CN115040695A

CN115040695A - Application of fusion protein active interface based on VE-cad-Fc/N-cad-Fc

Info

Publication number: CN115040695A
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: 2022-09-13
Anticipated expiration: 2041-03-09
Also published as: CN115040695B

Abstract

The invention relates to a vascular endothelial cell cadherin-linker fusion protein and nerve cell cadherin-linker fusion protein modified matrix, a preparation method thereof, and application thereof in promoting vascular repair or regeneration and preparing medicines 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 the application of a bioactive material in regulating stem cells to promote tissue engineering vascularization and regenerative medicine. Specifically, a combined bioactive interface of vascular endothelial cadherin-Fc fusion protein (VE-cad-Fc) and nerve 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 combined bioactive interface in improving ischemic diseases such as myocardial infarction, lower limb ischemia and the like is realized.

Background

With the development of tissue engineering and regenerative medicine, a variety of tissue engineering materials have been used in clinical applications, 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, steady-state maintenance and lesion repair of most tissues in human body highly depend on abundant vascular systems in the tissues to provide nutrition and oxygen, and the rapid and effective vascularization is always 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 synergistic effect of the physicochemical property of the material and the cell biological information molecules is utilized to promote the vascularization promotion function of the stem cells, so that the tissue engineering scaffold material has a good application prospect. Throughout recent years, based on regenerative medicine research of tissue engineering materials, bioactive materials of bionic extracellular matrix components (including proteins or functional polypeptides such as collagen, laminin and fibronectin) have been widely used, and the results show that the bioactive materials can remarkably promote the adhesion of stem cells, but the functions of promoting vascular repair or angiogenesis can be effectively realized only by the synergistic effect of sustained-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 of the slow release of the cell growth factor and the stability thereof are difficult to accurately regulate, for example, Vascular Endothelial Growth Factor (VEGF) is a well-known biological information 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 stem cells loaded by the material are implanted into the body, the survival time of the stem cells is short, the expression level of the biological function in the body is low, and the clinical application of the existing biological material in the medical field of angiogenesis transformation promotion is severely limited.

As the human mesenchymal stem cells (hMSCs) with the most clinical application potential in stem cells, the source is wide, the immunogenicity is low, and the hMSCs not only have a better secretion function, but also have certain multidirectional differentiation potential. In order to solve the bottleneck problem of research center for promoting vascularization by the cooperation of the biological materials and the stem cells, a VE-cad-Fc artificial extracellular matrix is developed, and the VE-cad-Fc is found to be used as an artificial cell matrix to be cooperated with VEGF to improve the differentiation efficiency of the stem cells to endothelial cells, and the effect of the VE-cad-Fc artificial extracellular matrix is obviously superior to that of a collagen matrix. Meanwhile, our earlier studies have found that, compared with the general biomaterial of collagen-type bionic extracellular matrix components, the composite aggregate of the modified biomaterial of epithelial cadherin (E-cad-Fc)) and the stem cells, which is developed by us, can remarkably prolong the survival time of the stem cells in vivo and improve the proliferation, differentiation and functional expression levels of the stem cells. Therefore, the VE-cad-Fc modified material and the stem cell composite aggregate have been systematically examined for the performance of promoting vascularization, and the research result shows that the VE-cad-Fc matrix has insufficient specific adhesion capacity for regulating stem cells, and the function of regulating stem cells to promote vascularization depends on the synergistic effect of the VE-cad-Fc matrix and exogenous VEGF.

On the other hand, it has been shown that stem cells, particularly mesenchymal stem cells, generally highly express cadherin (N-cadherin), and that intracellular N-cadherin mediates the formation of adhesive junctions between stem cells through extracellular domain homolog protein binding, promoting expression of the intercellular phenotype. In view of the classical theory that different subtype cadherin proteins mediate highly subtype-dependent selective cell recognition and adhesion and regulate the cell directed differentiation characteristics, researches have been carried out in recent years to show that the artificial extracellular biomimetic matrix derived from the neural cadherin can promote the adhesion and proliferation of stem cells and promote the differentiation of the stem cells to chondrocytes and nerve cells.

So far, no report that the cadherin-derived artificial extracellular matrix or the cadherin-derived artificial extracellular matrix combined with vascular endothelial cells can remarkably promote the differentiation and vascularization of stem cells to endothelial cells exists in the prior art. When the regulation and control of different subtype cadherin fusion protein matrixes on the fate of stem cells are systematically researched, the application potential of the VE-cad-Fc/N-cad-Fc combined biological activity interface in the medicine for promoting the vascularization of tissue engineering and treating ischemic disease transformation is creatively discovered and optimized.

Disclosure of Invention

The invention finds that the self-assembly of vascular endothelial cadherin-Fc fusion protein (for example, human endothelial cadherin-Fc fusion protein, abbreviated as hVE-cad-Fc) and neural cadherin-Fc fusion protein (for example, human neural cadherin-Fc fusion protein, abbreviated as hN-cad-Fc) on the surface of a polystyrene culture plate forms a VE/N-cad-Fc active protein interface; the compound is used as an artificial extracellular matrix, so that the adhesion and proliferation of stem cells can be enhanced, and the secretion function of mesenchymal stem cells can be obviously enhanced; more importantly, the VE/N-cad-Fc matrix obviously reduces the dependence of the directional differentiation of stem cells to endothelial cells on exogenous VEGF. Meanwhile, VE-cad-Fc and N-cad-Fc are combined to modify the biomaterial microspheres on the surface, and a composite assembly of the biomaterial microspheres and stem cells is developed, so that the secretion function of vascularization related factors of the stem cells and the differentiation efficiency of the stem cells to endothelial cells are obviously enhanced, the vascularization of the material/cell composite assembly is improved, and the function of repairing and regenerating ischemic disease tissue blood vessels is improved.

The main functions of the VE/N-cad-Fc substrate in promoting the vascular repair/regeneration function of ischemic tissues are as follows:

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

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

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

4. In the differentiation process, VE-cad-Fc and N-cad-Fc are combined to continuously up-regulate the secretion of vascularization related factors such as stem cell VEGF and the like, so as to replace the action of exogenous VEGF in the directional differentiation process of stem cells to endothelial cells, and effectively remove the dependence of the stem cells to the directional differentiation of the endothelial cells on the addition of VEGF to the exogenous source;

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

VE-cad-Fc and N-cad-Fc jointly mediate stem cell aggregation to assemble multicellular aggregates, which can effectively regulate and control the factor secretion function of the stem cell aggregates, improve the directional differentiation efficiency of the endothelium thereof and provide new ideas and technologies for the vascularization of tissue engineering materials and the treatment of ischemic diseases.

In particular, the invention relates to the application of the combined use of VE-cad-Fc and N-cad-Fc in the regulation of the directional differentiation and secretion function of stem cell endothelium.

One aspect of the invention relates to the proportion optimization of two cadherin fusion proteins when the PLGA composite microspheres are modified in a combined manner, and preferably the proportion of VE-cad-Fc to N-cad-Fc is 1: 1.

Yet another 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 N-cad-Fc are attached to a substrate.

One aspect of the 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 particular embodiment of each 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 application of the cadherin fusion protein in combination in mediating stem cell aggregates to repair ischemic diseases of lower limbs and promoting angiogenesis of tissues of the lower limbs. The cadherin fusion protein is VE-cad-Fc and N-cad-Fc.

The invention also relates to application of the cadherin fusion protein in combination in mediating the repair of the stem cell aggregate on myocardial ischemia diseases and promoting the regeneration of myocardial tissue blood vessels. The cadherin fusion protein is VE-cad-Fc and N-cad-Fc.

In particular embodiments of each 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 primarily functions to mediate cadherin ectodomain adhesion; in a specific embodiment of the present invention, the linker is not limited to Fc as long as the linker protein comprises a hydrophobic interaction domain, i.e., when the linker protein is linked to the cadherin extracellular domain via a hydrophobic interaction domain to form a fusion protein, it may function to mediate cadherin fusion protein adhesion; in particular embodiments of the invention, the linker protein may comprise a His linker, Fc of human IgG, Fc of rabbit IgG, or Fc of mouse IgG, etc.; in the present invention, the linker is preferably Fc of human IgG 1.

In one embodiment of the present 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 the N-cad-Fc promote the secretion of vascularization factors of 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 invention, the VE-cad-Fc and N-cad-Fc inhibit collagen deposition in the muscles of ischemic lower limbs and promote angiogenesis of the tissues of the lower limbs.

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

In particular embodiments of the above aspects of the invention, the cell is a mesenchymal stem cell, 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 a vascular endothelial cell cadherin-linker fusion protein and a neural cell cadherin-linker fusion protein, preferably the matrix is a polystyrene culture plate or hydrophobic microspheres.

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

preferably, the sequence of the vascular endothelial cell 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 cell cadherin-linker fusion protein and the neuronal cell cadherin-linker fusion protein are the same or different, preferably the same; preferably, the linker is a His-linker, Fc of human IgG, Fc of rabbit IgG, or Fc of mouse IgG; more preferably Fc of human IgG, most preferably Fc of human IgG 1;

more preferably, the vascular endothelial cadherin-linker fusion protein is preferably the sequence shown in SEQ ID NO 1; the nerve cell cadherin-linker fusion protein is preferably a 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 preparation method according to item 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 which is an 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 cells are mesenchymal stem cells, iPS cells or 14-day endocystic embryonic stem cells, preferably mesenchymal stem cells, preferably of mammalian origin, more preferably of human, porcine or murine origin.

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

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

(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

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

10. Use of a vascular endothelial cell 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 preparation of a medicament for the treatment of an ischemic disease; preferably, the ischemic disease includes, for example, myocardial infarction, lower limb ischemia, and the like.

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

12. The use of items 9-10, wherein the vascular endothelial cadherin-linker fusion protein and the neuronal 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 an endothelial-like cell, characterized by culturing a stem cell in the presence of a vascular endothelial cell cadherin-linker fusion protein and the neuronal cell cadherin-linker fusion protein.

14. The method of item 13, wherein the vascular endothelial cadherin-linker fusion protein and the neuronal 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 interaction.

15. The method of item 13, wherein the stem cell is a mesenchymal stem cell, an iPS cell or a 14-day endocystic embryonic stem cell, preferably a mesenchymal stem cell.

Reference data

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[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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Drawings

FIG. 1: construction and expression of a 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 is a schematic diagram: construction and expression of human neural cell cadherin-Fc fusion protein (hN-cad-Fc) plasmid, wherein FIG. 2A is construction of hN-cad-Fc expression vector; FIG. 2B shows the expression and detection of hN-cad-Fc expression vector.

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

FIG. 4: detecting the adhesion forms and proliferation conditions of the hMSCs on negative control (PS), positive control (Collagen), hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc substrates, wherein FIG. 4A is the light microscope observation of the adhesion forms of the hMSCs on different substrates; FIG. 4B is the CCK-8 absorbance measurements of hMSCs proliferation on different matrices.

FIG. 5: expression of VE-cadherin and N-cadherin of hMSCs on negative control (PS), positive control (Collagen), hVE-cad-Fc, hN-cad-Fc and 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: effect of control group (Collagen), hVE-cad-Fc, hN-cad-Fc substrate on hMSCs gene expression, wherein FIG. 6A is a transcriptome expression calorimetry map of hMSCs on different substrates; FIG. 6B is a statistics of the number of differentially expressed genes in hMSCs on different substrates; FIG. 6C is a statistic of up-and down-regulated genes of hMSCs on differentially expressed genes on different substrates.

FIG. 7: the hVE-cad-Fc and hN-cad-Fc matrices 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 is a KEGG analysis of cytokine-associated pathways of hMSCs activated by hN-cad-Fc substrate

FIG. 8: and (3) detecting the gene expression of the hMSCs vascularization cytokine activated by the negative control (PS), the positive control (Collagen), the hVE-cad-Fc, the hN-cad-Fc and the hVE/hN-cad-Fc substrate, wherein the detection time points are 3 days, 7 days and 14 days respectively.

FIG. 9: gene and protein expression detection of different matrixes promoting differentiation of hMSCs to endothelial cells, wherein fig. 9A is gene expression detection of vascular endothelial cell growth factor receptor-2, platelet-endothelial cell adhesion molecule and vascular endothelial cell cadherin surface marker of different matrixes promoting differentiation of hMSCs to endothelial cells; FIG. 9B shows the protein expression assay of VEGF receptor-2, platelet-endothelial cell adhesion molecule and vascular endothelial cell cadherin surface markers with different matrices to promote differentiation of hMSCs into endothelial cells.

FIG. 10: detecting intracellular signal pathways of different matrix-activated hMSCs, wherein fig. 10A shows protein expressions of phosphorylated vascular endothelial growth factor receptor-2, phosphorylated focal adhesion kinase, phosphatidylinositol kinase and phosphorylated protein kinase B in cells of different matrix-activated hMSCs at 1 week; FIG. 10B shows the intracellular phosphorylation of vascular endothelial growth factor receptor-2, phosphofocal adhesion kinase, phosphatidylinositol kinase, and phosphoprotein kinase B in hMSCs at 2 weeks with different substrates.

FIG. 11: the particle size of the PLGA composite microspheres is counted and characterized, wherein FIG. 11A is a surface topography observation SEM image of the PLGA composite microspheres; FIG. 11B is the 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: fixing and optimizing a cadherin fusion protein matrix on a PLGA composite microsphere, wherein FIG. 12A shows hVE-cad-Fc, hN-cad-Fc and immunofluorescence detection of the hVE-cad-Fc and hN-cad-Fc combined on the surface of the PLGA composite microsphere; FIG. 12B shows the optimal concentration detection of hVE-cad-Fc, hN-cad-Fc and their combination (hVE/N-cad-Fc) on the surface of PLGA composite microspheres.

FIG. 13: preparation and particle size statistics of negative controls (cell aggregates without microspheres), positive controls (cell aggregates with Collagen matrix-modified PLGA microspheres), MVP (cell aggregates with hVE-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 combination-modified PLGA microspheres), wherein FIG. 13A is a light mirror image prepared from different cell aggregates; FIG. 13B is a particle size statistic of negative control cell aggregates; FIG. 13C is the particle size statistics of the cell aggregates of each experimental group containing PLGA microspheres.

FIG. 14: the detection time points of the ratio of hVE-cad-Fc to hN-cad-Fc in the cell aggregate of the hVE/hN-cad-Fc combined modified PLGA microsphere and the detection of the microsphere cell ratio on the gene expression level of hMSCs paracrine angiogenin, transforming growth factor-beta, endothelial cell growth factor, basic fibroblast growth factor, hepatocyte growth factor and tumor necrosis factor-alpha expression are 3 days, 7 days and 14 days respectively, wherein FIG. 14A shows the effect of different ratios of hVE-cad-Fc/hN-cad-Fc on the paracrine function of hMSCs, and FIG. 14B shows the effect of different ratios of microspheres/cells on the paracrine function of hMSCs.

FIG. 15: and (3) detecting the expression of the vascularized cytokines of different cell aggregates at 3 days, 7 days and 14 days respectively.

FIG. 16: detecting hMSC secreted vascularization factor protein in the cell aggregates of the control group (cell aggregates without microspheres) and the cell aggregates containing hVE/hN-cad-Fc combined modified PLGA composite microspheres, wherein FIG. 16A is a heat map statistic for detecting the secretion amount of vascularization factor protein in different cell aggregates by using a human vascularization factor detection kit; FIG. 16B shows the detection of hMSC paracrine vascularization factor endothelial growth factor, transforming growth factor-beta, basic fibroblast growth factor, hepatocyte growth factor and the secretion of angiogenin in different cell aggregates by using protein quantitative kit.

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

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

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

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

FIG. 21: the different cell aggregates are used for evaluating the treatment effect of the mouse lower limb ischemia model and comparing the treatment effect with the effect of a sham operation group, wherein the figure 21A is the laser Doppler evaluation of samples of different experimental groups; fig. 21B is a blood flow statistics of blood flow recovery effect for different experimental group samples.

FIG. 22: different cell aggregates are used for histological evaluation of the treatment effect of the mouse lower limb ischemia model and comparison of the treatment effect with the effect of a sham operation group, wherein a graph 22A is the evaluation of microvascular H & E staining of samples of different experimental groups; FIG. 22B is a Mahalanobis staining evaluation of collagen deposition in samples from different experimental groups; figure 22C is a sample of different experimental groups evaluated by microvascular H & E staining for alpha-smooth muscle actin, von willebrand factor and platelet-endothelial cell adhesion molecule histological immunostaining.

FIG. 23: different cell aggregates are used for evaluating the treatment effect of the rat myocardial ischemia model and comparing the treatment effect with the treatment effect of a sham operation group and a PBS control group, wherein, FIG. 23A is the ultrasonic cardiogram evaluation of samples of different experimental groups; FIG. 23B is a left ventricular ejection fraction comparison of samples from different experimental groups; FIG. 23C is a comparison of the left ventricular short axis contraction rates of different experimental group samples; FIG. 23D is a left ventricular end-diastolic volume comparison of samples from different experimental groups; FIG. 23E is a comparison of the end-systolic volume of the rat left ventricle for different experimental group samples.

FIG. 24: different cell aggregates are used for the histological evaluation of the rat myocardial ischemia model treatment and the effect comparison with a sham operation group and a PBS control group, wherein, a graph 24A is the comparison of collagen deposition conditions of different experimental group samples evaluated by a Ma's triple staining method; FIG. 24B is a graph showing the proportion of fibrotic region 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 a sham operation group, and the gene expression level detection of the vascularization factor of a PBS control group sample is performed, wherein, a graph 25A is the gene level detection of the alpha-smooth muscle actin expression quantity of different experiment group samples, a graph 25B is the gene level detection of the cardiac troponin expression quantity of different experiment group samples, a graph 25C is the gene level detection of the alpha-myocardial skeleton protein expression quantity of different experiment group samples, a graph 25D is the gene level detection of the vascular endothelial growth factor expression quantity of different experiment group samples, a graph 25E is the gene level detection of the von willebrand disease growth factor expression quantity of different experiment group samples, and a graph 25F is the gene level detection of the tumor necrosis factor-alpha expression quantity of different experiment group samples.

FIG. 26: different cell aggregates are used for rat myocardial ischemia model treatment and a sham operation group, protein expression levels of vascular endothelial cell growth factors, von willebrand disease growth factors, cardiac troponin, alpha-accessory myokinetin and alpha-smooth muscle agonist proteins of PBS control group samples are detected, wherein, a graph 26A is the detection of the protein expression levels of different vascularization factors of different experimental group samples after 1 week of treatment, a graph 26B is the detection of the protein expression levels of different vascularization factors of different experimental group samples after 2 weeks of treatment, a graph 26C is the detection of the protein expression levels of different vascularization factors of different experimental group samples after 3 weeks of treatment, and a graph 26D is the detection of the protein expression levels of different vascularization factors of different experimental group samples after 4 weeks of treatment.

Detailed Description

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 illustrative purposes only and are not intended to limit the present invention.

The conventional chemical reagents used in the examples were purchased from Solebao Biotechnology, Inc., the cell culture consumables used were purchased from Corning, the mesenchymal stem cell culture medium used was purchased from Biotechnology, the mesenchymal stem cell differentiation medium used was purchased from Lorsha, Switzerland, the PLGA composite microspheres used were synthesized by the existing techniques in the laboratory (see Ge M, Sheng Y, Qi S et al, PLGA/chitosan-heparin composite microspheres prepared with the aid of microorganisms for the construction of hMSC aggregates Chemistry B,2020,8(43):9921 and 9932).

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

The construction method is described in Dufeng apparatus, doctor's thesis, southern Kai university, 11 months 2011. Mainly as follows.

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

Human VE cadherin protein sequences and functional partitions are recorded according to the UniProt database, specific PCR primers are designed by combining the sequences of genes recorded in GenBank (NCBI Reference Sequence: NM-001795.3), and the extracellular region of hVE cadherin protein (EC1-EC5) is amplified. An upstream primer (P1); 5'-CCGGATATCATGCAGAGGCTCATGATGCTCC-3' (SEQ ID NO.6), EcoRV cleavage site, downstream primer (P2) 5'-AAGCGGCCGCTCTGGGCGGCCATATC-3' (SEQ ID NO.7), Not I cleavage site. Both primer synthesis and sequencing were done by Invitrogen, Inc.

HUVEC cell (ScienCell) Total mRNA extraction: mRNA was extracted according to the conventional method of molecular cloning, A laboratory Manual (third edition). And measuring the O.D value to quantify the purity and degree of the RNA.

BD company kit based on purchase of reverse transcription

Manipulation in MicroRNA Assays

The reverse transcription system is as follows:

TABLE 1

The reverse transcription procedure was as follows:

TABLE 2

Using mRNA extracted from HUVEC as a template to amplify a VE-cad gene segment, wherein a PCR reaction system comprises the following steps:

TABLE 3

The amplification conditions were as follows: denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles, and final extension at 72 ℃ for 10 min. 380. mu.L of ddH was added to the reaction mixture ₂ O, extracting with phenol/chloroform/isoamyl alcohol once again in equal volume, adding 1/10 volume of 3M NaAc (pH5.0) and 2 volume times of absolute ethyl alcohol, and standing at-20 ℃ for 1 h; centrifuging at 12000rpm for 10min at 4 deg.C, washing the DNA precipitate with 70% ethanol twice, vacuum drying, and dissolving the precipitate in appropriate amount of TE.

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

(1) PCR product after Hind III and Not I double digestion purification

The enzyme digestion system is as follows:

TABLE 4

Reacting at 37 ℃ overnight, inactivating the enzyme at 65 ℃ for 15min, and supplementing 350 mu L of ddH into the reaction solution ₂ O, extracted once with an equal volume of phenol/chloroform/isoamyl alcohol, added with 1/10 volumes of 3M NaAc (pH5.0), 2 volumes of absolute ethanol, and left at-20 ℃ for 1 h. After centrifugation at 12000rpm for 10min at 4 ℃, the DNA precipitate was washed twice with 70% ethanol, vacuum-dried, and the precipitate was dissolved in 10. mu.L of TE.

(2) Cutting pcDNA/3.1 Hind III and Not I;

TABLE 5

The reaction was carried out overnight at 37 ℃. Separating the enzyme digestion product by electrophoresis in 1% agarose gel, cutting the target fragment under ultraviolet lamp, recovering with DNA agarose gel recovery kit (TaKaRa), dissolving the recovered fragment in 25 μ L ddH ₂ And (4) in O.

(3) Ligation and transformation of vector and target fragment

The reaction system is as follows:

TABLE 6

Reacting at 16 ℃ for 16 h. Then CaCl ₂ The transformation competent cells BL21(DE3) were cultured overnight at 37 ℃ for 16-18 h. Transformants were picked and a small amount of plasmid was extracted for detection.

The recovered VE-cadherin extracellular region of the target gene and the vector pcDNA3.1 carrying the Fc fragment were subjected to double digestion at a constant temperature of 37 ℃ (Hind III and Not I), respectively. After recovery of the gel, the recovered products were mixed and ligated overnight at 16 ℃ under the catalysis of T4 DNA ligase. Coli DH 5. alpha. competent cells were transformed with the ligation products, and ampicillin (Amp) was used ⁺ ) And (5) carrying out resistance screening. After plasmid extraction, double enzyme digestion identification is carried out, and DNA sequence analysis is carried out on the recombinant plasmid which is preliminarily identified to be correct (shown in figure 1A). The constructed recombinant plasmid was named pcDNA3.1/hVE-cad-Fc. The sequence is verified to be correct through sequencing. The sequence of hVE-cad-Fc is shown in SEQ ID NO. 1, wherein the sequence of hVE-cad is shown in SEQ ID NO. 2, and the sequence of Fc is shown in SEQ ID NO. 3.

1.3 cell transfection and protein purification

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

The target protein was purified by a Hitrap rProtein A FF column from GE Healthcare using specific binding of the immunoglobulin Fc fragment to rProtein A.

1.4 Western blotting analysis

The purified hVE-cad-Fc was electrophoresed on 10% SDS-PAGE and transferred to PVDF membrane, blocked with 5% skim milk for 2h, incubated with primary anti-rabbit anti-human VE-cadherin extracellular domain monoclonal antibody (RD, USA, 1: 400 dilution) overnight at 4 deg.C, incubated with HRP-labeled goat anti-rabbit secondary antibody (abcam, USA, 1:10000 dilution) for 1h at room temperature, washed with TBST, exposed to DAB reagent, developed and fixed for analysis. And when the formation of the Fc dimer is detected, the beta mercaptoethanol is not added into the loading buffer solution. The results are shown in FIG. 1B.

As can be seen from FIG. 1B, a band was observed at 240kD in the non-reduced state and at 120kD in the reduced state, suggesting that the hVE-cad-Fc fusion protein exists in the form of a dimer (FIG. 1B).

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

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

TABLE 7

Program setting of the PCR machine: 95 ℃ for 1min for 1 cycle, 95 ℃ for 5min for annealing temperature 1min for 72 ℃ for 45s for 35 cycles, 72 ℃ for 10min for 1 cycle, preserving heat at 4 ℃, and detecting the expression condition of the PCR product 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 hN-cad-Fc is shown as SEQ ID NO. 4, wherein the sequence of SEQ ID NO. 5 shows the sequence of hN-cad; the sequence of SEQ ID NO 3 indicates the sequence of Fc.

2.1 construction and expression of human hN-cad-Fc

2.1.1 cloning and sequence analysis of the Gene N-cadherin in the extracellular region of cadherin in nerve cells

According to the Sequence and functional partition of human N-cadherin protein recorded in UniProt database, designing specific PCR primer by combining with the Sequence of gene (NCBI Reference Sequence: NM-001795.3) recorded in GenBank, amplifyingIncreasing the extracellular domain of hN cadherin (EC1-EC 5). An upstream primer (P1); 5' -CCGGATATCATGCAGAGGCTCATGATGCTCC-3 '(SEQ ID NO.8), introduces EcoRV enzyme cutting site, and downstream primer (P2) 5' -AAGCGGCCGCTCTGGGCGGCCATATC-3' (SEQ ID NO.9), and a Not I cleavage site was introduced. Both primer synthesis and sequencing were done by Invitrogen, Inc.

Extraction of total mRNA of nerve cells (ScienCell): mRNA was extracted according to the conventional method of molecular cloning, A laboratory Manual (third edition). And measuring the O.D value to quantify the purity and concentration of the RNA. BD company kit based on purchase of reverse transcription

The reverse transcription system was operated in MicroRNA Assays as follows:

TABLE 8

The reverse transcription procedure was as follows:

TABLE 9

Using mRNA extracted from nerve cells as a template to amplify an N-cadherin gene fragment, wherein a PCR reaction system comprises the following steps:

watch 10

The amplification conditions were as follows: denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles, and final extension at 72 ℃ for 10 min. 380. mu.L of ddH was added to the reaction mixture ₂ O, then extracting once with phenol/chloroform/isoamyl alcohol with the same volume,1/10 volumes of 3M NaAc (pH5.0), 2 volumes of absolute ethanol, and standing at-20 ℃ for 1 h; centrifuging at 12000rpm for 10min at 4 deg.C, washing the DNA precipitate with 70% ethanol twice, vacuum drying, and dissolving the precipitate in appropriate amount of TE.

2.1.2 pcDNA3.1-hN-cad-Fc eukaryotic expression vector construction

(1) PCR product after EcoRV and Not I double enzyme digestion purification

The enzyme digestion system is as follows:

TABLE 11

Reacting overnight at 37 deg.C, inactivating enzyme at 65 deg.C for 15min, and adding 350 μ L ddH into the reaction solution ₂ O, extracted once with an equal volume of phenol/chloroform/isoamyl alcohol, added with 1/10 volumes of 3M NaAc (pH5.0), 2 volumes of absolute ethanol, and left at-20 ℃ for 1 h. Centrifugation was carried out at 12000rpm for 10min at 4 ℃ and the DNA precipitate was washed twice with 70% ethanol, vacuum-dried and dissolved in 10. mu.L of TE.

(2) pcDNA/3.1 EcoR V and Not I;

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

TABLE 12

The reaction was carried out at 37 ℃ overnight. Separating the enzyme digestion product by electrophoresis in 1% agarose gel, cutting the target fragment under ultraviolet lamp, recovering with DNA agarose gel recovery kit (TaKaRa), dissolving the recovered fragment in 25 μ L ddH ₂ And (4) in O.

(3) Ligation and transformation of vector and target fragment

The reaction system is as follows:

watch 13

The recovered target gene hN cadherin extracellular region and the vector pcDNA3.1 carrying Fc fragment were subjected to double digestion at a constant temperature of 37 ℃ (EcoR V and Not I), respectively. After recovery of the gel, the recovered products were mixed and ligated overnight at 16 ℃ under the catalysis of T4 DNA ligase. Coli DH5 α competent cells were transformed with the ligation products, followed by resistance selection with ampicillin (Amp +). After plasmid extraction, double enzyme digestion identification is carried out, and DNA sequence analysis is carried out on the recombinant plasmid which is preliminarily identified to be correct. The constructed recombinant plasmid was named pcDNA3.1/hN-cad-Fc (see FIG. 2A). The sequence is verified to be correct through sequencing.

2.1.3 cell transfection and protein purification

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

2.1.4 Western blotting analysis

The purified hN-cad-Fc was electrophoresed on 10% SDS-PAGE and transferred to PVDF membrane, 5% skim milk blocked for 2h, primary anti-rabbit anti-human N-cadherin extracellular domain monoclonal antibody (RD, 1: 400 dilution) incubated overnight at 4 ℃, HRP-labeled goat anti-rabbit secondary antibody (abcam,1: 10000 dilution) incubated for 1h at room temperature, TBST washed, DAB reagent exposed, developed and fixed for analysis. And when the formation of the Fc dimer is detected, the beta mercaptoethanol is not added into the loading buffer solution. A band was observed at 240kD in the case of non-reduced state and at 120kD in the case of 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 Joint modified matrices

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

The hVE-cad-Fc prepared in example 1 was dissolved in sterile PBS in a clean bench to a concentration of 100. mu.g/mL, and then stored in a cryofreezer for a long period. Taking out the subpackaged fusion protein, melting, and preparing each concentration gradient by using PBS: 3. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL and 30. mu.g/mL, and then incubated on a 96-well polystyrene plate, left overnight in a low temperature refrigerator or at room temperature for about 2 hours. Then, the supernatant was discarded, washed three times with PBS, and the optimum adsorption concentration of hVE-cad-Fc on the polystyrene surface was measured by ELISA. The modified polystyrene plate was added to 300mL of 5% BSA solution, and the mixture was placed on a shaker at 37 ℃ and blocked at a constant temperature of 100rpm for 2 hours. After blocking, add 1: a1000-part dilution of horseradish peroxidase-labeled human IgG antibody was incubated at 100rpm in a shaker at 37 ℃ for 2 h. After PBST solution washing 5 times, each tube was added with 300. mu.L of TMB color developing solution, shaking table at 37 ℃ and light-shielding reaction at 100rpm for 15min, added with stop solution, and absorbance was read at 452 nm. Each concentration set 5 replicates.

As shown in the attached FIG. 3.A, with the increase of the concentration of hVE-cad-Fc solution, the absorbance at 450nm also increased, and the absorbance value curve leveled off to 10. mu.g/mL. Therefore, when the protein concentration reaches 10 mu g/mL, the protein concentration fixed on the surface of the polystyrene flat plate reaches the maximum, the solution concentration is continuously increased, the amount of the protein fixed on the surface of the flat plate does not continuously increase, and the protein concentration of 10 mu g/mL is the most economical and effective fixed concentration for the fixation of the two-dimensional polystyrene flat plate. Therefore, the two-dimensional hVE-cad-Fc modified matrix in the subsequent experiment is prepared by using 10 mug/mL hVE-cad-Fc solution.

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

The hN-cad-Fc prepared in example 2 was dissolved in sterile PBS in a clean bench to a concentration of 100. mu.g/mL, and then stored in a cryofreezer for a long period. Taking out the subpackaged fusion protein, melting, and preparing each concentration gradient by using PBS: 3. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL and 30. mu.g/mL, and then incubated on a 96-well polystyrene plate, left overnight in a low temperature refrigerator or at room temperature for about 2 hours. Then, the supernatant was discarded, washed three times with PBS, and the optimum adsorption concentration of hN-cad-Fc on the polystyrene surface was measured by ELISA. The modified polystyrene plate was added to 300mL of 5% BSA solution, and the mixture was placed on a shaker at 37 ℃ and blocked at a constant temperature of 100rpm for 2 hours. After blocking, add 1: the human IgG antibody labeled with horseradish peroxidase diluted at a ratio of 1000 was incubated for 2 hours at a constant temperature of 100rpm in a shaker at 37 ℃. After PBST solution washing 5 times, each tube was added with 300. mu.L of TMB color developing solution, shaking table at 37 ℃ and light-shielding reaction at 100rpm for 15min, added with stop solution, and absorbance was read at 452 nm. Each concentration set 5 replicates.

As shown in the attached FIG. 3.B, with the increase of the concentration of hN-cad-Fc solution, the absorbance at 450nm increased, and the absorbance value curve leveled off to 10. mu.g/mL. Therefore, when the protein concentration reaches 10 mug/mL, the protein concentration fixed on the surface of the polystyrene flat plate reaches the maximum, the solution concentration is continuously increased, the amount of the protein fixed on the surface of the flat plate does not continuously increase, and the protein concentration of 10 mug/mL is the most economical and effective fixed concentration for the fixation of the two-dimensional polystyrene flat plate. Therefore, the two-dimensional hN-cad-Fc modified matrix in the subsequent experiments was prepared by using 10. mu.g/mL hN-cad-Fc solution.

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

Incubators with hVE-cad-Fc concentrations of 0. mu.g/mL, 3. mu.g/mL, 5. mu.g/mL, and 10. mu.g/mL were prepared as in example 3.1, respectively, and polystyrene plates were fixed, after which hN-cad-Fc prepared as in example 2 was formulated with PBS to each concentration gradient: 3 mu g/mL,5 mu g/mL,10 mu g/mL,20 mu g/mL, then incubated in a plate fixed with 0 mu g/mL,3 mu g/mL,5 mu g/mL,10 mu g/mL hVE-cad-Fc incubation solution, and left overnight in a low temperature refrigerator or at room temperature for about 2 h. Then, the supernatant was discarded, washed three times with PBS, and then the absorbance of the surface of the hVE/hN-cad-Fc conjugate-modified polystyrene plate was measured by ELISA. The jointly modified polystyrene plate was added to 300mL of 5% BSA solution, and the plate was placed on a shaker at 37 ℃ and blocked at a constant temperature of 100rpm for 2 hours. After blocking, add 1: a1000-part dilution of horseradish peroxidase-labeled human IgG antibody was incubated at 100rpm in a shaker at 37 ℃ for 2 h. After PBST solution washing 5 times, each tube was added with 300. mu.L of TMB color developing solution, and the reaction was carried out in a shaker at 37 ℃ for 15min in a dark place at 100rpm, and then stop solution was added thereto, and absorbance was read at 452 nm. Each concentration set 5 replicates.

As shown in FIG. 3.C, as the concentration of hN-cad-Fc solution increased, the absorbance of the plate immobilized with hVE-cad-Fc solution at 0. mu.g/mL, 3. mu.g/mL, 5. mu.g/mL also increased, while the absorbance of the plate immobilized with hVE-cad-Fc solution at 10. mu.g/mL did not substantially increase. Meanwhile, when the concentration of hN-cad-Fc reaches 5 mug/mL, the absorbance of the plate fixed by 5 mug/mL of hVE-cad-Fc solution basically reaches the maximum, and when the concentration of hN-cad-Fc reaches 10 mug/mL, the absorbance of the plate (blank plate) fixed by 0 mug/mL of hVE-cad-Fc solution basically reaches the maximum, so that it can be seen that when the concentration of the combined total protein reaches 10 mug/mL, the concentration of the protein fixed on the surface of the polystyrene plate reaches the maximum, the concentration of the fusion protein is continuously increased, and the amount of the protein fixed on the surface of the plate does not continuously increase. This result indicates that the 10. mu.g/mL protein concentration is the most economically efficient fixed concentration for the fixation of hVE/hN-cad-Fc in combination with modified polystyrene plates. Therefore, the subsequent experiment hVE/hN-cad-Fc combined modified two-dimensional matrix is prepared by using fusion protein solution with the total concentration of 10 mug/mL.

Example 4 two-dimensional hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc combination matrix promote adhesion and proliferation assays of hMSCs

Firstly, preparing different two-dimensional cell culture matrixes, wherein a negative control is an untreated PS plate; the positive control is a collagen matrix prepared by using a collagen diluent, and the preparation method comprises the following steps: diluting collagen to 1 μ g/mL with glacial acetic acid solution, adding 200 μ L per well of 96 cell culture plate, incubating for 2h, and washing with PBS for 3 times (30 s each time) before inoculating cells; see example 3.1 preparation of two-dimensional hVE-cad-Fc culture medium; preparation of hN-cad-Fc substrate see example 3.2 preparation of two-dimensional hN-cad-Fc culture substrate; the preparation method of the hVE/hN-cad-Fc combined medium is described in example 3.3. preparation of two-dimensional hVE/hN-cad-Fc combined culture medium. After the preparation of the two-dimensional cell culture medium is completed, the hMSCs obtained by digestion are mixed at a ratio of 1 × 10 ⁴ The density of each hole is respectively cultured on the surfaces of negative control, positive control, hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc substrate, the time point of observation by a light microscope is set as 24h, and the proliferation detection experiment: is divided intoCulture supernatants were aspirated at time points other than 4h, 24h, and 48h, washed 3 times with PBS for 30s each, and MTT medium (1mg/mL) in fresh medium was added. The culture was continued, after 3h, the supernatant was removed, 100. mu.L of DMSO was added to each well, and shaking was carried out for 10min in the dark. The absorbance was measured at 450nm after aspiration of the solution.

As shown in FIG. 4A, hVE-cad-Fc, hN-cad-Fc, and hVE/hN-cad-Fc substrate all significantly promoted adhesion of hMSCs compared to the negative control group, with adhesion efficiency close to that of the positive control group. As can be seen from FIG. 4B, hVE-cad-Fc, hN-cad-Fc, and hVE/hN-cad-Fc substrate significantly increased the proliferation efficiency of hMSCs at 4h, 24h, and 48h, compared to the negative control group. This result indicates that the hVE/hN-cad-Fc substrate can significantly promote the adhesion and proliferation efficiency of hMSCs.

Example 5 two-dimensional hVE/hN-cad-Fc Combined matrix-activated cadherin signaling pathway assay for hMSCs

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

RNA extraction: extracting total RNA of cells by using Trizol reagent, adding 1mL Trizol solution into each sample hole to crack the cells, shaking, standing for 5min at room temperature, centrifuging for 5min at 4 ℃ and 10000rpm, and absorbing supernatant and transferring the supernatant into a new centrifuge tube. To the transferred supernatant was added 200. mu.L of chloroform (per 1mL of Trizol), mixed by inversion, allowed to stand at room temperature for 5min, and centrifuged at 4 ℃ at 10000rpm for 10 min. After centrifugation, the solution was divided into three layers, top, middle and bottom, and the uppermost layer of clear inorganic aqueous phase was carefully pipetted into a fresh centrifuge tube (taking care not to pipette the white middle layer). Adding 500 mu L of isopropanol into the absorbed inorganic water phase, reversing and uniformly mixing, standing at room temperature for 5min, centrifuging at 4 ℃, 10000rpm for 5min, and obtaining colloidal precipitate at the tube bottom, wherein the colloidal precipitate is the extracted RNA sample. The supernatant was discarded, 1mL of 75% ethanol (prepared with DEPC water) was added, and the mixture was centrifuged at 7500rpm for 5min at 4 ℃. After centrifugation, the supernatant is discarded, the centrifuge tube is inverted, or the residual liquid is carefully sucked by a gun, kept stand and dried, 20-60 mu L DEPC water is added, and the mixture is subjected to warm bath at 55-60 ℃ for 10 min. After warm bath, a small amount of the solution can be taken out to detect the RNA concentration, and the rest is placed in a refrigerator at the temperature of minus 80 ℃ for storage.

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 addition amount of each component of the reverse transcription PCR System

Temperature system: 20 μ L, 42 deg.C [30min ], 85 deg.C [5min ], 4 deg.C [ long time ]. After the reaction is finished, a small amount of DNA is taken for DNA concentration detection, 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 using a 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 according to the steps in the Roche real-time fluorescent quantitative PCR kit, and the components are shown in table 16:

TABLE 16 addition amount of each RT-PCR component

Adding the components according to the steps, shaking, mixing uniformly, centrifuging, placing in a real-time fluorescent quantitative PCR instrument, and operating the steps of: 5min at 95 ℃; 30s at 95 ℃, 1min at 72 ℃ and 42 cycles of annealing temperature of each primer; 5min at 72 ℃; infinity at 4 ℃. Each set of experiments was set to 5 replicates, and an internal reference was added to each set as reference, 2 ^-ΔΔt The initial concentration of each sample was calculated.

Protein extraction: add 100 μ L of lysis solution to each sample well, blow with a gun and beat evenly, lyse for 1min, this step needs to be done on ice. After cracking, the sample is placed at 12000rpm and 4 ℃ for centrifugation for 10min, the centrifugation is completed, the supernatant is taken, at this time, protein concentration can be measured, 5 × loading Buffer is added, boiled water is boiled for 5min, and then the sample is placed at-20 ℃ for storage.

Western blot detection experiment: the addition amounts of the components of the separation gel are shown in table 17:

TABLE 17 addition of the components for the separation gel

After gelling, the ddH2O was decanted off and the remaining ddH2O was blotted dry using filter paper to make a concentrated gel. The addition of each component of the concentrated gum is shown in table 18:

TABLE 185% addition of the components for the concentrated gum formulation

The loading amount is determined by the concentration of the protein sample, and the loading amounts of different samples are ensured to be consistent. Electrophoresis procedure: 100V for 10 min; 120V, 40-70 min. And after electrophoresis is finished, performing a membrane transferring step. Firstly, 10 times of rotating membrane Buffer is configured: 30g Tris, 144g glycine. When used, the mixture was diluted 10-fold, and 200mL of methanol was added per 800mL of the dilution. Putting the rubber plate after electrophoresis into an electric rotating clamp, wherein the black clamping surface is arranged below, and the sequence is as follows: black gel → black mesh → three layers of filter paper → glue (Marker strip on the right) → PVDF film → three layers of filter paper → white mesh → white gel. Then, the electrotransfer solution is added, the electrode is connected, and 100V is carried out for 90 min. Sealing, sealing liquid preparation: skimmed milk 2g +40ml BST, closed at room temperature for 2 h. Performing antibody blocking experiment after the above steps are completed, respectively adding an endothelial cadherin primary antibody (Abcam,1:2000 dilution), a neural cadherin primary antibody (Abcam,1:2000 dilution), an internal reference primary antibody (Abcam,1:2000 dilution) and blocking overnight, and washing PBST for 5 times, 5min each time; after washing was completed, a 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, color development: and uniformly mixing the color developing solution A/B solution, adding the color developing solution A/B solution to the PVDF membrane, and carrying out exposure detection by using a chemiluminescence device.

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

Example 6 activation of factor secretion-related signalling pathways and vascularization of hMSCs by fusion protein matrices

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

And (3) detecting the secretion of different stroma-activated hMSCs (human mesenchymal stem cells) vascularization factors: first, hMSCs were cultured on PS surface (negative control), collagen surface (positive control), hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc jointly modified culture substrate surface, respectively, RNA of each sample was extracted at 3 days, 7 days and 14 days time points, respectively, according to the method in example 5, and PCR was performed, with the primer sequences shown in table 19, and after the PCR was completed, each PCR sample was taken for agarose gel electrophoresis.

Watch 19

As shown in FIG. 6A and FIG. 6B, hVE-cad-Fc and hN-cad-Fc can significantly alter the transcriptome of hMSCs compared to the control, and hVE-cad-Fc can alter the expression of 524 genes in the transcriptome of hMSCs, wherein 248 genes appear up-regulated and 276 genes appear down-regulated, and hN-cad-Fc can alter the expression of 172 genes in the transcriptome of hMSCs, wherein 54 genes appear up-regulated and 118 genes appear down-regulated. As can be seen from the analysis of the KEGG cell pathways for the differential genes in FIGS. 7A and 7B, both the hVE-cad-Fc substrate and the hN-cad-Fc substrate activated cytokine secretion-related pathways of hMSCs 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 surface of the hVE-cad-Fc stroma express angiogenin, the capabilities of transforming growth factor-beta, endothelial cell growth factor and hepatocyte growth factor are obviously improved, the hMSCs on the surface of the hN-cad-Fc stroma express angiogenin, the capability of basic fibroblast growth factor is obviously improved, and the capabilities of the hMSCs on the surface of the hVE/hN-cad-Fc stroma express all the factors are obviously improved. The results prove that both the hVE-cad-Fc matrix and the hN-cad-Fc matrix can activate cytokine secretion related channels of the hMSCs and activate factor secretion of the hMSCs, and the hVE/hN-cad-Fc matrix can simultaneously up-regulate cytokine secretion of the hMSCs activated by the hVE-cad-Fc matrix and the hN-cad-Fc matrix, which suggests that the hVE/hN-cad-Fc combined matrix has the strongest improvement effect on paracrine functions of the hMSCs and the strongest promotion effect on angiogenesis promoting capability of the hMSCs.

Example 7 fusion protein matrix activation of differentiation of hMSCs into endothelial cells and detection of related signaling pathways

First, 6-well plates of negative control, positive control, hVE-cad-Fc, hN-cad-Fc, and hVE/hN-cad-Fc combination-modified matrices were prepared according to the method of example 5, and then 10 wells were filled with the modified matrices ⁵ hMSCs are inoculated on the density of cells/holes, endothelial differentiation culture medium (EBM-2 medium, Lonza) is added for endothelial differentiation culture, samples of each group are taken for detecting endothelial differentiation indexes, namely vascular endothelial growth factor receptor-2, platelet-endothelial cell adhesion molecules and endothelial cadherin after differentiation culture for 1 week and 2 weeks respectively, the detection means mainly adopts PCR and immunoblotting detection, and the detection method comprises the following steps:

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

TABLE 20 endothelial differentiation index primer sequences

And (3) carrying out protein immunoblotting detection: first, protein samples of each differentiated sample group were extracted for 1 week and 2 weeks, respectively, according to the protein extraction method of example 5, and then polyacrylamide gel electrophoresis, membrane transfer, blocking solution incubation were performed, and after the above steps were completed, antibody blocking experiments were performed, wherein endothelial cell growth factor receptor-2 (VEGFR-2) primary antibody (Abcam,1:2000 dilution), platelet-endothelial cell adhesion molecule (CD31) primary antibody (Abcam,1:2000 dilution), endothelial cadherin (VE-cadherin) primary antibody (Abcam,1:2000 dilution), phosphorylated vascular endothelial cell growth factor receptor-2 (pVEGFR-2) primary antibody (Abcam,1:2000 dilution), phosphorylated focal adhesion kinase (pFAK) primary antibody (Abcam,1:2000 dilution), phosphatidylkinase (PI3K) primary antibody (Abcam,1:2000 dilution) were added, phosphorylated protein kinase b (pakt) primary antibody (Abcam,1:2000 dilution), β -actin primary antibody (Abcam,1:2000 dilution) was blocked overnight, PBST was washed 5 times for 5min each; after washing was completed, a 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, color development: and uniformly mixing the color developing solution A/B solution, adding the color developing solution A/B solution to the PVDF membrane, and carrying out exposure detection by using a chemiluminescence device.

As shown in figure 9, compared with the negative control group, the positive control group, hVE-cad-Fc group, hN-cad-Fc group, hVE/hN-cad-Fc combined modified matrix group can obviously up-regulate the expression of vascular endothelial-like cell markers such as VEGFR-2 and CD31 and VE-cadherin in the differentiation process of hMSCs, and the hVE/hN-cad-Fc matrix can obviously promote the differentiation of hMSCs into endothelial cells, and the effect of the hVE/hN-cad-Fc matrix is obviously stronger than the promotion effect of hMSCs on hMSCs by using the hVE-cad-Fc matrix and the hN-cad-Fc combined modified matrix alone. Secondly, as can be seen from the attached figure 10, compared with the negative control group, the positive control group, the hVE-cad-Fc group, the hN-cad-Fc group and the hVE/hN-cad-Fc combined modified matrix group can obviously activate the expression of pVEGFR-2, p-FAK, PI3K and pAKT, which proves that the hVE/hN-cad-Fc combined modified matrix can obviously promote the differentiation of hMSCs to endothelial cells by activating the pVEGFR-2/p-FAK/pAKT/PI3K signal channels.

Example 8 immobilization and optimized characterization of fusion protein matrices on the surface of PLGA composite microspheres

8.1 preparation of PLGA composite microspheres

40mg of PLGA (L: G: 50, Mw: 25000, Jinan Dai handle & Tibet, Ltd.) was weighed, added to 1mL of methylene chloride, and the mixture was dissolved sufficiently with magnetic stirring and filtered through an organic phase filter. And (3) resuspending the obtained chitosan-heparin compound in a sterile PBS solution, and performing high-speed ultrasound for 2min to obtain a uniform emulsion. 0.1mL of the above solution (containing 0mg, 10mg and 20mg of chitosan-heparin complex respectively) was added to 1mL of PLGA/DCM solution (40mg/mL), and rapidly placed on ice for 2min of high-speed ultrasonic emulsification 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, Aladdin) is weighed and dissolved in 100mL of distilled water to prepare 2% (w/v) of PVA solution, the PVA is slowly dissolved at room temperature and can be properly heated to accelerate dissolution, after being completely dissolved, the solution filtered by a polyether sulfone filtering membrane is used as a continuous phase of a microfluidic system, and the PVA solution is easy to generate white floccules after being stored for a long time, so the PVA solution is prepared at present when being used.

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

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

The dispensed hVE-cad-Fc in example 1 and the dispensed hN-cad-Fc in example 2 were thawed and then mixed with PBS to form 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 mixed solution are respectively used for incubating 1mg of PLGA composite microspheres, after fully shaking, the mixed solution is placed in a horizontal shaking table at 37 ℃ for incubation for 2h at 150rpm, the supernatant is discarded, and fusion proteins which are not stably fixed are washed (3 times) by shaking with PBS. The hVE/hN-cad-Fc combined modified PLGA composite microspheres are prepared by preparing incubation solutions with hVE-cad-Fc concentrations of 3. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL respectively, incubating 1mg PLGA composite microspheres respectively, placing the microspheres in a horizontal shaker at 37 ℃ after fully shaking for 2h at 150rpm, discarding supernatant, washing (3 times) fusion proteins which are not stably fixed by shaking with PBS, and then preparing hN-cad-Fc prepared in example 2 into each concentration gradient by PBS: 3 mu g/mL,7 mu g/mL,10 mu g/mL,20 mu g/mL solution, then incubating again 3 mu g/mL,5 mu g/mL,10 mu g/mL,20 mu g/mL hVE-cad-Fc immobilized PLGA composite microspheres, after shaking thoroughly, placing in a horizontal shaker at 37 ℃ to incubate for 2h at 150rpm, discarding the supernatant, and washing (3 times) the fusion protein that is not stably immobilized with PBS by shaking. Then, the amount of immobilized antibody was measured by adding 300. mu.L of 5% BSA solution, placing the mixture in a horizontal shaker at 37 ℃ for 2 hours at 150rpm, then adding 100. mu.L of diluted HRP-labeled goat anti-human IgG (Abcam, USA) at a dilution ratio of 1:10000, placing the mixture in a horizontal shaker at 37 ℃ for 1 hour at 150rpm in the dark, washing the mixture with 0.01M PBS for 5 times, adding 300. mu.L of TMB (Solibao, Cat. PR1200) color developing solution per tube, placing the mixture in a horizontal shaker at 37 ℃ for 30 minutes at 150rpm in the dark, adding 300. mu.L of stop solution, adding 200. mu.L of the solution to a 96-well plate, and measuring absorbance at 452nm to determine the amount of immobilized antibody bound to the surface of the microspheres.

The method is characterized by inspecting the fixed states of hVE-cad-Fc and hN-cad-Fc on the surface of the PLGA microsphere by an immunofluorescence staining method, and comprises the following specific steps: adding 300 mu L of 5% BSA solution into each tube of hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc combined modified PLGA composite microsphere samples prepared with the optimal adsorption concentration, placing the tubes in a shaker at 37 ℃ and sealing the tubes for 2 hours at constant temperature of 100 rpm. After blocking, add 1: rabbit anti-human VE-cadherin antibody (Abcam) and mouse anti-human N-cadherin antibody (Abcam) diluted at a ratio of 200 were incubated for 2h at a constant temperature of 100rpm in a shaker at 37 ℃. After 5 washes of PBST solution, the hVE-cad-Fc group was added with 1: FITC-labeled secondary goat anti-rabbit antibody (Beyotime) diluted at 200 ratios, hN-cad-Fc group was added to a 1: rhodamine labeled goat anti-mouse secondary antibody (Beyotime) diluted at 200 ratio, hVE/hN-cad-Fc group was added simultaneously with 1: a 200-ratio dilution of FITC-labeled secondary goat anti-rabbit antibody (Beyotime) and 1: rhodamine labeled secondary goat anti-mouse antibody (Beyotime) diluted at 200% ratio was incubated in a shaker at 37 ℃ for 2h in the dark at 100 rpm. After centrifugation, the PBST solution was washed 5 times and placed in a confocal dish. And (3) observing the sample under a confocal fluorescence microscope, setting the PLGA microspheres which are not incubated by the fusion protein as a control group, and processing and imaging through the steps.

As can be seen from the attached drawings 11A and 11B, the PLGA composite microspheres prepared by microfluidics have smooth surfaces, the particle size is 21 +/-1.5 microns on the total, the particle size uniformity is good, and as can be seen from the attached drawing 11C, the PLGA composite microspheres are obviously degraded within 14 days, which proves that the PLGA composite microspheres have good degradability, and the rapid degradation can be suggested when the PLGA composite microspheres are applied to in vivo treatment at the later stage, so that the safety of the PLGA composite microspheres in the in vivo application can be improved to a certain extent. As can be seen from FIG. 12A, compared with PLGA microspheres without incubation of fusion proteins, the hVE-cad-Fc matrix modified microspheres, hN-cad-Fc matrix modified microspheres, and hVE/hN-cad-Fc matrix combined modified microspheres all showed significant fluorescent staining, and the hVE/hN-cad-Fc combined modified microspheres simultaneously showed green fluorescent and red fluorescent staining of hVE-cad-Fc and hN-cad-Fc, which demonstrates that the hVE-cad-Fc matrix, hN-cad-Fc matrix can be simultaneously stably fixed on the surface of PLGA microspheres. 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 at 450nm along with the increase of the concentrations of the hVE-cad-Fc and hN-cad-Fc solutions, and the absorbance curve is flattened when the absorbance reaches 20 μ g/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 amount of the fusion protein fixed on the surface of the PLGA composite microsphere cannot be continuously increased, and the micro PLGA composite microsphere with the protein concentration of 20 mug/mL is the most economical and effective fixed concentration. When hVE-cad-Fc and hN-cad-Fc are simultaneously immobilized on the surface of the PLGA composite microsphere, the absorbance of the plate immobilized by hVE-cad-Fc solution of 3 mug/mL, 5 mug/mL and 10 mug/mL is increased along with the increase of the concentration of the hN-cad-Fc solution, while the absorbance of the plate immobilized by hVE-cad-Fc solution of 20 mug/m is not increased basically. Meanwhile, when the hN-cad-Fc concentration reaches 10 mug/mL, the absorbance of the PLGA composite microspheres modified by the hVE-cad-Fc solution of 10 mug/mL basically reaches the maximum, and when the hN-cad-Fc concentration reaches 20 mug/mL, the absorbance of the PLGA composite microspheres fixed without the hVE-cad-Fc solution basically reaches the maximum. Therefore, when the concentration of the combined total protein reaches 20 mug/mL, the concentration of the protein fixed 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 fixed on the surface of the flat plate does not continuously increase. This result indicates that the total protein concentration of 20. mu.g/mL is the most economical and effective fixed concentration for the hVE/hN-cad-Fc combination in the PLGA composite microspheres. Therefore, in subsequent experiments, the hVE/hN-cad-Fc combined two-dimensional matrix is prepared by using a fusion protein solution with the total concentration of 20 mug/mL, and in order to further determine the regulation effect of the ratio of the hVE-cad-Fc and the hN-cad-Fc on the hMSCs during combined modification, in the subsequent examples, the ratio is further optimized.

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

The hVE-cad-Fc, hN-cad-Fc and hVE/hN-cad-Fc co-modified PLGA composite microspheres were prepared according to the method of example 8, while 10. mu.g/mL collagen-modified PLGA composite microspheres were prepared as the positive control for this experiment. Then the hMSC: hMSC and microspheres were mixed in a 3:1 ratio of microspheres, 7.5 × 10 each ⁵ Mixing hMSC cells with PLGA composite microspheres, and adding into Aggrewell ^TM In a culture plate, after centrifugation at 1000rpm for 5 minutes, the plate was placed in a cell incubator and incubated at 37 ℃ overnight, the cell aggregate morphology was observed microscopically, the aggregate was subsequently blown out with a 1000. mu.L blunt pipette tip, placed in a 15mL centrifuge tube and allowed to settle naturally to collect the precipitate, the precipitate was resuspended with 200. mu.L of 2% alginic acid solution and added dropwise to 0.1M CaCl ₂ Incubating in the solution for 10min to form alginic acid hydrogel, removing supernatant, washing alginic acid hydrogel with physiological saline, placing cell aggregates containing microspheres in different proportions in 6-well culture plate, culturing in 10% FBS DMEM/F12 medium, and changing solution every 3 days. At the same time, aggregates without microspheres were prepared as negative controls as described above.

Specifically, (1) cell aggregates containing no microspheres, which is abbreviated as a negative control group; (2) cell aggregates containing collagen-modified microspheres, abbreviated as positive control group; (3) cell aggregates containing hVE-cad-Fc modified microspheres, abbreviated as MVP group; (4) a cell aggregate containing hN-cad-Fc modified microspheres, abbreviated as MNP group; (5) the cell aggregate containing the hVE + hN combined modified microspheres is abbreviated as MV/NP group. The prepared cell aggregate is photographed and observed through a microscope, and particle size statistics is carried out. The results are shown in FIG. 13. As can be seen in fig. 13A, the resulting aggregate was structurally complete and edge-defined. As can be seen from FIGS. 13B and 13C, the cell aggregates prepared without microspheres had a particle size of 119.92. + -. 6.74. mu.m (negative control), and the cell aggregates with microspheres had a particle size of 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 aggregates of hMSCs.

Example 10 study of the Effect of different fusion protein matrices and different Microspherulocyte ratios on the paracrine function of hMSCs aggregate vascularization factor

In order to further determine the regulation effect of the ratio of the hVE-cad-Fc and the hN-cad-Fc on the hMSCs when the PLGA composite microspheres are modified by the combination, the ratio is further optimized, and blending incubators containing different hVE-cad-Fc and hN-cad-Fc are respectively prepared according to the method in example 8, (1) hVE-cad-Fc: hN-cad-Fc ═ 3:1(15 mug/mL hVE-cad-Fc +5 mug/mL hN-cad-Fc); (2) hVE-cad-Fc hN-cad-Fc ═ 1:1 (10. mu.g/mL hVE-cad-Fc + 10. mu.g/mL hN-cad-Fc); (3) hVE-cad-Fc hN-cad-Fc ═ 1:3 (5. mu.g/mL hVE-cad-Fc + 15. mu.g/mL hN-cad-Fc); the different hVE-cad-Fc was then prepared as in example 8: the fusion protein modified PLGA composite microspheres with hN-cad-Fc ratio were then prepared according to the method in example 9, containing different hVE-cad-Fc: the cell aggregate of the fusion protein modified PLGA composite microsphere with the hN-cad-Fc ratio is prepared by mixing the following components in a ratio of hMSC: hmscs were prepared by mixing hmscs and microspheres in a ratio of 3:1, and after 3 days, 7 days, and 14 days in DMEM/F12 medium containing 10% FBS, total RNA was extracted according to the method of example 5, reverse transcription was performed to obtain cDNA, and the gene expression level was detected by PCR using the cDNA as a template. The primer sequences used were the same as those of the angiopoietin primer sequences of Table 19 in example 6.

To further identify the different microspheres: the cell ratio is further optimized, modified microspheres with the optimal combination of hVE-cad-Fc and hN-cad-Fc are selected, cell aggregates with different microsphere-cell ratios of 1:1,1:3 and 1:6 are prepared according to the method in example 9, the cell aggregates are cultured in a DMEM/F12 culture medium containing 10% FBS for 3 days, 7 days and 14 days, total RNA is extracted according to the method in example 5, cDNA is obtained by reverse transcription, and PCR detection is carried out on the gene expression level by taking the cDNA as a template. The primer sequences used were the same as the vascularization factor primer sequences in Table 19 of example 6.

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

The results are shown in FIG. 14, and when the ratio of hVE-cad-Fc: hN-cad-Fc is 1:1, the hVE/hN-cad-Fc co-modified microsphere cell aggregate secreted angiogenin, TGF- β, EGF, bFGF, and HGF, most strongly and the ability to secrete TNF-. alpha.was the weakest, indicating that hVE-cad-Fc: when the hN-cad-Fc ratio is 1:1, the capacity of regulating the paracrine vascularization factors of the hMSCs cell aggregates is strongest, and the quantity of the secreted proinflammatory factors is less, which indicates that in the hMSCs cell aggregates, the hVE-cad-Fc: hN-cad-Fc ratio is 1:1(10 mug/mL hVE-cad-Fc +10 mug/mL hN-cad-Fc), which is the optimal cadherin ratio combination for regulating the hMSCs cell aggregates. As can be seen from figure 14B compared to microspheres: when the cell ratio is 1:1 and 1:6, when the ratio of microspheres: the cell ratio is 1:3, the ability of hVE/hN-cad-Fc co-modified microsphere cell aggregate to secrete the vascularization factor is the strongest, and the amount of the secreted tumor necrosis factor-alpha is relatively less, which indicates that the microsphere: when the cell ratio is 1:3, the capacity of regulating the paracrine vascularization factors of the hMSCs cell aggregates is the strongest, the proinflammatory factor secretion amount is less, and the cell ratio is shown as follows: the cell ratio is 1:3, which is the optimal microsphere cell ratio combination for regulating and controlling the hMSCs cell aggregate.

As can be seen from fig. 15, compared to the negative control (cell aggregates without microspheres), the positive control (cell aggregates with collagen-modified microspheres), MVP, MNP and MV/NP components all have significantly improved ability to secrete the vascularization factor, which indicates that the modification of the fusion protein can significantly improve the paracrine ability of the vascularization factor of the hMSCs cell aggregates. Meanwhile, we can see that compared with the MVP and MNP group, the MV/NP group has obviously improved capability of secreting the vascularization factors, especially the secretion of endothelial growth factors, which shows that the joint use of hVE-cad-Fc and hN-cad-Fc is superior to the use of a single fusion protein matrix. As can be seen from fig. 16A, the relative protein secretion values of the endothelial growth factor, thrombopoietin, hepatocyte growth factor, transforming growth factor- β 1, and basic fibroblast growth factor of the MV/NP group were significantly increased compared to the control group (cell aggregates without microspheres); as can be seen from FIG. 16B, the absolute values of protein secretion of the endothelial growth factor, transforming growth factor-. beta.1, basic fibroblast growth factor, hepatocyte growth factor and angiogenin of the MV/NP group were all significantly increased compared to the control group; the result shows that the addition of the hVE/hN-cad-Fc co-modified PLGA composite microspheres can not only up-regulate the gene expression level of the vascularization factor of the hMSCs cell aggregates, but also can obviously up-regulate the protein expression level of the vascularization factor of the hMSCs cell aggregates, which implies that the angiogenesis promoting capability of the MV/NP cell aggregates is obviously improved, and the better angiogenesis promoting effect can be exerted when the hVE/hN-cad-Fc co-modified PLGA composite microspheres are applied to in-vivo research.

Example 11 detection of the Effect of different fusion protein matrices modified hMSCs aggregates secreting vascularization factors on the improvement of the angiogenic potential of endothelial cells

To further examine the effect of the angiogenic factors secreted by the aggregates of the fusion protein matrix-modified hMSCs on the angiogenic capacity of endothelial cells, different cell aggregates and cell aggregates of the fusion protein-modified microspheres with the optimal hVE/hN-cad-Fc combination were prepared as in example 9, and after culturing in DMEM/F12 medium containing 10% FBS for 7 days, the medium was aspirated, washed 3 times with PBS solution, and then 1mL of DMEM-free/F12 medium was added, and culturing was continued for 24h to collect the supernatant.

Examination of Matrigel surface Human Umbilical Vein Endothelial Cells (HUVECs) tubulogenic effect:

1. preparation of Matrigel: thawing the matrigel at 4 deg.c, and pre-cooling in required pipette head and 48-well plate at 4 deg.c. After the matrigel is melted for later use, the matrigel is uniformly spread in a 48-well plate pre-cooled in advance.

2. Preparing a cell suspension: the HUVECs (ScienCell) which are pre-amplified are digested into a single-cell suspension by pancreatin, and a cell precipitate is obtained after centrifugation of a centrifugal tube. After removing the supernatant, the cells were resuspended in endothelial differentiation medium (EBM-2 medium, Lonza) at 2X 10 ⁵ The density of cells/well was spread on the surface of the coagulated Matrigel, placed in an incubator and cultured for 24 hours, then the endothelial differentiation medium (EBM-2 medium, Lonza) was aspirated and washed 2 times with PBS, the collected supernatants of each group were added, and the culture was continued

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

Detection of the effect on the migration ability of HUVECs:

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

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

3. Inoculating cells: the pre-warmed medium on the upper layer of the chamber was aspirated, 200. mu.L of the cell suspension prepared above was added to the upper layer of the chamber, 500. mu.L of the collected supernatants of each group were added to the lower chamber, and the culture was continued for 24 hours.

4. And (3) cell staining counting: the chamber was removed, the upper medium was discarded, the lower chamber layer was fixed with 4% paraformaldehyde for 15min, PBS washed 3 times, the lower chamber layer was incubated with 1% crystal violet and stained for 30min, PBS washed 3 times, the non-migrated cells on the upper chamber layer were wiped off with a cotton ball, and the picture was taken under a microscope.

As shown in FIGS. 17 and 18, the culture supernatants of the 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 with the culture supernatants of the negative control and positive control groups, while the HUVECs cultured under the culture supernatants of the MV/NP group formed more mature reticular structures (FIG. 17) and stronger migration ability (FIG. 18) compared with the culture supernatants of the MVP and MNP groups, which indicates that the co-modification of hVE/hN-cad-Fc has the strongest ability to improve the angiogenesis of HUVECs, and suggests that the MV/NP cell aggregates can play a better angiogenesis promoting effect.

Example 12 detection of the Effect of different fusion protein matrix modification on promoting differentiation of hMSCs aggregates into endothelial cells

To examine the effect of modification of different fusion protein matrices on the differentiation of hMSCs aggregates into endothelial cells, different cell aggregates and cell aggregates of optimally modified microspheres with a combination of hVE-cad-Fc and hN-cad-Fc were prepared as in example 9, and then the aggregates were blown out with a 1000. mu.L blunt pipette tip, placed in a 15mL centrifuge tube for natural sedimentation to collect the precipitate, resuspended with 200. mu.L of 2% alginic acid solution and added dropwise to 0.1M CaCl ₂ Incubating in the solution for 10min to form alginic acid hydrogel, removing supernatant, washing alginic acid hydrogel with physiological saline, placing different cell aggregates in 6-well culture plate, adding endothelial differentiation medium (EBM-2 medium, Lonza) for continuous culture, and changing the solution once every 2 days.

Immunofluorescence staining of different cell aggregate differentiation culture samples: at different time points of 1 week, 2 weeks, 3 weeks, 4 weeks, the alginic acid hydrogel was lysed with 0.1M EDTA solution, after collecting cell aggregates, 1mL of freshly prepared 4% paraformaldehyde solution was added, fixed on ice for 30min, the supernatant was discarded and 1mL of pre-cooled PBS was added and placed on ice for 10min, and this was repeated twice. The supernatant was discarded and 1mL of PBSDT solution was added and blocked at room temperature for 3h, during which the tube was inverted 1 time every 30 min. After settling for 10min by gravity, the supernatant was discarded, primary antibody (Abcam,1:200 dilution) diluted with PBSDT was added, and the mixture was incubated at 4 ℃ for 24-48 h, during which the tube was inverted 8-10 times. And (4) performing gravity settling on ice for 10min, discarding the supernatant, washing with a precooled PBSB solution, performing gravity settling on ice for 10min, and repeating for 5 times. The supernatant was discarded, a fluorescent secondary antibody diluted with PBSB (Abcam,1:200 dilution) was added and incubated at room temperature in the dark for at least 3h, during which the tubes were inverted 1 time every 30 min. And (3) performing gravity settling on ice for 10min, removing the supernatant, washing liver organs for 5 times by using PBSB, removing the supernatant, adding 50-100 mu L of DAPI solution containing an anti-fluorescence quencher into each tube, and dyeing for 15 min. The samples were placed in confocal petri dishes and examined for layer scanning using a confocal laser microscope at 488nm, 561nm and uv excitation light, and photographed for observation.

And (3) climbing out endothelial cells on the surface of the Matigel by different cell aggregate differentiation culture samples and carrying out immunofluorescence staining detection: at the time point of 2 weeks, alginic acid hydrogel was lysed with 0.1M EDTA solution, and after cell aggregates were collected, each group of collected cell aggregate samples were resuspended in endothelial differentiation medium (EBM-2 medium, Lonza) according to the method for preparing Matrigel in example 11, and cultured on the surface of 24-well Matrigel, respectively, after culturing in an incubator for 48 hours, the medium was aspirated and washed 2 times with PBS, photographed under a microscope, and the field effect was observed. Then cytoskeleton staining and immunofluorescence staining are carried out. Cytoskeleton staining: fixing with 4% paraformaldehyde at room temperature for 30min, and washing with PBS for three times; the wells were incubated with 1% TritonX-100 for 10 min. Discarding 1% Triton X-100, washing with PBS three times for 10min each time; blocking with 5% BSA blocking solution at room temperature for 1 h; adding FITC-labeled phalloidin diluted with 5% BSA, and incubating for 1 h; washing unbound phalloidin with PBS for three times, each time for 10 min; adding DAPI (1: 1000) diluted with PBS and incubating for 10 min; PBS was washed three times and the distribution of the cell cytoskeleton was observed using a confocal laser microscope. And (3) immunofluorescence staining: fixing cells with 4% paraformaldehyde, fixing for 15min at room temperature, performing membrane rupture on cytoplasmic protein (1% TritonX-100) in the next step, and performing PBS (phosphate buffer solution) cleaning three times if cytoplasmic protein is cell membrane protein; adding goat serum blocking solution, and sealing at room temperature for 30-45 min; adding primary antibody (Abcam, diluted 1: 200) diluted by goat serum blocking solution, and incubating overnight in a shaking table at 4 deg.C; PBS wash three times, each time for at least 5min, then add 488 fluorescence labeled secondary antibody (Abcam,1:200 dilution), room temperature incubation 1-2 h; washing with PBS to remove unbound secondary antibody, adding prepared DAPI (1: 1000), and incubating for 2 min; after three times of PBS washing, the distribution of cell membrane proteins was observed by laser confocal microscopy.

As shown in fig. 19 and fig. 20, compared to the negative control and the positive control, the MVP, MNP and MV/NP groups showed more staining of endothelial cell markers (endothelial cadherin, platelet-endothelial adhesion molecule) at 1 week, 2 weeks, 3 weeks and 4 weeks, and the results of experiments on endothelial cell crawling on Matrigel surface by different cell aggregates differentiated for 2 weeks and immunofluorescence staining thereof showed that the crawling number of the endothelial cells of MVP, MNP and MV/NP is significantly higher than that of the negative control and the positive control, which indicates that the modification of the fusion protein can significantly up-regulate the differentiation of hMSCs cell aggregates into endothelial cells; meanwhile, we can see that the expression quantity of the endothelial cell markers and the quantity of the endothelial cell crawled out in the MV/NP group are obviously higher than those in the MVP and MNP groups, which indicates that the co-modification of hVE-cad-Fc + hN-cad-Fc can more effectively promote the differentiation process of the MV/NP cell aggregates to the endothelial cells compared with the modification of a single fusion protein.

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

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:

constructing a mouse lower limb ischemia model: 90 adult male Balb/c mice (the experimental animal center of the academy of military medical sciences, Beijing) with the age of 6 weeks are selected, and the weight is 20-25 g. Anesthesia was performed by intraperitoneal injection of 3.2mL/kg of 10% chloral hydrate. After anesthetizing, the mice were placed in a supine position and fixed on an operating plate. The skin was lifted with ophthalmologic forceps and a longitudinal incision was made approximately 5mm long along the blood vessel from the groin to the inner thigh. Slightly separating out the femoral artery by using surgical forceps under a dissecting microscope, performing high ligation on the proximal end of the starting point of the femoral artery by using a No.7 surgical suture and separating off a section of the femoral artery, and manufacturing a lower limb ischemia model of the high ligation and separation of the femoral artery. The experimental group division was (1) sham operation group (lower limb only skin suture) (2) PBS group (PBS solution injected in ischemia model lower limb muscle) (3) control group (PBS solution containing MCP cell aggregates injected in ischemia model lower limb muscle) (4) MVP group (PBS solution containing MVP cell aggregates injected in ischemia model lower limb muscle) (5) MNP group (PBS solution containing MNP cell aggregates injected in ischemia model lower limb muscle) (6) MV/NP group (PBS solution containing MNP cell aggregates injected in ischemia model lower limb muscle). After the operation, the body temperature of the animals is recovered, laser Doppler experiments are carried out at time points of 1 week, 2 weeks, 3 weeks and 4 weeks respectively, the blood reperfusion quantity of the lower limbs of the mice is detected, and statistical analysis is carried out. Meanwhile, the neck of the mouse is removed at the time points of 1 week, 2 weeks, 3 weeks and 4 weeks, the mouse is killed by taking the lower limb muscle, the muscle is washed clean and then is fixed in 4% paraformaldehyde, the mouse is dehydrated in a full-automatic dehydrator and embedded, paraffin section and H & E staining are carried out, and the histological change of the muscle of the lower limb of the mouse and the angiogenesis condition of the muscle of the lower limb are observed.

As shown in fig. 21, compared to the PBS group and the control group, the MVP, MNP and MV/NP groups showed better integrity of the lower limbs at 1 week, 2 weeks, 3 weeks and 4 weeks (fig. 21A), and significantly higher blood reperfusion amounts (fig. 21B), while the MV/NP groups showed significantly higher blood reperfusion amounts of the lower limbs than the MVP and MNP groups, and significantly lower muscle damage of the lower limbs than the MVP and MNP groups. As can be seen from FIG. 22, the MV/NP treated group had more new microvessels at 4 weeks as compared with the PBS group, the control group, the MVP group, and the MNP group (FIG. 22A), and also less collagen deposition (fig. 22B), results of immunostaining for alpha-smooth muscle actin, von willebrand factor, and platelet-endothelial cell adhesion molecules showed that MV/NP treated groups compared to other cell aggregate groups, the expression level of alpha-smooth muscle actin, von Willebrand factor and platelet-endothelial cell adhesion molecule is higher, this demonstrates that MV/NP cell aggregates can better promote angiogenesis and blood perfusion in the lower limb muscles of mice for the treatment of lower limb ischemia, and that the hVE/hN-cad-Fc co-modification can be more effective for the treatment of lower limb ischemic diseases than the modification of a single fusion protein.

Example 14 study 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 aggregate 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 according to the regulations and laws of biomedical engineering institute of Tianjin. SD rats (Experimental animals center of military medical academy of sciences, Beijing) were first weighed and injected with chloral hydrate into the abdominal cavity of the rat at an anesthetic dose of 0.024g/100 g. After the rats are completely anesthetized and lose mobility, the rat hairs on the neck and the chest are removed by using a special shaver. And then fixing the rat on an operating table by using a rubber band to ensure that the body of the rat stretches and the rat breathes smoothly. The chest shaves were disinfected with iodophors before starting the procedure. The skin and muscle were torn apart with a scalpel along the two or three ribs of the rat to obtain an opening of about 2 cm. The rat heart is squeezed out from a rib gap by hand, the left anterior descending coronary artery is ligated at the position 2cm below the pulmonary artery cone and the left auricle, the ligation depth is noticed during operation, then whether the color of the anterior wall of the left ventricle of the heart undergoes the change process of whitening firstly and then darkening is observed to determine whether the ligation is successful, after the coronary artery ligation is determined to be successful and no malignant arrhythmia is caused, the thoracic cavity is closed, after the rat can breathe autonomously within 1 minute, the fast second chest opening is matched with the injection of gel, and the second chest opening is required to expose the heart and the injection of cell aggregates to be closely matched for improving the success rate of the operation and the survival rate of the laboratory mouse. The wound is sutured layer by layer, grouping marks are made, the rat after the operation is placed on an electric blanket to help the rat to recover consciousness, the rat can be fed with water properly after waking up, the body temperature is kept, and the rat is transferred to an animal room for subsequent experiments. The experimental group division was (1) sham (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 body temperature of the animals is recovered, ultrasonic Doppler experiments are carried out at time points of 1 week, 2 weeks, 3 weeks and 4 weeks respectively, the myocardial function recovery of rats is detected, and statistical analysis is carried out. Meanwhile, the rats are killed by removing necks at the time points of 1 week, 2 weeks, 3 weeks and 4 weeks, the myocardial tissues are taken and washed clean, then the rats are placed in 4% paraformaldehyde for fixation, dehydrated in a full-automatic dehydrator, embedded, and then paraffin sections and mahalanobis staining are carried out, the histological pathological changes of the myocardial tissues of the rats are observed, and the fibrosis degree and the thickness of the wall of the left ventricle are counted.

Real-time fluorescent quantitative PCR analysis and immune protein blotting detection of the expression of the vascularization factor in the myocardial tissue. Real-time fluorescent quantitative PCR analysis of the expression of the vascularization factors in myocardial tissue rats were anesthetized and sacrificed 7, 14, 21, and 28 days after acute myocardial infarction surgery. Harvested hearts were loaded into enzyme-depleted cryopreservation tubes and quenched in liquid nitrogen. Total RNA was isolated using TRIzol kit (Invitrogen, usa). One microgram of RNA (1. mu.g) was reverse transcribed into cDNA. Using cDNA as a template, adding primers in the table 21 in sequence according to the Roche Realtine PCR kit specification, quickly centrifuging by a palm centrifuge, placing in a fluorescent quantitative PCR instrument, and multiplying by 1 at 95 ℃ for 5 minutes according to a program; 95 ℃ for 30s, annealing temperature for 1min, 72 ℃ for 1min × 35; 5min × 1 at 72 deg.C; and (4) carrying out detection at the temperature of 4 ℃. The data obtained were referenced to the internal reference beta-actin of each group, according to 2 ^-△△Ct And carrying out data statistics.

TABLE 21 myocardial tissue RT-PCR primer sequences

Western blot analysis of the expression of the vascularization factors in myocardial tissues for Western blot analysis, rats were anesthetized and sacrificed 7 days, 14 days, 21 days, 28 days after acute myocardial infarction surgery, hearts were harvested and quenched in liquid nitrogen. The liquid nitrogen-quenched tissue blocks were washed 2-3 times with pre-cooled PBS to remove blood stain, cut into small pieces, placed in a homogenization tube, and 10-fold volume of lysis buffer (10mM Tris, 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10mM EDTA and protease inhibitor cocktail, pH7.4) was 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 bicinchoninic acid (BCA) kit (ThermoScientific, usa). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis. The isolated protein was transferred to PVDF (polyvinylidene fluoride) membrane (Roche, Switzerland) and incubated with primary antibody (Abcam,1:2000 dilution) overnight at 4 ℃. The secondary antibody (Beyotime,1:2000 dilution) was diluted with TBST solution at a ratio of 1:3000 and then added to the incubation dish for incubation 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 statistical results.

As shown in fig. 23, compared to the PBS, control, MVP, and MNP groups, the ejection function of the myocardium was stronger in the MV/NP treated rats (fig. 23A), the left ventricular ejection fraction of the myocardium was higher in the MV/NP treated rats (fig. 23B), the left ventricular short axis shortening rate was higher (fig. 23C), the left ventricular end-diastolic volume was smaller (fig. 23D), and the left ventricular end-systolic volume was smaller (fig. 23E). 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 ventricular wall (fig. 24C) compared to the PBS, control, MVP, MNP groups. As can be seen from fig. 25, RNA expression levels of the vascularization factors, α -smooth muscle actin (fig. 25A), cardiac troponin (fig. 25B), α -cardiac skeletal protein (fig. 25C), vascular endothelial cell growth factor (fig. 25D), von willebrand disease growth factor (fig. 25E), and tumor necrosis factor- α (fig. 25F), were significantly increased in the myocardial tissues of the MV/NP treated rats compared to the PBS, control, MVP, MNP groups. As can be seen from fig. 26, compared to the PBS group, the control group, the MVP group, and the MNP group, the ability of the myocardial tissue of the MV/NP-treated rats to express α -smooth muscle actin, cardiac troponin, α -cardiac skeletal protein, vascular endothelial growth factor, von willebrand disease growth factor, and tumor necrosis factor- α, was significantly improved at 1 week (fig. 26A),2 weeks (fig. 26B),3 weeks (fig. 26C), and 4 weeks (fig. 26D). The results show that the MV/NP cell aggregate can obviously reduce the collagen deposition of the myocardium of a rat when being used for treating the rat myocardial ischemia model, inhibit the ventricular remodeling after ischemia, promote the expression of a vascularization factor in the myocardial tissue and promote the regeneration of blood vessels in the myocardium.

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

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

2. The modified matrix of claim 1, wherein said vascular endothelial cell cadherin is human vascular endothelial cell cadherin, and said neural cell cadherin is human neural cell cadherin;

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

3. The modified matrix according to claim 1, wherein said hydrophobic microspheres are PLGA microspheres, preferably PLGA/chitosan-heparin core-shell structured composite microspheres.

5. The preparation method according to 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 matrix of any one of claims 1-3 and cells.

7. The cell aggregate according to claim 6, wherein the cells are mesenchymal stem cells, iPS cells or 14-day endocystic embryonic stem cells, preferably mesenchymal stem cells, preferably of mammalian origin, more preferably of human, porcine or murine origin.

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

9. Use of a vascular endothelial cadherin-linker fusion protein and a neuronal cadherin-linker fusion protein or a modified matrix according to any one of claims 1 to 3 or a cell aggregate according to claims 6 to 8 in any one of:

(i) promoting vascular repair or regeneration in vitro;

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

10. Use of vascular endothelial cell cadherin-linker fusion protein and neuronal cell cadherin-linker fusion protein or the modified matrix of any one of claims 1 to 3 or the cell aggregate of claims 6 to 8 in the preparation of a medicament for the treatment of an ischemic disease; preferably, the ischemic disease includes, for example, myocardial infarction, lower limb ischemia, and the like.

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

12. The use of claims 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.

14. The method of claim 13, wherein the vascular endothelial cadherin-linker fusion protein and the neuronal 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 claim 13, wherein the stem cell is a mesenchymal stem cell, an iPS cell or a 14-day endocystic embryonic stem cell, preferably a mesenchymal stem cell.