A stem cell engineering transformation method based on polydopamine and VCAM1 antibody and its application

Abstract

The present invention belongs to the field of biotechnology and discloses a stem cell engineering method based on polydopamine and VCAM1 antibodies and its application. Specifically, a double-stranded DNA fragment and a VCAM1 antibody conjugate comprising the double-stranded DNA fragment are disclosed. The double-stranded DNA fragment comprises a DNA monomer 1 and a DNA monomer 2. The nucleotide sequence of the DNA monomer 1 is shown in SEQ ID NO: 1. The nucleotide sequence of the DNA monomer 2 is shown in SEQ ID NO: 2. The nucleotide sequences of the DNA monomer 1 and the DNA monomer 2 are complementary. The stem cell engineering method based on polydopamine and VCAM1 antibodies provided by the present invention modifies the polydopamine and VCAM1 antibodies on the cell surface, thereby greatly enhancing the retention and survival of stem cells in damaged tissues and promoting the repair of damaged tissues by stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a stem cell engineering transformation method based on polydopamine and VCAM1 antibody and an application thereof. Background Art

[0002] Stem cells possess numerous unique properties and capabilities, including sustained high self-renewal activity, multi-lineage differentiation capacity, host repopulation, and secretion of reparative paracrine factors. These properties and capabilities make them an ideal cell source for cell therapy, regenerative medicine, and tissue engineering. Transplanted stem cells primarily exert their therapeutic functions through paracrine pathways, improving the microenvironment of damaged tissues and demonstrating significant therapeutic potential in the treatment of major diseases such as tissue injury. However, studies have shown that the retention rate of transplanted stem cells within damaged tissue is extremely poor. For intravenously delivered stem cells, the percentage of transplanted cells that successfully colonizes the damaged tissue is typically less than 1%. Even when delivered to the injured site via local injection, the cells readily spread, with the percentage of cells successfully colonizing the damaged tissue typically less than 5%. Furthermore, the survival rate of transplanted stem cells within the damaged tissue is extremely low. Studies have shown that the majority of stem cells transplanted into the damaged myocardium die within one week, with a survival rate of only approximately 1% within 4-6 weeks. These low retention and survival rates severely limit the clinical efficacy of transplanted stem cells. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the shortcomings and defects of stem cells in clinical treatment mentioned in the above-mentioned prior art, and provide a new stem cell engineering modification method to modify particles and adhesion molecules that remove reactive oxygen species on the surface of stem cells. This engineering modification method is simple to operate and has little impact on cells. Compared with unmodified stem cells, the engineered stem cells prepared can be more efficiently retained and survive in damaged tissues, thereby better repairing damaged tissues.

[0004] To this end, the present invention provides a double-stranded DNA fragment.

[0005] The present invention also provides a VCAM1 antibody conjugate.

[0006] The present invention also provides a cell.

[0007] The present invention also provides a kit.

[0008] The present invention also provides the application of the double-stranded DNA fragment, VCAM1 antibody conjugate, polydopamine nanoparticles or kit.

[0009] The present invention also provides a cell engineering transformation method.

[0010] The present invention also proposes the application of the above-mentioned cells or cell engineering transformation method.

[0011] According to a first aspect of the present invention, a double-stranded DNA fragment is provided, comprising a DNA monomer 1 and a DNA monomer 2; the nucleotide sequence of the DNA monomer 1 is shown in SEQ ID NO: 1; the nucleotide sequence of the DNA monomer 2 is shown in SEQ ID NO: 2; and the nucleotide sequences of the DNA monomer 1 and the DNA monomer 2 are complementary.

[0012] In some embodiments of the present invention, the 5' end of the nucleotide sequence of the DNA monomer 1 is connected to an NH2C6 group.

[0013] In some embodiments of the present invention, a cholesterol group is linked to the 5' end of the nucleotide sequence of the DNA monomer 2.

[0014] According to a second aspect of the present invention, a VCAM1 antibody conjugate is provided, comprising a VCAM1 antibody and a polymer covalently linked; the polymer comprising at least one of the double-stranded DNA fragment described in the first aspect of the present invention, DSPE-PEG-NHS, DMPE-PEG-NHS, and DOPE-PEG-NHS;

[0015] In some embodiments of the present invention, the method for preparing the VCAM1 antibody conjugate comprises the following steps:

[0016] A1: DNA monomer 1 and cross-linker 1 are mixed and reacted to obtain DNA monomer 1-cross-linker 1;

[0017] A2: VCAM1 antibody and cross-linker 2 are mixed and reacted to obtain VCAM1 antibody-cross-linker 2;

[0018] A3: mixing the DNA monomer 1-crosslinker 1 described in step A1 with the VCAM1 antibody-crosslinker 2 described in step A2 to react, thereby obtaining a VCAM1 antibody-DNA monomer 1 covalent conjugate;

[0019] A4: The VCAM1 antibody-DNA monomer 1 covalent conjugate described in step A3 is mixed with DNA monomer 2 to react to obtain a VCAM1 antibody conjugate.

[0020] In some preferred embodiments of the present invention, the cross-linking agent 1 in step A1 includes at least one of succinimidyl-4-formylbenzoate (S-4FB), NHS-PEG-Azide, NHS-PEG-DBCO and NHS-PEG-BCN.

[0021] In some more preferred embodiments of the present invention, the cross-linking agent 1 in step A1 is S-4FB.

[0022] In some more preferred embodiments of the present invention, the NH2C6 group of the DNA monomer 1 in step A1 is linked to S-4FB.

[0023] In some preferred embodiments of the present invention, the reaction temperature in step A1 is 20°C to 37°C.

[0024] In some more preferred embodiments of the present invention, the reaction temperature in step A1 is 25°C.

[0025] In some preferred embodiments of the present invention, the reaction time in step A1 is 1 to 3 hours.

[0026] In some more preferred embodiments of the present invention, the reaction time in step A1 is 2 hours.

[0027] In some preferred embodiments of the present invention, the reaction in step A1 is carried out under an oscillation reaction at a rotation speed of 300 to 500 rpm.

[0028] In some more preferred embodiments of the present invention, the reaction in step A1 is carried out under an oscillation reaction at a rotation speed of 400 rpm.

[0029] In some preferred embodiments of the present invention, the cross-linking agent 2 in step A2 includes at least one of succinimidyl-6-hydrazinonicotinic acid acetone hydrazone (S-HyNic), NHS-PEG-Azide, NHS-PEG-DBCO and NHS-PEG-BCN.

[0030] In some more preferred embodiments of the present invention, the cross-linking agent 2 in step A2 is S-HyNic.

[0031] In some preferred embodiments of the present invention, the reaction temperature in step A2 is 20°C to 37°C.

[0032] In some more preferred embodiments of the present invention, the reaction temperature in step A2 is 25°C.

[0033] In some preferred embodiments of the present invention, the reaction time in step A2 is 1 to 3 hours.

[0034] In some more preferred embodiments of the present invention, the reaction time in step A2 is 2 hours.

[0035] In some preferred embodiments of the present invention, the reaction in step A2 is carried out under an oscillation reaction at a rotation speed of 300 to 500 rpm.

[0036] In some more preferred embodiments of the present invention, the reaction in step A2 is carried out under an oscillation reaction at a rotation speed of 400 rpm.

[0037] In some preferred embodiments of the present invention, the reaction in step A3 is a covalent linking reaction of S-HyNic and S-4FB.

[0038] In some preferred embodiments of the present invention, the reaction temperature in step A3 is 20°C to 37°C.

[0039] In some more preferred embodiments of the present invention, the reaction temperature in step A3 is 25°C.

[0040] In some preferred embodiments of the present invention, the reaction time in step A3 is 1 to 3 hours.

[0041] In some more preferred embodiments of the present invention, the reaction time in step A3 is 2 hours.

[0042] In some preferred embodiments of the present invention, the reaction in step A3 is carried out under an oscillation reaction at a rotation speed of 300 to 500 rpm.

[0043] In some more preferred embodiments of the present invention, the reaction in step A3 is carried out under an oscillation reaction at a rotation speed of 400 rpm.

[0044] In some preferred embodiments of the present invention, the reaction in step A4 is a base complementary pairing reaction between DNA monomer 1 and DNA monomer 2.

[0045] In some preferred embodiments of the present invention, the reaction temperature in step A4 is 20°C to 37°C.

[0046] In some more preferred embodiments of the present invention, the reaction temperature in step A4 is 25°C.

[0047] In some preferred embodiments of the present invention, the reaction time in step A4 is 10 to 50 minutes.

[0048] In some more preferred embodiments of the present invention, the reaction time in step A4 is 30 minutes.

[0049] In some preferred embodiments of the present invention, the reaction in step A4 is carried out under an oscillation reaction at a rotation speed of 400-600 rpm.

[0050] In some more preferred embodiments of the present invention, the reaction in step A4 is carried out under an oscillation reaction at a rotation speed of 500 rpm.

[0051] According to a third aspect of the present invention, a cell is provided, the surface of which is modified with: the VCAM1 antibody conjugate and polydopamine nanoparticles as described in the second aspect of the present invention.

[0052] In some embodiments of the present invention, the polydopamine nanoparticles are dopamine monomers in a Ca-containing 2+ polymerized in an alkaline buffer solution.

[0053] In some preferred embodiments of the present invention, the alkaline buffer comprises Tris-HCl buffer.

[0054] In some preferred embodiments of the present invention, the pH value of the alkaline buffer solution is 7.8-8.8.

[0055] In some more preferred embodiments of the present invention, the pH value of the alkaline buffer solution is 8.35.

[0056] In some preferred embodiments of the present invention, the Ca in the alkaline buffer is 2+ The content is 0.11~4.44wt%.

[0057] In some more preferred embodiments of the present invention, the Ca in the alkaline buffer is 2+ The content is 1.11wt%.

[0058] In some preferred embodiments of the present invention, the concentration of the dopamine monomer in the alkaline buffer solution is 0.05-5 mg / mL.

[0059] In some more preferred embodiments of the present invention, the concentration of the dopamine monomer in the alkaline buffer is 2 mg / mL.

[0060] In some embodiments of the invention, the cells comprise stem cells.

[0061] In some preferred embodiments of the present invention, the stem cells include mesenchymal stem cells.

[0062] According to a fourth aspect of the present invention, a kit is provided, comprising:

[0063] (1) the VCAM1 antibody conjugate according to the second aspect of the present invention; and

[0064] (2) The polydopamine nanoparticles as described in the third aspect of the present invention.

[0065] According to the fifth aspect of the present invention, a use of the double-stranded DNA fragment described in the first aspect of the present invention, the VCAM1 antibody conjugate described in the second aspect of the present invention, the polydopamine nanoparticles described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention in cell engineering is proposed.

[0066] In some embodiments of the invention, the cells comprise stem cells.

[0067] In some preferred embodiments of the present invention, the stem cells include mesenchymal stem cells.

[0068] According to a sixth aspect of the present invention, a cell engineering modification method is provided, comprising the following steps:

[0069] S1: mixing the cells to be modified with the dopamine monomer described in the third aspect of the present invention to react to obtain cells modified with polydopamine nanoparticles;

[0070] S2: The cells modified with the polydopamine nanoparticles described in step S1 are mixed with the VCAM1 antibody conjugate described in the second aspect of the present invention, and the engineered cells are obtained by reaction.

[0071] In some embodiments of the present invention, the cells to be transformed in step S1 include stem cells.

[0072] In some preferred embodiments of the present invention, the stem cells include mesenchymal stem cells.

[0073] In some embodiments of the present invention, the final concentration of the dopamine monomer in step S1 is 0.05-5 mg / mL.

[0074] In some preferred embodiments of the present invention, the final concentration of the dopamine monomer in step S1 is 2 mg / mL.

[0075] In some embodiments of the present invention, the cells to be modified in step S1 are mixed with dopamine monomers in a Ca-containing 2+ reacted in an alkaline buffer.

[0076] In some preferred embodiments of the present invention, the alkaline buffer is Tris-HCl buffer.

[0077] In some more preferred embodiments of the present invention, the concentration of the Tris-HCl buffer is 10 mM.

[0078] In some preferred embodiments of the present invention, the pH value of the alkaline buffer solution is 7.8-8.8.

[0079] In some more preferred embodiments of the present invention, the pH value of the alkaline buffer solution is 8.35.

[0080] In some preferred embodiments of the present invention, the Ca in the alkaline buffer is 2+ The content is 0.11~4.44wt%.

[0081] In some more preferred embodiments of the present invention, the Ca in the alkaline buffer is 2+ The content is 1.11wt%.

[0082] In some embodiments of the present invention, the reaction temperature in step S1 is 20-37°C.

[0083] In some preferred embodiments of the present invention, the reaction temperature in step S1 is 25°C.

[0084] In some embodiments of the present invention, the reaction time in step S1 is 5 to 15 minutes.

[0085] In some preferred embodiments of the present invention, the reaction time in step S1 is 8 minutes.

[0086] In some embodiments of the present invention, the reaction in step S1 is carried out under an oscillation reaction at a rotation speed of 400-600 rpm.

[0087] In some preferred embodiments of the present invention, the reaction in step S1 is performed under an oscillation reaction at a rotation speed of 500 rpm.

[0088] In some embodiments of the present invention, the density of the cells to be transformed in the reaction system in step S1 is 5×10 5 ~1.5×10 6 pieces / mL.

[0089] In some preferred embodiments of the present invention, the density of the cells to be transformed in the reaction system in step S1 is 1×10 6 pieces / mL.

[0090] In some embodiments of the present invention, the density of the polydopamine nanoparticle-modified cells in the reaction system in step S2 is 3×10 6 ~7×10 6 pieces / mL.

[0091] In some preferred embodiments of the present invention, the density of the polydopamine nanoparticle-modified cells in the reaction system in step S2 is 5×10 6 pieces / mL.

[0092] In some embodiments of the present invention, the final concentration of the VCAM1 antibody conjugate in the reaction system in step S2 is 0.5-1.5 μM.

[0093] In some preferred embodiments of the present invention, the final concentration of the VCAM1 antibody conjugate in the reaction system in step S2 is 1 μM.

[0094] In some embodiments of the present invention, the reaction temperature in step S2 is 20-37°C.

[0095] In some preferred embodiments of the present invention, the reaction temperature in step S2 is 25°C.

[0096] In some embodiments of the present invention, the reaction time in step S2 is 15 to 60 minutes.

[0097] In some preferred embodiments of the present invention, the reaction time in step S2 is 30 minutes.

[0098] In some embodiments of the present invention, the reaction in step S2 is carried out under an oscillation reaction at a rotation speed of 400-600 rpm.

[0099] In some preferred embodiments of the present invention, the reaction in step S2 is performed under an oscillation reaction at a rotation speed of 500 rpm.

[0100] According to the seventh aspect of the present invention, the use of the cell according to the third aspect of the present invention or the cell engineering method according to the sixth aspect of the present invention in at least one of (1) to (3) is proposed:

[0101] (1) Preparation of products for tissue repair;

[0102] (2) preparing products for enhancing the retention of cells in damaged tissues;

[0103] (3) Prepare products for enhancing the survival ability of cells in damaged tissues.

[0104] In some embodiments of the present invention, the product comprises a medicine or a health product.

[0105] In some preferred embodiments of the present invention, the dosage form of the drug is a liquid dosage form.

[0106] In some embodiments of the invention, the cells comprise stem cells.

[0107] In some preferred embodiments of the present invention, the stem cells include mesenchymal stem cells.

[0108] The present invention has at least the following beneficial effects:

[0109] (1) The double-stranded DNA fragment provided by the present invention comprises a pair of DNAs (DNA monomer 1 and DNA monomer 2) with complementary sequences. The double-stranded DNA fragment is designed with a desired DNA sequence by utilizing the programmability of DNA. The DNA fragment can be used to modify the VCAM1 antibody on the surface of stem cells, thereby improving the adhesion of stem cells to damaged tissues and promoting tissue repair.

[0110] (2) The VCAM1 antibody conjugate provided by the present invention can be modified on the surface of stem cells, thereby improving the adhesion ability of stem cells, allowing them to reside in damaged tissues for a long time and better play a repair role;

[0111] (3) The polydopamine nanoparticles provided by the present invention are formed by spontaneous polymerization of dopamine monomers in an alkaline buffer solution. 2+ The bridging effect of the glycosides binds to the surface of stem cells, thereby enhancing the resistance of stem cells to reactive oxygen species in damaged tissues, thereby increasing the survival rate of stem cells in damaged tissues and promoting tissue repair;

[0112] (4) The cells provided by the present invention are engineered cells whose surfaces are modified with the aforementioned VCAM1 antibody conjugate and polydopamine nanoparticles. Modification with the aforementioned functional molecules can simultaneously improve the retention rate and survival rate of stem cells in damaged tissues without affecting the viability and migration ability of the stem cells.

[0113] (5) The stem cell engineering transformation method proposed in the present invention only requires that the cells to be transformed be combined with dopamine monomers in a Ca-containing 2+ The invention relates to a method for preparing an engineered stem cell by mixing the DNA monomer 1 with an alkaline buffer solution to allow the dopamine monomer to self-polymerize on the cell surface; then linking the DNA monomer 1 with the VCAM1 antibody; finally, mixing the DNA monomer 1-VCAM1 antibody covalent conjugate, DNA monomer 2, and cells modified with polydopamine nanoparticles, and linking the VCAM1 antibody to the cell surface using a DNA hybridization chain reaction, thereby obtaining engineered stem cells. Compared with existing stem cell modification methods, the stem cell modification method provided by the present invention is simple to operate, time-saving, highly efficient, has no safety risks, has a wide range of administration routes, and can effectively improve the retention rate and survival rate of stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0115] Figure 1 Schematic diagram of the process of stem cell engineering transformation method in Example 1 of the present invention;

[0116] Figure 2The scanning electron microscopy results of the control group cells and the polydopamine-modified group mesenchymal stem cells in Example 1 of the present invention are shown, wherein the scale bar is 2 μm;

[0117] Figure 3 The transmission electron microscopy results of the control group cells and the polydopamine-modified group mesenchymal stem cells in Example 1 of the present invention are shown, wherein the scale bar is 2 μm;

[0118] Figure 4 This is a diagram showing the SDS-PAGE electrophoresis results of the VCAM1 antibody and the DNA1-VCAM1 antibody covalent conjugate in Example 1 of the present invention;

[0119] Figure 5 Figure 1 is a flow cytometry result graph showing affinity binding of the VCAM1 antibody and DNA1-VCAM1 antibody covalent conjugate to K562 and C166 cells in Example 1 of the present invention; wherein A and B are the flow cytometry histogram and the FITC channel fluorescence intensity statistical graph, respectively;

[0120] Figure 6 This is a flow cytometry result diagram of mesenchymal stem cells in the control group, polydopamine-modified group, and polydopamine-VCAM1 antibody-modified group in Example 1 of the present invention;

[0121] Figure 7 This is a laser confocal image of mesenchymal stem cells modified with polydopamine and VCAM1 antibody in Example 1 of the present invention, wherein the scale bar is 25 μm;

[0122] Figure 8 The scanning electron microscopy results of the control group cells and the polydopamine-modified group embryonic stem cells in Example 2 of the present invention are shown, wherein the scale bar is 2 μm;

[0123] Figure 9 This is a laser confocal image of embryonic stem cells modified with polydopamine and VCAM1 antibody in Example 2 of the present invention, wherein the scale bar is 25 μm;

[0124] Figure 10 Flow cytometry analysis of apoptosis of mesenchymal stem cells in the control group, polydopamine-modified group, and polydopamine and VCAM1 antibody-co-modified group in the experimental examples of the present invention;

[0125] Figure 11 Figure 2 is a graph showing the migration of mesenchymal stem cells in the control group and the engineered group in the experimental example of the present invention; wherein A is a micrograph of the mesenchymal stem cells in each group after migration, wherein the scale bar is 100 μm, and B is a statistical graph showing the number of cells that migrated in each group;

[0126] Figure 12 Statistical graph of the paracrine factor content of mesenchymal stem cells in the control group and the engineered transformation group in the experimental example of the present invention;

[0127] Figure 13 The fluorescence imaging images of intracellular reactive oxygen species in the control group and the polydopamine-modified group of mesenchymal stem cells treated with hydrogen peroxide in the experimental example of the present invention are shown, wherein the scale bar is 50 μm;

[0128] Figure 14 This is a graph showing the results of detecting the cell viability of mesenchymal stem cells in the control group and the engineered group treated with different concentrations of hydrogen peroxide using the CCK8 method in the experimental examples of the present invention; wherein, "ns" represents no significant difference, and "***" represents p < 0.001;

[0129] Figure 15 Live-dead fluorescence imaging of mesenchymal stem cells in the control group and the engineered group treated with different concentrations of hydrogen peroxide;

[0130] Figure 16 Figures 1 and 2 show the results of mesenchymal stem cell adhesion to C166 cells in the control group and the engineered group in the experimental examples of the present invention; Figure A shows the fluorescence imaging of mesenchymal stem cells adhesion to C166 cells in the control group and the engineered group, with a scale bar of 100 μm; Figure B shows the statistical graph of the number of mesenchymal stem cells adhering to C166 cells in each group;

[0131] Figure 17 Schematic diagram of the process of establishing a rat myocardial infarction model and injecting cells into the myocardium in the experimental example of the present invention;

[0132] Figure 18 Figure 1 shows the retention and survival results of mesenchymal stem cells in the control group and the engineered group three days after injection of mesenchymal stem cells into the infarcted myocardium of rats in a test example of the present invention; Figure A shows the in vivo imaging of mesenchymal stem cells in the infarcted myocardium of rats in the control group and the engineered group; Figure B shows the statistical graph of the bioluminescence intensity of mesenchymal stem cells in the infarcted myocardium of rats in each group;

[0133] Figure 19 Figure 1 is a graph showing the results of the engineered mesenchymal stem cells restoring cardiac function in rats with infarction in a test example of the present invention; wherein A is a cardiac ultrasound image of rats in each group after 28 days of treatment; B is a statistical graph of the left ventricular ejection fraction of the hearts in each group; and C is a statistical graph of the left ventricular fractional shortening of the hearts in each group;

[0134] Figure 20 Figures 1 and 2 show the results of the engineered mesenchymal stem cells repairing infarcted myocardium in rats in the experimental examples of the present invention; A is a Masson's trichrome staining image of heart tissue sections in each group after 28 days of treatment; B is a statistical graph of the infarct area size in the myocardial tissue sections in each group; and C is a statistical graph of the left ventricular wall thickness in the myocardial tissue sections in each group.

[0135] Figure 21Figure 1 is a fluorescence imaging image of reactive oxygen species in myocardial tissue of rats treated with engineered mesenchymal stem cells for three days after myocardial infarction in the experimental example of the present invention; Figure A is a fluorescence imaging image of reactive oxygen species in heart slices of each group, with a scale of 100 μm; Figure B is a statistical graph of the fluorescence intensity of reactive oxygen species in heart slices of each group;

[0136] Figure 22 These are CD31 vascular immunofluorescence staining images of rats after myocardial infarction repaired by engineered mesenchymal stem cells in the experimental examples of the present invention; A is a CD31 vascular fluorescence imaging image of heart sections of each group 28 days after treatment, with a scale bar of 100 μm; B is a statistical graph of the number of CD31 blood vessels in heart sections of each group;

[0137] Figure 23 These are images of α-SMA vascular immunofluorescence staining after the engineered mesenchymal stem cells repaired the infarcted myocardium in the experimental examples of the present invention; wherein, A is an α-SMA vascular fluorescence imaging image of the heart sections of each group after 28 days of treatment, with a scale of 100 μm; B is a statistical graph of the number of α-SMA vessels in the heart sections of each group. DETAILED DESCRIPTION

[0138] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0139] Example 1

[0140] This embodiment provides a stem cell engineering transformation method, and according to the method, an engineered mesenchymal stem cell is prepared. The flow chart of the stem cell engineering transformation method is as follows: Figure 1 As shown, the specific steps include:

[0141] 1. Preparation of polydopamine nanoparticle-modified mesenchymal stem cells:

[0142] (1) Dissolve 4 mL of 1 M CaCl2 and 0.4 mL of 1 M Tris-HCl solution in 35 mL of deionized water, and adjust the pH to 8.35 with 0.1 M NaOH solution to obtain a Tris-HCl alkaline buffer solution. 2+ The content is 1.11wt%;

[0143] (2) Weigh 10 mg of dopamine hydrochloride and dissolve it in 50 μL of Tris-HCl alkaline buffer prepared in step (1) to prepare a dopamine hydrochloride solution with a concentration of 200 mg / mL;

[0144] (3) Take 990 μL of Tris-HCl alkaline buffer and add it to the 1×10 6 10 μL of the dopamine hydrochloride solution prepared in step (2) was added to the cell suspension, and the cells were gently blown 5 times with a pipette to evenly blow the cells.

[0145] (4) Transfer the evenly dispersed cell suspension from step (3) to a shaking metal bath and shake at 500 rpm at 25°C for 8 min, gently pipetting 1 to 2 times in between.

[0146] (5) After the reaction is completed, the cell suspension is removed and centrifuged at 300 g for 5 min. The supernatant is removed to obtain a cell pellet.

[0147] (6) Add 1 mL of 1×PBS to the cell pellet obtained by centrifugation in step (5) to resuspend the cells, centrifuge again at 300 g for 5 min, and remove the supernatant to prepare polydopamine nanoparticle-modified mesenchymal stem cells.

[0148] The mesenchymal stem cells modified with polydopamine nanoparticles prepared in step 1 were characterized by scanning electron microscopy and transmission electron microscopy. Figure 2 (SEM) and Figure 3 (Transmission electron microscopy) shows that the control group cells are unmodified mesenchymal stem cells.

[0149] Depend on Figures 2 and 3 It can be seen that the cell surface of the control group is relatively smooth, while the surface of the mesenchymal stem cells modified with polydopamine nanoparticles is rough, and obvious nanoparticles can be seen, indicating that the present invention successfully modifies the surface of mesenchymal stem cells with polydopamine.

[0150] 2. Covalently link DNA monomer 1 and VCAM1 antibody:

[0151] (1) The nucleotide sequence of DNA monomer 1 covalently coupled to the VCAM1 antibody is: 5'-GGTAAACA AAGGCTGCTCCAGAAGCAGCCTTTGTTTACCGATTAGC-3' (SEQ ID NO: 1), and an NH2C6 group is connected to the 5' end. The DNA monomer 1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. In the subsequent steps, DNA monomer 1 is referred to as DNA1;

[0152] (2) DNA1 is linked to succinimidyl-4-formylbenzoate (S-4FB):

[0153] ① Prepare a DNA1 solution by vortexing 30 nmol DNA1 in 60 μL of Buffer M (i.e., 1× PBS, pH 8.0). The DNA1 solution was then annealed in a PCR instrument at 95°C for 5 min, 84°C for 2 min, 73°C for 2 min, 62°C for 2 min, 51°C for 2 min, 40°C for 2 min, 30°C for 2 min, and 25°C for 2 min.

[0154] ② Dissolve 1 mg of S-4FB (Solulink, Catalog No. S-1004-010) in 40 μL of anhydrous dimethyl sulfoxide (DMSO) to prepare a 25 mg / mL S-4FB solution. Add 30 μL of anhydrous DMSO and 7.2 μL of the S-4FB solution to the annealed DNA1 solution, vortex to mix thoroughly, and shake at 400 rpm at 25°C for 2 h.

[0155] ③ After the reaction is completed, transfer the reaction mixture to a 10 kDa ultrafiltration tube, add 400 μL of Buffer C (i.e., 1× PBS, pH 6.0), centrifuge at 13,000 g for 10 min, discard the waste liquid, and repeat this process four times; then, invert the ultrafiltration tube into a new centrifuge tube, centrifuge at 1,300 g for 3 min, and add Buffer C to the collected DNA1-S-4FB to a volume of 100 μL to obtain a DNA1-S-4FB solution;

[0156] (3) VCAM1 antibody linked to succinimidyl-6-hydrazinonicotinic acid acetone hydrazone (S-HyNic):

[0157] ① Transfer 100 μg of VCAM1 antibody (Biolegend, Cat. No. 105728) to a 10 kDa ultrafiltration tube, add Buffer M to a total volume of 500 μL, centrifuge at 13,000 g for 10 min, and discard the waste liquid; then add Buffer M again to a total volume of 500 μL, centrifuge at 13,000 g for 10 min, and discard the waste liquid; then invert the ultrafiltration tube into a new centrifuge tube, centrifuge at 1300 g for 3 min, and dilute the collected VCAM1 antibody to 100 μL with Buffer M to obtain a VCAM1 antibody solution;

[0158] ② Dissolve 100 μg of S-HyNic (Solulink, Catalog No. S-1002-105) in 35 μL of anhydrous DMSO to prepare a 2.86 mg / mL S-HyNic solution. Add 3 μL of the S-HyNic solution to the VCAM1 antibody solution and mix thoroughly by pipetting to obtain a VCAM1 antibody mixed solution. Shake at 400 rpm at 25°C for 2 h.

[0159] ③ After the reaction is completed, transfer the VCAM1 antibody mixed solution to a 30 kDa ultrafiltration tube, add Buffer C to a total volume of 500 μL, centrifuge at 13,000 g for 10 minutes, discard the waste liquid, and repeat this washing process three times; then, turn the ultrafiltration tube upside down in a new centrifuge tube, centrifuge at 1,500 g for 3 minutes, and add Buffer C to the collected S-HyNic-VCAM1 antibody to a volume of 100 μL to obtain the S-HyNic-VCAM1 antibody solution;

[0160] (4) Covalent coupling of DNA1-S-4FB and S-HyNic-VCAM1 antibody:

[0161] ① Take 11 μL of DNA1-S-4FB solution prepared in step (2) and 12 μL of 10× Turbolink Catalyst Buffer (Solulink, catalog number S-2006-105) and add them to 100 μL of S-HyNic-VCAM1 antibody solution prepared in step (3), pipette to mix evenly, and shake at 400 rpm at 25°C for 2 h;

[0162] ② After the reaction, the reaction system was transferred to a 50 kDa ultrafiltration tube, and 1× PBS (pH 7.4) was added to make the total volume 500 μL. The tube was centrifuged at 13,000 g for 10 min, the waste liquid was discarded, and the washing process was repeated 5 times. The ultrafiltration tube was then inverted in a new centrifuge tube and centrifuged at 1,500 g for 3 min. The collected DNA1-VCAM1 antibody was adjusted to 100 μL with 1× PBS (pH 7.4) to obtain the DNA1-VCAM1 antibody covalent conjugate.

[0163] In order to test whether DNA1 and VCAM1 antibody are successfully linked, the molecular weight of the DNA1-VCAM1 antibody covalent conjugate prepared in step 2 was detected by SDS-PAGE electrophoresis. Figure 4 shown.

[0164] Depend on Figure 4It can be seen that compared with the band migration rate of the VCAM1 antibody, the band migration rate of the DNA1-VCAM1 antibody covalent conjugate is significantly slower. The connection of DNA1 to the VCAM1 antibody increases the molecular weight of the VCAM1 antibody, indicating that DNA1 is successfully covalently coupled to the VCAM1 antibody.

[0165] To evaluate whether the connection of DNA1 would affect the specificity of VCAM1 antibody, the binding specificity of the DNA1-VCAM1 antibody covalent conjugate prepared in step 2 was detected by flow cytometry. Figure 5 As shown; the control group was unmodified K562 cells or C166 cells.

[0166] Depend on Figure 5 It can be seen that the binding of VCAM1 antibody and DNA1-VCAM1 antibody covalent conjugate to K562 (which does not express VCAM1) is very weak, while the binding of VCAM1 antibody and DNA1-VCAM1 antibody covalent conjugate to C166 (which overexpresses VCAM1) can be effectively bound, and there is no significant difference in the binding strength of VCAM1 antibody and DNA1-VCAM1 antibody covalent conjugate to C166, which indicates that the attachment of DNA1 to VCAM1 antibody does not affect the specificity of VCAM1 antibody.

[0167] 3. Preparation of mesenchymal stem cells engineered with polydopamine and VCAM1 antibodies:

[0168] (1) The nucleotide sequence of DNA monomer 2, which is complementary to DNA monomer 1, is: 5'-TTTTTTTTTTTTTTTTTTTTGCTAATCGGTAAACAAAGGCTGC-3' (SEQ ID NO: 2), and a cholesterol group is connected to the 5' end; DNA monomer 2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and DNA monomer 2 is referred to as DNA2 in subsequent steps;

[0169] (2) Take 1×10 6 The polydopamine-surface-modified mesenchymal stem cells prepared in step 1 above were resuspended in 200 μL of 1× PBS (pH 7.4), and cholesterol-labeled DNA2 was added to make the final concentration of DNA2 1 μM. At the same time, 3 μL of the DNA1-VCAM1 antibody covalent conjugate prepared in step 2 above was added and mixed by pipetting to obtain a cell mixture solution;

[0170] (3) Transfer the cell mixture solution from step (2) to a shaking metal bath and shake at 500 rpm at 25°C for 30 min, mixing once every 5 min with a pipette;

[0171] (4) After the reaction, 1 mL of 1× PBS (pH 7.4) was added to the cell mixture solution, centrifuged at 300 g for 5 min, the supernatant was removed, and the washing process was repeated twice to obtain mesenchymal stem cells engineered with polydopamine and VCAM1 antibodies.

[0172] In order to test whether the DNA-VCAM1 conjugate was successfully modified to the cell surface, the VCAM1 antibody (labeled with FITC) on the surface of the mesenchymal stem cells engineered with polydopamine and VCAM1 antibody prepared in step 3 was characterized by flow cytometry and laser confocal microscopy. The results were as follows: Figure 6 and Figure 7 shown.

[0173] Depend on Figure 6 It can be seen that the mesenchymal stem cells modified with polydopamine and VCAM1 antibody have obvious fluorescence signals compared with the control group (unmodified cells) and the polydopamine modified group (i.e., the cells prepared in step 1 of Example 1), indicating that the VCAM1 antibody was successfully modified on the mesenchymal stem cells.

[0174] Depend on Figure 7 It can be seen that the FITC fluorescence signal is located on the surface of mesenchymal stem cells, which indicates that the VCAM1 antibody is successfully modified on the cell membrane of mesenchymal stem cells.

[0175] Example 2

[0176] This embodiment provides a stem cell engineering method, and an engineered embryonic stem cell is prepared according to the method. The specific preparation steps are the same as those in Example 1.

[0177] The prepared polydopamine nanoparticles modified embryonic stem cells were characterized by scanning electron microscopy. Figure 8 The control group cells were unmodified embryonic stem cells.

[0178] Depend on Figure 8 It can be seen that the cell surface of the control group is relatively smooth, while the surface of the embryonic stem cells modified with polydopamine nanoparticles is rough, and obvious nanoparticles can be seen, indicating that the present invention successfully modifies the surface of embryonic stem cells with polydopamine.

[0179] In order to test whether the DNA-VCAM1 conjugate was successfully modified to the cell surface, the VCAM1 antibody (labeled with FITC) on the surface of embryonic stem cells engineered with polydopamine and VCAM1 antibody was characterized using laser confocal microscopy. Figure 9 shown.

[0180] Depend on Figure 9It can be seen that the FITC fluorescence signal is located on the surface of embryonic stem cells, which indicates that the VCAM1 antibody is successfully modified on the cell membrane of embryonic stem cells.

[0181] Test example

[0182] This test example tested the various characteristics and properties of the engineered mesenchymal stem cells prepared in Example 1. The specific test steps and results are as follows:

[0183] 1. Test the effect of surface modification on the activity of mesenchymal stem cells:

[0184] (1) Take 1×10 5 Unmodified mesenchymal stem cells (control group), 1×10 5 Polydopamine-modified mesenchymal stem cells (prepared in step 1 of Example 1) and 1×10 5 Each polydopamine-VCAM1 antibody-modified mesenchymal stem cell (prepared in step 3 of Example 1) was resuspended in 195 μL Annexin V-FITC conjugate (Beyotime, product number C1062S-2);

[0185] (2) Add 5 μL of Annexin V-FITC (Beyotime, product number C1062S-1) to each group and mix thoroughly by gently pipetting;

[0186] (3) Add 10 μL of propidium iodide staining solution (Beyotime, product number C1062S-3) to each tube and mix thoroughly by gently pipetting.

[0187] (4) Incubate in the dark at 25°C for 15 min, then place in an ice bath. Gently pipette 2–3 times during the incubation period.

[0188] (5) Flow cytometry test, the results are as follows Figure 10 shown.

[0189] Depend on Figure 10 It can be seen that there is no significant difference in the viability of mesenchymal stem cells modified with polydopamine and mesenchymal stem cells modified with polydopamine-VCAM1 antibody compared with unmodified mesenchymal stem cells, indicating that the polydopamine and VCAM1 antibody conjugate has almost no effect on the viability of mesenchymal stem cells when modified on the cell surface.

[0190] 2. Test the effect of surface modification on the migration ability of mesenchymal stem cells:

[0191] (1) 200 μL of unmodified mesenchymal stem cells (control group), polydopamine-modified mesenchymal stem cells (prepared in step 1 of Example 1), and polydopamine-VCAM1 antibody-modified mesenchymal stem cells (prepared in step 3 of Example 1) resuspended in serum-free DMEM were seeded into the upper chambers of different Transwell chambers (Corning, 3422) in a 24-well plate, with a cell concentration of 2.5×10 5 / mL, add 700 μL of DMEM medium containing 10% serum into the lower chamber of the Transwell chamber;

[0192] (2) Place the 24-well plate in a cell culture incubator at 37°C and 5% CO2 for 12 h;

[0193] (3) Remove the chamber from the 24-well plate, discard the culture medium, and then immerse the chamber in PBS containing 4% paraformaldehyde for 15 minutes to fix the cells. After fixation, wash the chamber twice with 1× PBS;

[0194] (4) Add crystal violet staining solution to the chamber, immerse the upper and lower sides of the chamber bottom in the staining solution, and stain for 20 min. After staining, wash the chamber twice with 1× PBS and wipe the cells on the upper side of the chamber bottom clean with a cotton swab;

[0195] (5) Use a microscope to observe and photograph the cells on the bottom side of the chamber, and use ImageJ software to count and quantify the number of cells. The results are as follows Figure 11 shown.

[0196] Depend on Figure 11 It can be seen that there is no significant difference in the number of migrating cells in the polydopamine modified group and the polydopamine-VCAM1 antibody modified group compared with the unmodified mesenchymal stem cells, indicating that the modification of the cell surface by the polydopamine and VCAM1 antibody conjugate has almost no effect on the migration ability of mesenchymal stem cells.

[0197] In addition, this experimental example also tested the effects of polydopamine modification and polydopamine and VCAM1 antibody modification on the paracrine secretion of mesenchymal stem cells. The specific steps are as follows:

[0198] Take 5×10 5 Unmodified mesenchymal stem cells (control group), 5×10 5 Polydopamine-modified mesenchymal stem cells (prepared in step 1 of Example 1) and 5×10 5Mesenchymal stem cells modified with polydopamine-VCAM1 antibodies (prepared in step 3 of Example 1) were seeded in three culture dishes, and 8 mL of complete culture medium was added to each culture dish to allow the three groups of cells to adhere and grow for 3 days. After 3 days, the complete culture medium was removed, and 8 mL of serum-free culture medium was added to each of the three groups of cells, and the cells were cultured for another 3 days. After the serum-free culture was completed, the serum-free culture medium from the three groups of cells was collected. ELISA kits (Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog numbers JLC3311, JLC3907, JLC3897, JLC3170, and JLC4130) were used to detect the concentrations of FGF2, VEGF, HGF, SDF-1, and TGF-β1 in the serum-free culture medium of the three groups. The results are shown in FIG. Figure 12 shown.

[0199] Depend on Figure 12 It can be seen that there is no significant difference in the concentration of various paracrine factors secreted by the control group cells, polydopamine modified group cells and polydopamine-VCAM1 antibody modified group cells, which indicates that polydopamine modification and polydopamine and VCAM1 antibody modification have no effect on the paracrine secretion of mesenchymal stem cells.

[0200] 3. Testing the ability of polydopamine-modified mesenchymal stem cells to scavenge hydrogen peroxide:

[0201] (1) Three groups were set up: control group, control group + hydrogen peroxide, and polydopamine modified group + hydrogen peroxide. The control group consisted of unmodified mesenchymal stem cells, and the polydopamine modified group consisted of cells prepared in step 1 of Example 1. Each group contained 1×10 6 Resuspend each mesenchymal stem cell in 500 μL 1× PBS; add 30 wt% hydrogen peroxide to a final concentration of 1 mM and mix thoroughly by gently pipetting.

[0202] (2) Incubate at room temperature for 1 h. After incubation, centrifuge at 300 g for 5 min, discard the supernatant, wash the cells once with 1× PBS, and resuspend the cells in 1 mL of 1× PBS.

[0203] (3) Add 1 μL of DCFH-DA probe (Beyotime, product number S0033S-1) to the cell suspension and incubate at 37°C for 20 min;

[0204] (4) After incubation, centrifuge at 300 g for 5 min, wash the cells twice with 1× PBS, and resuspend the cells in 1 mL of 1× PBS;

[0205] (5) Take 30 μL of the cell suspension obtained in step (4) and place it in a confocal dish. Use a laser confocal microscope to image the cells. The results are as follows: Figure 13 shown.

[0206] Depend on Figure 13 It can be seen that the intracellular reactive oxygen species fluorescence intensity of the polydopamine modified group + hydrogen peroxide is significantly lower than that of the control group + hydrogen peroxide, which indicates that polydopamine modification can effectively improve the reactive oxygen species scavenging ability of mesenchymal stem cells.

[0207] 4. CCK8 assay to test the cell viability of engineered mesenchymal stem cells after hydrogen peroxide treatment:

[0208] (1) Unmodified mesenchymal stem cells (control group), polydopamine-modified mesenchymal stem cells (prepared in step 1 of Example 1), and polydopamine-VCAM1 antibody-modified mesenchymal stem cells (prepared in step 3 of Example 1) were resuspended in complete DMEM medium to obtain a cell concentration of 2×10 5 Cell suspension of 100 cells / mL;

[0209] (2) 100 μL of cell suspension from each group was inoculated into a 96-well plate, with 9 replicate wells for each group, for a total of 27 wells. The 9 replicate wells of each group were then divided equally into 3 groups: a control group without hydrogen peroxide, a low-concentration treatment group with a final hydrogen peroxide concentration of 400 μM, and a high-concentration treatment group with a final hydrogen peroxide concentration of 800 μM.

[0210] (3) Place the 96-well plate in a 37°C, 5% CO2 incubator and incubate for 2 h;

[0211] (4) After incubation, add 10 μL of CCK8 solution to each well and continue incubation for 2 h;

[0212] (5) Use an enzyme-labeled instrument to measure the absorbance of cells in each well at 450 nm and calculate the cell viability. The results are as follows: Figure 14 shown.

[0213] Depend on Figure 14 It can be seen that after treatment with 400 μM and 800 μM hydrogen peroxide, the cell viability of polydopamine-modified mesenchymal stem cells or polydopamine-VCAM1 antibody-modified mesenchymal stem cells was significantly higher than that of unmodified mesenchymal stem cells, indicating that both polydopamine modification and polydopamine-VCAM1 antibody modification can effectively improve the survival rate of mesenchymal stem cells in the hydrogen peroxide microenvironment.

[0214] The three groups of cells treated with 800 μM hydrogen peroxide were stained with a live-dead staining kit and then imaged using laser confocal microscopy. The results are shown in Figure 2. Figure 15 shown.

[0215] Depend on Figure 15It can be seen that the number of living cells in the polydopamine-modified mesenchymal stem cells or the polydopamine-VCAM1 antibody-modified mesenchymal stem cells in the imaging field of view is significantly higher than that in the unmodified mesenchymal stem cell group, indicating that both polydopamine modification and polydopamine-VCAM1 antibody modification can effectively improve the survival of mesenchymal stem cells in the hydrogen peroxide microenvironment.

[0216] 5. Testing the Adhesion of Engineered Mesenchymal Stem Cells to Vascular Endothelial Cells C166:

[0217] (1) Equal amounts of C166 cells were seeded into three confocal microplates and cultured in a 37°C, 5% CO2 incubator. The cells were used for subsequent experiments when they grew to a confluence of more than 85%.

[0218] (2) Take 1×10 6 Unmodified mesenchymal stem cells (control group), polydopamine-modified mesenchymal stem cells (prepared in step 1 of Example 1), and polydopamine-VCAM1 antibody-modified mesenchymal stem cells (prepared in step 3 of Example 1) were resuspended in 1 mL of 1×PBS;

[0219] (3) Add 1 μL of Calcein AM (Beyotime, product number C2015S-1) to each tube, mix thoroughly by pipetting, and place in a 37°C incubator for staining for 40 min.

[0220] (4) After staining, centrifuge at 300 g for 5 min, remove the supernatant, wash the cells twice with 1× PBS, and finally resuspend in 1 mL of 1× PBS;

[0221] (5) The cell suspension was added to different confocal microplates seeded with C166 cells, and then the microplates were transferred to a 37°C incubator and incubated for 40 min;

[0222] (6) After incubation, remove the cell suspension from the dish and gently wash the dish three times with 1× PBS;

[0223] (7) Use laser confocal microscopy to observe the cells adhered to the dish and take pictures. Use ImageJ to quantify the number of adhered cells. The results are as follows: Figure 16 shown.

[0224] Depend on Figure 16 It can be seen that the number of mesenchymal stem cells modified with polydopamine and VCAM1 antibody adhering to C166 cells is about 4 times that of unmodified mesenchymal stem cells or mesenchymal stem cells modified with polydopamine, indicating that modification with VCAM1 antibody can significantly improve the adhesion ability of mesenchymal stem cells to vascular endothelial cells C166.

[0225] 6. Testing the retention and survival of engineered mesenchymal stem cells in a rat myocardial infarction model:

[0226] (1) Construction of rat myocardial infarction model:

[0227] The construction process of rat myocardial infarction model is as follows Figure 17 The specific steps are as follows:

[0228] Male SD rats weighing approximately 250 g were selected and intraperitoneally injected with 45 mg / kg of sodium pentobarbital. After the rats were anesthetized, their limbs were fixed, and an 18G indwelling needle was inserted into the rat's trachea. The indwelling needle was connected to a ventilator, which was turned on with the tidal volume set to 6.0 mL, the respiratory ratio set to 5:4, and the respiratory rate set to 80 breaths / min. The rat's chest cavity was opened to expose the heart, and the left anterior descending coronary artery of the rat was ligated with 5-0 silk suture at a position 2 to 3 mm below the pulmonary artery cone and the left atrial appendage. After ligation, mesenchymal stem cells were injected.

[0229] (2) Intramyocardial injection of engineered mesenchymal stem cells:

[0230] ① The mesenchymal stem cells used in this section were all luciferase-expressing SD rat bone marrow-derived mesenchymal stem cells (purchased from Nanjing Wan Muchun Co., Ltd.). Polydopamine-modified mesenchymal stem cells and polydopamine-VCAM1 antibody-modified mesenchymal stem cells were prepared according to the engineering modification method provided in Example 1;

[0231] ②1×10 6 Unmodified mesenchymal stem cells, 1×10 6 Polydopamine-modified mesenchymal stem cells and 1×10 6 Each polydopamine-VCAM1 antibody-modified mesenchymal stem cell was resuspended in 100 μL of 1× PBS. 100 μL of the cell suspension was injected into five sites at the border of the myocardial infarction using an insulin needle, with 20 μL injected into each site. The rat's chest was then closed, the air in the chest cavity was evacuated, and the skin and flesh were sutured with 5-0 silk sutures.

[0232] (3) Bioluminescence imaging:

[0233] D-luciferin potassium salt (Aladdin) was dissolved in sterile 1×PBS to a final concentration of 15 mg / mL. On the third day after the injection of mesenchymal stem cells, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital. After the rats were anesthetized, 2.5 mL of D-luciferin potassium salt solution was injected into the peritoneal cavity of each rat. The rats were then placed in a small animal in vivo imaging device for imaging. The results were as follows: Figure 18 shown.

[0234] Depend on Figure 18 It can be seen that the bioluminescence signal intensity of the polydopamine modified group was about 4 times that of the control group; the bioluminescence signal intensity of the polydopamine-VCAM1 antibody modified group was about 8 times that of the control group and about 2 times that of the polydopamine modified group, which shows that polydopamine modification can effectively improve the survival of mesenchymal stem cells in infarcted myocardium; compared with only polydopamine modification, modification of polydopamine and VCAM1 antibody can further promote the retention and survival of mesenchymal stem cells in damaged tissues.

[0235] 7. Testing the effect of engineered mesenchymal stem cells on myocardial repair in rats:

[0236] (1) Construction of rat myocardial infarction model and experimental grouping:

[0237] A rat myocardial infarction model was established according to the method described in step (1) in Part 6 of this experimental example. The experiment was divided into five groups: a sham operation group (without coronary artery ligation, and the rest of the steps were the same), a PBS group, a control group, a polydopamine-modified group, and a polydopamine-modified-VCAM1 antibody-modified group, with five rats in each group.

[0238] (2) Intramyocardial injection of engineered mesenchymal stem cells:

[0239] The mesenchymal stem cells used here are SD rat bone marrow-derived mesenchymal stem cells (purchased from Shanghai Biotechnology Co., Ltd.). The cells were injected into the myocardium according to the method described in step (2) of Part 6 of this experimental example;

[0240] Among them, the sham operation group was not injected with cells and PBS, the PBS group was injected with 100 μL 1×PBS intramyocardially, the control group was injected with unmodified mesenchymal stem cells, the polydopamine-modified group was injected with mesenchymal stem cells modified only with polydopamine (prepared according to the method of step 1 in Example 1), and the polydopamine-VCAM1 antibody-modified group was injected with mesenchymal stem cells modified with polydopamine-VCAM1 antibody (prepared according to the method of step 3 in Example 1);

[0241] (3) Small animal ultrasound detection of rat cardiac function:

[0242] After 4 weeks of treatment, the rats were placed in an anesthesia machine and anesthetized with isoflurane. Echocardiography was performed using the Vevo3100 imaging system. Left ventricular ejection fraction (LVEF) and fractional shortening (FS) were calculated based on M-mode ultrasound imaging. The calculation formula was: left ventricular ejection fraction = (left ventricular end-diastolic volume - left ventricular end-systolic volume) / left ventricular end-diastolic volume × 100%, fractional shortening = (left ventricular diastolic diameter - left ventricular systolic diameter) / left ventricular diastolic diameter × 100%. The results are shown in Figure 2. Figure 19 As shown;

[0243] (4) Pathological section examination of the repair effect of myocardial infarction in rats:

[0244] After 4 weeks of treatment, all rats were euthanized, and their hearts were removed. The hearts were fixed with paraformaldehyde, dehydrated with graded alcohol, and then embedded in paraffin to form myocardial tissue wax blocks. The hearts were cross-sectioned using a paraffin slicer with a thickness of 5 μm. The obtained heart sections were dewaxed with xylene and then rehydrated. The normal myocardium and fibrotic myocardium were stained using a Masson's trichrome staining kit (Solerbo). The stained sections were finally sealed with neutral resin and read using a pathology section scanner. The results are shown in the figure below. Figure 20 shown.

[0245] Depend on Figure 19 It can be seen that after 4 weeks of treatment, the left ventricular ejection fractions of the sham operation group, PBS group, control group, polydopamine modified group and polydopamine-VCAM1 antibody modified group were 86.64%, 37.55%, 51.01%, 63.53% and 75.61%, respectively. This indicates that compared with the sham operation group, the PBS group, control group, polydopamine modified group and polydopamine-VCAM1 antibody modified group have gradually enhanced effects on the recovery of cardiac function of rat infarcted heart.

[0246] Depend on Figure 20 It can be seen that compared with the sham operation group, the infarct size of the PBS group, control group, polydopamine modified group, and polydopamine-VCAM1 antibody modified group gradually decreased, while the left ventricular wall thickness gradually increased, which indicates that the repair effect increases successively, and the polydopamine-VCAM1 antibody modified group has the best therapeutic effect.

[0247] In addition, the tissue reactive oxygen species probe DHE was used to detect the reactive oxygen species level in the infarcted myocardial tissue after 3 days of treatment. Figure 21 shown.

[0248] Depend on Figure 21 It can be seen that compared with the sham operation group, the levels of reactive oxygen species in the myocardial tissue of the polydopamine modified group and the polydopamine and VCAM1 antibody modified group were lower, indicating that polydopamine modification can effectively improve the reactive oxygen species scavenging ability of mesenchymal stem cells.

[0249] After 4 weeks of treatment, immunofluorescence staining was performed on the myocardial tissues of each group. Figure 22 and 23 As shown. Figures 22-23Compared with the sham-operated group, the number of CD31- and α-SMA-positive vessels increased in the PBS, control, polydopamine-modified, and polydopamine and VCAM1 antibody-modified groups, indicating that the PBS, control, polydopamine-modified, and polydopamine and VCAM1 antibody-modified groups exhibited increasing effects in promoting angiogenesis. The main reason for the low retention of stem cells at the site of tissue injury is that the cells must undergo in vitro expansion and culture before being transplanted into the body. During this process, stem cells lose key cell surface biomolecules, such as adhesion molecules. Furthermore, the damaged tissue is hypoxic and contains excessive amounts of reactive oxygen species. This harsh microenvironment is the primary cause of transplanted stem cell death. Currently, some researchers have genetically engineered stem cells to overexpress relevant adhesion molecules on their cell surface, which has improved cell retention at the site of tissue injury. However, this method is complex, inefficient, time-consuming, costly, and poses safety risks. In addition, studies have reported that the use of hydrogel encapsulation of stem cells can increase cell retention time and survival rate, but excessive encapsulation volume is not conducive to the transmission of substances, and is likely to cause problems such as metabolite accumulation, insufficient supply of nutrients and hypoxia, and large-sized gel encapsulation is only suitable for local administration, which limits the cell administration route. Microgels can achieve single-cell encapsulation of stem cells. This encapsulation system does not limit the delivery route of cells and can effectively improve the survival rate of stem cells. However, microgel encapsulation will shield important biological functional molecules on the cell surface and may also affect the exchange of nutrients between cells and the outside world, affecting cell function. In order to overcome the shortcomings of the above method, the present invention provides a stem cell engineering transformation method, which is simple and safe to operate and has little effect on cells. The engineered stem cells prepared can be more efficiently retained and survive in damaged tissues and repair damaged tissues than untransformed stem cells.

[0250] The present invention also verified the above experimental results in induced pluripotent stem cells and cardiac stem cells, and the results confirmed that effects comparable to those of mesenchymal stem cells can be achieved, indicating that the stem cell engineering transformation method provided by the present invention is applicable to a variety of stem cell types.

[0251] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A method for engineering transformation of mesenchymal stem cells, characterized in that: The mesenchymal stem cell engineering transformation method comprises the following steps: S1: Mixing the mesenchymal stem cells to be modified with dopamine monomers to react and obtain cells modified with polydopamine nanoparticles; S2: mixing the cells modified with the polydopamine nanoparticles described in step S1 with the VCAM1 antibody conjugate, and reacting to obtain engineered cells; The final concentration of the dopamine monomer in step S1 is 0.05-5 mg / mL; The cells to be modified in step S1 are mixed with dopamine monomers in a solution containing 0.11-4.44 wt% Ca 2+ The reaction was carried out in Tris-HCl buffer; The reaction conditions in step S1 are 20-37° C. for 5-15 min; The VCAM1 antibody conjugate in step S1 is prepared by a preparation method comprising the following steps: A1: DNA monomer 1 and cross-linker 1 are mixed and reacted to obtain DNA monomer 1-cross-linker 1; A2: VCAM1 antibody and cross-linker 2 are mixed and reacted to obtain VCAM1 antibody-cross-linker 2; A3: mixing the DNA monomer 1-crosslinker 1 described in step A1 with the VCAM1 antibody-crosslinker 2 described in step A2 to react, thereby obtaining a VCAM1 antibody-DNA monomer 1 covalent conjugate; A4: mixing the VCAM1 antibody-DNA monomer 1 covalent conjugate described in step A3 with DNA monomer 2 to react to obtain a VCAM1 antibody conjugate; The DNA fragment includes a DNA monomer 1 and a DNA monomer 2; the nucleotide sequence of the DNA monomer 1 is shown in SEQ ID NO: 1; the nucleotide sequence of the DNA monomer 2 is shown in SEQ ID NO: 2; the nucleotide sequences of the DNA monomer 1 and the DNA monomer 2 are complementary; The 5' end of the nucleotide sequence of the DNA monomer 1 is connected to an NH2C6 group; The 5' end of the nucleotide sequence of the DNA monomer 2 is connected to a cholesterol group.

2. The method for engineering transformation of mesenchymal stem cells according to claim 1, characterized in that: The reaction conditions in step S2 are 20-37° C. for 15-60 min.

3. Use of the method for engineering transformation of mesenchymal stem cells according to claim 1 or 2 in at least one of (1) to (3): (1) Preparation of products for tissue repair; (2) Preparation of products for enhancing the retention of mesenchymal stem cells in damaged tissues; (3) Preparation of products for enhancing the survival ability of mesenchymal stem cells in damaged tissues.

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