Method for enhancing paracrine function of umbilical cord mesenchymal stem cells through gene modification and application of method

Abstract

The invention discloses a method for enhancing the paracrine function of umbilical cord mesenchymal stem cells through gene modification and application of the method, and relates to the technical field of biology. The method comprises the following steps: connecting a target gene with an oxygen-sensitive sequence, and introducing the connected target gene and oxygen-sensitive sequence into mesenchymal stem cells through a vector, the oxygen sensitive sequence comprises a hypoxia reaction element HRE and a hypoxia inducible factor HIF-1alpha enhancer sequence, the HRE sequence is TCACGTG, and the HIF-1alpha enhancer sequence is as shown in SEQ ID NO: 1. Through specific gene modification, the level of cytokines specifically generated by the mesenchymal stem cells in the ischemic tissue can be remarkably improved, the paracrine function of the mesenchymal stem cells is enhanced, and the treatment effect is improved, so that the effectiveness and safety of gene modified mesenchymal stem cell treatment are improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for enhancing the paracrine function of umbilical cord mesenchymal stem cells through gene modification and an application thereof. Background Art

[0002] Umbilical cord mesenchymal stem cells (UC-MSCs) are a type of multipotent stem cells isolated from the Wharton's jelly of the umbilical cord of newborns. UC-MSCs have been widely used in the field of regenerative medicine due to their easy access, low immunogenicity, high proliferation ability and multidirectional differentiation potential. They have been used to treat a variety of diseases, including but not limited to neurological diseases such as stroke, Parkinson's disease, spinal cord injury, etc.; cardiovascular diseases such as myocardial infarction, ischemic heart disease, etc.; autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, etc.; liver diseases such as cirrhosis, acute liver failure, etc.; orthopedic diseases such as osteoarthritis, fracture healing, etc. The therapeutic effect of UC-MSCs is mainly attributed to its powerful paracrine function, which can secrete a variety of growth factors, cytokines and exosomes, which can promote angiogenesis, inhibit inflammatory response, reduce cell apoptosis and promote the repair of damaged tissues. For example, in the treatment of stroke, UC-MSCs can promote neuronal survival and angiogenesis by secreting factors such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), thereby improving the patient's neurological function.

[0003] Although UC-MSCs show good prospects in clinical applications, there are still some shortcomings, such as limited paracrine function. In some cases, the paracrine function of UC-MSCs is not enough to achieve the desired therapeutic effect. Especially in cases where high levels of cytokine support are required, such as severe tissue damage or chronic diseases. For another example, the survival rate and functional maintenance after transplantation, UC-MSCs may be affected by the host microenvironment after transplantation, resulting in decreased survival rate or weakened function.

[0004] In order to overcome the shortcomings of primary MSCs, researchers have adopted a variety of strategies, mainly including gene modification. Gene modification is to introduce target genes into UC-MSCs through genetic engineering technology to enhance their paracrine function. For example, transfection of genes such as VEGF and HGF can significantly increase the secretion level of cytokines. However, this type of method has technical shortcomings, and the secretion level of cytokines is generally increased, which poses a safety hazard.

[0005] Therefore, providing a method with high safety and the ability to significantly enhance the paracrine function of umbilical cord mesenchymal stem cells is a technical problem that urgently needs to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide a method for enhancing the paracrine function of umbilical cord mesenchymal stem cells through gene modification and its application.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a method for enhancing the paracrine function of mesenchymal stem cells by gene modification, comprising: connecting a target gene to an oxygen-sensitive sequence and then introducing the resultant gene into the mesenchymal stem cells via a vector;

[0009] The oxygen sensitive sequence includes a hypoxia response element HRE and a hypoxia inducible factor HIF-1α enhancer sequence, the HRE sequence is TCACGTG, and the HIF-1α enhancer sequence is shown in SEQ ID NO:1.

[0010] Specifically, the target genes include but are not limited to VEGF and HGF.

[0011] Furthermore, the target gene is VEGF.

[0012] Specifically, the two ends of the HRE sequence and the HIF-1α enhancer sequence contain EcoRI and BamHI restriction endonuclease recognition sites.

[0013] Specifically, the mesenchymal stem cells include umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells and placental mesenchymal stem cells.

[0014] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0015] Specifically, the method comprises the following steps:

[0016] (1) Amplify the HRE sequence and HIF-1α enhancer sequence;

[0017] (2) digesting the amplified HRE sequence and HIF-1α enhancer sequence, and digesting the vector at the same time;

[0018] (3) inserting the digested sequence into the vector by ligase, and transfecting into competent cells for lentiviral packaging to obtain lentiviral particles;

[0019] (4) The mesenchymal stem cells are cultured under hypoxic conditions, and when the cells reach 70-80% confluence, a transfection mixture containing lentiviral particles is added for culture.

[0020] Further, step (1) is to amplify the HRE sequence and the HIF-1α enhancer sequence using PCR;

[0021] Furthermore, the HRE forward primer is shown as SEQ ID NO:2, and the HRE reverse primer is shown as SEQ ID NO:3.

[0022] Furthermore, the HIF-1α enhancer forward primer is shown in SEQ ID NO:4, and the HIF-1α enhancer reverse primer is shown in SEQ ID NO:5.

[0023] Specifically, the HRE forward primer contains an EcoRI site, and the HRE reverse primer contains a BamHI site.

[0024] Specifically, the HIF-1α enhancer forward primer contains an EcoRI site, and the HIF-1α enhancer reverse primer contains a BamHI site.

[0025] Specifically, in step (2), the HRE sequence and the HIF-1α enhancer sequence are double-digested using EcoRI and BamHI enzymes.

[0026] Specifically, the vector described in step (2) includes but is not limited to a lentiviral vector and an adeno-associated viral vector.

[0027] Furthermore, the vector is a lentiviral vector.

[0028] Specifically, in step (2), the vector is double-digested using EcoRI and BamHI enzymes.

[0029] According to some embodiments of the present invention, the vector is a pLenti-CMV-VEGF-IRES-GFP plasmid.

[0030] Specifically, the ligase in step (3) includes but is not limited to T4 DNA ligase and T7 DNA ligase.

[0031] Furthermore, the ligase is T4 DNA ligase.

[0032] Specifically, the competent cells described in step (3) include but are not limited to Escherichia coli, yeast or Bacillus subtilis.

[0033] Furthermore, the competent cells are Escherichia coli.

[0034] Specifically, in step (3), the lentiviral packaging is to co-transfect the constructed vector and packaging plasmid into cells.

[0035] Furthermore, the packaging plasmid includes psPAX2 plasmid and pMD2.G plasmid.

[0036] Furthermore, the cells are HEK293T cells.

[0037] Specifically, the hypoxic conditions described in step (4) contain 1-3% O2; further, the hypoxic conditions contain 3% O2.

[0038] Specifically, the content of mesenchymal stem cells in step (4) is 5×10 5 cells / well.

[0039] Specifically, the culture medium for culturing mesenchymal stem cells in step (4) includes, but is not limited to, DMEM / F12 medium, MRS medium, M17 medium, PDA medium, LB medium, TB medium, SOB medium, MEM medium, and RPMI1640 medium.

[0040] Furthermore, the culture medium for culturing mesenchymal stem cells in step (4) is DMEM / F12 culture medium (containing 10% FBS).

[0041] According to some embodiments of the present invention, the culture medium may contain specific growth factors, including but not limited to insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), etc.

[0042] Specifically, the transfection mixture in step (4) includes a culture medium and lentiviral particles; further, the culture medium is a DMEM / F12 culture medium, and the MOI of the lentiviral particles is 10.

[0043] Specifically, the culture time after adding the transfection mixture in step (4) is 48-72 hours; further, the culture time after adding the transfection mixture is 72 hours.

[0044] In another aspect, the present invention provides gene-modified mesenchymal stem cells prepared by the above method.

[0045] Specifically, the mesenchymal stem cells include umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells and placental mesenchymal stem cells.

[0046] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0047] Specifically, the mesenchymal stem cells have stronger paracrine function.

[0048] In another aspect, the present invention provides use of the above-mentioned gene-modified mesenchymal stem cells in the preparation of a drug for treating ischemic cardiovascular and cerebrovascular diseases.

[0049] Specifically, the ischemic cardiovascular and cerebrovascular diseases include but are not limited to ischemic stroke, coronary heart disease, and myocardial infarction.

[0050] Furthermore, the ischemic cardiovascular and cerebrovascular disease is ischemic stroke.

[0051] Specifically, the drug includes a pharmaceutically acceptable carrier.

[0052] In another aspect, the present invention provides a pharmaceutical composition, comprising mesenchymal stem cells prepared by the above method.

[0053] Specifically, the mesenchymal stem cells are gene-modified mesenchymal stem cells.

[0054] Specifically, the pharmaceutical composition can be used to promote tissue repair and regeneration.

[0055] Specifically, the pharmaceutical composition can be used to treat ischemic cardiovascular and cerebrovascular diseases.

[0056] Furthermore, the ischemic cardiovascular and cerebrovascular diseases include but are not limited to ischemic stroke, coronary heart disease, and myocardial infarction.

[0057] Furthermore, the ischemic cardiovascular and cerebrovascular disease is ischemic stroke.

[0058] Specifically, the pharmaceutical composition may further include a pharmaceutically acceptable carrier.

[0059] Furthermore, the pharmaceutically acceptable carrier includes, but is not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, adhesives, preservatives, lubricants, pH regulators, cryoprotectants, and flavoring agents.

[0060] Furthermore, the dosage form of the pharmaceutical composition is drops, mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, suspensions, powders, emulsions, solutions, pellets, pills, buccal tablets, lyophilized powder injections, gels, suppositories or aerosols.

[0061] The beneficial effects of the present invention are:

[0062] The present invention can significantly increase the level of cytokines produced by UC-MSCs through specific gene modification, enhance its paracrine function, and improve the therapeutic effect, especially significantly increase the expression level of cytokines in ischemic and hypoxic tissues, thereby improving the effectiveness and safety of gene-modified UC-MSC treatment. DETAILED DESCRIPTION

[0063] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0064] Example 1 Construction of tissue oxygen concentration sensitive VEGF gene expression vector

[0065] 1. Material preparation:

[0066] Plasmid pLenti-CMV-VEGF-IRES-GFP (carrying VEGF gene), derived from the China Plasmid Vector Strain Cell Line Gene Bank-National Type Culture Collection Center (BioVector NTCC Inc.); tissue oxygen concentration-sensitive promoter (such as HRE, Hypoxia-Responsive Element); enhancer sequence (such as HIF-1α enhancer); PCR primers; restriction endonucleases (such as EcoRI, BamHI); T4 DNA ligase; HEK293T cell transfection reagent (such as Lipofectamine 3000); culture medium (DMEM, containing 10% FBS).

[0067] 2. Experimental steps:

[0068] (1) Design and synthesis of HRE and enhancer sequences:

[0069] HRE sequence (Hypoxia-Responsive Element): 5'-TCACGTG-3';

[0070] HIF-1α enhancer sequence: 5′-GGCGTGATTTCCAGCCTCT-3′ (SEQ ID NO: 1);

[0071] HRE and enhancer sequences were synthesized, and EcoRI and BamHI restriction endonuclease recognition sites were introduced at both ends.

[0072] (2) Amplification of HRE and enhancer sequences:

[0073] Use PCR to amplify the HRE and enhancer sequences with the following primers:

[0074] HRE forward primer: 5'-GAATTCGCGGCCGCTCACGTG-3' (including EcoRI site), (SEQ ID NO: 2);

[0075] HRE reverse primer: 5'-GGATCCGCGGCCGCTCACGTG-3' (including BamHI site), (SEQ ID NO: 3);

[0076] Enhancer forward primer: 5'-GAATTCGGCGTGATTTCCAGCCTCT-3' (including EcoRI site), (SEQ ID NO: 4);

[0077] Enhancer reverse primer: 5'-GGATCCGGCGTGATTTCCAGCCTCT-3' (including BamHI site), (SEQ ID NO: 5).

[0078] (3) Cloning of HRE and enhancer sequences:

[0079] The amplified HRE and enhancer sequences were double-digested (EcoRI and BamHI). At the same time, the pLenti-CMV-VEGF-IRES-GFP plasmid was double-digested (EcoRI and BamHI). The HRE and enhancer sequences were inserted into the pLenti-CMV-VEGF-IRES-GFP plasmid using T4 DNA ligase. Transform competent Escherichia coli (such as DH5α) and select positive clones for sequencing verification.

[0080] The lentiviral packaging process is as follows: The constructed pLenti-HRE-Enhancer-VEGF-IRES-GFP plasmid, psPAX2 plasmid and pMD2.G plasmid (1:1:1) were co-transfected into HEK293T cells. The HRE and enhancer sequences were correctly inserted into the pLenti-CMV-VEGF-IRES-GFP plasmid by sequencing.

[0081] Example 2 Transfection of UC-MSCs and expression of VEGF protein

[0082] 1. Material preparation:

[0083] UC-MSCs were prepared by Beijing Regeneration Century Biotechnology Co., Ltd. and confirmed to be mesenchymal stem cells through cell morphology identification, membrane protein expression, and tri-lineage differentiation verification; constructed lentiviral vector (containing VEGF gene and GFP marker); culture medium (DMEM / F12, containing 10% FBS); ELISA kit for detecting VEGF (Beijing Yilairui Biotechnology Co., Ltd., product number: YA-10102); RT-PCR (Beijing Yita Biotechnology Co., Ltd., YT8381) and Western Blot reagent (Beijing Biolab Technology Co., Ltd., WE0297-RYP); flow cytometer.

[0084] 2. Experimental steps:

[0085] UC-MSCs were seeded in 6-well plates, with approximately 5×10^5 cells per well. The cells were cultured using DMEM / F12 medium (containing 10% FBS) and placed in a 37°C, 5% CO2 incubator overnight, or in a 3% oxygen concentration incubator (CO2 incubator with a hypoxic culture module). When the cells reached 70-80% confluence, lentiviral transfection was performed.

[0086] Prepare the transfection mixture first. Take 1 mL of serum-free DMEM / F12 medium and add an appropriate amount of lentiviral particles (MOI = 10). Incubate at room temperature for 10 minutes. Evenly add the mixture to the cell culture well. Replace the medium with fresh medium 24 hours after transfection. Screen GFP-positive cells. 72 hours after transfection, screen GFP-positive cells by flow cytometry. Collect GFP-positive cells, continue to amplify and perform subsequent experiments.

[0087] 3. Result analysis:

[0088] Flow cytometry confirmed that the GFP positive rate was over 85%. The results of ELISA test of culture supernatant showed that under low oxygen concentration (3%), UC-MSCs with VEGF gene with oxygen concentration dependent sequence had significantly increased VEGF level, which was statistically different from control UC-MSC and UC-MSC transfected with VEGF gene only. There was no significant change in cell proliferation ability, and the survival rate remained above 90%.

[0089] Table 1 ELISA results

[0090]

[0091] Note: Compared with ①, *p<0.05, **p<0.01; compared with ②, #p<0.05.

[0092] Table 2 Cell proliferation rate results

[0093]

[0094]

[0095] Example 3 Pharmacodynamics of Genetically Modified UC-MSCs Injected into Rats with Middle Cerebral Artery Occlusion

[0096] 1. Material preparation:

[0097] Genetically modified UC-MSCs (transfected with VEGF gene); rats (Sprague-Dawley rats); tools for establishing the middle cerebral artery occlusion (MCAO) model (syringes and microsurgical instruments, behavioral testing equipment, tissue sections and staining reagents).

[0098] 2. Experimental steps:

[0099] Healthy adult male Sprague-Dawley rats weighing 250-300 grams were selected. The experiment was divided into four groups, 8 rats in each group: sham operation control group, model animal injection PBS control group, model animal injection VEGF gene modified UC-MSC group (gene modification group), model animal injection VEGF gene modified UC-MSCs with oxygen concentration sensitive sequence group (regulatable gene modification group).

[0100] The MCAO model was established as follows: The middle cerebral artery occlusion (MCAO) model was established by thread embolism, and a nylon thread was inserted into the internal carotid artery until the origin of the middle cerebral artery to block blood flow for 90 minutes. The nylon thread was removed and blood flow was restored. 24 hours after MCAO, the two types of gene-modified UC-MSCs (1×10^6 cells) were suspended in PBS and injected into the corresponding groups of rats through the tail vein.

[0101] Behavioral test: Neurological Deficit Score (NDS) was performed on the 7th and 14th days after surgery. Scoring criteria (Longa scoring method): ① No neurological deficit: 0 points; ② The front paw on the paralyzed side cannot be fully extended: 1 point; ③ Turning in circles toward the paralyzed side when walking: 2 points; ④ Falling toward the paralyzed side when walking: 3 points; ⑤ Unable to walk automatically and loss of consciousness: 4 points.

[0102] Pathological examination: On the 14th day after surgery, the rats were killed and the brain tissues were taken for pathological examination. TTC staining was used to observe the infarct area, CD31 immunohistochemistry (angiogenesis marker) and VEGF immunohistochemistry.

[0103] TTC (triphenyltetrazolium) staining includes the following steps: Tissue processing: Take the ischemic infarction tissue immediately after the experimental animal is killed and freeze it at -80℃ for 10-15 minutes. Then, thaw the tissue slightly at room temperature and use a sharp blade to cut the tissue into slices with a thickness of 2-3 mm, ensuring that the cut surface is neat. Staining: Preheat the TTC dye solution to 37℃, and then place the tissue slices in a clean container to ensure that the tissue is completely immersed in the TTC dye solution. Place the container in a 37℃ constant temperature incubator and stain in the dark for about 30 minutes. During this period, gently shake the container every 10 minutes and gently turn the tissue to ensure uniform contact with the dye solution. Stop staining: When the tissue appears dark red, pour out the TTC dye solution, and slowly add formalin fixative to the container to immerse the tissue to terminate the staining reaction. The sample needs to be kept in the dark at 4℃ overnight.

[0104] Antibodies for immunohistochemistry were purchased from Wuhan Boster Biotechnology Co., Ltd., primary antibodies anti-mouse CD31 (Cat. No. A01513-3), VEGF (Cat. No. BA2090-1), SV ultrasensitive two-step kit (Cat. No. SV0002). The main steps were referred to the literature (Chen Yanming et al. Expression of VEGF and CD31 in skin squamous cell carcinoma, China Practical Medicine, 2012, 7(26): 1-2).

[0105] 3. Results Analysis

[0106] The therapeutic effect was observed by behavioral tests (neurological deficit score), cerebral infarction range detection, CD31 staining and VEGF staining. The results showed that compared with the PBS control group, the neurological function of the UC-MSC group modified with VEGF gene was significantly improved and angiogenesis was enhanced. However, the effect of the UC-MSC group modified with VEGF gene carrying oxygen concentration sensitive sequence was more significant, especially in the non-ischemic liver tissue, the VEGF expression level of the UC-MSC group modified with VEGF gene carrying oxygen concentration sensitive sequence was not higher than that of the UC-MSC group modified with VEGF gene.

[0107] Table 3 Pharmacodynamic results of gene-modified US-MSCs

[0108]

[0109] Note: Compared with the sham operation group, *p<0.05, **p<0.01, ***p<0.001; compared with the gene modification group, #p<0.05.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for enhancing the paracrine function of mesenchymal stem cells by genetic modification, characterized in that: It includes connecting the target gene with the oxygen-sensitive sequence and then introducing it into the mesenchymal stem cells through a vector; The oxygen sensitive sequence includes a hypoxia response element HRE and a hypoxia inducible factor HIF-1α enhancer sequence, the HRE sequence is TCACGTG, and the HIF-1α enhancer sequence is shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The target gene is VEGF.

3. The method according to claim 1, characterized in that The mesenchymal stem cells include umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells and placental mesenchymal stem cells.

4. The method according to claim 3, characterized in that The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

5. The method according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: (1) Amplify the HRE sequence and HIF-1α enhancer sequence; (2) digesting the amplified HRE sequence and HIF-1α enhancer sequence, and digesting the vector at the same time; (3) inserting the digested sequence into the vector by ligase, and transfecting into competent cells for lentiviral packaging to obtain lentiviral particles; (4) The mesenchymal stem cells are cultured under hypoxic conditions, and when the cells reach 70-80% confluence, a transfection mixture containing lentiviral particles is added for culture.

6. The method according to claim 5, characterized in that Step (1) is to use PCR to amplify the HRE sequence and the HIF-1α enhancer sequence; the HRE forward primer is shown in SEQ ID NO: 2, and the HRE reverse primer is shown in SEQ ID NO: 3; the HIF-1α enhancer forward primer is shown in SEQ ID NO: 4, and the HIF-1α enhancer reverse primer is shown in SEQ ID NO:

5.

7. The method according to claim 5, characterized in that The vectors described in step (2) include lentiviral vectors and adeno-associated viral vectors.

8. The method according to claim 7, characterized in that The vector described in step (2) is a lentiviral vector.

9. The method according to claim 5, characterized in that The hypoxic conditions described in step (4) contain 1-3% O2.

10. The method according to claim 9, characterized in that The hypoxic conditions contained 3% O2.

11. The method according to claim 5, characterized in that The culture time after adding the transfection mixture in step (4) is 48-72 hours.

12. Genetically modified mesenchymal stem cells prepared by the method according to any one of claims 1 to 11.

13. Use of the genetically modified mesenchymal stem cells according to claim 12 in the preparation of a drug for treating ischemic cardiovascular and cerebrovascular diseases.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the genetically modified mesenchymal stem cells according to claim 12.

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