CN110656091A

CN110656091A - Application of recombinant mesenchymal stem cells in preparation of medicine for treating myocardial infarction

Info

Publication number: CN110656091A
Application number: CN201911071310.1A
Authority: CN
Inventors: 陈月秋; 沈振亚; 余云生
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2019-11-05
Filing date: 2019-11-05
Publication date: 2020-01-07

Abstract

The invention discloses an application of IL-33 in preparation of a mesenchymal stem cell immunoregulation preparation, which constructs a recombinant allogeneic MSCs for regulating immunoreaction, modifies the MSCs by utilizing IL-33, improves the survival rate of the MSCs under a hypoxia condition and improves the immunosuppressive function of the MSCs by improving the influence of the allogeneic MSCs on macrophage polarization. The invention also discloses application of the recombinant mesenchymal stem cells in preparing a medicament for treating myocardial infarction, and the medicament can treat myocardial infarction by allogeneic cell transplantation.

Description

Application of recombinant mesenchymal stem cells in preparation of medicine for treating myocardial infarction

Technical Field

The invention relates to the field of biomedicine, in particular to application of a recombinant mesenchymal stem cell in preparing a medicament for treating myocardial infarction.

Background

Myocardial infarction is a cardiovascular disease that seriously endangers human health due to myocardial cell death and fibrosis caused by prolonged ischemia. With the development of socio-economic and the change of national lifestyle, the number of patients with cardiovascular diseases in our country is on an increasing trend, and the death rate of Acute Myocardial Infarction (AMI) is also continuously increasing. The existing means is difficult to radically cure the heart, the most thorough means is heart transplantation, but the heart transplantation is limited by donor sources. Acute myocardial infarction induces a vigorous innate immune response in heart cells, mobilizing a large number of neutrophils, monocytes and macrophages to the myocardium, thus enhancing early heart damage.

Stem cell transplantation therapy is a novel clinical leading-edge technology in recent years, and the principle of the stem cell transplantation therapy is that stem cells are transplanted into infarcted myocardial tissues in a homologous manner, and cardiac remodeling is reversed, biological functions of an injured heart are enhanced, and cardiac functions are improved by regulating and controlling apoptosis of the myocardial cells, proliferation of fibroblasts, chemotaxis of immune cells, increase of the number of heart muscle-like cells and the number of capillaries. Mesenchymal Stem Cells (MSCs), a type of stem cell with differentiation potential and immunoregulatory function, exhibit considerable therapeutic potential in regenerative medicine and immunotherapy. MSCs have the characteristics of wide sources, easy separation and culture, multidirectional differentiation potential, low immunogenicity and the like, so that the MSCs are widely applied to preclinical and clinical treatment of various diseases.

Interleukin 33(IL-33) is a novel member of the IL-1 superfamily, a novel immunocytokine with multiple functions. In allogeneic heart transplantation, IL-33 can promote a Th 2-type immune response by increasing the number of myeloid-derived suppressor cells (MDSCs) and Treg cells, and ultimately prolong allograft survival. The IL-33 recombinant protein can prolong the survival of donors in heart transplantation by reducing IFN gamma and increasing the cell number of regulatory T cells; IL-33 can reduce myocardial remodeling following myocardial infarction through the p38MAPK and NF-. kappa.B signaling pathways. In the article "correlation study of myeloperoxidase and interleukin 6 with coronary artery lesion of patient with acute myocardial infarction", only interleukin 6 is disclosed as a biological index for predicting the lesion degree of AMI patient, and the correlation study of the treatment effect of interleukin 6 on coronary artery lesion of patient with acute myocardial infarction is not involved. It is very interesting to investigate whether IL-33 modified MSCs have a stronger function of survival of the surviving myocardium and regulating immunity.

The currently applied MSCs transplantation for treating myocardial infarction is a promising therapeutic approach, but the following defects exist: 1) the early stage after acute myocardial infarction is mainly necrosis of myocardial cells and infiltration of inflammatory cells, and scar tissue is formed after 2 weeks, so the optimal time for cell transplantation after myocardial infarction is within 14 days after myocardial infarction. Acute myocardial infarction induces a vigorous innate immune response in heart cells, mobilizing a large number of neutrophils, monocytes and macrophages to the myocardium, thus enhancing early heart damage. Therefore, in order to ensure that the myocardial infarction can be effectively treated in time clinically, the mesenchymal stem cells from the foreign body are better choices. 2) Factors such as heterogeneity, donor-to-donor difference, cell aging and in vitro culture of the MSCs influence the immunoregulation function of the MSCs, myocardial infarction is generally acute, and early-stage over-intense inflammation plays an important role in hindering the treatment of the myocardial infarction. Under normal conditions, MSCs are in a quiescent state, i.e., their immunoregulatory function is in a weak state, resulting in a less than expected effect of MSCs in the treatment of immune-related diseases such as myocardial infarction. CN201780049088 discloses a composition for treating cancer, and CN201811589083 discloses a recombinant mesenchymal stem cell, wherein although interleukin modified MSCs are involved, the application in treating myocardial infarction is not involved.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an application of recombinant mesenchymal stem cells in preparing a medicament for treating myocardial infarction, the invention constructs recombinant allogeneic MSCs for regulating immunoreaction, and uses IL-33 to modify the MSCs, so that the survival rate of the MSCs under a hypoxia condition is improved, and the immunosuppressive function of the MSCs is improved by improving the influence of the allogeneic MSCs on macrophage polarization.

The first purpose of the invention is to disclose the application of IL-33 in preparing mesenchymal stem cell immunoregulation preparation.

Further, the immune regulation preparation comprises recombinant lentivirus particles, wherein the recombinant lentivirus particles are obtained after cells are transfected by a recombinant plasmid for expressing IL-33 and a lentivirus packaging vector, and the recombinant plasmid for expressing IL-33 is prepared by amplifying a front primer sequence and a back primer sequence of the IL-33 and an empty plasmid pCDH-CMV-MCS-EF 1-copGFP.

Further, the front primer sequence comprises a nucleotide sequence shown in SEQ ID No.1, and the rear primer sequence comprises a nucleotide sequence shown in SEQ ID No. 2.

Further, the preparation method of the recombinant plasmid for expressing the IL-33 comprises the following steps:

PCR amplification was performed using the front and rear primer sequences, and the resulting PCR amplified fragment and the empty plasmid were digested with XbaI and BamHI, and ligated with T4 ligase to give recombinant plasmid pCDH-IL33 expressing IL-33.

Further, the PCR amplification conditions were as follows: 95 ℃ for 5 min; (95 ℃, 30s-45 s; 60 ℃, 45 s; 72 ℃, 45s-60s) -35-40 cycles; 75 ℃ for 10 min.

Further, lentiviral packaging vectors include psPAX and pMD2. G.

Further, the preparation method of the recombinant lentivirus particle comprises the following steps:

transfecting cells with the recombinant plasmid for expressing the IL-33 and a lentivirus packaging vector together for packaging lentivirus, taking supernatant, filtering, mixing with PEG, incubating, and centrifuging to obtain the recombinant lentivirus particles.

Preferably, the recombinant plasmid for expressing IL-33 is pCDH-IL33, and the lentiviral packaging vectors are psPAX and pMD2.G, and the mass ratio of the pCDH-IL 33: psPAX: pmd2.g ═ 4:3: 2.

Further, the cells were HEK293NT cells.

Further, the molecular weight of PEG is 7000 9000.

The second purpose of the invention is to provide a preparation method of the recombinant mesenchymal stem cell, which comprises the following steps:

infecting mesenchymal stem cells by using recombinant lentivirus particles to obtain the recombinant mesenchymal stem cells; the recombinant lentiviral particles are obtained by transfecting cells by recombinant plasmids expressing IL-33 and lentiviral packaging vectors, and the recombinant plasmids expressing IL-33 are prepared by amplifying front and back primer sequences of IL-33 and empty plasmids pCDH-CMV-MCS-EF 1-copGFP.

Further, mesenchymal stem cells are infected in the presence of polybrene (polybrene). The concentration of polybrene is 8-10 mug/mL. Polybrene can improve the infection efficiency.

Further, the recombinant plasmid and recombinant lentiviral particles for expressing IL-33 were prepared in the same manner as described above.

Further, lentiviral packaging vectors include psPAX and pMD2. G.

The fourth purpose of the invention is to protect the recombinant mesenchymal stem cell prepared by the preparation method.

The fifth purpose of the invention is to protect the application of the recombinant mesenchymal stem cells in the preparation of the medicine for treating the myocardial infarction.

Further, the mesenchymal stem cells are all derived from foreign bodies.

By the scheme, the invention at least has the following advantages:

the invention discloses an application of IL-33 in preparation of a mesenchymal stem cell immunoregulation preparation, in order to improve the effect of allogeneic MSCs cell transplantation, the invention modifies MSCs by IL-33, edits transplanted cells, improves the survival rate of MSCs under a hypoxia condition and improves the immunosuppressive function of allogeneic MSCs by improving the influence of allogeneic MSCs on macrophage polarization.

The invention also discloses application of the IL-33 modified MSCs (namely recombinant MSCs) in preparing medicaments for treating myocardial infarction, and the medicaments can treat myocardial infarction by allogeneic cell transplantation. The recombinant MSCs can regulate immune response in the allograft transplantation process, have the capacity of resisting severe inflammatory environment of myocardial infarction, provide a set of better treatment scheme for treating acute inflammatory response in the early stage of myocardial infarction, and can become an important means for regulating and controlling the microenvironment of the heart and myocardial remodeling.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a diagram showing the result of identifying rat bone marrow mesenchymal stem cells;

FIG. 2 is a schematic diagram of the structure of an empty plasmid vector;

FIG. 3 is a graph of the efficiency of recombinant plasmid packaging and infection with MSCs;

FIG. 4 is a diagram showing the expression of genes involved in the immune regulation of MSCs;

FIG. 5 is a macrophage polarization map;

FIG. 6 is a graph showing the results of animal heart ultrasonography and Masson's Trichrome staining;

note: Vector-MSCs in the figure are empty plasmid control groups; IL33-MSCs are MSCs with high expression of IL-33; p < 0.05 indicated a difference; p < 0.0001 indicates significant differences.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Acute myocardial infarction results in a strong innate immune response by heart cells, mobilizing a large number of neutrophils and macrophages to the myocardium. The post-myocardial infarction mainly comprises the following three processes: firstly, an inflammation stage; repairing and proliferating stage; and the mature stage. It is noteworthy that the inflammatory phase in the early phase of myocardial infarction is extremely complex and plays an important role in the determination of the final infarct size. Therefore, the IL-33 high expression stimulates silent MSCs to obtain stronger immunoregulation function, overcomes the defect of low efficiency of MSCs in the existing clinical treatment of myocardial infarction, and provides a new treatment scheme for clinical treatment of cardiovascular diseases.

Example 1 isolation of rat mesenchymal stem cells:

1) under sterile conditions, the femur and tibia of the rat were isolated and the marrow in the medullary cavity was flushed with a 1mL syringe.

2) The washed bone marrow was filtered through a 70 μm filter and centrifuged at 1350rpm for 15 min.

3) Adding erythrocyte lysate, re-suspending, and lysing for 5min to obtain erythrocyte in bone marrow.

4) PBS was washed 1-2 times, then resuspended in freshly prepared DMEM/F12+ 10% FBS and plated onto petri dishes.

5) Nonadherent cells were aspirated after 72 hours, and after every 2-3 days of fluid change, passaging was performed until the cell density in the dish reached 80%, at which time the cells were labeled as passage P0.

Example 2 identification of rat mesenchymal stem cells:

1) taking P3-P5 generation cells with good growth state, digesting the cells by 0.25% pancreatin and EDTA, collecting the cells and counting.

2) 2 x 10 to⁵The cells were divided into aliquots to which antibodies CD105-PE (12-1051, eBioscience), CD73-PE (551123, BD), CD90-PE (551401, BD), CD29-FITC (555005, BD), CD45-PE (6828, BD) and CD11b/c-FITC (b141556, Biolegend) were added.

3) After mixing, incubating for 20min at 4 ℃ in the dark, washing twice with PBS, and detecting by using Guavaaesycyte flow cytometer. The results of the measurement are shown in FIG. 1, and the abscissa represents the fluorescence intensity and the ordinate represents the number of cells.

Example 3. editing IL-33 high expression plasmid design of mesenchymal stem cells:

1) the sequence of rat IL-33 was found on the NCBI website and it was found that the gene had only one transcript and that the CDS region was continuous in the mRNA. The nucleotide sequence of the IL-33CDS is shown as SEQ ID No. 3.

2) The CDS region was predicted to have no restriction sites for restriction enzymes BamHI and XbaI by primer5 software, so that the primers were designed to add BamHI and XbaI endonucleases at both ends.

The primer sequence is as follows:

the sequence of the pre-primer is as follows: 5 'TTAAGGATCCGCCACCATGAGACCTAGAATGAAGTATTCGAAC 3' (SEQ ID No. 1);

the rear primer sequence: 5 'TTTTTCTAGATTACATCTTAGAGAGCTTAAACATGATAT 3' (SEQ ID No. 2).

The complete sequence of IL-33 is obtained by PCR amplification by using the front primer sequence and the rear primer sequence, and the PCR amplification conditions are as follows: 95 ℃ for 5 min; (95 ℃, 30s-45 s; 60 ℃, 45 s; 72 ℃, 45s-60s) -35-40 cycles; 75 ℃ for 10 min.

3) pCDH-CMV-MCS-EF1-copGFP was used as a vector (see FIG. 2), and the vector had a catalog number of CD511B-1 and a length of 7544 bp. The IL-33 sequence was introduced into the vector to construct a recombinant plasmid. The position of the introduction was between BamHI and XbaI in the multiple cloning site and copGFP in the empty plasmid was used as an indicator protein for the construction of lentiviruses and transfection efficiency.

4) The recombinant plasmid pCDH-IL33 was transfected into HEK293NT cells together with psPAX, pMD2.G for lentiviral packaging. Using a 10cm dish as an example, pCDH-IL33, psPAX and pMD2.G were mixed at a ratio of 4:3:2 (24. mu.g DNA total) and HEK293NT cells were transfected by Lip 2000.

5) Collecting culture supernatant of 24 and 48 hr, filtering with 0.45 μm filter, adding PEG8000, mixing at 4 deg.C overnight, centrifuging at 4 deg.C for 20min the next day to obtain lentivirus particles, and storing at-80 deg.C; the empty vector pCDH-CMV-MCS-EF1-copGFP packaged virus served as a control.

6) The packaged virus was infected with MSCs and polybrene (8. mu.g/mL) was added to enhance the infection efficiency.

7) Lentivirus infection efficiency was observed by GFP green fluorescence in plasmids under an inverted fluorescence microscope (as shown in figure 3).

Example 4 high expression of IL-33 enhances immune modulatory function in mesenchymal stem cells:

1) after infecting the MSCs for 48 hours by the high expression plasmid (recombinant lentivirus particle) prepared in the step 6) of the embodiment 3, collecting cells, and then extracting RNA by a conventional method; QPCR detects MSCs with high IL-33 expression, and as shown in figure 4, the expression of the immune regulation related genes IDO and COX2 and pGE2 of the MSCs is increased.

2) MSCs with high IL-33 expression promote the polarization of M2 type macrophages.

Separating mononuclear-macrophage from bone marrow: bone marrow of rat femur and tibia was obtained, lysed 2 times for 5min each by adding 5mL of erythrocyte lysate, and then washed 3 times with RPMI1640 medium, followed by filtering with a 70 μ M sieve to obtain single cells, RPMI1640 (20% FBS) was cultured for about 2 hours, replaced with fresh culture medium and cultured for another 72 hours by adding 25ng/mL of macrophage colony stimulating factor (M-CSF), to obtain rat bone marrow-derived mononuclear-macrophages.

② the IL-33 high expression MSCs and mononuclear-macrophages are co-cultured for 48 hours, then flow detection is carried out by CD68(Bio-Rad, USA), iNOS (Abcam, USA) and CD206 (Proteintetech, USA) (as shown in figure 5), the result shows that the recombinant lentivirus particles can promote the polarization of M2 type macrophages.

Example 5 high expression of IL-33 enhances the effect of myocardial infarction treatment in mesenchymal stem cells:

firstly, constructing a myocardial infarction model: firstly, a rat myocardial infarction model is established through coronary artery left anterior descending ligation, and simultaneously, 1 x 10 is injected into the myocardium⁶(ii) the prevention and treatment of myocardial infarction by individual differently treated MSCs;

masson's trichrome staining for myocardial infarction area detection: after constructing the myocardial infarction model and injecting different cells for 28 days, Vevo2100 heart ultrasonography is used for detecting the Ejection Fraction (EF), the shortening Fraction (FS), the left ventricular end contraction and relaxation diameter (LVEDD, LVESD) and the like of the rat heart so as to evaluate the influence of IL33-MSCs on the myocardial function recovery of the myocardial infarction model rat; the heart was then dilated by injecting KCL solution into the apical area of the rat, and then fixed with PBS and 4% PFA, and cardiac fibrosis was detected using Masson's trichrome staining kit. As shown in FIG. 6, MSCs highly expressing IL-33 had a stronger effect of treating myocardial infarction.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

Application of IL-33 in preparing mesenchymal stem cell immunoregulation preparation.
2. Use according to claim 1, characterized in that: the immune regulation preparation comprises recombinant lentiviral particles, wherein the recombinant lentiviral particles are obtained after cells are transfected by recombinant plasmids expressing IL-33 and lentiviral packaging vectors, and the recombinant plasmids expressing IL-33 are prepared by amplifying pre-primer sequences and post-primer sequences of IL-33 and empty plasmids pCDH-CMV-MCS-EF 1-copGFP.
3. Use according to claim 2, characterized in that: the front primer sequence comprises a nucleotide sequence shown in SEQ ID No.1, and the rear primer sequence comprises a nucleotide sequence shown in SEQ ID No. 2.
4. The use according to claim 2, wherein the recombinant plasmid for the expression of IL-33 is prepared by a process comprising the steps of:

PCR amplification was performed using the front and rear primer sequences, and the resulting PCR amplified fragment and the empty plasmid were digested with XbaI and BamHI, and ligated with T4 ligase to give recombinant plasmid pCDH-IL33 expressing IL-33.
5. Use according to claim 2, characterized in that: the lentiviral packaging vector comprises psPAX and pMD2. G.
6. A preparation method of recombinant mesenchymal stem cells is characterized by comprising the following steps:

infecting mesenchymal stem cells by using recombinant lentivirus particles to obtain the recombinant mesenchymal stem cells; the recombinant lentiviral particles are obtained by transfecting cells by recombinant plasmids expressing IL-33 and lentiviral packaging vectors, and the recombinant plasmids expressing IL-33 are prepared by amplifying front and back primer sequences of IL-33 and empty plasmids pCDH-CMV-MCS-EF 1-copGFP.
7. The method of claim 6, wherein: the front primer sequence comprises a nucleotide sequence shown in SEQ ID No.1, and the rear primer sequence comprises a nucleotide sequence shown in SEQ ID No. 2.
8. The method of claim 6, wherein: the lentiviral packaging vector comprises psPAX and pMD2. G.
9. A recombinant mesenchymal stem cell prepared by the preparation method of any one of claims 6 to 7.
10. Use of the recombinant mesenchymal stem cell of claim 9 in the preparation of a medicament for treating myocardial infarction.