Umbilical cord mesenchymal stem cells for treating myocardial infarction and preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an umbilical cord mesenchymal stem cell, a preparation method thereof, and an application in treating myocardial infarction. The present invention provides an umbilical cord mesenchymal stem cell, which secretes a specific exosome, wherein the exosome contains a fusion protein, wherein the fusion protein is formed by the fusion of a peptide targeting ischemic myocardial tissue and an exosome transmembrane protein CD9, and is anchored on the surface of the exosome, thereby ensuring that the exosomes secreted by the umbilical cord mesenchymal stem cell have stronger targeting and activity. The present invention also provides a method for preparing umbilical cord mesenchymal stem cells, and further, the umbilical cord mesenchymal stem cells prepared by the present invention are used to prepare a drug for the treatment of myocardial infarction.

Description

Technical Field

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

[0002] Myocardial infarction (MI) is a serious cardiovascular disease. Although the dying myocardium can be saved by reconstructing blood circulation through interventional therapy, drug thrombolysis or coronary artery bypass grafting, it is still the second leading cause of death in my country. After myocardial infarction occurs, a large number of myocardial cells die, the inflammatory response intensifies, fibroblasts transform into myofibroblasts, and then induce myocardial fibrosis, which eventually leads to heart failure.

[0003] In recent years, stem cells and the exosomes they secrete have become a strategy for the treatment of myocardial infarction. Studies have shown that stem cells can play a repair role by differentiating cardiomyocytes. Other studies have shown that the repair of infarcted myocardium is mainly achieved through paracrine secretion. Stem cells secrete growth factors, cytokines, chemokines and exosomes in the form of cell vesicles and act on cells, promoting angiogenesis, anti-apoptosis, inhibiting inflammatory response, improving the microenvironment, and promoting damage repair.

[0004] Exosomes are natural carriers for transporting biomolecules between cells and are also effective tools for drug therapy. They can improve the water solubility of drug systems, reduce drug toxicity, and can be cultured in large quantities in vitro. They are very stable. Therefore, exosome transplantation is very popular for the treatment of myocardial infarction. However, the contents of exosomes are unclear, the targeting specificity is poor, and the residence time in the myocardial infarction area is short, which limits their application. Therefore, the targeting of exosomes is an urgent problem that needs to be solved. Summary of the invention

[0005] Based on the deficiencies in the prior art, the purpose of the present invention is to provide an umbilical cord mesenchymal stem cell that secretes exosomes, the surface of which expresses a fusion protein of a peptide targeting ischemic myocardial tissue and an exosomal transmembrane protein CD9, thereby improving the targeting of the exosomes and the residence time in the lesion area, thereby improving the effect of umbilical cord mesenchymal stem cells in treating myocardial infarction.

[0006] One aspect of the present invention is to provide a peptide T12 targeting ischemic myocardial tissue. Preferably, the peptide of the present invention is obtained by screening through phage display technology. Specifically, the present invention uses phage display technology to screen and obtain peptide segments that specifically bind to myocardial ischemic tissue from a polypeptide library. Specifically, the amino acid sequence of the peptide T12 of the present invention is shown in SEQ ID No. 1: CTSCRPPRK.

[0007] Among them, the steps to obtain T12 are as follows:

[0008] (1) Construction of phage library to screen cardiomyocyte homing peptides;

[0009] (2) After three rounds of washing, cardiomyocyte homing peptides were enriched;

[0010] (3) Collect positive clones and sequence them;

[0011] (4) Screening of the best homing peptides targeting ischemic cardiomyocytes.

[0012] Another aspect of the present invention further provides an exosome transmembrane protein CD9, which binds to the CD9 receptor anchored on the surface of the exosome membrane, and its amino acid sequence is shown in SEQ ID No. 2:

[0013] MMLVGFLGCCGAVQESQCMLGLFFGFLLVIFAIEIAAAIWGYSHKDEVIKEVQEFYKDTYNKLKTKDEPQRETLKAIHYALNCCGLAGGVEQFISDICPKKDVLETFTVKSCPDAIKEVFDNKFHIIGAVGIGIAVVMGMERQRGRCSDQSLSPPEWSI.

[0014] Another aspect of the present invention also provides a fusion protein, specifically, T12 and CD9 protein are directly connected to construct the fusion protein T12-CD9. The fusion protein of the present invention binds to CD9R on the surface of exosomes through CD9, anchors the surface of exosomes, and makes T12 tightly connected to exosomes in the form of binding to CD9 receptors, and then targets ischemic myocardial tissue through T12, and brings the exosomes secreted by umbilical cord mesenchymal stem cells to the lesion area for treatment, thereby improving the targeting of exosomes and the residence time in the lesion area, and improving the effect of umbilical cord mesenchymal stem cells in treating myocardial infarction.

[0015] The steps for preparing the fusion protein are as follows:

[0016] (1) Artificially synthesize the DNA sequences of T12 and CD9;

[0017] (2) Inserting lentiviral vector to construct a recombinant expression vector;

[0018] (3) Transfect 293T cells to express the fusion protein.

[0019] Another aspect of the present invention is to provide an umbilical cord mesenchymal stem cell. Specifically, the exosomes secreted by the umbilical cord mesenchymal stem cell are anchored on the membrane surface of the exosome through the CD9 of the fusion protein binding to the CD9 receptor on the membrane surface of the exosome, and then the exosomes are targeted to the ischemic myocardial tissue through the polypeptide, thereby treating myocardial infarction.

[0020] The steps for preparing umbilical cord mesenchymal stem cells are as follows:

[0021] (1) Transfect 293T cells with a lentiviral vector expressing the fusion protein, culture the 293T cells, and harvest the fusion protein;

[0022] (2) Cultivating umbilical cord mesenchymal stem cells;

[0023] (3) Infecting umbilical cord mesenchymal stem cells with a lentivirus expressing the fusion protein;

[0024] (4) Cultivate umbilical cord mesenchymal stem cells and collect them in large quantities.

[0025] Another aspect of the present invention is to provide a drug for treating myocardial infarction. Specifically, the drug comprises the aforementioned umbilical cord mesenchymal stem cells and a pharmaceutically acceptable carrier.

[0026] Another aspect of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the treatment of myocardial infarction. Specifically, umbilical cord mesenchymal stem cells secrete exosomes, and the exosomes anchor the fusion protein T12-CD9 through the surface of the CD9 receptor, and then target the ischemic myocardial tissue through T12, and bring the exosomes to the lesion area for treatment, thereby improving the targeting of the exosomes, prolonging the residence time of the exosomes in the lesion area, and improving the effect of umbilical cord mesenchymal stem cells in the treatment of myocardial infarction.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention screens a polypeptide T12 that specifically targets ischemic myocardial tissue through phage display technology, and constructs a fusion protein with CD9. The property of CD9 binding to the CD9 receptor on the surface of the exosome membrane is used to anchor the fusion protein on the surface of the exosomes secreted by umbilical cord mesenchymal stem cells, and further uses T12 to bring the exosomes to the ischemic myocardial tissue, ultimately treating myocardial infarction.

[0029] 2. Through the targeting of T12 and CD9 as a bridge, the exosomes secreted by umbilical cord mesenchymal stem cells are brought to the lesion area, which improves the targeting and residence time in the lesion area, thereby improving the effect of umbilical cord mesenchymal stem cells in treating myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Electron microscopy of exosomes secreted by umbilical cord mesenchymal stem cells Figure 1 ;

[0031] Figure 2 Electron microscopy of exosomes secreted by umbilical cord mesenchymal stem cells Figure 2 ;

[0032] Figure 3WB expression of apoptosis-inducing factor 3 (AIFM3) and myocardial LC3B. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0034] The human umbilical cord mesenchymal stem cells used in the following embodiments were purchased from ATCC.

[0035] Example 1: Screening of peptide T12 targeting ischemic myocardial tissue using phage display technology

[0036] (1) Screening of cardiomyocyte homing peptides: 40 minutes after myocardial infarction in rats, phage clones were injected into the left ventricle through the apex. After circulating in the body for 10 minutes, PBS was injected again through the apex to elute unbound phages. The rats were then killed and the myocardial infarction area was extracted.

[0037] (2) Enrichment of cardiomyocyte homing peptides: The extracted tissues were processed and homogenized to prepare a suspension. The precipitate was collected after centrifugation, and then transfected into E. coli and amplified to obtain the first round of enriched phages. The first round of enriched phages were amplified again and injected into a rat myocardial infarction model for the second round of screening. Similarly, the third round of screening was performed to obtain the third round of enriched phages.

[0038] (3) After three rounds of screening, 20 phage clones (T1-T20) that bind to the myocardial infarction site with high specificity were obtained, and the obtained peptides were sequenced.

[0039] (4) The 20 peptides synthesized above were labeled with the Cy5.5 fluorescent group and injected into a rat myocardial infarction model via the tail vein. After 10 minutes of in vivo circulation, the rats were killed and the strongest cardiomyocyte binding peptide T12 was screened based on the fluorescence intensity and co-localization with cardiomyocytes.

[0040] Example 2: Construction of fusion protein T12-CD9 by T12 and CD9

[0041] The DNA sequences of T12 and CD9 were artificially synthesized respectively and inserted into lentiviral vectors to construct recombinant expression vectors. The lentiviral vectors were transfected into 293T cells for culture to express fusion proteins. The fusion proteins were extracted and sequenced for identification.

[0042] DNA sequence of T12: tgcaccagctgccgcccgccgcgcaaa (SEQ ID No. 3);

[0043] DNA sequence of CD9: atgatgctggtgggctttctgggctgctgcggcgcggtgcaggaaagccagtgcatgctgggcctgttttttggctttctgctggtgatttttgcgattgaaattgcggcgg cgatttggggctatagccataaagatgaagtgattaaagaagtgcaggaattttataaagatacctataacaaactgaaaaccaaagatgaaccgcagcgcgaaaccctgaaagcgattcatt atgcgctgaactgctgcggcctggcgggcggcgtggaacagtttattagcgatatttgcccgaaaaaagatgtgctggaaacctttaccgtgaaaagctgcccggatgcgattaaagaagtgt ttgataacaaatttcatattattggcgcggtgggcattggcattgcggtggtgatgggcatggaacgccagcgcggccgctgcagcgatcagagcctgagcccgccggaatggagcatt (seq ID No.4).

[0044] Example 3: Preparation of umbilical cord mesenchymal stem cells

[0045] Umbilical cord mesenchymal stem cells were cultured according to conventional means in the art, and cells with good growth status were selected and subcultured in a new cell culture dish at a ratio of 1:2. The cells were cultured overnight, and the density was about 50% on the second day. Take out the cells from the incubator 2 hours before infection, remove the original culture medium of the cells, add 1 / 2 volume of fresh culture medium, and then add the lentivirus stock solution packaged by 293T cells prepared in Example 2 to the cells, and infect for 4 hours at 37°C and 5% CO2. On the second day after infection (about 24 hours), the culture medium containing the virus was removed and replaced with fresh complete culture medium at 37°C and 5% CO2 for continued culture. 48 hours after infection, the selection medium containing 2 μg / ml puromycin was replaced for pressure screening until no more cell death was observed. Take the transfected cells with good growth status, culture the cells with α-MEM medium containing 15% Exo-FBS for 48 hours, and then collect the umbilical cord mesenchymal stem cells.

[0046] Take part of the umbilical cord mesenchymal stem cell suspension and centrifuge at 2000g for 30 minutes to remove dead cells and cell debris. Transfer the supernatant to a new centrifuge tube and add 0.5 times the volume of exosome separation reagent, turn it upside down, mix it thoroughly, and incubate it at 4°C overnight. The next day, centrifuge it at 10000g for 60 minutes at 4°C, and white precipitate bands can be seen on the tube wall. After discarding the supernatant, resuspend the precipitate with an appropriate amount of PBS, and then identify the collected exosomes by electron microscopy.

[0047] Example 4: Treatment of myocardial infarction with umbilical cord mesenchymal stem cells

[0048] (1) Preparation of rat myocardial infarction model

[0049] Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at a ratio of 0.3 ml / 0.1 kg, and chest hair was removed with depilatory cream. The rats were then fixed on the operating table. The chest skin was disinfected with 75% alcohol, and the skin and muscles were bluntly separated to expose the ribs. The intercostal muscles were bluntly separated between the 3rd and 4th intercostals and the pericardial cavity was entered. After pulling and exposing the heart, the capsule was bluntly separated to completely expose the heart and ensure a good field of vision. Between the left atrial appendage and the pulmonary artery cone, the left anterior descending coronary artery (LAD) running in the myocardium and in the same direction as the great cardiac vein was found and ligated with silk suture. After LAD occlusion, the corresponding blood supply area turned white, and myocardial infarction symptoms appeared. A total of 12 rat myocardial infarction models were prepared and randomly divided into 3 groups, with 4 rats in each group, namely (1) no treatment group, (2) conventional umbilical cord mesenchymal stem cell treatment group, and (3) umbilical cord mesenchymal stem cell treatment group containing T12-CD9 fusion protein.

[0050] (2) Transplantation of umbilical cord mesenchymal stem cells for treatment

[0051] The umbilical cord mesenchymal stem cells prepared in Example 3 were transplanted into the rat myocardial infarction model group (3) via the tail vein, while group (1) was not treated and group (2) was transplanted with conventional umbilical cord mesenchymal stem cells via the tail vein.

[0052] (3) Detection of the expression of apoptosis-inducing factor 3 (AIFM3) and LC3B in myocardial tissue

[0053] On the third day after surgery, the rats were killed, and the hearts were taken out after lavage. The left ventricular myocardial tissue was taken to extract the total protein of the tissue, and the expression of apoptosis-inducing factor 3 (AIFM3) and LC3B in the myocardial tissue was detected by WB.

[0054] Results and Analysis of Examples 1-4

[0055] Example 1: Through three rounds of enrichment, 20 peptides with high specificity binding to myocardial ischemic tissue were obtained, among which T12 had the highest binding property.

[0056] Example 2: By sequencing the fusion protein, it can be seen that the constructed fusion protein is expressed correctly.

[0057] Example 3: Exosomes were identified by electron microscopy, and the exosomes were found to have a diameter of 30-100 nm and a typical cup-shaped shape.

[0058] Example 4: Through WB analysis, (1) the apoptosis-inducing factor in the myocardial tissue of the umbilical cord mesenchymal stem cell treatment group containing the fusion protein was significantly reduced compared with the untreated group, and it also showed a better therapeutic effect compared with the conventional umbilical cord mesenchymal stem cell treatment group; (2) the LC3B in the myocardial tissue of the umbilical cord mesenchymal stem cell treatment group containing the fusion protein was significantly increased compared with the untreated group, and it also showed a better therapeutic effect compared with the conventional umbilical cord mesenchymal stem cell treatment group. It can be seen that the umbilical cord mesenchymal stem cells containing the fusion protein of the present invention improve the therapeutic effect by utilizing the targeting of T12.

[0059] The above describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and the description only describe the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A polypeptide targeting ischemic myocardial tissue, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. A fusion protein, characterized in that: The fusion protein is formed by directly connecting and fusion of the polypeptide according to claim 1 and the CD9 protein, and the amino acid sequence of the CD9 protein is shown in SEQ ID No.

2.

3. The fusion protein according to claim 2, characterized in that The fusion protein is expressed by transfecting 293T cells with a lentiviral expression vector.

4. An umbilical cord mesenchymal stem cell, characterized in that: The membrane surface of the exosomes secreted by the umbilical cord mesenchymal stem cells is anchored with the fusion protein of claim 2, and the preparation method of the umbilical cord mesenchymal stem cells comprises the following steps: (1) Transfecting 293T cells with a lentiviral vector expressing the fusion protein of claim 2, culturing the 293T cells, and harvesting the fusion protein; (2) Cultivating umbilical cord mesenchymal stem cells; (3) Infecting umbilical cord mesenchymal stem cells with the lentivirus expressing the fusion protein of claim 2 packaged by the 293T cells obtained in step (1); (4) Cultivating and collecting the umbilical cord mesenchymal stem cells obtained in step (3).

5. The umbilical cord mesenchymal stem cells according to claim 4, characterized in that The fusion protein binds to the CD9 receptor on the membrane surface of the exosome through the CD9 protein, so that the fusion protein is anchored on the membrane surface of the exosome.

6. Use of the umbilical cord mesenchymal stem cells according to claim 4 in the preparation of a medicine for treating myocardial infarction.

7. The use according to claim 6, characterized in that: The treatment is achieved through exosomes secreted by umbilical cord mesenchymal stem cells. The secreted exosomes are anchored on the membrane surface of the exosomes through the CD9 protein of the fusion protein binding to the CD9 receptor on the surface of the exosome membrane, and then the exosomes are targeted to ischemic myocardial tissue through the polypeptide, thereby treating myocardial infarction.

8. A method for preparing umbilical cord mesenchymal stem cells according to claim 4, characterized in that: The preparation method comprises the following steps: (1) Transfecting 293T cells with a lentiviral vector expressing the fusion protein of claim 2, culturing the 293T cells, and harvesting the fusion protein; (2) Cultivating umbilical cord mesenchymal stem cells; (3) Infecting umbilical cord mesenchymal stem cells with the lentivirus expressing the fusion protein of claim 2 packaged by the 293T cells obtained in step (1); (4) Cultivating and collecting the umbilical cord mesenchymal stem cells obtained in step (3).

9. A drug for treating myocardial infarction, characterized in that: The medicine comprises the umbilical cord mesenchymal stem cells according to claim 4 and a pharmaceutically acceptable carrier.

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