CN115040695B

CN115040695B - Application of VE-cad-Fc/N-cad-Fc based fusion protein active interface

Info

Publication number: CN115040695B
Application number: CN202110254212.2A
Authority: CN
Inventors: 杨军; 谢敬辉; 李子薇
Original assignee: Nankai University
Current assignee: Nankai University
Priority date: 2021-03-09
Filing date: 2021-03-09
Publication date: 2023-10-13
Anticipated expiration: 2041-03-09
Also published as: CN115040695A

Abstract

The invention relates to vascular endothelial cell cadherin-linker fusion protein and nerve cell cadherin-linker fusion protein modified matrix, as well as preparation methods and application thereof in promoting vascular repair or regeneration and preparing medicaments for treating ischemic diseases.

Description

Application of VE-cad-Fc/N-cad-Fc based fusion protein active interface

Technical Field

The invention relates to the field of application of bioactive materials to control stem cells to promote tissue engineering vascularization and regeneration medicine. Specifically, a bioactive interface combining vascular endothelial cell cadherin-Fc fusion protein (VE-cad-Fc) and neural cell cadherin-Fc fusion protein (N-cad-Fc) is constructed, the secretion and differentiation characteristics of mesenchymal stem cells are regulated and controlled, tissue engineering vascularization is promoted, and the application of the vascular endothelial cell cadherin-Fc fusion protein and the neural cell cadherin-Fc fusion protein in the treatment of ischemic diseases such as myocardial infarction, lower limb ischemia and the like is improved.

Background

With the continued development of tissue engineering and regenerative medicine, various tissue engineering materials have been used in clinic, including tissue engineering skin, cornea, cartilage, etc., and these constructs are tissues or organs that are less dependent on blood supply. However, the occurrence, development, maintenance of homeostasis and lesion repair of most tissues in the human body are highly dependent on the abundant vascular system in the tissues to provide nutrition and oxygen, and rapid and effective vascularization has been a key bottleneck problem restricting the application of tissue engineering scaffold materials in biomedicine.

In recent years, the tissue engineering scaffold material is used for loading cell growth factors and/or stem cells, and the physical and chemical properties of the synergistic material and the cell biological information molecules are combined to promote the vascularization function of the stem cells, so that the tissue engineering scaffold material has a good application prospect. In recent years, based on regenerative medicine research of tissue engineering materials, bioactive materials of bionic extracellular matrix components (including collagen, laminin, fibronectin and other proteins or functional polypeptides) have been widely used, and the results show that the bioactive materials can significantly promote the adhesion of stem cells, but the functions of promoting vascular repair or angiogenesis can be effectively realized only by the synergistic action of slow-release series of cell growth factors (including vascular cell growth factors, hepatocyte growth factors, fibroblast growth factors and the like). However, a great deal of research has shown that the concentration and stability of the slow release of the cell growth factor are difficult to accurately regulate, for example, the Vascular Endothelial Growth Factor (VEGF) is a recognized bioinformatic molecule with the function of promoting angiogenesis, and long-term or excessive use in vivo can cause local inflammation, edema and even excessive generation of blood vessels with incomplete structures to cause malignant lesions. Meanwhile, the material loaded stem cells are implanted into the body, the survival time of the stem cells is short, the expression level of in-vivo biological functions is low, and the clinical application of the existing biological material in the field of angiogenesis promoting transformation medicine is severely limited.

The human mesenchymal stem cells (hMSCs) with the most clinical application potential in the stem cells have wide sources and low immunogenicity, have good secretion function and have certain multidirectional differentiation potential. In order to solve the bottleneck problem of the biological material cooperated with stem cells to promote vascularization research center, VE-cad-Fc artificial extracellular matrix is developed, and VE-cad-Fc is found to be used as artificial cell matrix to cooperate with VEGF to promote the differentiation efficiency of stem cells to endothelial cells, and the effect is obviously better than that of collagen matrix. Meanwhile, previous researches of the invention have found that compared with common biological materials containing collagen bionic extracellular matrix components, the composite aggregate of the epithelial cadherin fusion protein (E-cad-Fc)) modified biological materials and stem cells can remarkably prolong the in vivo survival time of the stem cells and improve the proliferation, differentiation and functional expression level of the stem cells. Therefore, we systematically examined the vascularization promoting performance of the VE-cad-Fc modified material and stem cell composite aggregate, and the research result shows that the VE-cad-Fc matrix has insufficient specific adhesion regulating capability of stem cells, and the function of regulating the stem cells to promote vascularization depends on the synergistic effect of the VE-cad-Fc matrix and exogenous VEGF.

On the other hand, studies have shown that stem cells, particularly mesenchymal stem cells, often highly express N-cadherin, and that cell endogenous N-cadheirn mediates the formation of an adhesive linkage between stem cells through extracellular domain homologous protein binding, promoting mesenchymal phenotype expression. In view of the classical theory that different subtypes of cadherins mediate highly subtype-dependent selective cell recognition, adhesion, and regulate cell directional differentiation characteristics, studies have been made in recent years to show that artificial extracellular biomimetic matrices derived from neurotensin are capable of promoting not only adhesion and proliferation of stem cells, but also differentiation of stem cells into chondrocytes and nerve cells.

Up to now, no report has been made in the prior art on the significant promotion of differentiation of stem cells into endothelial cells by any of the artificial outer matrices derived from neurocadherin or their combination with the artificial outer matrices derived from vascular endothelial cadherin. When stem cell fate regulation is performed by systematically researching different subtype cadherin fusion protein substrates, creatively discovers and optimizes the application potential of VE-cad-Fc/N-cad-Fc combined bioactive interface in tissue engineering vascularization promotion and ischemic disease transformation medicine treatment.

Disclosure of Invention

The invention discovers that vascular endothelial cell cadherin-Fc fusion proteins (for example, human endothelial cell cadherin-Fc fusion proteins, abbreviated as hVE-cad-Fc) and neurocyte cadherin-Fc fusion proteins (for example, human neurocyte cadherin-Fc fusion proteins, abbreviated as hN-cad-Fc) self-assemble on the surface of a polystyrene culture plate to form a VE/N-cad-Fc active protein interface; as an artificial extracellular matrix, the cell can not only enhance the adhesion and proliferation of stem cells, but also remarkably enhance the secretion function of mesenchymal stem cells; more importantly, the VE/N-cad-Fc matrix significantly reduces the dependence of directed differentiation of stem cells into endothelial cells on exogenous VEGF. Meanwhile, VE-cad-Fc and N-cad-Fc are combined to surface modify biological material microspheres, and a composite assembly of the microspheres and stem cells is developed, so that the secretion function of vascularization related factors of the stem cells and the efficiency of differentiation to endothelial cells are obviously enhanced, the vascularization of the material/cell composite assembly and the function of improving the vascular repair and regeneration of ischemic disease tissues are improved.

The VE/N-cad-Fc matrix plays the following main roles in promoting the vascular repair/regeneration function of ischemic tissues:

The VE-cad-Fc and the N-cad-Fc are combined to form a VE/N-cad-Fc active protein interface by self-assembly on the surface of a polystyrene culture plate, so that the VE/N-cad-Fc combined dependent cell adhesion effect is realized;

the VE-cad-Fc and the N-cad-Fc are used together to activate the expression of endogenous VE-cadherin and N-cadherin of stem cells, further continuously activate the phosphorylation of focal adhesion kinase/phosphatidylinositol kinase/protein kinase B, and promote the adhesion and survival of the stem cells, thereby being beneficial to the regulation and control of the secretion function and differentiation of the stem cells;

the combination of VE-cad-Fc and N-cad-Fc significantly enhances the factor secretion function of stem cells, further regulating the differentiation of stem cells into endothelial cells.

4. In the differentiation process, the VE-cad-Fc and the N-cad-Fc are used together to continuously up-regulate the secretion of vascularization related factors such as stem cell VEGF and the like, thereby replacing the effect of exogenous VEGF in the directional differentiation process of stem cells to endothelial cells, and effectively removing the dependence of the directional differentiation of the stem cells to the endothelial cells on exogenous VEGF addition;

the VE-cad-Fc and the N-cad-Fc can continuously activate the phosphorylation of VEGF receptor, so that the differentiation efficiency of stem cells to endothelial cells is obviously improved;

VE-cad-Fc and N-cad-Fc are combined to mediate stem cell aggregation to assemble multicellular aggregate, which can effectively regulate and control the factor secretion function of the stem cell aggregate and improve the directional differentiation efficiency of the endothelium, thus providing a new idea and technique for vascularization of tissue engineering materials and treatment of ischemic diseases.

In particular, the invention relates to the use of VE-cad-Fc and N-cad-Fc in combination for the regulation of endothelial differentiation and secretory function of stem cells.

One aspect of the invention relates to the optimization of the ratio of two cadherin fusion proteins when combined to modify PLGA composite microspheres, preferably the ratio of VE-cad-Fc to N-cad-Fc is 1:1.

A further aspect of the invention relates to the use of VE-cad-Fc and N-cad-Fc for culturing stem cells or for promoting the paracrine function of stem cell vascularization factors. Preferably, the VE-cad-Fc and the N-cad-Fc are attached to a substrate.

One aspect of the present invention relates to the use of VE-cad-Fc and N-cad-Fc to promote differentiation of stem cells into endothelial cells. The VE-cad-Fc and the N-cad-Fc are used in combination.

In a specific embodiment of the above aspects of the invention, the VE-cad-Fc and the N-cad-Fc are immobilized on a substrate, preferably on the same substrate.

One aspect of the invention relates to the use of a cadherin fusion protein in combination to mediate the repair of lower limb ischemic disease by stem cell aggregates, promoting revascularization of lower limb tissue. The cadherin fusion proteins are VE-cad-Fc and N-cad-Fc.

In yet another aspect, the invention relates to the use of a cadherin fusion protein in combination to mediate the repair of myocardial ischemia disease by stem cell aggregates, promoting revascularization of myocardial tissue. The cadherin fusion proteins are VE-cad-Fc and N-cad-Fc.

In a specific embodiment of the above aspects of the invention, the vascular endothelial cell cadherin is human vascular endothelial cell cadherin; the neural cell cadherin is human neural cell cadherin;

in the present invention, fc is a linker protein that serves primarily to mediate cadherin extracellular domain adhesion; in particular embodiments of the invention, the linker is not limited to Fc, so long as the linker protein comprises a hydrophobic interaction domain, i.e., can function to mediate the adhesion of a cadherin fusion protein when the linker protein is linked to the cadherin extracellular domain via the hydrophobic interaction domain to form a fusion protein; in particular embodiments of the invention, the linker protein may comprise a His linker, an Fc of human IgG, an Fc of rabbit IgG, or an Fc of mouse IgG, or the like; in the present invention, the linker is preferably an Fc of human IgG 1.

In one embodiment of the invention described above, the ratio of said VE-cad-Fc to said N-cad-Fc is preferably 3:1,1:1 and 1:3, preferably 1:1.

In one embodiment of the invention, the VE-cad-Fc and N-cad-Fc promote the secretion of vascularization factors from mesenchymal stem cells.

In one embodiment of the invention, the VE-cad-Fc and N-cad-Fc promote expression of endothelial cell surface markers of mesenchymal stem cells.

In one embodiment of the present invention, the VE-cad-Fc and N-cad-Fc inhibit collagen deposition in ischemic lower limb muscles, promoting revascularization of lower limb tissue.

In one embodiment of the present invention, the VE-cad-Fc and N-cad-Fc inhibit collagen deposition and ventricular remodeling in myocardial tissue, promote angiogenesis and recovery of myocardial function in myocardial tissue.

In a particular embodiment of the above aspects of the invention, the cells are mesenchymal stem cells, preferably of mammalian origin, more preferably of human, porcine or murine origin.

In summary, the present invention provides the following embodiments:

1. a modified matrix comprising vascular endothelial cell cadherin-linker fusion proteins and neural cell cadherin-linker fusion proteins, preferably the matrix is a polystyrene culture plate or a hydrophobic microsphere.

2. The modified matrix of item 1, wherein the vascular endothelial cell cadherin is human vascular endothelial cell cadherin and the neuronal cell cadherin is human neuronal cell cadherin;

preferably, the sequence of the vascular endothelial cadherin is shown as SEQ ID NO. 2, and the sequence of the neural cell cadherin is shown as SEQ ID NO. 5;

Preferably, the linkers in the vascular endothelial cadherin-linker fusion protein and the neurocadherin-linker fusion protein are the same or different, preferably the same; preferably, the linker is a His linker, an Fc of human IgG, an Fc of rabbit IgG, or an Fc of mouse IgG; more preferably, it is the Fc of human IgG, most preferably that of human IgG 1;

more preferably, the vascular endothelial cell cadherin-linker fusion protein is preferably a sequence as set forth in SEQ ID NO. 1; the neurocyte cadherin-linker fusion protein is preferably the sequence shown in SEQ ID NO. 4.

3. The modified matrix of item 1, wherein the hydrophobic microspheres are PLGA microspheres, preferably PLGA/chitosan-heparin core-shell structure composite microspheres.

4. A method of preparing a modified matrix comprising modifying a matrix by mixing a vascular endothelial cell cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein with the matrix.

5. The method of claim 4, wherein the matrix is a hydrophobic microsphere, preferably a PLGA microsphere, more preferably a PLGA composite microsphere modified with chitosan and heparin.

6. A cell aggregate formed from the modified substrate of any one of items 1 to 3 and cells.

7. The cell aggregate according to item 6, wherein the cell is a mesenchymal stem cell, an iPS cell or an embryonic stem cell of 14 days or less blasts, preferably a mesenchymal stem cell, preferably derived from a mammal, more preferably from a human, pig or mouse.

8. The cell aggregate according to any of items 6 to 7, wherein the ratio of the cells to the modified matrix is 1:1 to 6:1, preferably 3:1.

9. Use of vascular endothelial cadherin-linker fusion proteins and neuronal cadherin-linker fusion proteins or the modified matrix of any one of items 1-3 or the cell aggregates of items 6-8 in any one of the following:

(i) Promoting vascular repair or regeneration in vitro;

(ii) Promoting differentiation of stem cells into endothelial-like cells;

(iii) Promoting adhesion and survival of stem cells to activate intracellular cadherin signaling pathways; or (b)

(iv) Promoting the directional differentiation of stem cell endothelium and the regulation of the secretion function of vascularization factors.

10. Use of a vascular endothelial cadherin-linker fusion protein and a neural cell cadherin-linker fusion protein or the modified matrix of any one of items 1-3 or the cell aggregate of items 6-8 in the manufacture of a medicament for treating an ischemic disease; preferably, the ischemic diseases include, for example, myocardial infarction, ischemia of lower limbs, and the like.

11. The use of clauses 9-10, wherein the vascular endothelial cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are used in combination; preferably, the ratio is 1:3 to 3:1, preferably 1:1.

12. The use of clauses 9-10, wherein the vascular endothelial cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are immobilized on a substrate, preferably on the same substrate or on different substrates, more preferably on the same or different substrates by hydrophobic interactions.

13. A method for preparing endothelial-like cells, characterized in that stem cells are cultured in the presence of vascular endothelial cadherin-linker fusion proteins and said neuronal cadherin-linker fusion proteins.

14. The method of item 13, wherein the vascular endothelial cell cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are immobilized on a substrate, preferably on the same substrate or on different substrates, more preferably on the same or different substrates by hydrophobic interactions.

15. The method of item 13, wherein the stem cells are mesenchymal stem cells, iPS cells or embryonic stem cells at a blastula stage within 14 days, preferably mesenchymal stem cells.

Reference material

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[4]Egger,D,Tripisciano,C,Weber,V.Dynamic Cultivation of Mesenchymal Stem Cell Aggregates[J].Bioengineering(Basel),2018,5(2):1-15.

[5]S.M.Incorporation of Retinoic Acid Releasing Microspheres into Pluripotent Stem Cell Aggregates for Inducing Neuronal Differentiation[J].Cellular and Molecular Bioengineering 2015,8(3):307-319.

[6]Shapiro L,Fannon AM,Kwong PD.Structural basis of cell-cell adhesion by cadherins[J].Nature.1995；374(6520):327–37.

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[8]Haque A,Yue S,Motazedian A.Characterization and neural differentiation of mouse embryonic and induced pluripotent stem cells on cadherin-based substrata[J].Biomaterials,2012,33(20):5094-5106.

[9]Zhang Y,Mao H,Qian M.Surface modification with E-cadherin fusion protein for mesenchymal stem cell culture[J].J.Mater.Chem.B,2016,4(24):4267-4277.

Drawings

Fig. 1: construction and expression of human vascular endothelial cell cadherin-Fc fusion protein (hVE-cad-Fc) plasmid, wherein FIG. 1A is the construction of an hVE-cad-Fc expression vector; FIG. 1B shows the expression and detection of hVE-cad-Fc expression vector.

Fig. 2: construction and expression of human neural cell cadherin-Fc fusion protein (hN-cad-Fc) plasmid, wherein FIG. 2A is construction of an hN-cad-Fc expression vector; FIG. 2B shows the expression and detection of hN-cad-Fc expression vector.

Fig. 3: detection of immobilized optimum concentration of hVE-cad-Fc on PS (polystyrene) surface, wherein FIG. 3A shows immobilized optimum concentration of hVE-cad-Fc on PS surface; FIG. 3B shows the detection of the immobilized optimum concentration of hN-cad-Fc on the PS surface; FIG. 3C shows the combined immobilized optimal concentration detection of hVE-cad-Fc and hN-cad-Fc on PS surface.

Fig. 4: detection of adhesion morphology and proliferation of hMSCs on negative control (PS), positive control (Collagen), hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc substrates, wherein FIG. 4A shows the optical observation of adhesion morphology of hMSCs on different substrates; FIG. 4B shows the CCK-8 absorbance detection for hMSCs proliferation on different substrates.

Fig. 5: expression of hMSCs on negative control (PS), positive control (Collagen), hVE-cad-Fc, hN-cad-Fc and VE-cadherin and N-cadherin on hVE/hN-cad-Fc substrates, wherein FIG. 5A shows mRNA expression of VE-cadherin and N-cadherin of hMSCs on different substrates; FIG. 5B shows the protein expression of VE-cadherin and N-cadherin of hMSCs on different substrates.

Fig. 6: control (Collagen), effects of hVE-cad-Fc, hN-cad-Fc matrices on hMSCs gene expression, wherein FIG. 6A is a transcriptome expression profile of hMSCs on different matrices; FIG. 6B is a graph showing the quantitative statistics of differentially expressed genes of hMSCs on different substrates; FIG. 6C is a graph showing statistics of up-and down-regulated genes of hMSCs differentially expressed on different substrates.

Fig. 7: the hVE-cad-Fc and hN-cad-Fc matrix activate cytokine-related pathways of hMSCs, wherein FIG. 7A is a KEGG analysis of cytokine-related pathways of hVE-cad-Fc matrix activated hMSCs; FIG. 7B KEGG analysis of cytokine-related pathways of hN-cad-Fc matrix activated hMSCs

Fig. 8: negative control (PS), positive control (Collagen), hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc matrix activated gene expression of hMSCs vascularized cytokines at 3 days, 7 days and 14 days, respectively.

Fig. 9: the gene and protein expression detection of different matrixes for promoting the differentiation of hMSCs to endothelial cells, wherein FIG. 9A shows the gene expression detection of different matrixes for promoting the differentiation of hMSCs to endothelial cells, vascular endothelial growth factor receptor-2, platelet-endothelial cell adhesion molecules and vascular endothelial cell cadherin surface markers; FIG. 9B shows the protein expression assay of vascular endothelial cell growth factor receptor-2, platelet-endothelial cell adhesion molecules and vascular endothelial cell cadherin surface markers for different substrates promoting differentiation of hMSCs into endothelial cells.

Fig. 10: intracellular signaling pathway detection of different stroma-activated hMSCs fig. 10A shows protein expression of phosphorylated vascular endothelial growth factor receptor-2, phosphorylated focal adhesion kinase, phosphatidylinositol kinase, and phosphorylated protein kinase B in hMSCs cells activated by different stroma at 1 week; FIG. 10B shows protein expression of phosphorylated vascular endothelial growth factor receptor-2, phosphorylated focal adhesion kinase, phosphatidylinositol kinase, and phosphorylated protein kinase B in hMSCs activated by different substrates at 2 weeks.

Fig. 11: the particle size statistics and characterization of the PLGA composite microspheres, wherein FIG. 11A is an SEM image of the surface morphology of the PLGA composite microspheres; FIG. 11B is a graph showing particle size statistics of PLGA/chitosan-heparin composite microspheres; FIG. 11C is an SEM morphological analysis of the degradation degree of PLGA composite microspheres at 14 days.

Fig. 12: immobilization and optimization of a cadherin fusion protein matrix on PLGA composite microspheres, wherein FIG. 12A is an immunofluorescence detection of hVE-cad-Fc, hN-cad-Fc and a combination of both on the surface of PLGA composite microspheres; FIG. 12B shows the detection of optimal concentrations of hVE-cad-Fc, hN-cad-Fc, and combinations thereof (hVE/N-cad-Fc) on the surface of PLGA composite microspheres.

Fig. 13: preparation and particle size statistics of negative control (cell aggregates without microspheres), positive control (cell aggregates with Collagen matrix modified PLGA microspheres), MVP (cell aggregates with hN-cad-Fc matrix modified PLGA microspheres), MNP (cell aggregates with hN-cad-Fc matrix modified PLGA microspheres), and MV/NP (cell aggregates with hVE/hN-cad-Fc combined modified PLGA microspheres), wherein fig. 13A is a photomicrograph of different cell aggregates preparations; FIG. 13B is a particle size statistic of negative control cell aggregates; FIG. 13C is a graph showing particle size statistics of cell aggregates from each experimental group containing PLGA microspheres.

Fig. 14: the ratio of hVE-cad-Fc to hN-cad-Fc in the cell aggregates of the hVE/hN-cad-Fc combined modified PLGA microspheres was measured for the paracrine angiopoietin, transforming growth factor-beta, endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, and gene expression level of tumor necrosis factor-alpha expression of hVE/hN-cad-Fc, at 3 days, 7 days, and 14 days, respectively, wherein FIG. 14A shows the effect of the ratio of different hVE-cad-Fc/hN-cad-Fc on the paracrine function of hMSCs, and FIG. 14B shows the effect of different microsphere/cell ratios on the paracrine function of hMSCs.

Fig. 15: the vascularized cytokine expression of different cell aggregates was detected at 3 days, 7 days, and 14 days, respectively.

Fig. 16: detection of hMSC secreted vascularization factor protein in control group (cell aggregate without microsphere), cell aggregate with hme/hN-cad-Fc combined modified PLGA composite microsphere, wherein fig. 16A is a thermogram statistic of the amount of secreted vascularization factor protein in different cell aggregates detected using human vascularization factor detection kit; FIG. 16B shows the measurement of the amount of hMSC, transforming growth factor-beta, basic fibroblast growth factor, hepatocyte growth factor and angiogenin secreted by hMSC in different cell aggregates using protein quantification kit.

Fig. 17: effect of negative control, positive control, MVP, MNP and MV/NP culture supernatants on the ability of HUVEC cells to tube on Matrigel surface.

Fig. 18: the effect of culture supernatants of different cell aggregates on HUVEC cell migration capacity (Transwell migration assay).

Fig. 19: vascular endothelial cell index after 1 week, 2 weeks, 3 weeks and 4 weeks of culture of different cell aggregates in vascular endothelial cell differentiation medium: vascular endothelial cell cadherin staining and platelet-endothelial cell adhesion molecule staining detection.

Fig. 20: after different cell aggregates are cultured for 2w in an endothelial differentiation medium, the cell aggregates are subjected to tube formation and cytoskeleton on the surface of Matrigel, and platelet-endothelial cell adhesion molecules are subjected to immunofluorescence staining evaluation.

Fig. 21: different cell aggregates were used for evaluation of the therapeutic effect of the mouse lower limb ischemia model and compared with the effect of the sham surgery group, wherein fig. 21A is a laser doppler evaluation of different experimental group samples; fig. 21B is a blood flow statistic of the blood flow restoration effect of different experimental group samples.

Fig. 22: different cell aggregates were used for histological evaluation of the therapeutic effect of the mouse lower limb ischemia model and compared with the effect of the sham surgery group, wherein fig. 22A is a microvascular H & E staining evaluation of different experimental group samples; FIG. 22B is a Markov staining evaluation of collagen deposition for samples from different experimental groups; FIG. 22C is an evaluation of H & E staining of microvessels from different experimental groups for evaluation of tissue immunostaining for alpha-smooth muscle actin, von Willebrand factor and platelet-endothelial cell adhesion molecules.

Fig. 23: different cell aggregates were used for evaluation of the therapeutic effect of the rat myocardial ischemia model and compared with the effect of the sham operation group, PBS control group, wherein fig. 23A is an echocardiographic evaluation of different experimental group samples; FIG. 23B is a left ventricular ejection fraction comparison of samples from different experimental groups; FIG. 23C is a graph showing the left ventricular short axis shortening ratio comparison for different experimental group samples; FIG. 23D is a left ventricular end-diastole volume comparison for different experimental group samples; fig. 23E is a comparison of end-systole volumes of rat left ventricle for different experimental group samples.

Fig. 24: the different cell aggregates were used for histological evaluation of the rat myocardial ischemia model treatment and effect comparison with sham surgery group, PBS control group, wherein FIG. 24A is a comparison of collagen deposition of samples of different experimental groups evaluated by Marshall's trichromatic method; FIG. 24B is a graph showing statistics of the proportion of fibrotic regions in samples from different experimental groups; fig. 24C is left ventricular wall thickness statistics for different experimental group samples.

Fig. 25: the different cell aggregates are used for rat myocardial ischemia model treatment and detection of gene expression level of vascularization factors of PBS control group samples, wherein FIG. 25A is gene level detection of alpha-smooth muscle actin expression level of different experimental group samples, FIG. 25B is gene level detection of myocardial troponin expression level of different experimental group samples, FIG. 25C is gene level detection of alpha-myocardial skeleton protein expression level of different experimental group samples, FIG. 25D is gene level detection of vascular endothelial growth factor expression level of different experimental group samples, FIG. 25E is gene level detection of von Willebrand growth factor expression level of different experimental group samples, and FIG. 25F is gene level detection of tumor necrosis factor-alpha expression level of different experimental group samples.

Fig. 26: the different cell aggregates were used for rat myocardial ischemia model treatment and its treatment with sham surgery, vascular endothelial growth factor, von willebrand growth factor, cardiac troponin, alpha-actin and alpha-smooth muscle agonistic protein expression level assays of samples from PBS control group, wherein FIG. 26A is protein expression level assay of different vascularization factors of samples from different experimental group after 1 week of treatment, FIG. 26B is protein expression level assay of different vascularization factors of samples from different experimental group after 2 weeks of treatment, FIG. 26C is protein expression level assay of different vascularization factors of samples from different experimental group after 3 weeks of treatment, and FIG. 26D is protein expression level assay of different vascularization factors of samples from different experimental group after 4 weeks of treatment.

Detailed Description

Hereinafter, the present invention will be described more specifically with reference to examples. However, it will be understood by those skilled in the art that the following examples are for the purpose of illustrating the invention only and are not intended to limit the invention.

The conventional chemical reagents used in the examples were all purchased from Soy Biotechnology Co., ltd, the cell culture consumables were all purchased from Corning, USA, the mesenchymal stem cell culture medium was purchased from Biotechnology Co., USA, the mesenchymal stem cell differentiation medium was purchased from Switzerland, and the PLGA composite microspheres were synthesized in the present laboratory using the existing techniques (see Ge M, eng Y, qi S et al PLGA/chitosan-heparin composite microparticles prepared with microfluidics for the construction of hMSC aggregates [ J ]. Journal of Materials Chemistry B,2020,8 (43): 9921-9932.).

Example 1: construction and expression of human vascular endothelial cell cadherin-Fc fusion protein (hVE-cad-Fc)

Construction methods see Du Fengyi, doctor's paper, university of south China, 11 months 2011. Mainly as follows.

1.1 cloning and sequence analysis of the endothelial cell cadherin extracellular region Gene VE-cad

Specific PCR primers were designed based on UniProt database listing human VE cadherin protein sequences and functional partitions, in combination with GenBank listing gene (NCBI Reference Sequence:NM-001795.3) sequences, to amplify the extracellular domains of hVE cadherin proteins (EC 1-EC 5). An upstream primer (P1); 5'-CCGGATATCATGCAGAGGCTCATGATGCTCC-3' (SEQ ID NO. 6), ecoRV cleavage site, downstream primer (P2) 5'-AAGCGGCCGCTCTGGGCGGCCATATC-3' (SEQ ID NO. 7), notI cleavage site. Primer synthesis and sequencing were all done by Invitrogen Inc.

HUVEC cell (scientific) total mRNA extraction: mRNA was extracted according to the conventional method of the molecular cloning Experimental guidelines (third edition). The o.d. value was measured to quantify the RNA purity and degree.

Reverse transcription was performed according to the BD company kit purchasedManipulation in MicroRNA Assays

The reverse transcription system is as follows:

TABLE 1

The reverse transcription procedure is as follows:

TABLE 2

The mRNA extracted by HUVEC is used as a template to amplify VE-cad gene fragments, and a PCR reaction system is as follows:

TABLE 3 Table 3

The amplification conditions were as follows: denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s, total of 35 cycles, and extension at 72℃for 10min. 380. Mu.L of ddH was added to the reaction solution ₂ O, extracting once again with equal volume of phenol/chloroform/isoamyl alcohol, adding 1/10 volume of 3M NaAc (pH 5.0), 2 times volume of absolute ethanol, and standing at-20deg.C for 1 hr; centrifugation at 12000rpm at 4℃for 10min, washing the DNA precipitate twice with 70% ethanol, vacuum drying, and dissolving the precipitate in an appropriate amount of TE.

1.2 Construction of pcDNA3.1-hVE-cad-Fc eukaryotic expression vector

(1) HindIII and NotI double-enzyme-cut purified PCR product

The enzyme digestion system is as follows:

TABLE 4 Table 4

Overnight reaction at 37℃and enzyme inactivation at 65℃for 15min, and addition of 350. Mu.L of ddH to the reaction solution ₂ O was extracted once with an equal volume of phenol/chloroform/isoamyl alcohol, 1/10 volume of 3M NaAc (pH 5.0) was added, 2 volumes of absolute ethanol were added, and the mixture was left at-20℃for 1 hour. 4 ℃,12000rpm releaseThe heart was left for 10min, the DNA pellet was washed twice with 70% ethanol, dried under vacuum and the pellet was dissolved in 10. Mu.L TE.

(2) HindIII and NotI cleavage of pcDNA/3.1;

TABLE 5

The reaction was carried out overnight at 37 ℃. Separating the digested product by electrophoresis in 1% agarose gel, cutting the target fragment under ultraviolet lamp, recovering with DNA agarose gel recovery kit (TaKaRa), and dissolving the recovered fragment in 25 μl ddH ₂ O.

(3) Ligation and transformation of a vector with a fragment of interest

The reaction system is as follows:

TABLE 6

The reaction was carried out at 16℃for 16h. CaCl then ₂ The transformed competent cells BL21 (DE 3) were cultured overnight at 37℃for 16-18 h. Transformants were picked and the plasmids were extracted in small amounts for detection.

The recovered extracellular region of the desired gene VE-cadherin and the vector pcDNA3.1 carrying the Fc fragment were subjected to double cleavage (HindIII and NotI) at a constant temperature of 37℃respectively. After recovery of the electrophoresis gel, the recovered products were mixed and ligated overnight at 16℃under the catalysis of T4 DNA ligase. After transformation of E.coli DH 5. Alpha. Competent cells with the ligation product, the ligation product was transformed with ampicillin (Amp ⁺ ) Resistance screening was performed. After plasmid extraction, double digestion was carried out to identify the recombinant plasmid which was initially identified as correct for DNA sequence analysis (FIG. 1A). The constructed recombinant plasmid was designated pcDNA3.1/hVE-cad-Fc. The sequence was verified to be correct by sequencing. The sequence of the hVE-cad-Fc is shown as SEQ ID NO. 1, wherein the sequence of the hVE-cad is shown as SEQ ID NO. 2, and the sequence of the Fc is shown as SEQ ID NO. 3.

1.3 cell transfection and protein purification

The pcDNA3.1/hVE-cad-Fc was transfected into 293F cells (China academy of sciences typical culture Collection Committee cell Bank).

Purification of the target protein was performed by means of a Hitrap rProtein A FF column from GE Healthcare, using specific binding of the immunoglobulin Fc fragment to rProtein a.

1.4 Western blotting assay

Purified hVE-cad-Fc was transferred to PVDF membrane after electrophoresis in 10% SDS-PAGE gel, blocked with 5% skim milk for 2h, incubated overnight at 4℃for a primary anti-rabbit anti-human VE-cadherin monoclonal antibody (RD, U.S. 1:400 dilution), incubated for 1h at room temperature for HRP-labeled goat anti-rabbit secondary antibody (abcam, U.S. 1:10,000 dilution), washed with TBST, and subjected to DAB reagent exposure development and fixing analysis. The loading buffer was tested for Fc dimer formation without beta mercaptoethanol. The results are shown in FIG. 1B.

From FIG. 1B, it can be seen that in the non-reduced state one band is seen at 240KD and in the reduced state one band is seen at 120KD, suggesting that the hVE-cad-Fc fusion protein exists as a dimer (FIG. 1B).

In the following examples, unless otherwise indicated, the PCR reactions involved are as follows:

the following components were added to RNase/DNase-free PCR tubes using a PCR kit supplied by TransGen Biotech according to the following table:

TABLE 7

Program setting of the PCR instrument: 95 ℃ for 5min 1 cycle, 95 ℃ for 5min annealing temperature for 1min 72 ℃ for 45s 35 cycles, 72 ℃ for 10min 1 cycle, 4 ℃ heat preservation, and detecting the expression condition of PCR products by using 1% agarose gel electrophoresis

EXAMPLE 2 construction and expression of human neural cell cadherin-Fc fusion protein (hN-cad-Fc)

The sequence of the hN-cad-Fc is shown as SEQ ID NO. 4, wherein the sequence of SEQ ID NO. 5 represents the sequence of the hN-cad; the sequence of SEQ ID NO. 3 represents the sequence of Fc.

2.1 construction and expression of human hN-cad-Fc

2.1.1 cloning and sequence analysis of the N-cadherin gene from the extracellular region of the neurocyte cadherin

Specific PCR primers were designed based on UniProt database listing human N-cadherin protein sequences and functional partitions, in combination with GenBank listing gene (NCBI Reference Sequence:NM-001795.3) sequences, to amplify the extracellular domains of hN cadherin proteins (EC 1-EC 5). An upstream primer (P1); 5' -CCGGATATCATGCAGAGGCTCATGATGCTCC-3 '(SEQ ID NO. 8), introducing an EcoRV cleavage site, downstream primer (P2) 5' -AAGCGGCCGCTCTGGGCGGCCATATC-3' (SEQ ID NO. 9), a NotI cleavage site was introduced. Primer synthesis and sequencing were all done by Invitrogen Inc.

Neural cell (scientific) total mRNA extraction: mRNA was extracted according to the conventional method of the molecular cloning Experimental guidelines (third edition). The o.d. values were measured to quantify RNA purity and concentration. Reverse transcription was performed according to the BD company kit purchasedThe reverse transcription system was operated in MicroRNA Assays as follows:

TABLE 8

The reverse transcription procedure is as follows:

TABLE 9

The mRNA extracted from nerve cells is used as a template to amplify the N-cadherin gene fragment, and a PCR reaction system is as follows:

table 10

2.1.2 Construction of pcDNA3.1-hN-cad-Fc eukaryotic expression vector

(1) EcoR V and NotI double enzyme-cut purified PCR product

The enzyme digestion system is as follows:

TABLE 11

Overnight reaction at 37℃and enzyme inactivation at 65℃for 15min, and addition of 350. Mu.L of ddH to the reaction solution ₂ O was extracted once with an equal volume of phenol/chloroform/isoamyl alcohol, 1/10 volume of 3M NaAc (pH 5.0) was added, 2 volumes of absolute ethanol were added, and the mixture was left at-20℃for 1 hour. The DNA precipitate was washed twice with 70% ethanol, dried in vacuo and dissolved in 10. Mu.L TE at 4℃and 12000rpm for 10min.

(2) EcoR V and NotI cleavage of pcDNA/3.1;

the double cleavage system (3X 50. Mu.L) of pcDNA/3.1 (ThermoFisher) is as follows:

Table 12

(3) Ligation and transformation of a vector with a fragment of interest

The reaction system is as follows:

TABLE 13

The recovered extracellular domain of the gene hN cadherin and the vector pcDNA3.1 carrying the Fc fragment were subjected to double digestion (EcoRV and NotI) at a constant temperature of 37℃respectively. After recovery of the electrophoresis gel, the recovered products were mixed and ligated overnight at 16℃under the catalysis of T4 DNA ligase. After transformation of E.coli DH 5. Alpha. Competent cells with the ligation products, resistance selection was performed with ampicillin (Amp+). And (3) extracting the plasmid, carrying out double enzyme digestion identification, and carrying out DNA sequence analysis on the recombinant plasmid which is initially identified to be correct. The constructed recombinant plasmid was designated pcDNA3.1/hN-cad-Fc (see FIG. 2A). The sequence was verified to be correct by sequencing.

2.1.3 cell transfection and protein purification

The pcDNA3.1/hN-cad-Fc was transfected into 293F cells (China academy of sciences typical culture Collection Committee cell Bank).

2.1.4 Western blotting assay

Purified hN-cad-Fc was transferred to PVDF membrane after electrophoresis in 10% SDS-PAGE gel, blocked with 5% skim milk for 2h, incubated overnight at 4℃with primary anti-rabbit anti-human N-cadherin extracellular domain monoclonal antibody (RD, 1:400 dilution), incubated with HRP-labeled goat anti-rabbit secondary antibody (abcam, 1:10,000 dilution) for 1h at room temperature, washed with TBST, and subjected to DAB reagent exposure development and fixing analysis. The loading buffer was tested for Fc dimer formation without beta mercaptoethanol. One band can be seen at 240KD in the non-reduced state and at 120KD in the reduced state, suggesting that hN-cad-Fc exists as a dimer. (FIG. 2B)

Example 3 preparation and optimization of two-dimensional hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc combination modified substrates

3.1 preparation of two-dimensional hVE-cad-Fc modified substrate and detection of optimal concentration

The hVE-cad-Fc prepared in example 1 was dissolved in sterile PBS in an ultra clean bench to a concentration of 100. Mu.g/mL, and then stored in a sub-package low temperature refrigerator for a long period of time. Melting the split fusion protein, and preparing the fusion protein into various concentration gradients by using PBS: 3. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL and 30. Mu.g/mL, then incubated on 96-well polystyrene plates, and left overnight in a low temperature refrigerator or at about 2h at ambient temperature. The supernatant was then discarded, washed three times with PBS and the optimal adsorption concentration of hVE-cad-Fc on the polystyrene surface was detected by ELISA. The modified polystyrene plate was added to 300mL of 5% BSA solution, placed on a shaking table at 37℃and blocked at 100rpm for 2h. After blocking 1 is added: the 1000-ratio diluted horseradish peroxidase-labeled human IgG antibody was placed on a shaking table at 37℃and incubated at a constant temperature of 100rpm for 2 hours. After washing 5 times with PBST solution, 300. Mu.L of TMB color development solution was added to each tube, and the reaction was carried out at 100rpm in a shaking table at 37℃for 15 minutes in the absence of light, and a stop solution was added to read absorbance at 452 nm. Each set of concentrations was set with 5 parallel samples.

As shown in FIG. 3.A, as the concentration of hVE-cad-Fc solution increased, the absorbance at 450nm increased, and the absorbance curve leveled off to 10. Mu.g/mL. As can be seen, when the protein concentration reached 10. Mu.g/mL, the protein concentration immobilized on the surface of the polystyrene plate reached the maximum, the solution concentration continued to be increased, and the amount of protein immobilized on the surface of the plate did not continue to be increased, indicating that the protein concentration of 10. Mu.g/mL was the most cost-effective immobilization concentration for immobilization of two-dimensional polystyrene plates. Thus, the two-dimensional hVE-cad-Fc modified substrates for this subsequent experiment were prepared using 10. Mu.g/mL of hVE-cad-Fc solution.

3.2 preparation of two-dimensional hN-cad-Fc modified substrate and detection of optimal concentration

The hN-cad-Fc prepared in example 2 was dissolved in sterile PBS in an ultra clean bench to a concentration of 100. Mu.g/mL, and then stored in a low temperature refrigerator for a long period of time. Melting the split fusion protein, and preparing the fusion protein into various concentration gradients by using PBS: 3. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL and 30. Mu.g/mL, then incubated on 96-well polystyrene plates, and left overnight in a low temperature refrigerator or at about 2h at ambient temperature. The supernatant was then discarded, washed three times with PBS, and the optimal adsorption concentration of hN-cad-Fc on the polystyrene surface was detected by ELISA. The modified polystyrene plate was added to 300mL of 5% BSA solution, placed on a shaking table at 37℃and blocked at 100rpm for 2h. After blocking 1 is added: the 1000-ratio diluted horseradish peroxidase-labeled human IgG antibody was placed on a shaking table at 37℃and incubated at a constant temperature of 100rpm for 2 hours. After washing 5 times with PBST solution, 300. Mu.L of TMB color development solution was added to each tube, and the reaction was carried out at 100rpm in a shaking table at 37℃for 15 minutes in the absence of light, and a stop solution was added to read absorbance at 452 nm. Each set of concentrations was set with 5 parallel samples.

As shown in FIG. 3.B, as the concentration of hN-cad-Fc solution increased, the absorbance at 450nm increased, and the absorbance curve leveled off to 10. Mu.g/mL. As can be seen, when the protein concentration reached 10. Mu.g/mL, the protein concentration immobilized on the surface of the polystyrene plate reached the maximum, the solution concentration continued to be increased, and the amount of protein immobilized on the surface of the plate did not continue to be increased, indicating that the protein concentration of 10. Mu.g/mL was the most cost-effective immobilization concentration for immobilization of two-dimensional polystyrene plates. Thus, the two-dimensional hN-cad-Fc modified substrates for this subsequent experiment were prepared using 10. Mu.g/mL of hN-cad-Fc solution.

3.3 preparation of two-dimensional hVE/hN-cad-Fc Combined modified matrix and detection of optimal concentration

Preparation of hVE-cad-Fc by the method of example 3.1 was performed by fixing polystyrene plates with incubation solutions having hVE-cad-Fc concentrations of 0. Mu.g/mL, 3. Mu.g/mL, 5. Mu.g/mL, and 10. Mu.g/mL, respectively, followed by preparing the hN-cad-Fc prepared in example 2 with PBS in respective concentration gradients: 3. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, and then incubated in a plate fixed with an hVE-cad-Fc incubation of 0. Mu.g/mL, 3. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, and left in a cryorefrigerator overnight or at about 2h. The supernatant was then discarded, washed three times with PBS, and the absorbance of the hVE/hN-cad-Fc combination modified polystyrene plate surface was measured by ELISA. The jointly modified polystyrene plates were added to 300mL of 5% BSA solution and incubated at 100rpm for 2h on a shaking table at 37 ℃. After blocking 1 is added: the 1000-ratio diluted horseradish peroxidase-labeled human IgG antibody was placed on a shaking table at 37℃and incubated at a constant temperature of 100rpm for 2 hours. After washing 5 times with PBST solution, 300. Mu.L of TMB color development solution was added to each tube, and the reaction was carried out at 100rpm in a shaking table at 37℃for 15 minutes in the absence of light, and a stop solution was added to read absorbance at 452 nm. Each set of concentrations was set with 5 parallel samples.

As shown in FIG. 3.C, as the concentration of hN-cad-Fc solution increased, the absorbance of the plate fixed with the hVE-cad-Fc solution at a concentration of 0. Mu.g/mL, 3. Mu.g/mL, and 5. Mu.g/mL increased, whereas the absorbance of the plate fixed with the hVE-cad-Fc solution at a concentration of 10. Mu.g/mL did not substantially increase. Meanwhile, when the hN-cad-Fc concentration reached 5. Mu.g/mL, the absorbance of the plate immobilized with the hVE-cad-Fc solution of 5. Mu.g/mL was substantially maximized, and when the hN-cad-Fc concentration reached 10. Mu.g/mL, the absorbance of the plate immobilized with the hVE-cad-Fc solution of 0. Mu.g/mL (blank plate) was substantially maximized, whereby it was seen that when the combined total protein concentration reached 10. Mu.g/mL, the protein concentration immobilized on the surface of the polystyrene plate reached the maximum, the fusion protein concentration was continuously increased, and the amount of protein immobilized on the surface of the plate did not continuously increase. This result demonstrates that a protein concentration of 10. Mu.g/mL is the most cost effective immobilization concentration for the immobilization of hVE/hN-cad-Fc combination modified polystyrene plates. Thus, the preparation of the jointly modified two-dimensional matrix for the subsequent experiment hVE/hN-cad-Fc used a fusion protein solution with a total concentration of 10. Mu.g/mL.

Example 4 two-dimensional hVE-cad-Fc, hN-cad-Fc, and hVE/hN-cad-Fc combined matrix to promote adhesion and proliferation assay of hMSCs

Firstly, preparing different two-dimensional cell culture matrixes, wherein negative control is an untreated PS plate; the positive control is collagen matrix prepared by using collagen diluent, and the preparation method comprises the following steps: diluting collagen to a concentration of 1 mug/mL by using glacial acetic acid solution, adding 200 mug/hole of a 96 cell culture plate for incubation for 2 hours, and washing with PBS for 3 times each time for 30s before inoculating cells; preparation of hVE-cad-Fc matrix see example 3.1 preparation of two-dimensional hVE-cad-Fc culture matrix; the preparation method of the hN-cad-Fc matrix is shown in example 3.2 for the preparation of a two-dimensional hN-cad-Fc culture matrix; hVE/hN-cad-Fc combined matrix preparationThe preparation method is described in example 3.3 for the preparation of a two-dimensional hVE/hN-cad-Fc co-culture substrate. After completion of the preparation of the two-dimensional cell culture medium, the hMSCs obtained by digestion were grown at 1X 10 ⁴ The density of the wells was cultured on the surface of the negative control, positive control, hVE-cad-Fc, hN-cad-Fc, hVE/hN-cad-Fc matrix, respectively, and the light-microscopic time point was set to 24h time point, proliferation assay: the culture supernatants were aspirated at 4h,24h, and 48h time points, washed 3 times with PBS, 30s each, and fresh medium-prepared MTT working solution (1 mg/mL) was added. After 3h, the supernatant was removed, 100. Mu.L DMSO was added to each well and shaking was performed in the dark for 10min. The solution was aspirated and absorbance was measured at 450 nm.

As shown in FIG. 4A, the hVE-cad-Fc, the hN-cad-Fc, and the hVE/hN-cad-Fc matrices significantly promoted the adhesion of hMSCs compared to the negative control group, and the adhesion efficiency was similar to that of the positive control group. As can be seen from FIG. 4B, the hVE-cad-Fc, hN-cad-Fc, hVE/hN-cad-Fc matrix significantly improved the proliferation efficiency of hMSCs at 4h,24h,48h, as compared to the negative control group. This result suggests that hVE/hN-cad-Fc matrix can significantly promote the adhesion and proliferation efficiency of hMSCs.

Example 5 two-dimensional hVE/hN-cad-Fc combination matrix activated hMSCs calyx signaling pathway assay

Firstly, preparing different two-dimensional cell culture matrixes, wherein negative control is an untreated PS plate; the positive control is collagen matrix prepared by using collagen diluent, and the preparation method comprises the following steps: diluting collagen to a concentration of 1 mug/mL by using glacial acetic acid solution, adding 1.5mL into each hole of a 6-hole cell culture plate, incubating for 2 hours, and washing with PBS for 3 times each time for 30s before inoculating cells; preparation of hVE-cad-Fc matrix see example 3.1 preparation of two-dimensional hVE-cad-Fc culture matrix; the preparation method of the hN-cad-Fc matrix is shown in example 3.2 for the preparation of a two-dimensional hN-cad-Fc culture matrix; the preparation method of hVE/hN-cad-Fc combined matrix is described in example 3.3, preparation of two-dimensional hVE/hN-cad-Fc combined culture matrix. After completion of the preparation of the two-dimensional cell culture medium, the hMSCs obtained by digestion were grown at 1X 10 ⁵ The density of the wells is respectively cultured on the surfaces of negative control, positive control, hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc substrates, and the culture medium is changed every two days and extracted at time points of 1 day, 3 days and 7 days respectivelymRNA and protein of cell samples cultured on different substrate surfaces are subjected to fluorescent quantitative PCR and Western immunoblotting detection by the expression levels of endothelial cadherin (VE-cadherin) and neurocadherin (N-cadherin).

RNA extraction: extracting total RNA of cells by using Trizol reagent, adding 1mL of Trizol solution into each sample hole to lyse the cells, shaking, standing for 5min at room temperature, centrifuging at 4 ℃ at 10000rpm for 5min, and sucking the supernatant into a new centrifuge tube. To the transferred supernatant was added 200. Mu.L of chloroform (1 mL of Trizol) and mixed by inversion, and the mixture was allowed to stand at room temperature for 5min and centrifuged at 10000rpm at 4℃for 10min. After centrifugation, the solution was separated into upper, middle and lower layers, and the uppermost transparent inorganic aqueous phase was carefully aspirated into a fresh centrifuge tube (note that no white middle layer was aspirated). Adding 500 mu L of isopropanol into the sucked inorganic water phase, reversing and uniformly mixing, standing at room temperature for 5min, and centrifuging at 10000rpm for 5min to obtain a colloidal precipitate at the bottom of the tube, wherein the colloidal precipitate is the extracted RNA sample. The supernatant was discarded, 1mL of 75% ethanol (DEPC water preparation) was added, and the pellet was not blown down and centrifuged at 7500rpm at 4℃for 5min. After centrifugation, the supernatant is discarded, the centrifuge tube is inverted, or the residual liquid is carefully sucked by a gun, and the mixture is left to stand and air-dried, 20-60 mu L of DEPC water is added, and the mixture is subjected to a warm bath at 55-60 ℃ for 10min. After the warm bath, a small amount of solution can be taken out to detect the concentration of RNA, and the rest is placed in a refrigerator at the temperature of minus 80 ℃ for preservation.

Reverse transcription PCR: the PCR tubes were taken and the following components (shown in Table 14) were added according to the Roche reverse transcription kit instructions:

TABLE 14 reverse transcription PCR System addition of Each component

And (3) a temperature system: 20 mu L,42℃ [30min ],85℃ [5min ],4℃ [ long time ]. After the reaction is completed, a small amount of DNA concentration detection is carried out, water is added for dilution until the final concentration of the sample DNA is 200 ng/. Mu.L, and the rest samples can be placed in a refrigerator at 4 ℃ for temporary storage.

Real-time fluorescent quantitative PCR: and (3) performing fluorescent quantitative PCR amplification by taking the DNA sample obtained by reverse transcription as a template. The primer sequences used are shown in Table 15:

TABLE 15 RT-PCR primer sequences

The reaction system was configured with reference to the steps in the Roche real-time fluorescent quantitative PCR kit, the components are shown in table 16:

TABLE 16 RT-PCR addition amounts of the components

After the components are added according to the steps, the components are vibrated, evenly mixed and centrifuged, and then the mixture is placed in a real-time fluorescence quantitative PCR instrument, and the operation steps are as follows: 95 ℃ for 5min; 30s at 95℃and 1min at 72℃for 42 cycles of annealing temperature of each primer; 72 ℃ for 5min;4 ℃ is infinity. Each group of experiments was set with 5 replicates, and internal references were added as reference, 2 ^-ΔΔt The initial concentration of each sample was calculated.

Protein extraction: 100 mu L of lysate is added to each sample hole, the mixture is blown by a gun uniformly, and the mixture is cracked for 1min, and the step is operated on ice. After completion of the lysis, the sample was centrifuged at 12000rpm at 4℃for 10min, and the supernatant was collected, at this time, the protein concentration was measured, 5 Xloading Buffer was added, boiled in water for 5min, and then stored at-20 ℃.

Western blot detection assay: the addition amounts of the components of the separator are shown in Table 17:

table 17 separator gum configuration for addition of each component

After gel formation, ddH2O was poured off, and the residual ddH2O was blotted off with filter paper to prepare a concentrated gel. The addition amounts of the components of the concentrated glue preparation are shown in table 18:

TABLE 18 5% concentrated gum formulation addition of each component

The loading amount is determined according to the concentration of the protein sample, and the loading amounts of different samples are consistent. Electrophoresis procedure: 100V,10min;120V,40-70min. After the electrophoresis is completed, a transfer step is performed. First, a 10 x transfer Buffer is configured: 30g Tris,144g glycine. When the solution is diluted 10 times, 200mL of methanol is added to each 800mL of the diluted solution. Placing the electrophoresed rubber plate into an electric rotating clamp, wherein the black clamp surface is arranged below, and the sequence is as follows: black glue plate, black net, three layers of filter paper, glue (Marker strip on right), PVDF film, three layers of filter paper, white net and white glue plate. Then adding electrotransfer liquid, connecting electrodes, and performing 100V for 90min. Sealing, preparing a sealing liquid: skimmed milk 2g+40mLPBST, and sealed at room temperature for 2h. After the steps are finished, performing an antibody blocking experiment, respectively adding the primary endothelial cadherin antibody (Abcam, 1:2000 dilution), blocking the primary nerve cadherin antibody (Abcam, 1:2000 dilution) and the primary reference antibody (Abcam, 1:2000 dilution) overnight, and cleaning the PBST for 5 times for 5 minutes each time; after washing, secondary antibody (Beyotime, 1:2000 dilution) was added for incubation at 37℃for 2h, and PBST was washed 5 times for 5min each. And finally, developing: and uniformly mixing the color development liquid A/B, adding the mixture onto a PVDF film, and performing exposure detection by a chemiluminescent device.

As shown in FIGS. 5A and 5B, the results of the real-time fluorescent quantitative PCR and the Western blotting showed that the hMSCs cultured on the hVE-cad-Fc surface highly expressed VE-cadherein, the hMSCs cultured on the hN-cad-Fc substrate surface highly expressed N-cadherein, and the hMSCs cultured on the hVE/hN-cad-Fc substrate surface highly expressed VE-cadherein and N-cadherein, compared to the negative control and positive control samples. This demonstrates that the hVE-cad-Fc matrix and the hN-cad-Fc matrix can activate VE-cadherin and N-cadherin expression, respectively, in hMSCs cells, while only the hVE/hN-cad-Fc combined matrix can activate VE-cadherin and N-cadherin high expression of hMSCs simultaneously, activating the cadherin signaling pathway of hMSCs.

Example 6 fusion protein matrix activates factor secretion associated Signal pathway of hMSCs and vascularization factor secretion

RNA sequencing analysis was first performed on hMSCs samples cultured on different substrate surfaces: hMSCs were cultured on type I collagen (control group), hVE-cad-Fc and hN-cad-Fc modified culture medium surfaces, respectively, for 48h, the supernatant was aspirated and the cells were washed 3 times with sterile 0.01M PBS, followed by 10cm each ² Adding a proper amount of Trizol in a proportion of 1mL Trizol on a plane, fully mixing, standing at room temperature for 10min until cells are completely lysed, transferring the solution into a clean centrifuge tube, marking the name of a sample, sending to a company for detection, and analyzing the obtained data.

Different substrates activate hMSCs angiogenic factor secretion assay: first, hMSCs were cultured on PS surface (negative control), collagen surface (positive control), hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc, respectively, and RNA was extracted from each sample at 3 days, 7 days and 14 days, respectively, according to the method of example 5, and PCR was performed, with the primer sequences shown in Table 19, and after the PCR was completed, agarose gel electrophoresis was performed on each PCR sample.

TABLE 19

As shown in FIGS. 6A and 6B, hVE-cad-Fc and hN-cad-Fc significantly altered the transcriptome of hMSCs, hVE-cad-Fc altered the expression of 524 genes in the transcriptome of hMSCs, 248 genes were up-regulated, 276 genes were down-regulated, and hN-cad-Fc altered the expression of 172 genes in the transcriptome of hMSCs, 54 genes were up-regulated, and 118 genes were down-regulated, as compared to the control. From the analysis of the KEGG cell pathways for the differential genes in fig. 7A and 7B, it can be seen that both the hVE-cad-Fc matrix and the hN-cad-Fc matrix activate cytokine secretion-related pathways of hMSCs as compared to the control group. As can be seen from FIG. 8, compared with the negative control group and the positive control group, the hMSCs on the hVE-cad-Fc matrix surface express angiogenin, transforming growth factor-beta, endothelial cell growth factor and hepatic cell growth factor, the hMSCs on the hN-cad-Fc matrix surface express angiogenin, the alkaline fibroblast growth factor and all factors on the hVE/hN-cad-Fc matrix surface express the same. This demonstrates that both the hVE-cad-Fc matrix and the hN-cad-Fc matrix activate the cytokine secretion-related pathways of hMSCs, activate the factor secretion of hMSCs, and that the hVE/hN-cad-Fc matrix can simultaneously up-regulate the cytokine secretion of hMSCs activated by both the hVE-cad-Fc matrix and the hN-cad-Fc matrix, suggesting that the combined hVE/hN-cad-Fc matrix has the strongest improving effect on the paracrine function of hMSCs and the strongest promoting effect on the angiogenesis promoting ability of hVE/hN-cad-Fc.

Example 7 fusion protein matrix activated hMSCs differentiation towards endothelial cells and related Signal pathway detection

A6-well plate of a negative control, a positive control, hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc combined modified substrate was first prepared as described in example 5, followed by 10 ⁵ The density of cells/holes is inoculated with hMSCs, and endothelial differentiation culture medium (EBM-2 medium, lonza) is added for endothelial differentiation culture, and after differentiation culture for 1 week, each group of samples are taken for detection of endothelial differentiation index vascular endothelial growth factor receptor-2, platelet-endothelial cell adhesion molecules and endothelial cadherin respectively after 2 weeks, and the detection means mainly adopts PCR and immunoWestern blotting detection, and the detection method is as follows:

and (3) PCR detection: mRNA samples of each of the differentiation sample groups were extracted for 1 week and 2 weeks, respectively, and subjected to PCR according to the RNA extraction method of example 5, the primer sequences used are shown in Table 20, and after the completion of PCR, each PCR sample was subjected to agarose gel electrophoresis.

TABLE 20 endothelial differentiation index primer sequences

Western blot detection: firstly, respectively extracting protein samples of each differentiation sample group for 1 week and 2 weeks according to the protein extraction method in the example 5, then performing polyacrylamide gel electrophoresis, membrane transfer and sealing liquid incubation according to the method in the example 5, performing an antibody sealing experiment after the above steps are completed, respectively adding an endothelial growth factor receptor-2 (VEGFR-2) primary antibody (Abcam, 1:2000 dilution), a platelet-endothelial adhesion molecule (CD 31) primary antibody (Abcam, 1:2000 dilution), an endothelial cadherin (VE-cadherin) primary antibody (Abcam, 1:2000 dilution), a phosphorylated vascular endothelial growth factor receptor-2 (pVEGFR-2) primary antibody (Abcam, 1:2000 dilution), a phosphorylated focal adhesion kinase (pF) primary antibody (Abcam, 1:2000 dilution), a phosphatidylinositol kinase (PI 3K) primary antibody (Abcam, 1:2000 dilution), a phosphorylated protein kinase B (pAKT) primary antibody (Abcam, 1:2000 dilution), a β -actin primary antibody (Abcam, 1:2000 dilution), and sealing 5:5 washing overnight each time; after washing, secondary antibody (Beyotime, 1:2000 dilution) was added for incubation at 37℃for 2h, and PBST was washed 5 times for 5min each. And finally, developing: and uniformly mixing the color development liquid A/B, adding the mixture onto a PVDF film, and performing exposure detection by a chemiluminescent device.

As shown in FIG. 9, the hVE/hN-cad-Fc group, the hVE/hN-cad-Fc combination modified matrix group significantly upregulated expression of vascular endothelial-like cell markers such as VEGFR-2 and CD31 and VE-cadherein during differentiation of hMSCs compared to the negative control group, the positive control group, the hVE-cad-Fc group, and the hVE/hN-cad-Fc matrix significantly promoted differentiation of hMSCs into endothelial cells, and the effect thereof was significantly stronger than the effect of the hVE-cad-Fc matrix and the hN-cad-Fc combination modified matrix alone. Second, as can be seen from fig. 10, compared with the negative control group, the positive control group, the hVE-cad-Fc, the hN-cad-Fc, the hVE/hN-cad-Fc combined modified matrix group can significantly activate the expression of pvgfr-2, p-FAK, PI3K, pAKT, which proves that the hVE/hN-cad-Fc combined modified matrix can significantly promote the differentiation of hMSCs into endothelial cells by activating the pvgfr-2/p-FAK/pAKT/PI 3K signaling pathway.

Example 8 immobilization and optimized characterization of fusion protein matrix on PLGA composite microsphere surface

8.1 preparation of PLGA composite microspheres

40mg PLGA (L: G=50:50, mw=25000, jinan Dai Bioengineering Co., ltd.) was weighed into 1mL of methylene chloride, and after magnetic stirring to dissolve sufficiently, was filtered through an organic phase filter. The obtained chitosan-heparin complex is resuspended in sterile PBS solution and subjected to high-speed ultrasound for 2min to obtain uniform emulsion. 0.1mL of the above solution (0 mg,10mg,20mg of solution containing chitosan-heparin complex, respectively) was added to 1mL of PLGA/DCM solution (40 mg/mL), and rapidly placed on ice for high-speed ultrasonic emulsification for 2min to obtain colostrum as the dispersed phase of the microfluidic system. 2g of polyvinyl alcohol (alcoholysis degree: 87.0-89.0%, viscosity: 4.6-5.4, aladin) is weighed and dissolved in 100mL of distilled water to prepare 2% (w/v) PVA solution, PVA is slowly dissolved at room temperature, the PVA solution can be properly heated to accelerate dissolution, and after complete dissolution, a polyether sulfone filtering membrane filtering solution is used as a continuous phase of a microfluidic system, and the PVA solution is easy to produce white floccules after long-term storage, so that the PVA solution is prepared immediately when in use.

And respectively adding the disperse phase and the continuous phase into a 20mL syringe, conveying the two phases into a microfluidic device by using a syringe pump, generating liquid drops through the shearing force action of the continuous relative disperse phase after the two phases are contacted in a chip, collecting the liquid drops by using a 2% PVA solution after the liquid drops are generated stably, volatilizing DCM through magnetic stirring, and solidifying the liquid drops after the organic reagent is completely volatilized to obtain the composite microspheres. Repeatedly washing the composite microsphere with distilled water to remove PVA, completely removing, drying in a freeze dryer to obtain composite microsphere, vacuum drying the prepared PLGA composite microsphere sample, fixing on the surface of an objective table, performing a gold spraying method to obtain a sample, and observing under an electron scanning microscope; meanwhile, 1mg of the prepared PLGA composite microsphere is taken, added with PBS solution, placed on a shaking table at room temperature, sampled at the time points of 1 day, 3 days, 7 days, 10 days and 14 days respectively, dried in vacuum, fixed on the surface of a stage, sampled by a gold spraying method, and observed for degradability under an electron scanning microscope, and the scanning analysis result of the electron microscope is shown in figure 11.

8.2 immobilization and optimized characterization of fusion protein matrix on PLGA composite microsphere surface

The hVE-cad-Fc prepared in example 1 and the hN-cad-Fc prepared in example 2 were thawed and then prepared in PBS at various concentration gradients: 3. Mu.g/mL, 7. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL and 30. Mu.g/mL of the above mixed solution were incubated with 1mg of PLGA composite microsphere, respectively, after sufficient shaking, the mixture was placed in a horizontal shaker at 37℃and incubated at 150rpm for 2 hours, the supernatant was discarded, and the non-stabilized fusion protein was washed (3 times) with PBS. The preparation method of the hVE/hN-cad-Fc combined modified PLGA composite microsphere comprises the steps of preparing incubation solutions with hVE-cad-Fc concentrations of 3 mug/mL, 5 mug/mL, 10 mug/mL and 20 mug/mL respectively, incubating 1mg of PLGA composite microsphere respectively, sufficiently shaking, placing the mixture in a horizontal shaking table at 37 ℃ for incubation at 150rpm for 2 hours, discarding the supernatant, washing (3 times) the fusion protein which is not stably immobilized by PBS, and preparing the hN-cad-Fc prepared in the example 2 into concentration gradients by PBS: 3. Mu.g/mL, 7. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, then incubating again 3. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL of hVE-cad-Fc immobilized PLGA composite microspheres, incubating in a horizontal shaker at 37℃for 2h after sufficient shaking, discarding the supernatant, washing (3 times) the unstabilized fusion protein with PBS. Then, a fixed amount of detection was performed by adding 300. Mu.L of 5% BSA solution, blocking at 150rpm for 2 hours in a 37℃horizontal shaker, then adding 100. Mu.L of diluted HRP-labeled goat anti-human IgG (Abcam, USA) at a dilution ratio of 1:10000, blocking at 150rpm for 1 hour in a 37℃horizontal shaker, washing with 0.01M PBS for 5 times, adding 300. Mu.L of TMB (Soilebao, cat# PR 1200) color development solution per tube, reacting at 150rpm for 30 minutes in a 37℃horizontal shaker, adding 300. Mu.L of stop solution, adding 200. Mu.L of solution into a 96-well plate, and measuring absorbance at 452nm to determine a fixed amount of fusion protein association on the surface of microspheres.

The fixed state of hVE-cad-Fc and hN-cad-Fc on the surface of PLGA microspheres is examined by using an immunofluorescence staining method, and the specific steps are as follows: the prepared hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc combined modified PLGA composite microsphere samples with optimal adsorption concentration were added with 300. Mu.L of 5% BSA solution per tube, and placed in a shaking table at 37℃and blocked at constant temperature of 100rpm for 2 hours. After blocking 1 is added: 200 ratio of diluted rabbit anti-human VE-cadherin antibody (Abcam) and murine anti-human N-cadherin antibody (Abcam) were incubated at 100rpm for 2h on a shaker at 37 ℃. After 5 washes with PBST solution, hVE-cad-Fc group was added 1:200 ratio dilution of FITC-labeled goat anti-rabbit secondary antibody (Beyotime), hN-cad-Fc group added 1:200 ratio of diluted Rhodamine labeled goat anti-mouse secondary antibody (Beyotime), hVE/hN-cad-Fc group was added simultaneously with 1:200 ratio of diluted FITC-labeled goat anti-rabbit secondary antibody (Beyotime) and 1:200 ratio of diluted Rhodamine labeled goat anti-mouse secondary antibody (Beyotidme) was incubated at 100rpm in a shaking table at 37℃for 2h under constant temperature and light protection. After centrifugation, the PBST solution was washed 5 times and placed in a confocal dish. The sample was observed under a confocal fluorescence microscope, and PLGA microspheres not incubated with fusion protein were set as control group and also imaged by the procedure described above.

From fig. 11A and 11B, it can be seen that the PLGA composite microsphere prepared by using the microfluidic method has a smooth surface, a particle diameter of 21±1.5 μm and a good uniformity of particle diameter, and from fig. 11C, it can be seen that the PLGA composite microsphere has obvious degradation within 14 days, which proves that the PLGA composite microsphere has good degradability, suggesting that the PLGA composite microsphere can be rapidly degraded when being applied to in vivo treatment, which can improve the safety of the PLGA composite microsphere applied in vivo to a certain extent. As can be seen from FIG. 12A, compared with the PLGA microspheres without incubating the fusion protein, the hVE-cad-Fc matrix modified microspheres, the hN-cad-Fc matrix modified microspheres and the hVE/hN-cad-Fc matrix combined modified microspheres all show obvious fluorescent staining, and the hVE/hN-cad-Fc combined modified microspheres show green fluorescent staining and red fluorescent staining of both the hVE-cad-Fc and the hN-cad-Fc, which proves that the hVE-cad-Fc matrix and the hN-cad-Fc matrix can be stably immobilized on the surface of the PLGA microspheres at the same time. As can be seen from FIG. 12B, when the single fusion protein is immobilized on the surface of the PLGA composite microsphere, the absorbance of the PLGA composite microsphere immobilized by the fusion protein is increased along with the increase of the concentration of the hVE-cad-Fc and the hN-cad-Fc solution, and the absorbance value curve is leveled when the absorbance is 20 mug/mL. Therefore, when the protein concentration reaches 20 mug/mL, the protein concentration fixed on the surface of the PLGA composite microsphere reaches the maximum, the solution concentration is continuously increased, the fusion protein fixed on the surface of the PLGA composite microsphere cannot be continuously increased, and the PLGA composite microsphere with the protein concentration of 20 mug/mL is the most economical and effective fixed concentration. When both hVE-cad-Fc and hN-cad-Fc were immobilized on the surface of PLGA composite microspheres, the absorbance of the plate immobilized with the hN-cad-Fc solution increased by 3. Mu.g/mL, 5. Mu.g/mL, and 10. Mu.g/mL, while the absorbance of the plate immobilized with the hVE-cad-Fc solution did not substantially increase. Meanwhile, when the concentration of the hN-cad-Fc reaches 10 mug/mL, the absorbance of the PLGA composite microsphere modified by the hVE-cad-Fc solution of 10 mug/mL basically reaches the maximum, and when the concentration of the hN-cad-Fc reaches 20 mug/mL, the absorbance of the PLGA composite microsphere fixed by the hVE-cad-Fc solution does not basically reach the maximum. As can be seen, when the combined total protein concentration reaches 20 mug/mL, the concentration of the protein immobilized on the surface of the PLGA composite microsphere reaches the maximum, the concentration of the solution is continuously increased, and the amount of the protein immobilized on the surface of the flat plate is not continuously increased. This result demonstrates that a total protein concentration of 20. Mu.g/mL is the most cost effective immobilization for hVE/hN-cad-Fc combination in PLGA composite microspheres. Therefore, in the subsequent experiments of hVE/hN-cad-Fc combined two-dimensional matrix preparation all adopted a total concentration of 20 mug/mL fusion protein solution, in order to further determine the regulation effect of the ratio of hVE-cad-Fc and hN-cad-Fc on hMSCs in the combined modification, in the subsequent examples, we further optimized the ratio.

Example 9 preparation and particle size statistics of different fusion protein matrix modified microsphere mediated hMSC aggregates.

The method of example 8 was followed to prepare hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc combined modified PLGA composite microspheres, respectively, while 10. Mu.g/mL of collagen modified PLGA composite microspheres was used as a positive control for this experiment. Followed by hMSC in proportion: microsphere = 3:1 ratio hMSC and microsphere were mixed, 7.5 x 10 each ⁵ Mixing hMSC cells with PLGA composite microsphere, and adding into Aggreewell ^TM In a culture plate, centrifuging at 1000rpm for 5 min, culturing overnight at 37deg.C in a cell incubator, observing cell aggregate morphology with a microscope, blowing out the aggregate with 1000 μl of blunt pipette head, placing in a 15mL centrifuge tube, naturally settling to collect precipitate, re-suspending the precipitate with 200 μl of 2% alginic acid solution, and dripping into 0.1M CaCl ₂ Incubating in solution for 10min to form alginic acid hydrogel, and discardingAfter the washing, the alginate hydrogel was washed with physiological saline, and the cell aggregates containing microspheres in different proportions were placed in 6-well plates and cultured in DMEM/F12 medium with 10% fbs, changing the fluid every 3 days. Aggregates without microspheres were also prepared as described above as negative controls.

Specifically, the cell aggregate without microspheres is (1) abbreviated as a negative control group; (2) Cell aggregates containing collagen-modified microspheres, abbreviated as positive control; (3) Cell aggregates containing hVE-cad-Fc modified microspheres, abbreviated MVP group; (4) Cell aggregates containing hN-cad-Fc modified microspheres, abbreviated MNP group; (5) Cell aggregates containing hve+hn combined modified microspheres, abbreviated MV/NP panel. The prepared cell aggregate was observed by photographing with a microscope, and particle size statistics was performed. The results are shown in FIG. 13. As can be seen in FIG. 13A, the aggregates produced are structurally complete and sharp-edged. As can be seen from FIGS. 13B and 13C, the cell aggregate particle size without microspheres was 119.92.+ -. 6.74 μm (negative control), and the cell aggregate particle size with microspheres was 128.35.+ -. 8.46. Mu.m, indicating that the addition of modified microspheres did not have a large effect on the formation and size of hMSCs aggregates.

Example 10 investigation of the Effect of different fusion protein matrices and different microsphere cell ratios on the paracrine function of hMSCs aggregate vascularization factor

To further determine the modulation effect of the ratio of hVE-cad-Fc and hN-cad-Fc on hMSCs when the PLGA composite microspheres were modified in combination, we further optimized the ratio, and prepared the blending incubations with different hVE-cad-Fc and hN-cad-Fc according to the method of example 8, (1) hVE-cad-Fc: hN-cad-Fc=3:1 (15. Mu.g/mL hVE-cad-Fc+5. Mu.g/mL hN-cad-Fc); (2) hVE-cad-Fc: hN-cad-Fc=1:1 (10 μg/mL hVE-cad-Fc+10 μg/mL hN-cad-Fc); (3) hVE-cad-Fc: hN-cad-Fc=1:3 (5 μg/mL hVE-cad-Fc+15 μg/mL hN-cad-Fc); different hVE-cad-Fc were then prepared according to the method in example 8: fusion protein modified PLGA composite microsphere with hN-cad-Fc ratio, then different hVE-cad-Fc was prepared as in example 9: cell aggregate of the hN-cad-Fc proportional fusion protein modified PLGA composite microsphere is prepared by the following steps of: microsphere = 3:1 ratio hMSC and microsphere preparation were mixed, cultured in DMEM/F12 medium containing 10% fbs for 3 days, 7 days, and after 14 days, total RNA was extracted and reverse transcribed to obtain cDNA according to the method of example 5, and PCR detection was performed on gene expression level using the cDNA as a template. The primer sequences used were identical to those of the vascularization factor primer sequences of table 19 in example 6.

To further determine the different microspheres: cell ratio control of hMSCs we further optimized the ratio, selected modified microspheres of optimal hVE-cad-Fc and hN-cad-Fc combinations, prepared cell aggregates with cell ratio = 1:1,1:3,1:6 according to the method in example 9, cultured in DMEM/F12 medium containing 10% fbs for 3 days, 7 days, 14 days later total RNA was extracted according to the method in example 5, reverse transcribed to cDNA, and PCR was performed on gene expression levels using cDNA as template. The primer sequences used were identical to those of the vascularization factor primer sequences of table 19 in example 6.

To investigate the effect of different fusion protein matrices on the paracrine function of the vascularization factors of hMSCs aggregates, different cell aggregates, and optimally modified microsphere cell aggregates of both hVE-cad-Fc and hN-cad-Fc combinations were prepared as in example 9, and cultured in DMEM/F12 medium containing 10% fbs for 3 days, 7 days, after 14 days total RNA was extracted as in example 5, reverse transcribed to cDNA, and PCR detection of gene expression levels was performed using cDNA as template. Primer sequences used example 6 the vascularization factor primer sequences of table 19. To further detect the relative secretion of hMSCs aggregate angiogenic factor protein, the detection of individual cell aggregates (control group) cultured for 7 days and cell aggregates (MV/NP) containing hVE/hN-cad-Fc modified microspheres were performed using a human angiogenic factor detection kit, the specific method as follows: collecting sample aggregate of control group and MV/NP group cultured to 7 days, sucking out the culture medium, adding PBS solution for cleaning for 3 times, adding 1mL of DMEM/F12 culture medium without FBS, continuously culturing for 24 hours, collecting the supernatant, and detecting the secretion amount of the vascularization factor according to the instruction steps of the human vascularization factor detection kit. Meanwhile, in order to further detect the absolute secretion of typical vascularization factor proteins of hMSCs aggregate, the detection kit is used for detecting the single cell aggregate (control group) cultured for 7 days and the culture supernatant of the cell aggregate (MV/NP) containing hVE/hN-cad-Fc combined modified microsphere by respectively using the ELISA detection kit of endothelial cell growth factor, transforming growth factor-beta 1, alkaline fibroblast growth factor, hepatocyte growth factor and angiopoietin, and the specific method is as follows: collecting sample aggregates of a control group and an MV/NP group which are cultured until 7 days, sucking out the culture medium, adding a PBS solution for cleaning for 3 times, adding 1mL of DMEM/F12 culture medium which does not contain FBS, continuously culturing for 24 hours, collecting the supernatant, respectively adding the supernatant into each factor ELISA detection kit, and detecting the absolute secretion amount of the protein of the factor according to the specification steps of the ELISA detection kit.

As a result, as shown in FIG. 14, when the hVE-cad-Fc was 1:1 as compared to the hVE-cad-Fc at a ratio of 3:1 and 1:3, the hVE/hN-cad-Fc co-modified microsphere cell aggregates secreted angiogenin, transforming growth factor-. Beta., endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor were the strongest and their ability to secrete tumor necrosis factor-. Alpha.was the weakest, indicating that hVE-cad-Fc: the ability to regulate the paracrine vascularization factor of the hMSCs cell aggregate is strongest and the quantity of the secreted pro-inflammatory factor is smaller when the hN-cad-Fc ratio is 1:1, which indicates that the hVE-cad-Fc ratio of 1:1 (10 mug/mL hVE-cad-Fc+10 mug/mL hN-cad-Fc) is the optimal cadherin ratio combination for regulating the hMSCs cell aggregate. It can be seen from fig. 14B that compared to the microspheres: when the cell ratio is 1:1 and 1:6, the microspheres: the cell ratio was 1:3, the hVE/hN-cad-Fc co-modified microsphere cell aggregates were the strongest in their ability to secrete vascularization factors, and their amount to secrete tumor necrosis factor- α was relatively small, indicating microspheres: at a cell ratio of 1:3, the ability to regulate the paracrine vascularization factor of hMSCs cell aggregates is strongest, and the quantity of secreted proinflammatory factors is smaller, which indicates that in hMSCs cell aggregates, the ratio microspheres: cell ratios of 1:3 are optimal microsphere cell ratio combinations for modulating hMSCs cell aggregates.

As can be seen from fig. 15, the positive control (cell aggregate containing collagen-modified microspheres) has significantly improved vascularization factor secretion ability of MVP, MNP and MV/NP components compared to the negative control (cell aggregate without microspheres), which suggests that modification of the fusion protein can significantly improve the paracrine ability of vascularization factor of hMSCs cell aggregate. At the same time, we can see that the MV/NP group also showed a significant increase in the ability to secrete vascularization factors, especially endothelial growth factors, compared to the MVP and MNP groups, indicating that the co-use of hVE-cad-Fc and hN-cad-Fc was superior to the use of the fusion protein matrix alone. As can be seen from FIG. 16A, the relative protein secretion values of the MV/NP group endothelial growth factor, thrombopoietin, hepatocyte growth factor, transforming growth factor-. Beta.1, and basic fibroblast growth factor were all significantly increased compared to the control group (without microsphere-containing cell aggregates); as can be seen from fig. 16B, the absolute values of protein secretion of MV/NP endothelial cell growth factor, transforming growth factor- β1, basic fibroblast growth factor, hepatocyte growth factor, angiogenin all appeared to be significantly increased compared to the control group; the addition of the hVE/hN-cad-Fc co-modified PLGA composite microsphere can not only up-regulate the gene expression level of the vascularization factors of the hMSCs cell aggregate, but also obviously up-regulate the protein expression level of the vascularization factors of the hMSCs cell aggregate, and the angiogenesis promoting capability of the MV/NP cell aggregate is obviously improved, so that the composite microsphere can play a better role in promoting angiogenesis when being applied to in vivo research.

Example 11 detection of the effects of different fusion protein matrix-modified hMSCs aggregates on the improvement of the angiogenic Capacity of endothelial cells

To further examine the effect of the vascularization factors secreted by the fusion protein matrix-modified hMSCs aggregates on endothelial cell angiogenesis capacity, different cell aggregates, as well as cell aggregates of fusion protein-modified microspheres of optimal hVE/hN-cad-Fc combinations, were prepared as in example 9, and after 7 days of incubation in DMEM/F12 medium with 10% FBS, the medium was aspirated, washed 3 times with PBS solution, after which 1mL of DMEM/F12 medium without FBS was added, and incubation was continued for 24h, and the supernatant was collected.

Detection of the influence of Matrigel surface on the ability of Human Umbilical Vein Endothelial Cells (HUVECs) to form tubes:

1. preparation of Matrigel: the matrigel is placed at 4 ℃ for thawing, and the required pipette tip and 48-well plate are precooled at 4 ℃. Waiting until the matrigel is melted for later use, and uniformly spreading in a pre-cooled 48-well plate.

2. Preparing a cell suspension: and (3) degrading the pre-amplified HUVECs (ScienCell) into single-cell suspension by using pancreatin, and centrifuging by using a centrifuge tube to obtain cell sediment. After removal of the supernatant, it was resuspended in endothelial differentiation medium (EBM-2 medium, lonza) according to a 2X 10 protocol ⁵ Spreading the cell/pore density on the surface of coagulated Matrigel, culturing in incubator for 24 hr, sucking off endothelial differentiation medium (EBM-2 medium, lonza), washing with PBS for 2 times, adding the collected supernatants, and culturing

3. Taking pictures in a tube: after incubation for 24h, the cells were taken out for observation and photographed.

Detection of the influence of migration ability of HUVECs:

1. preparation of a Transwell cell: the cell culture chamber was placed in a 24-well plate, 300. Mu.L of preheated serum-free medium was added to the chamber, and the mixture was allowed to stand at 37℃for 15 minutes for use.

2. Preparing a cell suspension: pre-amplified HUVECs were pancreatin digested into single cell suspensions. After centrifugation, the supernatant was removed and resuspended in serum-free medium. Adjusting cells to 1×10 ⁵ /mL。

3. Inoculating cells: the preheated medium in the upper layer of the cell was aspirated, 200. Mu.L of the cell suspension prepared above was added to the upper layer of the cell, 500. Mu.L of each of the collected supernatants was added to the lower layer, and the culture was continued for 24 hours.

4. Cell staining count: taking out the cell, discarding the culture medium at the upper layer, fixing the lower layer of the cell with 4% paraformaldehyde for 15min, washing with PBS for 3 times, incubating and dyeing the lower layer of the cell with 1% crystal violet for 30min, washing with PBS for 3 times, wiping off the non-migrated cells at the upper layer of the cell with cotton balls, and photographing under a microscope.

As shown in FIGS. 17 and 18, the culture supernatants of MVP, MNP and MV/NP groups all promoted the tube formation of HUVECs on the Matrigel surface and enhanced the migration ability of HUVECs compared to the culture supernatants of the negative and positive control groups, while HUVECs cultured under the culture supernatant of MV/NP groups formed a more mature network structure (FIG. 17) and a stronger migration ability (FIG. 18) compared to the culture supernatants of MVP and MNP groups, suggesting that the co-modification of hVE/hN-cad-Fc had the strongest ability to improve the angiogenesis of HUVECs, suggesting that MV/NP cell aggregates could exert a better pro-angiogenic effect.

Example 12 detection of the effect of different fusion protein matrix modifications on promotion of differentiation of hMSCs aggregates into endothelial cells

To examine the effect of different fusion protein matrix modifications on the endothelial cell differentiation of hMSCs aggregates, different cell aggregates, and optimally the cell aggregates of both hVE-cad-Fc and hN-cad-Fc combination modified microspheres were prepared as in example 9, followed by blowing the aggregates out with a 1000 μl blunt pipette head, placing in a 15mL centrifuge tube, naturally settling to collect the pellet, re-suspending the pellet with 200 μl 2% alginic acid solution and dripping into 0.1M CaCl ₂ Incubating in the solution for 10min to form alginic acid hydrogel, discarding supernatant, washing with physiological saline, placing different cell aggregates in 6-well culture plate, adding endothelial differentiation medium (EBM-2 medium, lonza), and continuously culturing, and changing liquid every 2 days.

Immunofluorescent staining of different cell aggregate differentiation culture samples: at various time points of 1 week, 2 weeks, 3 weeks, 4 weeks, the alginate hydrogel was lysed with 0.1M EDTA solution, after which cell aggregates were collected, 1mL of freshly prepared 4% paraformaldehyde solution was added, fixed on ice for 30min, the supernatant was discarded and 1mL of pre-chilled PBS was added and placed on ice for 10min and repeated twice. After discarding the supernatant, 1mL of PBSDT solution was added and the mixture was blocked at room temperature for 3 hours, during which the centrifuge tube was inverted 1 time every 30 min. The supernatant was discarded after 10min of gravitational settling, primary antibody diluted with PBSDT (Abcam, 1:200 dilution) was added and incubated at 4℃for 24-48 h, during which time the centrifuge tube was inverted 8-10 times. Gravity settling on ice for 10min, discarding supernatant, cleaning with pre-cooled PBSB solution, gravity settling on ice for 10min, and repeating for 5 times. The supernatant was discarded, and a fluorescent secondary antibody diluted with PBSB (Abcam, 1:200 dilution) was added and incubated at room temperature for at least 3h in the dark, during which the tubes were inverted 1 time every 30 min. The liver organoid is washed 5 times by PBSB after the supernatant is discarded after the ice is settled for 10min, 50 to 100 mu L of DAPI solution which is also provided with anti-fluorescence quenching agent is added into each tube after the supernatant is discarded for 15min. Samples were placed in a confocal dish and examined by scanning the samples with a confocal laser microscope at 488nm, 561nm and ultraviolet excitation light, and photographed under observation.

Endothelial cells of different cell aggregate differentiation culture samples climb out on the surface of Matigel and immunofluorescence staining detection: at the time point of 2 weeks, the alginic acid hydrogel was lysed with 0.1M EDTA solution, and after collecting the cell aggregates, the collected cell aggregate samples of each group were resuspended using endothelial differentiation medium (EBM-2 medium, lonza), cultured on the surface of 24-well Matrigel, respectively, placed in an incubator for 48 hours, the medium was aspirated, washed with PBS for 2 times, photographed under a microscope, and the bright field effect was observed. Then, cytoskeletal staining and immunofluorescence staining were performed. Cytoskeletal staining: fixing with 4% paraformaldehyde at room temperature for 30min, and cleaning with PBS for three times; the wells were incubated with 1% TritonX-100 for 10min. 1% Triton X-100 was discarded and washed three times with PBS for 10min each; blocking with 5% BSA blocking solution at room temperature for 1h; FITC-labeled phalloidin diluted with 5% BSA was added and incubated for 1h; PBS washes unbound phalloidin, three washes for 10min each; DAPI diluted with PBS (1:1000) was added for 10min incubation; PBS was washed three times and the distribution of the cytoskeleton was observed with a laser confocal microscope. Immunofluorescent staining: 4% paraformaldehyde to fix cells for 15min at room temperature, and for cytoplasmic proteins, membrane rupture (1% TritonX-100) is needed in the next step, and if the cytoplasmic proteins are cell membrane proteins, membrane rupture is not needed, and PBS is cleaned for three times; adding goat serum sealing liquid to seal for 30-45min at room temperature; then adding the primary antibody diluted by goat serum blocking solution (Abcam, diluted 1:200) and standing in a shaker at 4 ℃ for incubation overnight; washing with PBS three times for at least 5min each time, adding 488 fluorescence labeled secondary antibody (Abcam, 1:200 dilution), and incubating at room temperature for 1-2h; washing with PBS to remove unbound secondary antibody, adding formulated DAPI (1:1000), and incubating for 2min; after three washes with PBS, the distribution of cell membrane proteins was observed with a laser confocal microscope.

As shown in fig. 19 and 20, compared with the negative control group and the positive control group, the MVP, MNP and MV/NP groups showed more endothelial cell marker staining (endothelial cadherin, platelet-endothelial cell adhesion molecule) at 1 week, 2 weeks, 3 weeks and 4 weeks, while the endothelial cells of different cell aggregates differentiated for 2 weeks were climbed out on the surface of Matrigel and the immunofluorescence staining test results showed that the number of endothelial cells of MVP, MNP and MV/NP was significantly higher than that of the negative control group and the positive control group, which indicated that the modification of the fusion protein could significantly up-regulate the differentiation of hMSCs cell aggregates to endothelial cells; meanwhile, we can see that the expression level of endothelial cell markers in the MV/NP group and the amount of endothelial cells climbing out are obviously higher than those in the MVP and MNP groups, which shows that the co-modification of hVE-cad-Fc+hN-cad-Fc can more effectively promote the differentiation process of MV/NP cell aggregates to endothelial cells compared with the modification of single fusion protein.

Example 13 study of different fusion protein matrix modified hMSCs cell aggregates for treatment of mouse lower limb ischemia model

In order to detect the angiogenesis promoting function of different fusion protein matrix modified hMSCs cell aggregates in vivo experiments, a mouse lower limb ischemia model is further constructed for evaluating the angiogenesis promoting effect of different fusion protein matrix modified hMSCs cell aggregates. The method comprises the following steps:

Construction of a mouse lower limb ischemia model: 90 adult male Balb/c mice (Experimental animal center of the national academy of military medical science, beijing) with the age of 6 weeks are selected, and the weight is 20-25g. Anesthesia was given by intraperitoneal injection of 3.2mL/kg of 10% chloral hydrate. After the mice were anesthetized, they were placed in a supine position and fixed to the surgical plate. The skin was lifted using an ophthalmic forceps and a longitudinal incision was made along the vascular path from the groin to the inner thigh for a length of about 5mm. The deep femoral artery is gently separated under an dissecting microscope by using surgical forceps, and a section of femoral artery is high-level ligated and separated from the proximal end of the initial point of the deep femoral artery by using a No. 7 surgical suture, so as to manufacture a low limb ischemia model of the high-level ligated and separated femoral artery. The experimental group percentages were (1) sham operation group (suture of lower limb skin only) (2) PBS group (PBS solution injected in lower limb muscle of ischemia model) (3) control group (PBS solution containing MCP cell aggregate injected in lower limb muscle of ischemia model) (4) MVP group (PBS solution containing MVP cell aggregate injected in lower limb muscle of ischemia model) (5) MNP group (PBS solution containing MNP cell aggregate injected in lower limb muscle of ischemia model) (6) MV/NP group (PBS solution containing MNP cell aggregate injected in lower limb muscle of ischemia model). After the operation, the animals were noted for body temperature recovery, laser Doppler experiments were performed at time points of 1 week, 2 weeks, 3 weeks and 4 weeks, respectively, and the blood reperfusion amount of the lower limbs of the mice was detected and statistically analyzed. Meanwhile, the mice are sacrificed by cervical removal at the time points of 1 week, 2 weeks, 3 weeks and 4 weeks, the lower limb muscles are taken out, washed cleanly and then fixed in 4% paraformaldehyde, dehydrated in a full-automatic dehydrator, paraffin sections are carried out after embedding, H & E staining is carried out, and histological lesions of the lower limb muscles and the vascular regeneration condition of the lower limb muscles of the mice are observed.

The results are shown in FIG. 21, in which the lower limbs of the mice had better integrity at 1 week, 2 weeks, 3 weeks, and 4 weeks (FIG. 21A) and significantly higher blood reperfusion (FIG. 21B) than in the PBS and control groups, and the lower limbs of the mice had significantly higher blood reperfusion than in the MVP and MNP groups, and the lower limbs of the mice had significantly lower muscle damage than in the MVP and MNP groups. As can be seen from fig. 22, the MV/NP treated group had more new micro-vessels at 4 weeks (fig. 22A) than the PBS, control, MVP, MNP groups, and also had less collagen deposition (fig. 22B), and the result of immunostaining with α -smooth muscle actin, von willebrand factor, and platelet-endothelial cell adhesion molecules showed that the MV/NP treated group had higher expression levels of α -smooth muscle actin, von willebrand factor, and platelet-endothelial cell adhesion molecules than the other cell aggregate groups, demonstrating that MV/NP cell aggregates used for treating lower limb ischemia in mice better promoted angiogenesis and blood perfusion in lower limb muscles in mice, and that hVE/hN-cad-Fc co-modification could be more effective for treating lower limb ischemic disease than modification with single fusion protein.

EXAMPLE 14 research of different fusion protein matrix modified hMSCs cell aggregates for treatment of rat myocardial ischemia model

In order to further detect the angiogenesis promoting function of the fusion protein matrix modified hMSCs cell aggregates in vivo experiments, a rat myocardial ischemia model is constructed for evaluating the angiogenesis promoting effect of different fusion protein matrix modified hMSCs cell aggregates. The method comprises the following steps:

construction of rat myocardial ischemia model: all experimental procedures were performed in accordance with regulations and laws of the biomedical engineering institute in Tianjin. SD rats (Experimental animal center of the national academy of sciences, beijing) were first weighed and the rats were intraperitoneally injected with chloral hydrate at an anesthetic dose of 0.024g/100 g. After the rats were fully anesthetized and lost their ability to move, the cervical and thoracic bristles were removed using a special razor. And then the rat is fixed on an operating table by using the rubber band, so that the body of the rat is ensured to stretch and breathe smoothly. Prior to starting the procedure, the chest shaved area was disinfected with iodophor. The skin and muscle were torn with a scalpel along two or three ribs of the rat to give an opening of about 2 cm. The heart of the rat is extruded from a rib gap by hands, the left anterior descending coronary artery is ligated at the position of 2cm of the lower edge of the pulmonary artery cone and the left auricle, the ligation depth is noted during operation, then whether the color of the front wall of the left ventricle of the heart is subjected to the change process of whitening firstly and darkening secondly is observed to determine whether ligation is successful or not, after the success of coronary ligation and no malignant arrhythmia are determined, the chest is closed, after the rat can breathe spontaneously in 1 minute, the gel is injected by matching with the rapid secondary chest opening, and the secondary chest opening exposure heart and the injected cell aggregate are required to be matched closely for improving the success rate of operations and the survival rate of the experimental rat. The wounds are sutured layer by layer, grouping marks are made, the rats after operation are placed on an electric blanket to help the rats recover consciousness, the rats can be properly fed with water after awakening, the rats keep the body temperature, and the rats are transferred to animal houses for subsequent experiments. The experimental group percentages were (1) sham surgery group (skin suture only) (2) PBS group (PBS solution injected in ischemic myocardium) (3) control group (PBS solution containing MCP cell aggregates injected in ischemic myocardium) (4) MVP group (PBS solution containing MVP cell aggregates injected in ischemic myocardium) (5) MNP group (PBS solution containing MNP cell aggregates injected in ischemic myocardium) (6) MV/NP group (PBS solution containing MNP cell aggregates injected in ischemic myocardium). After the operation, the animal body temperature is recovered, ultrasonic Doppler experiments are respectively carried out at time points of 1 week, 2 weeks, 3 weeks and 4 weeks, and the myocardial function recovery of the rat is detected and is statistically analyzed. Meanwhile, the rats are sacrificed at 1 week, 2 weeks, 3 weeks and 4 weeks, the myocardial tissues are washed cleanly and then fixed in 4% paraformaldehyde, the rats are dehydrated in a full-automatic dehydrator, paraffin sections are carried out after embedding, mahalanobis staining is carried out, the histological lesions of the myocardial tissues of the rats are observed, and the fibrosis degree and the thickness of the left ventricle wall are counted.

Real-time fluorescent quantitative PCR analysis and immune western blot detection of expression of vascularization factors in myocardial tissues. Real-time fluorescent quantitative PCR analysis of vascularization factor expression in myocardial tissue rats were anesthetized and sacrificed 7 days, 14 days, 21 days, 28 days after acute myocardial infarction surgery. The harvested hearts were loaded into enzyme-depleted cryopreservation tubes and quenched in liquid nitrogen. Total RNA was isolated using the TRIzol kit (Invitrogen, USA). One microgram of RNA (1 μg) was reverse transcribed into cDNA. Sequentially adding primers in the table 21 by taking cDNA as a template according to the instructions of a Roche Realtine PCR kit, rapidly centrifuging by a palm centrifuge, and placing the primers in a fluorescent quantitative PCR instrument at 95 ℃ for 5 minutes multiplied by 1 according to a program; 30s at 95 ℃,1 minute at annealing temperature, 1 minute x 35 at 72 ℃; 5 min×1 at 72 ℃;4 ℃ infinity, and detection. The obtained data are based on the reference beta-actin of each group according to 2 ^-△△Ct And carrying out data statistics.

TABLE 21 RT-PCR primer sequences for myocardial tissue

Western blot analysis of vascularization factor expression in myocardial tissue for western blot analysis rats were anesthetized and sacrificed 7 days, 14 days, 21 days, 28 days after acute myocardial infarction surgery, hearts harvested and quenched in liquid nitrogen. The liquid nitrogen quenched tissue pieces were washed 2-3 times with pre-chilled PBS to remove blood traces, cut into small pieces, placed in a homogenization tube, and 10 volumes of lysis buffer (10mM Tris,150mM NaCl,1%Triton X-100, 1% sodium deoxycholate, 0.1%SDS,10mM EDTA and protease inhibitor cocktail, pH 7.4) were added. Then, the tube was placed on ice for 30 minutes and the supernatant was collected by centrifugation at 13000rpm for 10 minutes at 4 ℃ and the protein concentration was determined by using a bicinchoninic acid (BCA) kit (ThermoScientific, usa). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis. The isolated proteins were transferred to PVDF (polyvinylidene fluoride) membrane (Roche, switzerland) and incubated with primary antibody (Abcam, 1:2000 dilution) overnight at 4 ℃. Secondary antibodies (Beyotime, 1:2000 dilution) were diluted with TBST solution at a ratio of 1:3000 and then added to the incubation dishes for 30 minutes. Finally, the film was imaged by a chemiluminescent imaging analysis system (Tanon 5500, tanon, china). All bands were quantified using Image J software to obtain statistics.

As a result, as shown in FIG. 23, the cardiac muscle of the rats in the MV/NP treated group had a higher ejection function (FIG. 23A), the left ventricular ejection fraction of the cardiac muscle of the rats in the MV/NP treated group was higher (FIG. 23B), the left ventricular short axis shortening rate was higher (FIG. 23C), the left ventricular end diastole volume was smaller (FIG. 23D), and the left ventricular end systole volume was smaller (FIG. 23E) than those in the PBS group, the control group, the MVP group and the MNP group. As can be seen from fig. 24, the MV/NP treated rats had less collagen deposition in the myocardial tissue (fig. 24A), the lowest proportion of fibrotic regions (fig. 24B), and the greatest thickness of the left ventricle wall (fig. 24C) compared to the PBS, control, MVP, and MNP groups. As can be seen from FIG. 25, the RNA expression levels of these vascularization factors, namely, alpha-smooth actin (FIG. 25A), cardiac troponin (FIG. 25B), alpha-cardiac skeletal protein (FIG. 25C), vascular endothelial growth factor (FIG. 25D), von Willebrand growth factor (FIG. 25E), tumor necrosis factor-alpha (FIG. 25F) were significantly increased in the cardiac tissue of the MV/NP-treated rats compared to the PBS group, the control group, the MVP group, and the MNP group. As can be seen from FIG. 26, the capacity of the myocardial tissue of the MV/NP treated rats showed a significant increase in the protein levels of α -smooth muscle actin, cardiac troponin, α -myocardial skeletal protein, vascular endothelial growth factor, von Willebrand growth factor, tumor necrosis factor- α, etc. in 1 week (FIG. 26A), 2 weeks (FIG. 26B), 3 weeks (FIG. 26C), and 4 weeks (FIG. 26D) compared to the PBS group, the control group, the MVP group, and the MNP group. These results demonstrate that MV/NP cell aggregates used to treat rat myocardial ischemia models can significantly reduce collagen deposition in rat myocardium, inhibit ventricular remodeling after ischemia, and promote expression of vascularization factors in myocardial tissue, and promote revascularization in myocardium.

The above results indicate that the hVE-cad-Fc+hN-cad-Fc co-modification can more effectively promote angiogenesis in ischemic tissues and promote tissue repair compared with the modification of single fusion protein, which indicates the great application potential of hVE-cad-Fc+hN-cad-Fc co-modified hMSCs cell aggregates in the field of ischemic disease treatment.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

1. A modified matrix comprising a vascular endothelial cell cadherin-linker fusion protein and a neural cell cadherin-linker fusion protein.

2. The modified substrate of claim 1, wherein the substrate is a polystyrene culture plate or hydrophobic microspheres.

3. The modified matrix of claim 1, wherein the vascular endothelial cadherin is human vascular endothelial cadherin and the neural cell cadherin is human neural cell cadherin.

4. The modified matrix of claim 1, wherein the vascular endothelial cadherin has a sequence shown in SEQ ID No. 2 and the neuronal cadherin has a sequence shown in SEQ ID No. 5.

5. The modified matrix of claim 1, wherein the linkers in the vascular endothelial cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are the same or different.

6. The modified substrate of claim 5, wherein the linker is a His linker, an Fc of human IgG, an Fc of rabbit IgG, or an Fc of mouse IgG.

7. The modified substrate of claim 5 wherein the linker is an Fc of human IgG.

8. The modified substrate of claim 5 wherein the linker is human IgG1 Fc.

9. The modified matrix of claim 1, wherein the vascular endothelial cadherin-linker fusion protein is of the sequence shown in SEQ ID No. 1; the neurocyte cadherin-linker fusion protein is a sequence shown in SEQ ID NO. 4.

10. The modified matrix of claim 2, wherein the hydrophobic microspheres are PLGA microspheres.

11. The modified matrix of claim 2, wherein the hydrophobic microspheres are PLGA/chitosan-heparin core-shell structured composite microspheres.

12. A method of preparing a modified matrix comprising modifying a matrix by mixing a vascular endothelial cell cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein with the matrix.

13. The method of claim 12, wherein the matrix is a hydrophobic microsphere.

14. The method of claim 12, wherein the matrix is PLGA microspheres.

15. The method of claim 12, wherein the matrix is PLGA composite microsphere modified with chitosan and heparin.

16. A cell aggregate formed from the modified substrate of any one of claims 1-11 and cells.

17. The cell aggregate of claim 16, wherein the cell is a mesenchymal stem cell, an iPS cell, or an embryonic stem cell at a blastula stage within 14 days.

18. The cell aggregate of claim 16, wherein the cells are mesenchymal stem cells.

19. The cell aggregate of claim 16, wherein the cells are derived from a mammal.

20. The cell aggregate of claim 16, wherein the cells are derived from human, porcine, or murine.

21. The cell aggregate of any of claims 16-20, wherein the ratio of cells to modified matrix is 1:1 to 6:1.

22. The cell aggregate of any of claims 16-20, wherein the ratio of cells to modified matrix is 3:1.

23. Use of vascular endothelial cell cadherin-linker fusion proteins and neuronal cell cadherin-linker fusion proteins or modified matrices according to any of claims 1-11 or cell aggregates according to any of claims 16-22 in any of the following:

(i) Preparing a medicament for promoting vascular repair or regeneration;

(ii) Promoting differentiation of stem cells into endothelial-like cells;

24. Use of vascular endothelial cadherin-linker fusion proteins and neurocyte cadherin-linker fusion proteins or modified matrices according to any one of claims 1-11 or cell aggregates according to any one of claims 16-22 in the manufacture of a medicament for the treatment of ischemic diseases.

25. The use of claim 24, wherein the ischemic disease comprises myocardial infarction, lower limb ischemia.

26. The use of any one of claims 23-25, wherein the vascular endothelial cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are used in combination.

27. The use of claim 26, wherein the ratio of vascular endothelial cadherin-linker fusion protein and the neuronal cadherin-linker fusion protein is from 1:3 to 3:1.

28. The use of claim 26, wherein the ratio of vascular endothelial cadherin-linker fusion protein to neurocyte cadherin-linker fusion protein is 1:1.

29. The use of any one of claims 23-25, wherein the vascular endothelial cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are immobilized on the same substrate or on different substrates.

30. The use of claim 29, wherein the vascular endothelial cadherin-linker fusion protein and the neuronal cadherin-linker fusion protein are immobilized on the same or different substrates by hydrophobic interactions.

31. A method for preparing endothelial-like cells, characterized in that stem cells are cultured in the presence of vascular endothelial cadherin-linker fusion proteins and said neuronal cadherin-linker fusion proteins.

32. The method of claim 31, wherein the vascular endothelial cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are immobilized on the same substrate or on different substrates.

33. The method of claim 32, wherein the vascular endothelial cadherin-linker fusion protein and the neural cell cadherin-linker fusion protein are immobilized on the same or different substrates by hydrophobic interactions.

34. The method of claim 31, wherein the stem cells are mesenchymal stem cells, iPS cells, or embryonic stem cells at a blastula stage within 14 days.

35. The method of claim 31, wherein the stem cells are mesenchymal stem cells.