVIFPILAVLLSWGQFGIVTIKYKSSIMKEKTIFLFVGGLVLTIVVIVGAILFVPGEYSTKNSCGLGLIVIPTVILTLLQYCVFMIGVWMSPFTIAILILQVLGYVLSVVGLSLCVSECTPVHGPLLISGLGIIALAELLGLVYMKLVASNQKTIQPPRSN

[0021] Preferably, the sequence of the CD47 gene is as shown in SEQ ID NO: 1. SEQ ID NO: 1 is: ATGTGGCCCCTGGCGGCGCTGCTGCTGCTGGGCTCGGCGAGCTGCGGTTCGGCTCAGCTAATATTTAACATAACCAAATCTGTAGAATACACTGCTTGCAATGAAAGTGTTATCATCCCATGCTTTGTTAATAATGTGGAGGCCACAAACATTAATGAGATGTATGTAAAGTGGAAATTTAGGAAAAGACATCTTCACCTTTGATGGAGCTGTACAAAAGACCACT CACGGTGATAAGTTTAAGAGTACAAAAATCGTACCACAGAAATTACTGAATGGCATTGCCTCTTTGGAGATGAGTAAGGAAGAAGCTGTTGTAGGAAACTATACTTGTGAAGTTACTGAATTAAGCAGAGAAGGCGAAACCATCATAGAATTAAAATATCGAATTGTTTCATGGTTTTCTCCAAATGAAAATATCCTCATTGTTATTTTCCCAATTTTGGCTGTACTT CTGTCTTGGGGACAGTTTGGTATTGTGACAATTAAATATAAATCCAGTATTATGAAGGAGAAAACCATTTTTTATTTGTTGGTGGACTAGTGCTCACTATAGTTGTCATTGTCGGAGCCATTCTTTTCGTCCCAGGTGAATATTCAACAAAGAATTCTTGTGGACTTGGTTTAATTGTAATTCCTACAGTAATATTAACATTACTTCAGTACTGTGTGTTTATGATA GGTGTTTGGATGTCTCCTTTCACCATTGCCATATTAATCCTTCAGGTACTGGGCTATGTGCTCTCTGTGGTTGGACTAAGCCTCTGTGTCTCAGAGTGCACCCCAGTGCATGGCCCTCTTCTGATTTCAGGTTTGGGTATTATAGCTCTAGCAGAATTACTTGGATTAGTTTATATGAAACTTGTTGCTTCCAATCAGAAGACTATACAACCTCCTAGGAGTAACTGA

[0022] In the present invention, the mesenchymal stem cells are primary mesenchymal stem cells. Preferably, the mesenchymal stem cells are human or canine adipose mesenchymal stem cells.

[0023] In the present invention, the source of the mesenchymal stem cells is an organ or tissue commonly used in the art that can produce mesenchymal stem cells, preferably at least one of placenta, umbilical cord and adipose tissue.

[0024] The second aspect of the present invention provides a gene modification method for preparing a mesenchymal stem cell line, the method comprising: introducing a gene encoding CD47 into the mesenchymal stem cells.

[0025] In the present invention, the gene encoding CD47 (integrin-associated protein) is introduced into mesenchymal stem cells by any common gene introduction method in the art, preferably by lentiviral transfection or recombinant plasmid.

[0026] In the present invention, the amino acid sequence, gene sequence, mesenchymal stem cells and sources of the CD47 (integrin-associated protein) are as described above and will not be described in detail here.

[0027] The third aspect of the present invention provides mesenchymal stem cells prepared by the above preparation method.

[0028] The fourth aspect of the present invention provides that the above-mentioned mesenchymal stem cells enhance their ability to escape from macrophage phagocytosis or enhance their survival efficiency in vivo after transplantation.

[0029] Example 1 (1) Cloning of CD47 gene. CD47 gene (Reference Sequence: NM_001080721.1) predicted the location of CD47 in the genome. Primers were designed using the gene coding region as a template to amplify the CD47 gene. The coding region (CDs region) sequence is shown in SEQ ID NO: 1. The 5′-3′ sequence of the upstream primer is GGAACTGCTGTTGGCGGT (SEQ ID NO: 3), and the 5′-3′ sequence of the downstream primer is AGCAACTCCAACTCCCAGT (SEQ ID NO: 4).

[0030] The PCR reaction program for CD47 amplified fragments was as follows: 32 cycles, 98°C→10 s, 62°C→15 s, 72°C→10 s, 10 min→72°C. The amplification system is shown in Table 1.

[0031] Table 1 PCR reaction procedure for CD47 amplification Components system <![CDATA[Autoclaved distilled water H2O]]> 21 µL TAKARA 2×master mix 25 µL CD47 cDNA Template 2 µL CD47 upstream primer 1 µL CD47 downstream primers 1 µL

[0032] (2) CD47 gene transduction into mesenchymal stem cells 1) Extraction of primary human or canine mesenchymal stem cells ① Take fresh canine adipose tissue containing primary mesenchymal stem cells and cut small pieces; wash it with phosphate buffer on a culture dish until the washing solution is transparent, and then cut it into pieces; ② Transfer the chopped tissue pieces to a 50 ml centrifuge tube, add 25 ml of collagenase IV with a mass / volume concentration of 0.1%, and shake and digest on a shaker at 37°C for 30 minutes; ③ Add 20 ml of phosphate buffer to the digested tissue, mix well and filter through a 100-micron mesh; centrifuge the filtrate at 1200 rpm for 5 minutes, remove the supernatant and retain the cell pellet; ④ Add 5 ml of a-MEM culture medium containing 20 volume % fetal bovine serum to the cell pellet, mix well and inoculate into a 6-well culture plate; then place in an incubator with 5 volume % CO2 and a constant temperature of 37°C; replace with fresh a-MEM culture medium containing 10 volume % fetal bovine serum every 3 days.

[0033] 2) Using lentiviral transfection method to specifically overexpress CD47 gene on the above cells, construct CD47 overexpressing human or canine adipose-derived mesenchymal stem cells: ① Recombining the CDS region of the CD447 gene into the lentiviral backbone pCDH-CMV-EF1-NEO by homologous recombination to obtain an overexpression vector, such as Figure 1 ; ② Premix the vector, packaging element PAX2, and VSVG in a molar mass ratio of 4:2:2 to a total volume of 8 μl, then add 20 μL PEI and 500 μL Opti-MEM, mix well, add 293T cells cultured to 85%, and culture at 37°C and 5% CO2; ③ After 12 hours, the supernatant was discarded and replaced with complete culture medium. After culturing at 37°C and 5% CO2 by volume for 48 hours, the supernatant was collected to obtain the lentivirus packaging medium; ④ The lentiviral packaging solution collected in the previous step was mixed with complete culture medium at a ratio of 1:1, then added to canine adipose-derived mesenchymal stem cells cultured to 10%, and then added with 1:1000 PB and mixed. After culturing at 37°C and 5% CO2 by volume for 12 hours, the complete culture medium was replaced, and the medium was changed every 24 hours.

[0034] 3) Screening of mesenchymal stem cell lines overexpressing CD47.

[0035] When the cell confluence reached 85%, G418 (Geneticin, final concentration was 600 µg / mL) was added, and the cells were cultured at 37°C, 5% CO2 by volume for 12 hours, and then the complete culture medium was replaced. The medium was changed every 24 hours to obtain CD47-overexpressing adipose-derived mesenchymal stem cells (ADMSC / CD47).

[0036] 4) Detection of protein and mRNA levels The above cells were selected by dilution method, expanded and cultured, and then the mRNA levels were detected by real-time quantitative PCR, such as Figure 2 , Western blot analysis of CD47 results Figure 3 As shown (ADMSC on the left and ADMSC / CD47 on the right), it was found that compared with the control group (ADMSC), the screened CD47 overexpressing single cell clones overexpressed CD47 at both the protein level and the mRNA level.

[0037] 5) The survival of cells after co-culture with mouse macrophages RAW 264.7 for 96 hours was further detected. ADMSCs and ADMSC / CD47 cultured to the logarithmic growth phase were stained with PKH 26, and the steps were as follows: 0.25% trypsin was used to digest the cells, the trypsin was discarded after centrifugation, and the cells were resuspended in α-MEM culture medium to form a cell suspension. According to the instructions of the kit, the PKH26 dye was diluted with diluent C at a certain ratio, usually 1:250 to 1:500. Mix well to avoid bubbles. After the staining time is over, 5-10 times the volume of α-MEM is immediately added to terminate the staining reaction. Mix well. Centrifuge to remove excess dye and resuspend the cells in a new culture dish for culture. RWA 264.7 cells in good condition and undifferentiated are added to the stained ADMSCs at a ratio of 1:1 and cultured together. The medium is changed every day and the cell status is observed under fluorescence. Figure 4 As shown, the results showed that ectopic overexpression of CD47 enhanced the ability of ADMSCs to evade macrophage clearance.

[0038] 5) The survival efficiency of ADMSCs in vivo after intravenous transplantation was further tested. ADMSCs with GFP green fluorescence and ADMSC / CD47 were transplanted into mice. After 14 days, the livers of mice were collected and frozen sections were made to observe the survival of ADMSCs with green fluorescence. Figure 5 The results showed that ectopic overexpression of CD47 improved the survival efficiency of ADMSCs in the host after transplantation.

[0039] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A mesenchymal stem cell, characterized in that: An exogenous gene encoding CD47 gene is introduced into the mesenchymal stem cells.

2. The mesenchymal stem cell according to claim 1, wherein The amino acid sequence of CD47 is shown in SEQ ID NO: 2; and / or, the sequence of the gene encoding CD47 is shown in SEQ ID NO: 1; And / or, the mesenchymal stem cells are primary mesenchymal stem cells; And / or, the mesenchymal stem cells are mesenchymal stem cells of species such as humans or dogs.

3. A method for preparing mesenchymal stem cells, characterized in that: The method comprises: introducing a gene encoding CD47 into mesenchymal stem cells.

4. The method according to claim 3, wherein: The amino acid sequence of CD47 is shown in SEQ ID NO:

2.

5. The method according to claim 3, wherein: The sequence of the gene encoding CD47 is shown in SEQ ID NO: 1; And / or, the mesenchymal stem cells are primary mesenchymal stem cells; And / or, the mesenchymal stem cells are human or canine adipose-derived mesenchymal stem cells.

6. Mesenchymal stem cells prepared by the method according to any one of claims 3 to 5.

7. Use of the mesenchymal stem cells according to claim 1, 2 or 6 in the preparation of a material for escaping macrophage phagocytosis or prolonging its survival efficiency in vivo after transplantation.

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