CN115105583A - Preparation and application of cadherin fusion protein improved composite microsphere - Google Patents
Preparation and application of cadherin fusion protein improved composite microsphere Download PDFInfo
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- CN115105583A CN115105583A CN202110253084.XA CN202110253084A CN115105583A CN 115105583 A CN115105583 A CN 115105583A CN 202110253084 A CN202110253084 A CN 202110253084A CN 115105583 A CN115105583 A CN 115105583A
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Abstract
Preparation and application of cadherin fusion protein improved composite microspheres. The invention relates to an epithelial cell cadherin-Fc fusion protein and a nerve cell cadherin-Fc fusion protein modified matrix, a preparation method thereof and application thereof in promoting a cellular immune regulation function and/or promoting cartilage repair.
Description
Technical Field
The invention relates to the field of biomedical foundation and application development of preparation of modified biomaterial composite microspheres and regulation and control of stem cell function maintenance, in particular to the field of stem cell immune regulation and control and cartilage repair. More particularly, the invention relates to preparation of a biomaterial shell-core structure composite microsphere, and relates to cooperation of epithelial cell cadherin (hE-cadherin) -Fc fusion protein and/or nerve cell cadherin (hN-cadherin) -Fc fusion protein and new application of the biomaterial composite microsphere in promotion of functions of immune regulation and cartilage repair of mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) have become a major candidate for cell therapy and tissue repair due to their extensive source, self-renewal and paracrine capacity. The MSCs and the microspheres are combined to prepare the engineered cells, so that the innate characteristics of the MSCs can be better kept, and the treatment effect of the MSCs is enhanced. Wherein the uniformity of the microsphere particle size is critical to the homogeneity of the engineered cell internal structure and the release of its loaded soluble proteins and small molecules.
The traditional microsphere preparation technology is mainly an emulsion polymerization method, and the microspheres are prepared by depending on different shearing forces brought by different rotating speeds generated by magnetic stirring or ultrasound, so that the degradation of the microspheres is inconsistent and the stability of released medicines is reduced easily due to uneven particle size and batch-to-batch difference of the generated microspheres. The micro-fluid droplet technology applies stable shearing force between immiscible fluids, can effectively prepare monodisperse microspheres with uniform height without secondary purification, and can prepare microspheres with different structures by adjusting the structure and the device of a micro-fluidic chip and the micro-fluidic process, but the chip processing cost is high, the process is complex and the operation is not easy.
The bionic construction of extracellular microenvironment by using biological materials guides the behavior and function of cells, and plays an important role in modern regenerative medicine and tissue engineering. At present, researches generally consider that the immune regulation and control ability of the MSCs has plasticity, that is, the immune regulation and control ability of the MSCs can be obtained or enhanced in the process of in vitro culture, so that how to obtain stem cells with high biological functions through artificially regulating the interaction between cells is a key problem that researchers are constantly dedicated to solve. Cadherin (herein abbreviated cad) proteins are a class of Ca 2+ The cell surface-dependent transmembrane glycoprotein is an important member of a cell adhesion molecule family, the classical cadherins are transmembrane glycoproteins with strict cell specificity, the extracellular domain of the transmembrane glycoprotein mediates homologous cell connection (namely adhesive connection), the intracellular domain is connected with intracellular alpha, beta and gamma catenin, actin fibers are regulated and participate in various signal transduction, and the transmembrane glycoprotein has the functions of establishing cell polarity, maintaining cell morphology and tissue organ integrity and is also an important regulatory factor for cell differentiation and morphogenesis. Epithelial cadherins (E-cad) are a class of transmembrane glycoproteins that mediate intercellular adhesion in epithelial cells and embryonic stem cells and are essential for early epithelialization, cellular rearrangement, histomorphogenesis, establishment of cellular polarity, and maintenance of tissue architecture in mouse embryos. N-cadherin (N-cad) is one of important markers of the coagulation process of the cartilage morphogenesis development intermediate, the high expression of the N-cadherin is proved to promote the mesenchymal stem cells to generate the mesenchymal coagulation process, but the continuous enhancement of the expression of the N-cadherin can also inhibit the subsequent cartilage differentiation process of the mesenchymal coagulation process, so that the cell is subjected to the N-cadherin surfaceThe spatiotemporal regulation of the achievement is of great importance. In addition, it is difficult to maintain the characteristics of stable hyaline cartilage by directionally inducing and differentiating mature chondrocytes, so that the chondrocytes are easily enlarged and calcified, thereby losing the ability of synthesizing a chondrocyte-specific matrix and physiological functions.
Various fusion proteins based on IgG Fc segment and cadherin extracellular domain have been applied to the research of tissue engineering and regenerative medicine, for example, E-Cad-Fc, N-Cad-Fc and the like are used as one of the modification components of extracellular matrix for researching the influence on the regulation of cell behavior.
However, no report on the human epithelial cell cadherin-Fc fusion protein and human neural cell cadherin-Fc promoting mesenchymal stem cell immune regulation function exists in the prior art so far. The research on E-cadherin focuses more on promoting epithelial cell functions and the like, and at present, the research on inhibiting mature chondrocyte calcification and repairing articular cartilage through regulation and control of relevant signal paths by promoting stem cell differentiation to chondrocyte through the combination of human epithelial cadherin-Fc fusion protein and human neural cadherin-Fc is not available.
Disclosure of Invention
The inventor finds that the application of mesenchymal stem cell immune regulation and cartilage repair can be facilitated by using core-shell structure composite microspheres (such as PLGA, PCL, chitosan, heparin, hyaluronic acid and collagen) and by using epithelial cell cadherin-Fc fusion protein and nerve cell cadherin-Fc fusion protein for modification.
For example, the functional modified composite microspheres of the epithelial cell cadherin-Fc fusion protein and the nerve cell cadherin-Fc fusion protein can combine the dual advantages of fusion protein and three-dimensional culture, a matrix with the fusion protein fixed on the surface is introduced into a stem cell aggregate at the three-dimensional level, the paracrine and immunoregulation capability of the stem cells on the surface of the matrix are regulated and controlled by combining the cell surface cadherin and the same type of cadherin-Fc on the surface of the matrix, and the extracellular microenvironment, the immunoregulation function, the cell differentiation and the cartilage repair function of the stem cells are biomimetically constructed by further cooperating with exogenous cell inflammatory factors.
One aspect of the invention relates to the use of an epithelial cell cadherin-Fc fusion protein and a neural cell cadherin-Fc for culturing stem cells or promoting an immunomodulatory function of mesenchymal stem cells. Preferably, the epithelial cell cadherin-Fc fusion protein and the neuronal cell cadherin-Fc fusion protein are attached to a substrate.
Another aspect of the invention relates to the use of an epithelial cell cadherin-Fc fusion protein and a neuronal cell cadherin-Fc for chondrocyte differentiation, inhibition of mature chondrocyte calcification and repair of joint defects.
In another aspect, the invention relates to the preparation of a modified matrix comprising modifying said matrix with an epithelial cadherin-Fc fusion protein and a neuronal cadherin-Fc, wherein said matrix is a microsphere, preferably said microsphere has a particle size of 10-50 microns (preferably 20 ± 5 microns), preferably a surface hydrophobic microsphere, more preferably said microsphere is selected from the group consisting of PLGA/chitosan-heparin core-shell structure composite microspheres.
In particular embodiments of the above aspects of the invention, the epithelial cadherin is human epithelial cadherin; the nerve cell cadherin is human nerve cell cadherin; fc is that of human IgG (preferably IgG 1).
In a particular embodiment of each of the above aspects of the invention, the sequence of the epithelial cell cadherin consists of SEQ ID NO:2, the sequence of Fc consists of SEQ ID NO:3 is represented by the sequence; preferably, the sequence of the epithelial cell cadherin-Fc fusion protein consists of SEQ ID NO:1 is represented by the sequence of seq id no; the sequence of the neural cell cadherin consists of SEQ ID NO:5, the sequence of Fc consists of SEQ ID NO:3 is represented by the sequence; preferably, the sequence of the neural cell cadherin-Fc fusion protein consists of SEQ ID NO:4 in sequence list.
In a specific embodiment of each of the above aspects of the invention, the hE-cad-Fc is used in combination with the hN-cad-Fc.
In a particular embodiment of each of the above aspects of the invention, the hE-cad-Fc and the hN-cad-Fc are immobilized on a substrate, preferably on the same substrate.
In one aspect, the invention relates to the use of hE-cad-Fc and hN-cad-Fc and their modified matrices for the repair of cartilage layers and subchondral bone at cartilage defects following the mediated aggregation of stem cells into stable multicellular aggregates.
In one embodiment of the invention, the hE-cad-Fc and hN-cad-Fc and their modified substrates promote the phosphorylation of YAP protein and inhibit the entry of phosphorylated YAP protein into the nucleus.
In one embodiment of the invention, the hE-cad-Fc and hN-cad-Fc and their modified substrates inhibit β -catenin from entering the nucleus.
In one embodiment of the invention, the hE-cad-Fc and hN-cad-Fc and their modified matrices are effective in inhibiting mature chondrocyte calcification.
In one embodiment of the invention, the hE-cad-Fc and hN-cad-Fc and their modified matrices promote the repair of cartilage and subchondral bone in cartilage injuries by stem cell multicellular aggregates.
In one embodiment of the invention, the hE-cad-Fc and hN-cad-Fc fusion proteins promote differentiation of stem cell multicellular aggregates into chondrocytes at the site of joint defect and in situ fill the site of injury.
In one embodiment of the invention, the hE-cad-Fc and hN-cad-Fc fusion proteins promote secretion of anti-inflammatory factors by stem cell multicellular aggregates under conditions of joint injury inflammation.
Specifically, in the invention, the epithelial cell cadherin-Fc fusion protein and the neural cell cadherin-Fc enhance the paracrine function of the mesenchymal stem cells by up-regulating the expression of endogenous cadherin of the mesenchymal stem cells, thereby remarkably improving the treatment effect on colitis diseases. Mesenchymal stem cells promote the polarization of macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype by producing the immunosuppressive molecule prostaglandin E2, tumor necrosis factor-stimulating protein 6, stanniocalcin, etc., which is believed to be the basis for MSCs to treat inflammatory diseases. The mesenchymal stem cells directly influence T cells by secreting indoleamine 2, 3-dioxygenase, transforming growth factor, leukemia inhibitory factor, hepatocyte growth factor, interleukin 6 and the like, and the mesenchymal stem cells play an important role in preventing autoimmune cells and inflammation from damaging normal tissues and cells and maintaining immune homeostasis.
The epithelial cadherin-Fc fusion protein and the neural cell cadherin-Fc fusion protein accelerate the initial interstitial coagulation process and enhance the directional differentiation capability of the mesenchymal stem cells to chondrocytes by temporally and spatially regulating the expression of endogenous cadherins of the mesenchymal stem cells. Meanwhile, the expression of osteoblast related genes is inhibited by regulating and controlling the positioning of beta-catenin and YAP protein in cells, so that the calcification of differentiated and mature chondrocytes is inhibited, the differentiated and mature chondrocytes can be filled in situ at the cartilage defect part, and the repair of the soft cartilage defect part is promoted; and by promoting the paracrine function of mesenchymal stem cells and secreting immune regulatory factors, the effect of restoring the early inflammatory environment steady state of the cartilage defect part is achieved, and the repair of the cartilage defect model is accelerated.
In summary, the present invention provides the following embodiments:
1. a modified substrate comprising an epithelial cadherin-Fc fusion protein and a neuronal cadherin-Fc fusion protein, and said substrate is a hydrophobic microsphere.
2. The modified substrate of item 1, wherein said epithelial cadherin is human epithelial cadherin and the Fc is that of an IgG (preferably IgG 1); the neural cell cadherin is human neural cell cadherin, and Fc is Fc of IgG (preferably IgG 1);
preferably, the sequence of the epithelial cell cadherin consists of SEQ ID NO:2, the sequence of the neural cell cadherin consists of SEQ ID NO: as shown in figure 5, the first and second,
more preferably, the Fc of the epithelial cell cadherin-Fc fusion protein and the neuronal cell cadherin-Fc fusion protein are the same or different, preferably the same, more preferably SEQ ID NO:3 in the sequence shown in the sequence table (3),
more preferably, the epithelial cell cadherin-Fc fusion protein is preferably SEQ ID NO: 1; the nerve cell cadherin-Fc fusion protein is preferably SEQ ID NO:4, or a sequence shown in the figure.
3. The modified matrix of item 1, wherein the matrix is a PLGA microsphere, preferably a PLGA/chitosan-heparin core-shell structure composite microsphere.
4.A method of preparing a modified substrate comprising modifying a substrate by mixing an epithelial cell cadherin-Fc fusion protein and the neuronal cell cadherin-Fc fusion protein with the substrate.
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 matrix 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 from 3:1 to 10:1, preferably 3:1 or 5: 1.
9. Use of an epithelial cell cadherin-Fc fusion protein and a neural cell cadherin-Fc fusion protein or a modified matrix according to any of items 1 to 3 or a cell aggregate according to any of items 6 to 8 for promoting a cellular immunoregulatory function and/or for promoting cartilage repair, preferably the epithelial cell cadherin-Fc fusion protein and the neural cell cadherin-Fc fusion protein are immobilized on the same substrate or on different substrates, preferably the substrates are hydrophobic microspheres (preferably PLGA microspheres, more preferably PLGA composite microspheres modified with chitosan and heparin).
10. Use of an epithelial cell cadherin-Fc fusion protein and a neuronal cell cadherin-Fc fusion protein or a modified matrix according to any one of items 1 to 3 or a cell aggregate according to items 6 to 8 in the manufacture of a medicament for the treatment of: acute and chronic inflammation of colitis, arthritis, soft tissue, etc., and acute and chronic articular cartilage defect, etc.
11. The use according to item 9, wherein, when used for promoting cellular immunoregulatory function, the ratio of said epithelial cell cadherin-Fc fusion protein to said neuronal cell cadherin-Fc fusion protein is in the range of 1:5 to 5:1, preferably 1: 3.
12. The use according to item 11, wherein the promotion of immune regulation comprises enhancement of the ability to paracrine related factors (e.g., VEGF, TGF- β, IL-6, EGF, HGF, FGF-2, etc.), enhancement of the expression of genes for anti-inflammatory factors (e.g., IL-1, IL-10, TGF β, etc.), enhancement of the regulatory ability on macrophages.
13. The use of item 9, wherein, when used to promote cartilage repair, the ratio of the epithelial cadherin-Fc fusion protein to the neuronal cadherin-Fc fusion protein is from 1:1 to 1:3, preferably 1: 2.
14. The use of item 13, wherein the promotion of cartilage repair comprises promotion of chondrocyte differentiation, inhibition of mature chondrocyte calcification, joint defect repair, subchondral bone repair, and the like.
Reference data
1.An oxygen-releasing device to improve the survival of mesenchymal stem cells in tissue engineering,Biofabrication,11(2019).
2.Effect of acidic pH on PLGA microsphere degradation and release,Journal of Controlled Release,122(2007)338-344.
3.Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing,Nature Communications,9(2018).
4.Nanosphere-microsphere assembly:methods for core-shell materials preparation,Chemistry of materials,13(2001)2210-2216.
5.Biomimetic design of affinity peptide ligands for human IgG based on protein A-IgG complex,Biochemical Engineering Journal,88(2014(1-11).
6.FYWHCLDE-based affinity chromatography of IgG:Effect of ligand density and purifications of human IgG and monoclonal antibody,Journal of Chromatography A,1355(2014)107-114.
7.Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media,Journal of Chromatography A,1218(2011)1691-1700.
8.Purification of human immunoglobulin G via Fc-specific small peptide ligand affinity chromatography,Journal of Chromatography A,1216(2009)910-918.
9.Mesenchymal stem cells:mechanisms of inflammation,Annual review of pathology:mechanisms of disease,6(2011)457-478.
Drawings
FIG. 1: constructing chitosan heparin complexes, wherein FIG. 1A is an infrared diagram of the chitosan heparin complexes in different proportions; FIG. 1B is a graph showing the residual chitosan and heparin in the solution after the chitosan heparin complexes react at different ratios; FIG. 1C is zeta potential diagram of chitosan heparin complex solution with different proportions.
FIG. 2: hydrophilic treatment of the microfluidic system chip, wherein fig. 2A is a graph of the change in contact angle before and after hydrophilic treatment of the glass chip; fig. 2B is a light mirror image of the droplet prepared after the hydrophilic treatment.
FIG. 3: the microfluidic system regulates and controls the droplet size experiment by changing the flow rate of the continuous phase, wherein fig. 3A is an SEM image of the influence of the particle size of the dispersed phase on the microsphere when the fixed continuous phase is 0.5 mL/h; and 3B is a particle size statistical chart of the influence of the dispersion phase on the particle size of the microspheres when the fixed continuous phase is 0.5 mL/h.
FIG. 4: the microfluidic system regulates and controls the droplet size experiment by changing the dispersed phase flow rate, wherein fig. 4A is an SEM image of the continuous relative microsphere size effect when the fixed dispersed phase is 0.4 mL/h; FIG. 4B is a particle size histogram of the effect of continuous phase versus microsphere particle size for a fixed dispersed phase of 0.4 mL/h.
FIG. 5 is a schematic view of: the appearance characterization of the P/C-h composite microsphere prepared by microfluidics, wherein FIG. 5A is a surface appearance observation SEM image of the P/C-h composite microsphere; FIG. 5B is a SEM image of a section of the P/C-h composite microsphere.
FIG. 6: and (3) an experiment for detecting the internal structure of the P/C-h composite microsphere by using a fluorescent label.
FIG. 7: the characterization of the components of the P/C-h composite microspheres prepared by the microfluidic system is shown in FIG. 7A as an infrared detection diagram; fig. 7B thermal re-inspection map.
FIG. 8: degradation performance characterization of P/C-h composite microspheres prepared by the microfluidic system, wherein fig. 8A is a pH change diagram of physiological saline for soaking the composite microspheres in the degradation process; FIG. 8B is a graph of the change in mass loss of the composite microspheres during degradation; FIG. 8C is an SEM image of the morphology of the composite microspheres during degradation.
FIG. 9: research on release of growth factors of P/C-h composite microspheres prepared by a microfluidic system, wherein FIG. 9A is a release curve of the composite microspheres to FGF-2; figure 9B is a release profile of composite microspheres versus VEGF.
FIG. 10: research on cytotoxicity and hemolysis of P/C-h composite microspheres prepared by a microfluidic system, wherein fig. 10A is detection of cytotoxicity after culturing the composite microspheres and hMSCs on a TCPS plate for 1, 3, and 7 days; FIG. 10B is a hemolytic assay of the degradation solution after 14 days of microsphere degradation.
FIG. 11: a cell aggregate morphology prepared from cells and P/C-h composite microspheres, wherein FIG. 11A is a light microscope picture; FIG. 11B is a hematoxylin eosin staining diagram.
FIG. 12: a cell aggregate fluorescence staining pattern prepared by cells and P/C-h composite microspheres.
FIG. 13: cell aggregates containing no microspheres (control group), cell aggregates containing PLGA microspheres (MSC/PLGA group), cell aggregates containing P/C-h 2: 1 composite microsphere (MSC/P/C-h 2: 1 group) and a microsphere containing P/C-h 4: 1 cell aggregate Activity Studies of composite microspheres (MSC/P/C-h 4: 1 group), where FIG. 13A is dead-live staining; FIG. 13B is a CCK-8 experiment; FIG. 13C shows the expression of the aggregate apoptosis markers Bax, Bcl-2 and β -actin genes (PCR assay).
FIG. 14 is a schematic view of: cell aggregates containing no microspheres (control group), cell aggregates containing PLGA microspheres (MSC/PLGA group), cell aggregates containing P/C-h 2: 1 composite microsphere (MSC/P/C-h 2: 1 group) and a microsphere containing P/C-h 4: 1 composite microsphere (MSC/P/C-h 4: 1 group) cell aggregate dryness related genes Sox-2, Nanog and beta-actin gene expression (PCR detection).
FIG. 15: cell aggregates containing no microspheres, cell aggregates containing PLGA microspheres, cell aggregates containing P/C-h 2: 1 composite microsphere and a composite microsphere containing P/C-h 4: 1 detecting the gene level of the cell aggregate paracrine related factor of the composite microsphere (PCR detection).
FIG. 16: construction schematic diagram and identification of pcDNA3.1 expression vector of hE-cad-Fc. Constructing a schematic diagram and identifying hE-cad-Fc. In which FIG. 16A shows the process by which cDNA encoding the hE-cad and Fc domains in hE-cad-Fc was cloned into an expression vector. FIG. 16B shows the restriction enzyme identification of hE-cad-Fc fusion protein expression vector, M: protein standard; 1: enzyme digestion of hE-cad-Fc fusion protein gene fragment; 2: enzyme digestion of hE-cad-Fc ectodomain gene fragment; 3: carrying out enzyme digestion on the Fc gene fragment; 4: hE-cad-Fc plasmid vector which is not digested; FIG. 16C shows WB identification of hE-cad-Fc fusion protein, M: protein standard; p: human anti-human IgG 1; 1: reduced hE-cad-Fc fusion protein; 2: non-reduced hE-cad-Fc fusion protein.
FIG. 17: construction schematic diagram and identification of pcDNA3.1 expression vector of hN-cad-Fc. Including a schematic structural diagram of pcDNA3.1-hN-cad-Fc and SDS-PAGE and WB analysis of purified hN-cad-Fc, where M represents a protein standard; p represents human IgG (positive control, Sigma catalog No. m 15154); r represents the hN-cad-Fc fusion protein purified in a reduction state; n represents purified hN-cad-Fc fusion protein in a non-reduced state.
FIG. 18: P/C-h 2: 1 fixed quantity, stability and distribution of hE-cad-Fc/hN-cad-Fc on the surface of the composite microsphere.
FIG. 19: P/C-h 2: release curve of 1-IL-1 beta composite microsphere to IL-1 beta factor
FIG. 20: collagen, hE-cad-Fc, hN-cad-Fc and hE-cad-Fc modify P/C-h 2: 1-IL-1 β composite microsphere biocompatibility evaluation, wherein fig. 20A is CCK-8 detection evaluation of the effect of different surface-modified microspheres on the proliferation of hMSCs; FIG. 20B is a blood compatibility evaluation of different surface-modified microspheres; fig. 20C is a scanning electron microscope used to evaluate the platelet adhesion of different surface-modified microspheres.
FIG. 21: the different ratios of hMSCs cells to fusion protein surface modified P/C-h 2: the 1-IL-1 beta microsphere is used for preparing cell aggregate morphology and related gene expression.
FIG. 22: the modified P/C-h2 containing different ratios of hE-cad-Fc to hN-cad-Fc: gene expression of cell aggregates of 1-IL-1 beta composite microspheres.
FIG. 23: cell aggregates without microspheres, collagen-modified P/C-h 2: 1-IL-1 beta composite microsphere, hE-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microspheres (MEP group), hN-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microspheres (MNP group) and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 3) jointly modify P/C-h 2: and (3) observing the stem cell aggregate morphology of the 1-IL-1 beta composite microspheres (ME/NP group).
FIG. 24: cell aggregates without microspheres, collagen-modified P/C-h 2: 1-IL-1 beta composite microsphere, hE-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microsphere, hN-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microspheres and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 3) are combined to modify P/C-h 2: the stem cell aggregate of the 1-IL-1 beta composite microsphere has the factor secretion function after being cultured for 72 hours.
FIG. 25 is a schematic view of: cell aggregates without microspheres, collagen-modified P/C-h 2: 1-IL-1 beta composite microsphere, hE-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microsphere, hN-cad-Fc modified P/C-h 2: 1, combined modification of P/C-h2 by using composite microspheres and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 3): and culturing the stem cell aggregate of the 1-IL-1 beta composite microsphere for 72 hours to obtain the expression condition of the cell immune regulation related gene (PCR detection).
FIG. 26: cell aggregates without microspheres, collagen-modified P/C-h 2: 1-IL-1 beta composite microsphere, hE-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microsphere, hN-cad-Fc modified P/C-h 2: the 1-IL-1 beta composite microspheres and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 3) jointly modify P/C-h 2: 1-IL-1 β composite microsphere stem cell aggregates, and after 72h culture, expression of intracellular cadherins, wherein fig. 26A is expression of intracellular cadherin genes of the aggregates (PCR detection); fig. 26B is the aggregate endogenous cadherin protein expression (WB assay).
FIG. 27 is a schematic view showing: cell aggregates without microspheres, collagen-modified P/C-h 2: 1-IL-1 beta composite microsphere (collagen group), hE-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microspheres (MEP group), hN-cad-Fc modified P/C-h 2: 1-IL-1 beta composite microspheres (MNP group) and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 3) jointly modify P/C-h 2: stem cell aggregates of 1-IL-1 β composite microspheres (ME/NP group) with effects of aggregate cells on macrophage polarization after 72h of culture, wherein fig. 27A is a co-culture schematic; FIG. 27B shows TNF- α secretion by macrophages; FIG. 27C, D is macrophage M1 and M2 type specific protein expression; FIG. 27E, F is gene expression (PCR assay) associated with the regulation of macrophage polarization process by aggregate cells.
FIG. 28: PBS injection (blank), cell aggregates without microspheres (group M), hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 3) in combination with the modifications P/C-h 2: stem cell aggregates of 1-IL-1 β composite microspheres (ME/NP group) were intraperitoneally implanted for colitis mouse model treatment, where fig. 28A is colitis mouse weight change; figure 28B is disease activity index in colitis mice; fig. 28C is colitis mouse colon morphology; fig. 28D is colitis mouse colon length statistics.
FIG. 29: PBS injection (blank), cell aggregate without microspheres (group M), hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 3) in combination with modified P/C-h 2: evaluation of degree of colon lesions in colitis mice treated with stem cell aggregates of 1-IL-1 β composite microspheres (ME/NP group), wherein fig. 29A is HE staining of colitis mouse colon sections; fig. 29B is the pathology score of colon sections.
FIG. 30: PBS injection (blank), cell aggregate without microspheres (group M), hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 3) in combination with modified P/C-h 2: characterization of stem cell aggregate of 1-IL-1 beta composite microsphere (ME/NP group) on immune regulation function of colitis mouse model, FIG. 30A shows colitis mouse spleen CD4 + And CD8 + The T cell flow assay scatter plot of (a); FIG. 30B shows spleen CD8 of colitis mouse + A T cell histogram of (a); FIG. 30C shows colitis mouse spleen CD4 + A T cell histogram of (a); FIG. 30D is CD4 in spleen of colitis mouse + The T cell Th1 differentiation flow detection scatter diagram; FIG. 30E shows CD4 in spleen of colitis mouse + The T cell Th17 differentiation flow detection scatter diagram; FIG. 30F is CD4 in spleen of colitis mouse + A T cell Th1 differentiation histogram of (a); FIG. 30G shows CD4 in spleen of colitis mouse + T cell Th17 differentiation statistical map of (a).
FIG. 31: P/C-h 2: release profile of 1-TGF β 1 composite microspheres to TGF β 1 factor.
FIG. 32: on the surface of a Polystyrene (PS) culture plate modified by collagen, hE-cad-Fc/hN-cad-Fc with the concentration ratios of 1:1, 1:2 and 1:3 respectively and hN-cad-Fc, hMSCs are subjected to cartilage oriented induction differentiation culture to obtain the cell morphology.
FIG. 33: effect of different ratios of hE-cad-Fc/hN-cad-Fc combined matrix on cartilage differentiation of hMSCs. The concentrations of collagen (MCP group) and hE-cad-Fc/hN-cad-Fc were 1: 1. 1:2, 1:3 and hN-cad-Fc (MNP group) modified P/C-h 2: expression of cartilage markers after 1 week of directed induced differentiation of cell aggregates prepared from 1-TGF β 1 composite microspheres into cartilage, wherein fig. 33A is gene expression (PCR detection) of cartilage markers; fig. 33B is protein expression of cartilage markers (WB assay).
FIG. 34 is a schematic view of: n-cadherin of cell aggregates is expressed over time. Cell aggregates without microspheres (group M), P/C-h2 with collagen (group MCP), hN-cad-Fc (group MNP) and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (group MENP) combined modifications: expression of endogenous N-cadherin of cells in cell aggregate cartilage directed differentiation culture of 1-TGF β 1 composite microspheres, wherein fig. 34A is gene expression (PCR detection) of endogenous N-cadherin; FIG. 34B is the protein expression of endogenous N-cadherin (WB assay).
FIG. 35: differentiation efficiency of cell aggregates into cartilage. Cell aggregates without microspheres (group M), collagen (MCP group), hN-cad-Fc (MNP group), and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (MENP group) in combination with modified P/C-h 2: 1-TGF beta 1 composite microsphere cell aggregate differentiation culture 1, 2,3 and 4 weeks later, and then relevant genes and protein expression.
FIG. 36: efficiency of differentiation of cell aggregates into cartilage. Cell aggregates without microspheres (group M), containing collagen (group MCP), hN-cad-Fc (group MNP) and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (group MENP) in combination with the modifications P/C-h 2: results of alcian blue staining after 1-2 weeks of differentiation culture of cell aggregates of the 1-TGF β 1 composite microspheres.
FIG. 37: cell aggregates without microspheres (group M), containing collagen (group MCP), hN-cad-Fc (group MNP) and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (group MENP) in combination with the modifications P/C-h 2: and (3) performing differentiation culture on the cell aggregate of the 1-TGF beta 1 composite microsphere for 1 week and 2 weeks, and then performing safranine fast green staining.
FIG. 38: cell aggregates without microspheres (group M), containing collagen (group MCP), hN-cad-Fc (group MNP) and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (group MENP) in combination with the modifications P/C-h 2: and carrying out directional differentiation culture on the cell aggregate of the 1-TGF beta 1 composite microsphere for 2 weeks to obtain the expression and statistics of the osteogenesis related genes.
FIG. 39: cell aggregates without microspheres (group M), collagen (MCP group), hN-cad-Fc (MNP group), and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (MENP group) in combination with modified P/C-h 2: expression of endogenous E-cadherin and beta-catenin after 4 weeks of differentiation culture of cell aggregates of the 1-TGF beta 1 composite microspheres. Wherein FIG. 39A is an immunostaining for E-cadherin and β -catenin; FIG. 39B nuclear and cytoplasmic beta-catenin expression and statistics.
FIG. 40: cell aggregates without microspheres (group M), containing collagen (group MCP), hN-cad-Fc (group MNP) and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (group MENP) in combination with the modifications P/C-h 2: evaluating E-cadherin and YAP after cell aggregate differentiation culture of the 1-TGF beta 1 composite microspheres for 4 weeks; wherein FIG. 40A is an immunostaining of E-cadherin and YAP; fig. 40B is protein expression and statistics of YAP and phosphorylated YAP in the nucleus and cytoplasm. hE-cad-Fc inhibits YAP entry into the nucleus by promoting expression of cellular E-cadherin to enhance phosphorylation of YAP.
FIG. 41: injecting PBS into joint cavities after rat articular cartilage defect, wherein cell aggregates without microspheres (M group) and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 2) which respectively contain collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc/hN-cad-Fc (MENP group) are combined to modify P/C-h 2: cell aggregates of 1-TGF beta 1 composite microspheres, and results of arthroscopic observation after 3 weeks and 6 weeks of implantation.
FIG. 42: injecting PBS into joint cavities after rat articular cartilage defect, wherein cell aggregates without microspheres (M group) and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 2) which respectively contain collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc/hN-cad-Fc (MENP group) are combined to modify P/C-h 2: cell aggregates of 1-TGF beta 1 composite microspheres, results of safranin fast green staining of femoral tissue sections after 3 weeks and 6 weeks of repair.
FIG. 43: rat articular cartilage defect was injected with PBS in the articular cavity, cell aggregates without microspheres (group M) and collagen (group MCP), hN-cad-Fc (group MNP) and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (MENP group) were included respectively to modify P/C-h 2: the repairing effect of the subchondral bone is detected by MicroCT after 3 weeks and 6 weeks of repairing the cell aggregate of the 1-TGF beta 1 composite microsphere. Wherein 43Aa is 3-week 3D imaging, 43Ab is 3-week injury part cross section, and 43Ac is 3-week bone density and trabecular width statistical chart; 43Ba was 6-week 3D imaging, 43Bb was 6-week lesion cross-section, and 43Bc was a 6-week bone density and trabecular width histogram.
FIG. 44: rat articular cartilage defect was injected with PBS in the articular cavity, cell aggregates without microspheres (group M) and collagen (group MCP), hN-cad-Fc (group MNP) and hE-cad-Fc/hN-cad-Fc (concentration ratio 1: 2) (MENP group) were included respectively to modify P/C-h 2: 1-TGF beta 1 composite microsphere cell aggregate, 3 weeks and 6 weeks after repair, human protein Ku80 and human, mouse type 2 collagen immunostaining map.
FIG. 45: by adding IL-1 beta into a cartilage induced differentiation medium, an arthritis microenvironment is simulated in vitro, and cell aggregates without microspheres (M group) and combined modified P/C-h2 respectively containing collagen (MCP group), hN-cad-Fc (MNP group) and hE-cad-Fc/hN-cad-Fc (concentration ratio is 1: 2) (MENP group) are examined: the gene expression of anti-inflammatory factors IL-1, IL-10 and TGF beta in the cell aggregate of the 1-TGF beta 1 composite microsphere.
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 Ltd; the related chip is manufactured by Dalian Miao Limited company, and the related peptide is synthesized by Nanjing Kinsrui Biotech.
Example 1 Synthesis of Chitosan complexes with heparin
Chitosan and heparin complexes are prepared by ionic interactions. The details are as follows.
Accurately weighed 5mg heparin (from porcine intestinal mucosa, 185units/mg, Aladdin) was added to 1mL ddH 2 And in O, stirring and fully dissolving. Accurately weighing 5mg of chitosan (deacetylation degree is more than or equal to 95 percent, viscosity is 100-one power 200mPa.s, Aladdin) and adding 1mL of chitosan with the concentration of 1 percentFully dissolving the acetic acid solution; and used to titrate 5mg/mL heparin solution, according to chitosan: reacting heparin at volume ratio of 1:1, 2: 1, 3:1, 4: 1, and 5:1, incubating at room temperature for 30min, centrifuging at 2000 Xg for 5min, discarding supernatant, and treating with ddH 2 Cleaning twice, wherein the obtained white precipitate is chitosan-heparin compound; freeze-drying for later use and infrared spectrum detection. FIG. 1A shows the infrared spectrum of chitosan-heparin complex, wherein 1560 and 2818cm -1 Chitosan-NH stretching vibration characteristic peak, 1236, 1027 and 820cm -1 The peak is the S ═ O and C-O-S stretching vibration characteristic peak of heparin, which shows that chitosan-heparin compound is formed by the combination of anion and cation.
The content of unreacted heparin and chitosan was measured using dimethyl sub-blue (DMB) and orange red II, respectively. 50 mu L of chitosan heparin reaction supernatant liquid with different proportions is added into 100 mu L of DMB working solution containing 10.7 mu g, and the mixture is fully mixed, and the absorbance at 520nm is measured. And taking a heparin solution with a known concentration as a standard curve, and calculating the content of the heparin in the supernatant. 50 μ L of chitosan heparin reaction supernatant with different proportions is added into 20 μ L of orange red II (1mM), mixed well, centrifuged at 12000 Xg for 5min, and the supernatant is taken to measure the absorbance at 484 nm. And taking a chitosan solution with a known concentration as a standard curve, and calculating the content of the chitosan in the supernatant. FIG. 1B shows the amount of unreacted chitosan and heparin in the reaction supernatant, i.e. the content of unreacted heparin in the solution is less and less with the addition of chitosan, and the heparin content is the lowest at a heparin-to-heparin ratio of 3:1, indicating that heparin is completely titrated by the added chitosan.
Respectively taking 100 mu L, 200 mu L, 300 mu L, 400 mu L and 500 mu L of 5mg/mL chitosan solution, adding the chitosan solution into 100 mu L of 5mg/mL heparin solution for full reaction, then adding distilled water until the total volume is 750 mu L, and carrying out Zeta potential detection after fully and uniformly mixing. As shown in FIG. 1C, as the reaction ratio of chitosan heparin increases, the potential of the composite changes from the negative potential when the initial heparin content is higher to the positive potential when the initial heparin content is higher, and the potential tends to be stable at 3:1, and the composite microsphere is prepared by selecting the composite when the reaction ratio of chitosan and heparin is 3:1 according to the above results.
EXAMPLE 2 hydrophilic treatment of microfluidic chips
Firstly, sequentially infiltrating a micro-channel of a processing chip for 30min by using 1M HCl and 1M NaOH solutions filtered by a 0.22 mu M polyethersulfone filter membrane, and then washing the micro-channel for 30min by using distilled water to carry out hydrophilic treatment on the micro-channel. And after natural drying, carrying out contact angle detection. As shown in FIG. 2A, the contact angle of the chip surface before hydrophilic treatment is 82.2 degrees, the contact angle of the chip surface after hydrophilic treatment is 13.4 degrees, and the hydrophilicity of the chip is obviously improved. As shown in fig. 2B, after hydrophilic treatment, droplets with good dispersibility and uniform size can be generated in the microfluidic system, and the droplets are observed under an optical microscope to be arranged in a matrix structure. Namely, the hydrophilic treatment is beneficial to the stable generation of uniform monodisperse liquid drops by the microfluidic system.
Example 3 preparation of P/C-h composite microspheres by microfluidic technology
The preparation process of the composite microsphere is optimized by taking PLGA/chitosan-heparin (P/C-h) composite emulsion as a disperse phase and 2% polyvinyl alcohol solution as a continuous phase:
3.1 preparation of two-phase solution for microfluidic System
40mg of PLGA (L: G: 50, Mw: 25000, Jinan Dai handle bioengineering Co., Ltd.) was weighed out and dissolved in 1mL of Dichloromethane (DCM), and the solution was thoroughly dissolved by magnetic stirring and filtered through a microporous membrane of polyethersulfone for further use. And (3) resuspending the obtained chitosan-heparin compound in a sterile PBS solution, and performing high-speed ultrasonic treatment for 2min to obtain a uniform suspension for later use. 0.1mL of the chitosan-heparin complex suspension (containing 0mg, 10mg and 20mg of the complex respectively) is added into 1mL of the PLGA/DCM solution, and the mixture is quickly placed on ice for 2min of ultrasonic emulsification to be used as a dispersion phase of a 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, and after complete dissolution, the PVA solution is filtered by a polyether sulfone filter membrane to be used as a continuous phase of a microfluidic system for later use (for use in preparation).
3.2 preparation of P/C-h composite microspheres by microfluidic System
Respectively adding the dispersed phase and the continuous phase into a 20mL syringe (discharging bubbles), conveying the two phases to the cross-shaped microfluidic chip by using a syringe pump, collecting droplets by using a 2% PVA solution after the droplets generated by the system are stable, volatilizing DCM by magnetic stirring, and solidifying the droplets to obtain the composite microspheres; and repeatedly washing the composite microspheres with distilled water to remove PVA, freeze-drying to obtain the composite microspheres, and carrying out SEM detection. Liquid drops with different particle sizes can be prepared by regulating the flow rates of the two phases, as shown in figure 3, when the flow rate of the continuous phase is fixed to be 0.5mL/h, the flow rate of the disperse phase is changed, and the particle size of the microsphere is increased along with the increase of the flow rate of the disperse phase; meanwhile, as shown in FIG. 4, when the flow rate of the continuous phase was changed while the flow rate of the dispersed phase was fixed at 0.4mL/h, the particle size of the resulting microspheres decreased with the increase in the flow rate of the continuous phase within a certain range. The composite microspheres obtained when the flow rates of the continuous phase and the dispersed phase were maintained at 1.1 and 0.4mL/h, respectively, had a particle size of 23.5. mu.m. By the same method, the solution of the dispersed phase is changed to obtain PLGA/chitosan-heparin compound composite microspheres (P/C-h 2: 1 and P/C-h 4: 1, namely the proportion of PLGA to chitosan-heparin compound is 40 mg: 20mg and 40 mg: 10mg respectively) with different proportions; replacing chitosan and heparin compounds with PBS solution with the same volume, and preparing hollow PLGA microspheres according to the same method; firstly, the growth factor is combined with heparin, then heparin/chitosan compound is prepared, and the P/C-h composite microsphere (namely, the microsphere loaded with the growth factor) of the slow-release growth factor is prepared according to the same method.
Example 4 characterization of physical and chemical Properties of P/C-h composite microspheres
The system evaluates the appearance, components, internal structure, degradability and growth factor release characteristics of the P/C-h composite microspheres with different compositions.
Resuspending the lyophilized microspheres (PLGA, P/C-h 4: 1, P/C-h 2: 1) with distilled water, blowing uniformly, dropping on the surface of a clean silicon wafer, naturally drying, fixing on a stage by conductive adhesive, spraying gold for 30 seconds, and observing by SEM (scanning Electron microscope) as shown in FIG. 5A: the P/C-h composite microspheres have uniform size and smooth surfaces.
The lyophilized microspheres (PLGA, P/C-h 4: 1, P/C-h 2: 1) were resuspended in OCT and after complete freezing at-20 deg.C, the sections were sectioned in a cryomicrotome, 8 μm thick, and applied to a cover glass. After the OCT is cleaned by distilled water, the OCT is naturally dried, fixed to an objective table by conductive adhesive, and observed by SEM. As shown in fig. 5B: the P/C-h composite microsphere has a shell-core structure, and the shell thickness is uniform and is about 1-2 mu m.
Preparing FITC labeled chitosan, namely weighing 2mg of chitosan, dissolving the chitosan in 1% acetic acid solution, adding 10 mu L of 1mg/mL FITC solution while stirring, keeping the mixture away from light, and reacting the mixture for 24 hours on ice at 4 ℃; the reaction solution is put into a dialysis bag (molecular weight is 8000), dialyzed in distilled water (dialysate is changed every two days), dialyzed for two weeks, taken out, freeze-dried and stored at 4 ℃ for later use. The oil-soluble dye nile red is used for PLGA labeling, i.e. nile red is directly dissolved in DCM solution of PLGA (1mg/mL), chitosan/heparin complex is prepared using chitosan labeled with FITC and heparin, and the fluorescence-labeled composite microspheres are prepared according to the method in example 3. And (3) dropwise adding the prepared fluorescent labeled composite microspheres into a confocal special vessel, and observing by using a laser confocal microscope. As shown in fig. 6: FITC-labeled chitosan/heparin complexes were located inside the microspheres and Nile Red-labeled PLGA was located outside the microspheres with increasing distribution within the microspheres as the chitosan/heparin complexes were increased.
At room temperature, completely dry microspheres (PLGA, P/C-h 4: 1, P/C-h 2: 1) were mixed with potassium bromide and ground completely, then tabletted, and scanned by an infrared spectrometer, and the potassium bromide tabletted was used as a blank control. FIG. 7A shows the IR spectrum of microspheres from P/C-h 4: 1, P/C-h 2: 1 the characteristic peaks of chitosan, heparin and PLGA can be obviously observed in the infrared spectrogram of the composite microsphere. Wherein the thickness is 1236, 1027 and 820cm -1 Is a characteristic peak of heparin S ═ O, C-O, C-O-S, 1560cm -1 Is a characteristic peak of chitosan-NH, 1755cm -1 Is the stretching vibration peak of PLGA.
Weighing equal amount of completely dried microspheres (PLGA, P/C-h 4: 1, P/C-h 2: 1) at room temperature, adding into a thermogravimetric analyzer sample groove, and detecting. As shown in fig. 7B: all samples exhibited a typical two-stage or two-step decomposition trend, corresponding to two endothermic peaks. One of the small mass losses that occurs before 100 ℃ is the decomposition of the adsorbed moisture in the sample, and in the second degradation step a relatively large mass loss occurs, due to the decomposition of the polymer itself. Pure PLGA decomposed at around 280 ℃ and completely at 370 ℃ with almost 100% weight loss. The thermal decomposition of chitosan starts at 220 ℃, the weight loss is about 50% at 370 ℃, and the thermal decomposition of pure heparin starts at about 215 ℃. The results show that as the amount of chitosan/heparin in the composition increases, the mass loss at 450 ℃ is reduced in order, P/C-h 4: 1 was most reduced, P/C-h 2: one 1 time, i.e., during the thermal decomposition process, only PLGA completely decomposed, leaving more chitosan and heparin.
Weighing microspheres (PLGA, P/C-h 4: 1 and P/C-h 2: 1) in equal amount, respectively placing into a 10mL centrifuge tube, adding physiological saline in equal amount, incubating in a constant temperature shaking table at 37 ℃, sampling every three days, detecting the pH value of the leaching solution, weighing the microspheres after being cooled to dry, and observing the shape by using SEM. The mass loss was calculated by the following equation: mass loss (%) ═ (M) 0 -M d )/M 0 X 100%. Wherein M is 0 Is the initial mass of the microspheres, M d Is the mass of the degraded microspheres. Fig. 8A is a pH variation curve of physiological saline in which the microspheres are soaked, and it can be seen that the pH of the leaching solution of pure PLGA is decreased most rapidly, that is, the pH of the leaching solution is decreased rapidly due to lactic acid and glycolic acid generated by the degradation of PLGA, and the pH of the leaching solution of pure PLGA is decreased more slowly due to the presence of the composite microspheres, which indicates that the presence of chitosan relieves the acidic environment generated by the degradation of PLGA. On day 15 of the observation period, the pH value of the PLGA leach liquor is reduced to 6.23, while the leach liquor of the composite microspheres is still neutral, especially when the ratio of PLGA to chitosan heparin complex is P/C-h 2: 1, the pH value of the leaching solution is 7.20, namely, the amino group in the chitosan structure relieves the acidity generated by PLGA degradation to a certain extent.
FIG. 8B is a graph of the mass loss of microsphere degradation, which shows that the mass loss of pure PLGA microspheres degraded in the same time is greater than P/C-h 4: 1, P/C-h 2: 1 composite microspheres. Fig. 8C is an SEM image of degradation at different time points, as shown, the PLGA microspheres started to appear rough on the surface at day 3 of degradation, appeared to have significant voids on the surface at day 6, appeared to collapse and lost spherical structure at day 9, and had no spherical structure at day 12; the P/C-h composite microspheres degrade more slowly than PLGA microspheres, and the microsphere structure can be observed in 12 days, so that the improvement of the acidic microenvironment in the composite microspheres is considered to partially slow down the acid accelerated disintegration speed in the PLGA degradation process.
2mg of the growth factor-loaded microspheres (PLGA, P/C-h 4: 1, P/C-h 2: 1) were suspended in 0.5mL of PBS (0.02% Tween 20 and 10mg/mL of BSA), incubated in a 37 ℃ incubator, and the concentrations of the growth factors (FGF-2, VEGF) in the extracts were continuously determined. After centrifugation every 3 days, 10. mu.L of supernatant was collected, 10. mu.L of PBS (0.02% Tween 20 and 10mg/mL BSA) was added for further incubation, the supernatant was stored at-20 ℃ and the content of growth factors was measured using an enzyme-linked immunosorbent assay kit (ELISA) after the collection. The concentration of growth factor was obtained by comparison with a standard curve and all experiments were repeated three times. As shown in fig. 9: PLGA microsphere-loaded growth factors all exhibited a significant burst (40%) initially and had been substantially completely released on day 9. And P/C-h 4: 1.P/C-h 2: the 1 composite microsphere group shows a slow release trend, and the factors are released continuously within 15 days.
Example 5 two-dimensional Co-culture of P/C-h composite microspheres with cells
And (3) carrying out two-dimensional co-culture on the composite microspheres and umbilical cord mesenchymal stem cells (hMSCs) and researching the cytotoxicity and hemolysis of the composite microspheres under a two-dimensional condition.
hMSCs (Guangzhou Seisai Biotech Co., Ltd.) in good growth state were treated with trypsin at 37 ℃ for 1min, digested with DMEM/F12 medium containing 10% fetal bovine serum (FBS, BI, USA), centrifuged at 1000 Xg for 5min, the supernatant was discarded, and 200. mu.L MEM/F12 medium was added to prepare a high density cell suspension.
According to the ratio of 1:1, mixing hMSCs with PLGA, P/C-h 4: 1.P/C-h 2: 1, adding the mixed microspheres into a TCPS culture plate for co-culture, adding CCK-8 working solution (10: 1) on days 1, 3 and 5, incubating for 4h at 37 ℃, sufficiently shaking and uniformly mixing, and absorbing 100 mu L to measure the absorbance value at 450 nm. As shown in FIG. 10A, the results of two-dimensionally cultured hMSCs as a control group under the same conditions are shown in the following graph, in which the ratio of P/C-h 2: group 1 showed a better cell proliferation promoting effect. Taking fresh blood of ICR mice for 4 weeks into a centrifugal tube treated by EDTA, fully centrifuging and cleaning by using sterile normal saline until the supernatant is clear, and diluting red blood cells into 1 × 10 9 The suspension of red blood cells of (1). Respectively taking PLGA and P/C-h 4: 1.P/C-h 2: 1 composite micronMixing 800 μ L of degradation liquid of the ball for 15 days with 200 μ L of erythrocyte suspension, placing in a constant temperature incubator at 37 ℃ for incubation for 4h, taking ultrapure water with the same amount as a positive control, and taking normal saline as a negative control. The tube was then centrifuged at 10000 Xg for 5min, gently removed, observed and photographed. 100 μ L of the supernatant was placed in a clean 96-well plate and the OD at 577nm was measured under a microplate reader. The hemolysis rate (%) ═ OD sample-OD negative)/(OD positive-OD negative) was calculated according to the following equation]X 100. FIG. 10B shows the results of the hemolysis experiment, in which the PLGA group showed slight hemolysis when pure water was used as the positive control, and the ratio P/C-h 4: 1, P/C-h 2: the supernatant of group 1 was colorless and transparent, and no hemolysis occurred. The above results indicate that P/C-h 2: 1, the acidic microenvironment generated by PLGA degradation in the composite microspheres is relieved, the blood and cell compatibility of the composite microspheres is improved, and the cell proliferation is promoted.
Example 6 preparation of P/C-h composite microspheres and cell aggregates
Using in a low adhesion Aggrewell TM Principle of hMSCs spontaneously aggregating to aggregate in culture plate to sterile Aggrewell TM An Anti-Adherence centrifugation solution was added to the plate, centrifuged at 2000 Xg for 5min, the solution was discarded, and the plate was rinsed with PBS for future use. The sixth generation hMSCs in good state were digested from the culture flask, and 8X 10 5 Mixing cell and microsphere (PLGA, P/C-h 4: 1, P/C-h 2: 1) at ratio of 3:1, adding cell and microsphere suspension (600 cells/micropore) into treated Aggrewell TM In the plate, the plate was centrifuged at 1000 Xg for 5min at room temperature, and then placed in an incubator at 37 ℃ for incubation for 12 hours, and the state of formation of cell aggregates was observed under a microscope. Aggregate of hMSCs with PLGA microspheres added (expressed as MSC/PLGA), P/C-h 4: 1 microsphere hMSCs aggregates (expressed as MSC/P/C-h 4: 1), addition of P/C-h 2: 1 aggregate of hMSCs (expressed as MSC/P/C-h 2: 1) of composite microspheres, and aggregate of hMSCs (expressed as MSC) without microspheres added as a control group. FIG. 11A shows the results of an inverted phase contrast microscope, see agrewell TM Culturing in-plate MSC, MSC/PLGA, MSC/P/C-h 4: 1. MSC/P/C-h 2: group 1 can form aggregates with clear edges and complete structures. The aggregates have substantially the same size between different groups, as shown inThe composition of the microspheres in the pre-optimized proportion does not affect the formation and morphology of cell aggregates.
Suspending the prepared aggregate in 1.5% (w/v%) sodium alginate solution, and adding dropwise into 0.1M sterile CaCl 2 Incubating in solution at room temperature for 5min to form stable gel, and removing CaCl 2 The solution was washed 3 times with physiological saline and then fixed for more than 2 days by adding 4% paraformaldehyde. Placing the fixed aggregate into an automatic dehydrator for dehydration, embedding in a paraffin embedding machine, slicing in a slicer (the thickness is 3.5 mu m), baking the slices for 4h at 65 ℃, dewaxing the baked slices by dimethylbenzene for 10min, rehydrating by gradient ethanol, staining hematoxylin for 2min, washing by flowing water for 5min, differentiating by hydrochloric acid and alcohol for 2s, staining by eosin for 1min, washing by flowing water for 5min, dehydrating by gradient ethanol, transparentizing the dimethylbenzene for 10min, after the dimethylbenzene is completely dried in the air, sealing the slices by neutral gum, and observing on a machine. The results are shown in FIG. 11, where the microspheres are encapsulated by the cells and uniformly distributed within the aggregate.
Composite microspheres were prepared as in example 3 by adding nile red (1 μ g/mL) to DCM to prepare red fluorescently labeled composite microspheres, which were lyophilized for use. Preparing cell aggregates by using red fluorescence-labeled composite microspheres and hMSCs according to the method in the embodiment 5, soaking the prepared cell aggregates in 4% paraformaldehyde for fixing for 2 days, adding 1% TritonX-100 for incubation for 1h, using 1% BSA for room temperature blocking for 1h, after the blocking is finished, adding FITC-labeled phalloidin diluted by 1% BSA, incubating for 1h at room temperature in a dark place, then adding DAPI staining solution for incubation for 30min at room temperature in a dark place, washing for 3 times by using PBS after the incubation is finished, and observing under a laser confocal microscope. The results are shown in fig. 12, consistent with paraffin sectioning results, the microspheres were able to be uniformly distributed within the aggregates.
Example 7 investigation of the Effect of P/C-h composite microspheres on the Activity of aggregate cells
Cell aggregates were prepared according to the method of example 6, suspended in calcium alginate hydrogel, added with 10% FBS-containing DMEM/F12 solution, changed every 3 days in an incubator at 37 ℃, discarded on days 1, 3 and 7 of the culture, added with 0.1M sterile EDTA solution, shaken slightly, centrifuged at 1000 Xg for 5min after the calcium alginate hydrogel was lysed, discarded the supernatant, washed 3 times with PBS and recovered. First, the survival of cells in aggregates was examined by the dead-live staining method, FDA/PI cell dead-live staining solution was prepared according to the instruction, 5mg/mL of the mother solution was prepared by dissolving FDA in acetone, and 100. mu.g/mL of the mother solution was prepared by dissolving PI in PBS for further use. To the cell aggregates, FDA was added at 1: 100 dilution simultaneously with 1: and incubating the PI working solution diluted by 200 for 5min by a light-proof shaking table at room temperature, standing, sucking off the dye solution when the aggregates completely sink to the bottom of the centrifuge tube, washing twice with PBS (phosphate buffer solution) for 5min each time, transferring to a culture dish, and observing on a machine. Results are shown in FIG. 13A, where a small amount of apoptosis was observed inside the MSC group aggregates and MSC/PLGA group aggregates at day 3 of culture, and a large amount of PI-stained cells were observed at day 7, indicating that significant apoptosis had occurred, whereas MSC/P/C-h 2: no significant apoptosis was observed for group 1 aggregates.
Cell viability was measured in aggregates by the CCK-8 method by culturing the recovered cell aggregates in low adhesion plates and measuring cell viability on days 1, 3 and 7 as described in example 5. The results are shown in FIG. 13B. Consistent with FDA/PI staining results, MSC/P/C-h 4: 1 and MSC/P/C-h 2: the cells in the aggregate of group 1 show the tendency of promoting cell proliferation, and the activity of the MSC/PLGA aggregate cells is reduced, which indicates that the acidic environment for PLGA degradation is not beneficial to the activity maintenance of the cells in the three-dimensional aggregate.
Aggregates at 1, 3 and 7 days of culture cell aggregates were obtained per 500 aggregates (3X 10) according to the method in example 6 5 Cells) were added with 1mL Trizol to extract total RNA. The specific operation steps are as follows: adding 200 mu L of chloroform solution into 1mL of Trizol for treating cells, reversing, uniformly mixing, standing at room temperature for 3min, centrifuging at 4 ℃ by 12000 Xg for 15min, carefully sucking a colorless water layer by a pipette into a new centrifuge tube, adding 500 mu L of isopropanol into each tube, reversing, uniformly mixing, standing at room temperature for 10min, and centrifuging at 4 ℃ by 12000 Xg for 15 min. Removing supernatant, adding 1mL 75% ethanol, vortexing briefly, centrifuging at 7500 Xg for 5min, removing supernatant, air drying at room temperature, and adding 20 μ L of RNase-free ddH 2 O dissolved RNA (55-60 ℃ water bath). After the RNA was completely dissolved, the concentration was measured in the BioDrop. mu. LIFE and the RNA was immediately inverted to cDNA. Specifically, the following procedure was performed by placing the system shown in Table 1 in a centrifugal tube without RNase:
TABLE 1
And (3) mixing the reagents in the above table, quickly centrifuging, setting the degrees of 55 ℃ for 30min, 85 ℃ for 5min and 4 ℃ for infinity in a PCR instrument, carrying out reverse transcription, and storing the obtained cDNA in a refrigerator at-20 ℃ for later use. Then, semi-quantitative PCR detection is carried out, and a reaction system is configured according to the following table 2:
TABLE 2
After the components in the table are added in sequence, the mixture is quickly centrifuged and reacts in a PCR instrument according to the program to obtain a PCR product. Adding DNA loading buffer into the reaction product, and fully and uniformly mixing. 1g of agarose powder was weighed, dissolved in 100mL of TAE solution, and heated in a microwave oven to obtain agarose gel. After the Gel is cooled slightly, 10 mu L Gel Red is added and mixed evenly, then the mixture is poured into a Gel tank, and after the Gel is solidified completely, the sample is applied. The voltage is adjusted to 140V, after 30min electrophoresis, the gel is taken out and imaged, and the PCR primer sequences are shown in Table 3. As a result, as shown in FIG. 13C, on day 3 of culture, expression of the pro-apoptotic gene Bax could already be detected in the MSC group, and a small amount of Bax was also expressed in the MSC/PLGA group, whereas the P/C-h 2: group 1 had no expression of pro-apoptotic genes, while its anti-apoptotic gene Bcl-2 was significantly expressed. Showing that cell apoptosis occurs in the MSC group and the MSC/PLGA group at the moment, the ratio of MSC/P/C-h 2: group 1 did not show significant apoptosis. On day 7 of culture, the expression of the pro-apoptotic genes of the MSC and MSC/PLGA groups was further enhanced, MSC/P/C-h 2: no expression of the pro-apoptotic gene was detected within group 1, and the anti-apoptotic gene was consistently expressed at high levels.
TABLE 3
Example 8 investigation of the Effect of P/C-h composite microspheres on the biological function of aggregate cells
The system characterizes the influence of the composite microspheres on the dryness and paracrine performance of cell aggregates.
A series of cell aggregates were prepared according to the method of example 6, cultured normally for 7 days, and RNA was extracted according to the method of example 7 on days 1, 3 and 7 of the culture to detect the dry related gene expression (Sox-2, Nanog) by PCR, and as a result, the dry gene expression of the aggregates of MSC group and MSC/PLGA group was gradually decreased as the in vitro culture time was prolonged, as shown in FIG. 14. MSC/P/C-h 2: the sternness gene for group 1 aggregates was consistently highly expressed, indicating that MSC/P/C-h 2: 1 the composite microspheres can better maintain the dryness of the hMSCs in the aggregate.
Cell aggregates were prepared according to the method of example 6, and the aggregates were recovered according to the method of example 6 at 1, 3 and 7 days of culture. Performing gene level on the paracrine factors of the cells in the aggregate, detecting to extract total RNA according to the method of example 7, performing reverse transcription to obtain cDNA, taking the cDNA as a template, sequentially adding the components in the table 4 according to the Roche Realtine PCR kit specification, performing rapid centrifugation, placing in a fluorescence quantitative PCR instrument, and performing 5min multiplied by 1 at 95 ℃ according to the 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 are referenced to the internal parameter beta-actin in each group, according toData statistics were performed and the PCR primer sequences are shown in Table 5.
TABLE 4
The results are shown in FIG. 15, with the culture time extended, MSC/P/C-h 2: the expression level of the paracrine factors of the group 1 cells is continuously improved, which shows that the composite microspheres can maintain the high-level expression of the paracrine factors of the cells in the aggregate.
TABLE 5
Example 9: construction and expression of human hE-cad-Fc fusion protein
The construction method is described in xu-build bin, doctrine, south kayak university, 12 months in 2013 (xu-build bin, research on the construction of stem cell microenvironment by human E-cadherin fusion protein matrix [ D ]. tianjin. south kayak university life science college, 2013: 1-145.). Mainly as follows.
9.1 cloning and sequence analysis of E-cadherin gene in the extracellular region of cadherin of epithelial cells
Human E-cadherin protein sequences and functional partitions were recorded in the UniProt database, specific PCR primers were designed in combination with GenBank-recorded gene (NCBI Reference Sequence: NM 004360.3) sequences to amplify E-cadherin protein extracellular domains, and the primer sequences are shown in Table 6.
TABLE 6
L-02 (purchased from the cell bank of the China academy of sciences type culture Collection) cell 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 concentration of the RNA. Reverse transcription was performed according to the method in example 7. L-02mRNA was used as a template for reverse transcription to form cDNA, and E-cadherin gene fragments were amplified in a PCR system as shown in Table 7:
TABLE 7
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 4 deg.C and 12000g for 10min, washing DNA precipitate with 70% ethanol twice, vacuum drying, and dissolving the precipitate in appropriate amount of TE.
9.2 construction of pcDNA3.1-hE-cad-Fc eukaryotic expression vector
(1) PCR product after Hind III and NotI double digestion purification
The enzyme digestion system is as follows:
TABLE 8
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. Centrifugation was carried out at 12000 Xg for 10min at 4 ℃ and the DNA precipitate was washed twice with 70% ethanol, dried under vacuum and dissolved in 10. mu.L of TE.
(2) HindIII and NotI of pcDNA/3.1;
TABLE 9
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 10
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 E-cadherin extracellular region of the target gene and the vector pcDNA3.1 carrying the Fc fragment were subjected to double digestion (Hind III and NotI) at a constant temperature of 37 ℃ 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 product, followed by ampicillin (Amp) + ) 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 16A). The constructed recombinant plasmid was named pcDNA3.1/hE-cad-Fc. The sequencing verifies that the sequence is correct. The hE-cad-Fc fusion protein has the sequence of SEQ ID NO:1, wherein the sequence of hE-cad is shown as SEQ ID NO:2, the sequence of Fc is shown as SEQ ID NO:3, respectively.
9.3 cell transfection and protein purification
pcDNA3.1/hE-cad-Fc was transfected into 293F cells (purchased from the China academy of sciences tissue 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.
9.4 Western Blotting (WB) analysis
The purified hE-cad-Fc fusion protein was electrophoresed on 10% SDS-PAGE and transferred to PVDF membrane, blocked with 5% skim milk for 2h, incubated with primary rabbit anti-human E-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, and subjected to DAB reagent exposure, development and fixation analysis. And when the formation of the Fc dimer is detected, the beta mercaptoethanol is not added into the loading buffer solution. The results, as shown in FIG. 16B, suggest that the hE-cad-Fc fusion protein exists as a dimer, with one band being visible at-240 kD in the non-reduced state and one band being visible at-120 kD in the reduced state.
Example 10 construction and expression of human hN-cad-Fc fusion protein
The sequence of the hN-cad-Fc fusion protein is SEQ ID NO:4, wherein SEQ ID NO:5 represents the sequence of hN-cad; SEQ ID NO:3 represents the sequence of Fc.
TABLE 11
Human hN-cad-Fc fusion protein was constructed and expressed according to the method in example 9, and the cloning and Sequence analysis of N-cadherin gene in the extracellular region of neural cadherin were performed sequentially, and human N-cadherin protein Sequence and functional domains were included in UniProt database, and specific PCR primers were designed according to the Sequence of GenBank-included gene (NCBI Reference Sequence: NM-001795.3), and the extracellular region of hN cadherin protein (EC1-EC5) was amplified, and the upstream primer introduced with EcoR V cleavage site (underlined), and the downstream primer introduced with NotI cleavage site (underlined). Total mRNA extraction from neural cells (ScienCell), construction of pcDNA3.1-hN-cad-Fc eukaryotic expression vector, cell transfection and protein purification, WB analysis, and the results are shown in FIG. 17, where a band was seen at 240KD in the non-reduced state and at 120KD in the reduced state, suggesting that the hN-cad-Fc fusion protein is in the form of a dimer.
Example 11.P/C-h 2: 1 fixation and stability detection of composite microsphere surface fusion protein
The microspheres in this example were all P/C-h2 prepared according to the method in example 3:1 composite microspheres. Collagen type I (collagen, BD, usa, cat # 354249) was diluted with PBS buffer (pH 7.20) to a final concentration of 20 μ g/mL, the hE-cad-Fc, hN-cad-Fc and hE-cad-Fc/hN-cad-Fc mixed solutions (the ratio of hE-cad-Fc to hN-cad-Fc was 1: 3) were diluted to a final concentration of 20 μ g/mL, 1mg of P/C-h composite microsphere powder was soaked with the above mixed solutions, after shaking sufficiently, the powder was incubated at 37 ℃ for 2 hours at 150 × g, the supernatant was discarded, and the fusion protein that was not stably immobilized was washed (3 times) with PBS buffer. Then, a fixed amount of assay was performed by adding 300. mu.L of 5% BSA solution and shaking horizontally at 37 ℃Blocking for 2h at 150 Xg in bed, adding 100 μ L diluted goat anti-human IgG labeled with HRP (Abcam, USA) at a dilution ratio of 1: 10000, placing in a horizontal shaking table at 37 ℃ for blocking for 1h at 150 Xg in the dark, washing with PBS buffer for 5 times, adding 300 μ L of TMB (Profibus, Inc. PR1200) color developing solution into each tube, placing in a horizontal shaking table at 37 ℃ for reaction for 30min at 150 Xg in the dark, adding 300 μ L of stop solution, adding 200 μ L of solution into a 96-well plate, and measuring the absorbance value at 452nm to determine the maximum fixed amount of 3 fusion proteins co-immobilized on the microsphere surface. The hE-cad-Fc and hN-cad-Fc were then mixed at the maximum fixed amount P/C-h 2: 1, soaking the microspheres in PBS buffer solution, DF12 culture medium and DF12+ 10% FBS for 28 days, and detecting the absorbance of the supernatant at 0, 3 and 5 days to evaluate the fixation time of the fusion protein on the microsphere surface. Using DyLight respectively TM 550Antibody laboratory Kit (ThermoFisher, cat # 84530) and DyLight TM 488Microscale Antibody Labeling Kit (ThermoFisher, cat No. 53025) labels hE-cad-Fc and hN-cad-Fc respectively (the Labeling process is as follows), and then co-immobilizing three fluorescently labeled proteins in P/C-h2 according to the maximum immobilization amount: 1, compounding the microsphere surface, and observing the distribution of the fusion protein on the microsphere surface by using a laser confocal microscope. As a result, as shown in FIG. 18, two kinds of fusion proteins can be simultaneously immobilized on the surface of the microsphere; when the microspheres are soaked in a common PBS buffer solution, a culture medium or a culture medium containing serum for a period of time, the change of the absorbance of the supernatant is not large, which indicates that the fusion protein can stably exist on the surfaces of the microspheres for 5 days; the fusion protein with fluorescein is fixed on the surface of the microsphere and is detected by laser confocal detection, so that the two fusion proteins can be uniformly distributed on the surface of the microsphere.
Wherein the fusion protein fluorescence labeling step comprises:
1. mixing 40 μ L of 0.67M borate buffer solution with 500 μ L (2mg/mL) of fusion protein solution;
2. adding the solution into a test tube pre-filled with 50 mu g of fluorescent dye, gently vortexing, blowing and uniformly mixing by using a pipettor, and incubating for 60min at room temperature in a dark place;
3. placing 250 μ L of the uniformly mixed adsorption resin in a centrifuge tube containing a centrifugal column, centrifuging for 1min at 1000 Xg, discarding the supernatant, and transferring the centrifugal column to a new centrifuge tube;
4. adding 250 mu L of solution reacted in the step 2 into the centrifugal column of the step 3, and slightly whirling to mix the reaction solution with the adsorption resin;
5.1000g, centrifuging for 1min, collecting the purified solution to complete the labeling of the fusion protein, and storing at 4 ℃ or-80 ℃ in the dark.
Example 12 fusion protein surface modification P/C-h 2: detection of IL-1 beta releasing capacity of 1-IL-1 beta composite microsphere
IL-1. beta. loaded P/C-h2 was prepared as in example 4: 1 composite microspheres (P/C-h 2: 1-IL-1. beta.), prepared as described in example 4 for P/C-h 2: the IL-1 beta release performance of the 1-IL-1 beta microspheres was examined. The surface modification of the fusion protein P/C-h2 is shown in FIG. 19: the IL-1 beta loaded by the 1 composite microsphere presents a slow release trend, and the factor can be controllably released within 15 days.
Example 13 fusion protein surface modification P/C-h 2: cytotoxicity detection of 1-IL-1 beta composite microspheres
Firstly, the hMSCs cell and fusion protein surface modified P/C-h 2: 1-IL-1 beta composite microspheres are fully mixed in a ratio of 1:1, and each hole is divided into 1 multiplied by 10 4 The cells were seeded at a density in 96-well plates and the cells were assayed for viability and hemolysis by adding CCK-8 solution at 24, 48 and 72h respectively as described in example 5. The two-dimensional cultured hMSCs were used as the control group under the same conditions, and the results are shown in fig. 20, when hMSCs were co-cultured with the microspheres surface-modified with the fusion protein, the microspheres surface-modified with the fusion protein had no significant effect on the proliferation activity of cells, while the microspheres surface-modified with the fusion protein did not cause significant hemolysis, as compared to the control group. In addition, the microspheres with the surface modified by the fusion protein can inhibit the adhesion and activation of the blood platelets. The above comprehensively shows that the microsphere with the surface modified by the fusion protein has good compatibility between cells and blood.
Example 14 fusion protein surface modification P/C-h 2: determination of mixing ratio of 1-IL-1 beta composite microsphere and cell
Respectively mixing 8 × 10 5 The surface modification P/C-h2 of hMSCs cells and fusion protein: 1-IL-1. beta. composite microspheres were mixed at a ratio of (3: 1, 5:1, 8: 1, 10: 1), aggregates were prepared according to the method of example 6, cultured in 10% FBS DMEM/F12 medium, the solution was changed every 3 days, and on the 7 th day, aggregates were recovered according to the method of example 7 to detect changes in the cell secretion factors of different groups. As a result, as shown in FIG. 21, when the amount of microspheres was too high (the ratio of microspheres to cells was 3: 1), many microspheres were not able to completely enter the aggregate and a complete aggregate was not able to be formed. Further, from the quantitative statistical chart (21B, C), the actual ratios of hMSCs to microspheres before and after the formation of aggregates were 3.88: 1 (in this case, the addition ratio was 3: 1), 6.19: 1 (in this case, the addition ratio was 5: 1), 9.05: 1 (in this case, the addition ratio was 8: 1), and 10.52: 1 (in this case, the addition ratio was 10: 1), respectively, which indicates that the addition amounts of both affect the morphology of the cell aggregates and the final ratio of both in the aggregates. As shown in FIG. 21D, the dryness markers (Nanog and Sox-2), proliferation marker (Ki67), paracrine factors (TGF-. beta., HGF, and IDO), and the like of the cell aggregate were most strongly expressed when the ratio of cells to microspheres was 5:1, and thus the cell aggregate was prepared in the following examples at a ratio of cells to microspheres of 5: 1.
Example 15.P/C-h 2: determination of concentration of fusion protein of different subtypes on surface of 1-IL-1 beta composite microsphere
hE-cad-Fc and hN-cad-Fc were mixed at concentrations of 5:1 (hE-cad-Fc: 16.6. mu.g/mL, hN-cad-Fc: 3.3. mu.g/mL), 3:1 (hE-cad-Fc: 15. mu.g/mL, hN-cad-Fc: 5. mu.g/mL), 1:1 (hE-cad-Fc: 10. mu.g/mL, hN-cad-Fc: 10. mu.g/mL) and 1:3 (hE-cad-Fc: 15. mu.g/mL, hN-cad-Fc: 5. mu.g/mL), 1:5 (hE-cad-Fc: 3.3. mu.g/mL, hN-cad-Fc: 16.6. mu.g/mL), and the above solutions were used to prepare surface modified P/C-h2 of different fusion proteins according to the method of example 11: 1 composite microspheres and 20 μ g/mL collagen-modified P/C-h 2: 1-IL-1 beta composite microspheres. Subsequently, cell aggregates were prepared and cultured according to the method of example 14 by mixing hMSCs and microspheres in a ratio of 5:1, and RNA was extracted at day 5 according to the method of example 7, subjected to reverse transcription and examined for expression of the relevant gene. As shown in FIG. 22, when the ratio of hE-cad-Fc to hN-cad-Fc is 1:3, the immunomodulation-associated markers (STC-1, TSG-6) of hMSCs aggregates are expressed most strongly, and the dry markers (Sox-2) and paracrine factors (HGF, TGF-. beta.) are expressed more than the other ratios. Therefore, we found that the ratio of the fusion protein immobilized on the surface of the composite microsphere is hE-cad-Fc: hN-cad-Fc is 1:3 for subsequent studies.
Example 16 fusion protein modified P/C-h 2: preparation and characterization of 1-IL-1 beta composite microsphere mediated hMSCs aggregate
Fusion protein modified P/C-h2 was prepared according to the optimized ratio of example 14: 1-IL-1 beta composite microspheres and 20 μ g/mL collagen-modified P/C-h 2: 1-IL-1 beta composite microspheres. Cell aggregates were prepared according to the optimized ratio in example 15 and the method in example 6, placed in 6-well culture plates, cultured in DMEM/F12 medium containing 10% FBS, and changed every 3 days, specifically, (1) cell aggregates containing hMSCs without microspheres, abbreviated as control group; (2) contains only hE-cad-Fc modified P/C-h 2: cell aggregates of 1-IL-1. beta. microspheres, abbreviated as MEP group; (3) contains hN-cad-Fc modification P/C-h2 only: cell aggregates of 1-IL-1. beta. microspheres, abbreviated as MNP group; (4) the P/C-h2 modified by hE-cad-Fc and hN-cad-Fc according to the ratio of 1: 3: cell aggregates of 1-IL-1. beta. microspheres, abbreviated to the ME/NP group; (5) type I collagen-containing modified P/C-h 2: 1-IL-1. beta. microspheres, abbreviated as collagenous groups. And (3) photographing and observing the prepared cell aggregate through a microscope, collecting the cell aggregate, washing the cell aggregate twice by using PBS buffer solution, then placing the cell aggregate in a special fixing solution for the microscope for fixing overnight, dehydrating the cell aggregate by using freshly prepared gradient ethanol (100%, 95%, 90%, 80%, 70% and 60% for 10min respectively), adhering the cell aggregate to a conductive gel, spraying gold, and performing machine observation by using an SEM (scanning electron microscope). As a result, as shown in FIG. 23, the prepared aggregates had a complete structure and a clear edge, and the presence of microspheres in the aggregates could be clearly observed. And the diameters of the prepared different aggregates are basically consistent, which shows that the fusion protein surface modified microspheres have no significant influence on the formation and the size of the hMSCs aggregates.
Example 17 Effect of fusion protein matrices on paracrine and immunoregulatory function of hMSCs aggregates
After preparing various groups of cell aggregates by the method of example 14, preculture in DMEM/F12 medium containing IL-1. beta.10 ng/mL and 10% FBS for 3 days, total RNA was extracted by the method of example 7, and cDNA was obtained by reverse transcription, and quantitative PCR detection and data statistics were performed by the method of example 8 using cDNA as a template, and the primer sequences and annealing temperatures of the relevant genes are shown in Table 5. As shown in FIG. 24, the expression levels of factors such as aggregate HGF, IDO, IL-6, LIF and TGF-beta in the ME/NP group are significantly higher than those in other control groups, and hE/N-cad-Fc promotes the up-regulation of genes related to paracrine factors of the hMSCs aggregate, indicating that hE/N-cad-Fc plays a role in organogenesis, angiogenesis and immune response by promoting the paracrine function of the hMSCs and improving the hMSCs aggregate. As can be seen from FIG. 25, the expression levels of the TSG-6, STC-1 and PGE-2 genes of the ME/NP group cells are all significantly higher than those of other control groups, and the hMSCs play anti-inflammatory, anti-apoptotic and immune regulation roles by secreting related factors, which indicates that the hE/N-cad-Fc combined modified microspheres have the effects of significantly improving anti-inflammatory and immune regulation functions on hMSCs aggregates. Example 18 study of fusion protein matrix-mediated endogenous cadherin expression of aggregates of hMSCs
After preparing various groups of cell aggregates according to the method of example 14, pre-culturing the aggregates in DMEM/F12 medium containing IL-1. beta.10 ng/mL and 10% FBS for 3 days, total RNA was extracted according to the method of example 7, and the expression levels of E-cadherin and N-cadherin genes of hMSCs were measured. The protein was extracted according to the method in example 9, and the change in the protein level was detected by WB. As shown in FIG. 26, hE/N-cad-Fc could jointly enhance the expression of endogenous E-cadherin and N-cadherin of cells inside the aggregates of hMSCs. The up-regulation of the expression of endogenous cadherins of hMSCs, particularly E-cadherin and N-cadherin, is closely related to the cell proliferation, migration, dryness maintenance, anti-apoptosis and paracrine capacity of the hMSCs. The cadherin fusion protein can achieve the aim of further activating related functions of the hMSCs by enhancing the expression level of the cadherin of cells.
Example 19 Effect of fusion protein matrix-mediated aggregates of hMSCs on macrophages
Digesting well-conditioned macrophage, and measuring cellCounting according to 4 × 10 5 The ratio of each cell/well was inoculated into the upper layer of a 24-well transwell chamber (0.4 μm pore size), and after the cells were completely attached to the wall, the medium was changed to a medium containing 0.1 μ g/mL of LPS, and placed in an incubator to stimulate for 90 min. After the stimulation was complete, the LPS-containing medium was discarded and the ratio 2X 10 was adjusted 5 The density of hMSCs/well was added to the fusion protein matrix-mediated cell aggregates in the sub-chamber and placed in the incubator. After 5h of co-culture, the cell upper macrophage medium was collected, centrifuged at 500 Xg for 10min, and assayed for mTNFa content by Elisa according to the method described in the specification. The upper macrophage proteins were collected as in example 9 and macrophage polarization during WB assay. And (3) discarding the culture medium from the lower-layer different groups of hMSCs aggregates, extracting RNA according to the method in the embodiment 7, and detecting the expression of hMSCs related genes in the process. As shown in FIG. 27, it can be seen from FIG. 27B that the ME/NP group aggregate can significantly reduce the secretion of TNF- α from mouse macrophages, and from FIG. 27C, D that the ME/NP group aggregate can promote the conversion of more mouse macrophages from M1 type to M2 type. Meanwhile, in the expression of relevant regulatory genes (PLA2, COX2 and PGEs) of the hMSCs, the expression of the ME/NP group is strongest, which indicates that hE/N-cad-Fc promotes the hMSCs to play an anti-inflammatory role in the process.
Example 20 fusion protein matrix-mediated aggregation of hMSCs improves the therapeutic efficacy of hMSCs on colitis in mice
The mouse colitis model experiment is carried out by referring to a method of Matam Vijay-Kumar and the like, and C57BL/6J mice are adopted, and the week age is 6-8 weeks. Dextran Sodium Sulfate (DSS) was dissolved in sterilized water to prepare a 3% DSS solution, the drinking water for mice was changed to the 3% DSS solution every two days, and the mice were continuously fed for 7 days, after which they were changed to normal water. The method comprises the following specific steps:
1. 20 healthy C57BL/6J mice were weighed and randomly assigned to healthy (Control), PBS, M and ME/NP groups.
2. The first day when mice were drinking the 3% DSS solution (day 0 noted as treatment) and the second day when mice were drinking the 3% DSS solution (day 1 noted as treatment), cells were injected, and the prepared cell aggregates of each group were resuspended in PBS buffer and injected into the abdominal cavity of mice at a cell implantation amount of 100 ten thousand cells/mouse.
3. And (4) observing the state of the mouse every day, measuring the weight of the mouse, observing the shape of the excrement, and detecting the occult blood condition of the excrement. The mouse disease activity index was scored according to the following table:
TABLE 12
4. On day 14, the mice were sacrificed by removing their necks, the colons were taken out and photographed, and then the intestine was washed out repeatedly with 1640 medium and fixed in 4% paraformaldehyde for two weeks or more, dehydrated, embedded, sliced, and H & E stained according to the method of example 6, and the histological lesions of the colons of the mice were observed. The spleen of the mouse was taken out, added with PBS buffer (containing 2% FBS), sufficiently ground, filtered (200 mesh sieve), and centrifuged at 1000g at 4 ℃ for 5min to obtain spleen cells. 4mL of erythrocyte lysate was added and incubated for 10min, the supernatant was discarded by centrifugation, and the cells were resuspended in PBS buffer (containing 2% FBS) and counted. Single cells within lymph nodes were obtained following the same procedure.
Taking 1X 10 6 Spleen cells were plated in 96-well plates, centrifuged, supernatant discarded, 100. mu.L cell stimulating agent (1: 100 dilution) added, resuspended, and incubated at 37 ℃ for 4 h. After washing once with PBS buffer (containing 2% FBS), 100. mu.L of flow antibodies CD3 and CD8a (PBS buffer diluted 1: 500) were added to resuspend the cells and incubate at 4 ℃ for 30min in the absence of light. After the incubation is finished, washing once with PBS (2% FBS) buffer, centrifuging for 5min at 1800 Xg, adding 100 mu L of membrane breaking agent, incubating for 20min at 4 ℃, washing once with PBS buffer, centrifuging for 5min at 2000 Xg, adding flow antibodies IFNr-PerCP, IL4 and IL17(PBS buffer is diluted at 1: 500), mixing uniformly, incubating for 30min at 4 ℃ in the dark, washing once with PBS buffer, and detecting by a flow cytometer. Spleen cells were labeled with CD4-FITC and Foxp3 in the same manner. The results are shown in FIG. 28, in which the body weight of the PBS group mice was measured on the first day from the injection of the aggregates and on the 5 th day of treatmentThe weight of the ME/NP group begins to show a rising trend at the 8 th day, the weight of the M group of mice drops to the 9 th day, the rising trend appears, the weight of the ME/NP group is obviously higher than that of the M group at the 14 th day of the observation period, and the ME/NP group of the mice is similar to that of normal mice, is active in emotion and is normal in diet. As can be seen from FIG. 28B, the weight loss condition, the fecal hardness and the fecal occult blood condition of the mice are comprehensively graded, the overall grade of the mice in the ME/NP group is lowest, the disease degree of the mice in the ME/NP treatment group is proved to be the lowest, and the hE/N-cad-Fc modified microspheres have the function of improving the aggregate immunoregulation capability of hMSCs. As can be seen in FIG. 28C, the colon of the PBS group mice showed severe lesions with marked necrosis, atrophy and shortening of the colon, and no macroscopically intact stool was visible in the colon. The colon of the ME/NP treated mice is complete in shape, complete granular excrement exists inside the colon, and the length of the colon is closest to that of the normal mice.
As can be seen from fig. 29A, in the colon section of the normal mouse, there are intact lamina propria, mucosal layer, submucosa layer and muscular layer, a large number of epithelial cells and goblet cells, and lymphocyte infiltration was not substantially observed. In the colon sections of the mice in the PBS group, the colon epithelium is severely ulcerated, epithelial cells and goblet cells are largely lost, and a large amount of lymphocyte infiltration exists. This was slightly relieved in the colon of mice in the M treatment group, but a massive lymphocytic infiltration was still observed, indicating that aggregates in the M group had a slight inhibitory effect on colonic epithelial destruction. The colon structure of mice in the ME/NP injection treatment group is more complete, the damage degree of colon epithelial cells is minimum, the goblet cell loss is minimum, the lymphocyte infiltration is minimum, the ME/NP aggregate is proved to effectively relieve the colitis of the mice, and the hE/N-cad-Fc combined modification P/C-h 2: the 1-IL-1 beta composite microsphere obviously improves the anti-inflammatory and immune regulation functions of the hMSCs aggregate.
As can be seen in FIG. 30A, a large amount of CD4 was detected in the PBS group + And CD8 + T cell infiltration of (3). While injection of ME/NP group aggregates was effective in inhibiting CD4 in spleen + And CD8 + T cell infiltration of (3). As can be seen from FIG. 30D, hMSCs in the ME/NP group maximally inhibited T cell Th1 andth17 differentiation was also concluded in mesenteric lymph nodes. The results thus indicate that hE/N-cad-Fc modifies P/C-h2 in combination: the hMSCs aggregate formed by the mediation of the 1-IL-1 beta composite microspheres (ME/NP group) has higher immunoregulation activity, and inhibits the proliferation and differentiation of T cells to proinflammatory Th1 and Th17 cells.
Example 21 fusion protein surface modification P/C-h 2: 1-TGF beta 1 composite microsphere slow-release TGF beta 1
Modified P/C-h2 carrying TGF-beta 1 (TGF-beta 1, Peprotech, USA) fusion protein was prepared as in example 4: 1-TGF beta 1 composite microspheres prepared by the method of example 4 for P/C-h 2: the release performance of TGF beta 1 of the 1-TGF beta 1 microsphere is tested. The results are shown in FIG. 31, where in the first 12 days, P/C-h 2: 1 TGF-beta 1 factor in the composite microsphere is gradually released, and the factor is continuously released in the next 15 days.
Example 22 optimization of hE-cad-Fc and hN-cad-Fc fusion proteins use ratio and their impact on endogenous N-cadherin expression in aggregates of hMSCs
hE-cad-Fc and hN-cad-Fc were prepared as 10. mu.g/mL protein solutions. 1.5mL of a 10. mu.g/mL collagen solution, hE-cad-Fc and hN-cad-Fc were mixed in a 1:1 (hE-cad-Fc: 5. mu.g/mL, hN-cad-Fc: 5. mu.g/mL), 1:2 (hE-cad-Fc: 3.3. mu.g/mL, hN-cad-Fc: 6.6. mu.g/mL) and 1:3 (hE-cad-Fc: 2.5. mu.g/mL, hN-cad-Fc: 7.5. mu.g/mL) and a protein solution containing only 10. mu.g/mL hN-cad-Fc were added to six-well PS plates, respectively. The six-well plate is placed in a cell culture box and incubated for 2h at 37 ℃, and after being taken out, the supernatant is discarded and washed for 3 times by PBS buffer solution.
Followed by 1X 10 cell density 5 Cells/well, hMSCs were inoculated into six-well PS plates incubated with the protein mixtures of different subtypes and different concentrations, and cartilage differentiation-oriented induction medium (DF 12 medium containing 5% FBS, 10ng/mL TGF β 1, 200nM 2-phospho-ascorbic acid, 100nM dexamethasone) was added and cultured in a cell culture incubator. After 1 week of culture, the medium was discarded, the cells were washed with PBS buffer for 3 times and observed by photographing, as shown in FIG. 32, after 1 week of cartilage differentiation culture, cell aggregation occurred to various degrees in all groups except the collagen group. When hE-cad-Fc and hN-The concentration ratio of cad-Fc fusion protein is 1:2, the cell aggregates generated by spontaneous aggregation are more uniform in size and higher in density, and are more favorable for forming high cell density required by differentiation of the hMSCs to chondrocytes.
Protein solutions of 20. mu.g/mL collagen, hE-cad-Fc and hN-cad-Fc in the ratio of 1:1 (hE-cad-Fc: 10. mu.g/mL, hN-cad-Fc: 10. mu.g/mL), 1:2 (hE-cad-Fc: 6.5. mu.g/mL, hN-cad-Fc: 13. mu.g/mL) and 1:3 (hE-cad-Fc: 5. mu.g/mL, hN-cad-Fc: 15. mu.g/mL) and 20. mu.g/mL hN-cad-Fc were prepared as in example 11 and used to prepare P/C-h2 with different surface modifications, respectively: 1-TGF beta 1 composite microsphere. 9X 10 preparation of a mixture of two or more of the following compounds according to example 6 6 P/C-h 2: 1-TGF beta 1 composite microspheres are prepared according to the following steps of 1:3 to prepare cell aggregates. The 3D culture was performed using alginate hydrogel according to the method in example 6. Continuously inducing differentiation culture in cartilage differentiation culture medium for one week, changing the solution every two days, and lysing the alginate hydrogel spheres according to the method in example 6 on the seventh day to recover cell aggregates. mRNA was extracted according to the method of example 7 and subjected to RT-PCR using the primers shown in Table 13. The protein was extracted for WB assay according to the procedure of example 9. The results of the experiments are shown in FIG. 33, and when the ratio of hE-cad-Fc and hN-cad-Fc is 1:2, both at the gene level and at the protein level, the expression of Sox9, collagen type 2 and endogenous E-cadherin in cells is significantly higher than that of the other groups, demonstrating that the combined modification of P/C-h2 when the ratio of hE-cad-Fc and hN-cad-Fc is 1: 2: the 1-TGF beta 1 composite microspheres can better promote hMSCs to express chondrocyte marker proteins, and maintain high expression of E-cadherin.
Collagen solution (20. mu.g/mL), hN-cad-Fc (20. mu.g/mL), hE-cad-Fc and hN-cad-Fc (1: 2, hE-cad-Fc: 6.5. mu.g/mL, hN-cad-Fc: 13. mu.g/mL) were used to incubate P/C-h 2: 1-TGF beta 1 composite microsphere. P/C-h2 modified from different protein solutions according to the method of example 6: preparing 1-TGF beta 1 composite microspheres and hMSCs into cell aggregates, and marking as P/C-h2 containing collagen modification: 1-TGF beta 1 composite microsphere hMSCs aggregates (MCP), hN-cad-Fc modified P/C-h 2: 1-TGF beta 1 composite microsphere hMSCs aggregate (MNP), P/C-h2 modified by hE-cad-Fc and hN-cad-Fc protein according to the ratio of 1: 2: 1-TGF beta 1 composite microsphere hMSCs aggregates (MENPs). Additional preparations were made without P/C-h 2: pure cell aggregates (M) of 1-TGF beta 1 composite microspheres served as a control group. Cartilage-inducing differentiation medium (group M: DF12+ 5% FBS +10ng/mL TGF β 1+200nM 2-P-asc +100nM Dex, group aggregate containing P/C-h 2: 1-TGF β 1 composite microspheres: DF12+ 5% FBS +200nM 2-P-asc +100nM Dex) was used for continuous culture for 7 days with replacement every two days. mRNA was extracted according to the method of example 7 and detected by RT-PCR, the relevant primers are shown in Table 13, and protein was extracted according to the method of example 9 and the expression of endogenous N-cadherin in cells was detected over time. As a result, as shown in FIG. 34, on day 3 of differentiation, the gene and protein expression levels of N-cadherin in the MENP group reached a peak, and during the subsequent continued differentiation, the gene and protein expression of N-cadherin was sharply reduced; while the N-cadherin gene and protein expressed by the cells of group M peaked at day 5. And the peak values of the N-cadherins of the M group, the MCP group and the MNP group are all lower than the peak value of the MENP group. This shows that the combination of hE-cad-Fc/hN-cad-Fc (1: 2) in MENP can accelerate the up-regulation of N-cadherin expression in hMSCs, promote the process of interstitial coagulation of hMSCs, and improve the process of directional differentiation of hMSCs into chondrocytes.
Watch 13
Example 23 hE/N-cadherin-Fc combination modification of P/C-h-TGF beta 1 microsphere-mediated hMSCs aggregates to promote cartilage differentiation in vitro
Different sets of hMSCs aggregates were prepared according to the method of example 22 and cultured for 4 weeks in alginate hydrogel for chondrogenic differentiation, with samples taken every 7 days. mRNA was extracted according to the method of example 7 and RT-PCR was performed, and the relevant primers are shown in Table 13 and protein was extracted according to the method of example 9 and WB detection was performed. The following antibodies, Sox9(Abcam, usa), proteoglycan (Abcam, usa), type 2 collagen (Abcam, usa), and β -actin (Abcam, usa) were used to detect the expression of genes and proteins of Sox9, type 2 collagen and proteoglycan in different groups of aggregates after directed induced differentiation for 1 week, 2 weeks, 3 weeks, and 4 weeks as shown in fig. 35. After 1-week differentiation, the expression level of proteoglycan of the MENP group is obviously enhanced compared with that of the other two groups; after 2 weeks of differentiation, the expression levels of Sox9, type 2 collagen and proteoglycan in the MENP group are obviously higher than those of M, MCP and MNP groups; after 3 weeks and 4 weeks of differentiation, proteoglycan expression amount in the MENP group was still significantly higher than that in the other three groups. This indicates that, at P/C-h 2: after hE-cad-Fc/hN-cad-Fc (1: 2) is modified on the surface of the 1-TGF beta 1 composite microspheres, the expression of chondrocyte-related marker proteins including Sox9, type 2 collagen and proteoglycan can be continuously enhanced, and the method is also shown to be capable of accelerating the differentiation process of hMSCs to chondrocytes.
hMSCs aggregates of different compositions were prepared according to the method of example 22, cultured in alginic acid hydrogel spheres for 4 weeks for induced cartilage differentiation, samples were taken every 2 weeks, aggregates were recovered according to the method of example 6, fixed in 4% paraformaldehyde for more than 2 days, paraffin-embedded, sectioned and stained with alcian blue (solibao). The specific process of alcian blue staining is as follows:
baking at 1.60 deg.C until paraffin is melted, soaking in xylene I and xylene II for 5min respectively, and dewaxing;
soaking in 2.100%, 95%, 90%, 80% and 70% alcohol for 5min respectively;
3. washing with distilled water for 5min, and incubating with acidified solution for 3 min;
4, dyeing with an Alcian staining solution for 30min, and washing with running water for 5 min;
soaking 5.70%, 80%, 90%, and 100% alcohol respectively for 5s for decolorizing;
6. carrying out transparent treatment on the xylene I and the xylene II for 5min respectively;
7. and sealing the neutral gum into a piece, and observing the piece by using an upright microscope.
As shown in FIG. 36, after 2 weeks of differentiation, P/C-h2 in MCP, MNP and MENP groups: 1-TGF beta 1 composite microspheres are degraded, and the positive staining of the alcian blue of the MENP group is strongest; after 4 weeks of differentiation, P/C-h2 in MCP, MNP and MENP groups: the 1-TGF beta 1 composite microspheres are basically completely degraded, and the alcian blue staining effect in the MENP group is still strongest. This indicates that hE-cad-Fc/hN-cad-Fc (1: 2) in the MENP group could promote the differentiation of hMSCs into chondrocytes, and that chondrocytes obtained by the MENP grouping could maintain the functional properties of chondrocytes for a longer period of time.
Example 24 hE/N-cadherin-Fc combination modified P/C-h-TGF beta 1 composite microsphere mediated hMSCs aggregate inhibits calcification in late stage of cartilage differentiation
Different sets of hMSCs aggregates were prepared according to example 22 and cultured for 4 weeks in alginate hydrogel for induced cartilage differentiation, fixed with 4% paraformaldehyde for more than 2 days according to example 6, paraffin embedded and sectioned for safranin fast green staining (solibao) as follows:
baking at 1.60 deg.C until paraffin is melted, soaking in xylene I and xylene II for 5min respectively, and dewaxing;
soaking in 2.100%, 95%, 90%, 80%, 70% alcohol for 5min respectively;
3. washing with distilled water for 5min, counting in Weigert staining solution, and staining for 3 min;
4. differentiating the acidic ethanol differentiation solution for 15 s;
5. washing with distilled water for 5min, and dip-dyeing with fast green dyeing solution for 7 min;
6. washing with distilled water for 5min, and dip-dyeing with Safranin O stain for 3 min;
7. washing with distilled water for 5min, washing with acetic acid solution for 1min, and washing with distilled water for 5 min;
soaking 8.70%, 80%, 90% and 100% alcohol respectively for 5s for decolorizing;
9. carrying out transparent treatment on the xylene I and the xylene II for 5min respectively;
10. sealing neutral gum into a sheet; and (5) observing by an upright microscope.
As shown in fig. 37, after 2 weeks of differentiation, safranin staining was strongest in the MENP group compared to M, MCP and MNP groups, and no fast-green staining was evident in any of the four groups; after 4 weeks of differentiation, the cells of group M began to appear in loose morphology; and contains P/C-h 2: MCP, MNP and MENP groups of the 1-TGF beta 1 composite microspheres can still maintain the aggregate state, obvious fast green coloring appears in the MCP group, slight fast green coloring appears in the MNP group, and strong safranine coloring effect is still maintained in the MENP group without fast green coloring. This indicates that after 4 weeks of differentiation, the cells in the M group were dead and exfoliated, the cells in the MCP and MNP groups were calcified to different extents, and the MENP group was still able to maintain the chondrocyte characteristics without calcification.
Different sets of hMSCs aggregates were prepared according to the method of example 22, cultured in alginate hydrogel for 4 weeks for inducing differentiation of cartilage, RNA was extracted according to the method of example 7, gene expression of MMP13, ALP, collagen 10 and collagen type 2 was measured by PCR, and the ratio of gene expression of collagen type 2 and collagen type 10 was counted, using the primer sequences shown in table 14.
TABLE 14
As shown in fig. 38, after the chondrocytes after 4 weeks of oriented induction and differentiation, compared with MNP and MENP groups, the expression levels of MMP13, ALP and type 10 collagen in M group and MCP group were higher, and the ratio of type 2 collagen/type 10 collagen was lower, indicating that stronger chondrogenic phenomena in M group and MCP group occurred, while the expression levels of MMP13, ALP and type 10 collagen in MENP group were significantly reduced, indicating that the chondrocytes obtained by this grouping were more stable, and the combined modification of hE-cad-Fc/hN-cad-Fc (1: 2) could better inhibit the chondrocytes that were differentiated and matured from calcification.
Example 25 hE/N-cadherin-Fc combination modified P/C-h-TGF beta 1 microsphere mediated regulation mechanism for inducing differentiation of hMSCs aggregates into chondrocytes in vitro
Different groups of hMSCs aggregates were prepared according to the method of example 22, and cartilage-induced differentiation culture was performed in alginate hydrogel for 4 weeks, aggregates were recovered according to the method of example 6, fixed with 4% paraformaldehyde for more than 2 days, and washed three times with PBS buffer for 5min each. Then 100. mu.L goat serum (Biyun day) was added to each group of aggregates (diluted 1: 200 in PBS buffer) and blocked for 30min at room temperature. Then, the beta-catenin antibody (1: 500, Abcam) and the E-cadherin antibody (1: 500, Abcam) were diluted with PBS buffer, respectively, 50. mu.L of each was added to each group of aggregates, and after incubation overnight at 4 ℃, the aggregates were washed 3 times with PBS buffer, each for 5 min. FITC-labeled goat anti-mouse IgG antibody (Invitrogen, USA, cat # A-10680) and Rhodamine-labeled goat anti-rabbit secondary antibody (Invitrogen, USA, cat # 35560) were diluted 1: 500 with PBS buffer, 50. mu.L of each was added to each group of aggregates, incubated at room temperature for 2h, washed 3 times with PBS buffer, 5min each. Then 20. mu.L of DAPI-containing anti-fluorescence quencher (southern Biotech, USA, cat. No. 0100-20) was added to each aggregate group, and photographed using a confocal laser microscope.
Different groups of hMSCs aggregates were prepared according to the method of example 22 and cultured for 4 weeks in alginate hydrogel for cartilage-induced differentiation, the aggregates were recovered according to the method of example 6, then placed in 1.5ml ep tubes, 500 μ L of 0.25% trypsin was added, digestion was stopped by adding 500 μ L of DF12 medium containing 10% FBS after shaking in water bath at 37 ℃ for 5min, centrifugation was carried out at 4 ℃ for 5min at 1000 × g, supernatant was discarded and resuspended in PBS buffer, and centrifugation was carried out at 4 ℃ for 5min at 1000 × g. Then, proteins in the nucleus and cytoplasm were extracted with a cell nucleus extraction kit (Solarbio, U.S. Pat. No. R0050), and the nucleoplasmin was separated and subjected to WB detection by the method of example 9. The main steps of nucleoplasmin separation are as follows:
washing single cells with PBS buffer solution, centrifuging for 5min at 500g, and collecting cells;
2. adding 200 μ L plasma protein extraction reagent into every 20 μ L cell precipitate, fully vortexing for more than 15s to completely disperse cells, ice-bathing for 10min, centrifuging at 4 deg.C at 12000 Xg for 10 min;
3. collecting cell nucleus precipitate at the bottom of the centrifugal tube, and collecting protein solution in cytoplasm in supernatant for subsequent use;
4. after the supernatant was aspirated, 50. mu.L of a nucleoprotein extraction reagent was added to the nuclear sediment;
5. blowing or swirling for more than 15s by a pipettor until the mixture is completely dispersed, and centrifuging for 10min at 12000g at 4 ℃ in an ice bath mode for 10 min;
6. the supernatant was pipetted into a pre-chilled sample tube for intracellular proteins and stored at-80 ℃.
As a result, as shown in fig. 39, the expression of E-cadherin was significantly higher in the MENP group than in the other three groups, while β -catenin of the MENP group was mainly localized in cytoplasm; m, MCP and MNP group, and the beta-catenin is mainly located in nucleus, indicating that the combined modification of hE-cad-Fc/hN-cad-Fc (1: 2) in MENP group can effectively promote the differentiation of hMSCs to chondrocytes and promote the expression of E-cadherin of cells, so that the beta-catenin is more located in cytoplasm to inhibit entering nucleus to initiate chondrocytosis.
Different groups of hMSCs were prepared according to the method of example 22, and cultured for cartilage-induced differentiation in alginic acid hydrogel spheres for 4 weeks, the aggregates were recovered according to the method of example 6, fixed for more than 2 days by adding 4% paraformaldehyde, blocked according to the method of the above example, YAP antibody (PBS buffer 1: 500 dilution, Abcam) and E-cadherin antibody (PBS buffer 1: 500 dilution, Abcam) were incubated, washed, FITC-labeled goat anti-mouse IgG antibody and Rhodamine-labeled goat anti-rabbit secondary antibody were incubated, and then 20 μ L of DAPI-containing anti-fluorescence quencher was added, and photographed using a laser confocal microscope, with the results shown in fig. 40A.
hMSCs aggregates of different compositions were prepared according to the method of example 22, cultured for 4 weeks in alginate hydrogel spheres for induced cartilage differentiation, nucleoplasmin was separated according to the above procedure, and WB assay was performed according to the method of example 9. The results are shown in FIG. 40B, the total expression of YAP in MENP group is significantly lower than M, MCP and MNP group, and the expression of E-cadherin in MENP group is significantly higher than M, MCP and MNP group. The MCP group had the least cytoplasmic and the most nuclear phosphorylated YAP; the MENP group has the most YAP expression amount in cytoplasm and the least YAP expression amount in nucleus. This indicates that YAPs in the MENP group are more in a phosphorylated state and more localized in the cytoplasm to inhibit their entry into the nucleus to initiate chondrocyte calcification; m, MNP and the YAP in MCP group are more located in nucleus, and can be combined with DNA to promote the proliferation of chondrocyte, so that the cartilage is enlarged and calcified.
Example 26 study of hE/N-cadherin-Fc combination modified P/C-h-TGF beta 1 microsphere mediated hMSCs aggregates on treatment of rat knee cartilage injury
72 female SD rats (11 weeks, 320-340g) were purchased from the Nanjing Qinglongshan animal farm and used in this in vivo experiment. What is needed isAfter general anesthesia of animals, 60 cartilage defect models (diameter 2.5mm and height 1.5mm) were randomly prepared at the femoral groove of knee joint, and 12 wounds were directly sutured without modeling treatment, and the models were used as a control group. Then M, MCP, MNP and MENP aggregates (650 cell aggregates, about 4X 10) prepared in example 22 were loaded with PBS buffer as a vehicle 5 Cells) were injected into the joint cavities of cartilage-deficient rats, and an equal volume of PBS buffer was injected as a blank, and 6 replicates were set up for each group. Gross observations, histology and MicroCT analyses were performed at weeks 3 and 6, respectively.
The femur of SD rats was removed at 3 and 6 weeks after the operation and was observed in general, and the results are shown in fig. 41, and at 3 weeks after the operation, the depressed trace of the cartilage defect site was significantly reduced compared to the PBS group, M, MCP group and MENP group, and a small amount of transparent cartilage layer wrapping was observed on the surface of the defect site; at week 6 after surgery, the boundary traces of the cartilage defect sites of the MENP group disappeared, and the defect sites were covered with uniform hyaline cartilage tissue; while the indentations at the PBS, M and MCP group defects were still significant.
And (3) adding 4% paraformaldehyde into the taken femur, fixing for more than two days, adding 10mL of 10% EDTA decalcification solution, and replacing the decalcification solution every two days for 4 weeks. Paraffin embedding and sectioning are carried out according to the method in the example 6, safranine fast green histological staining is carried out according to the method in the example 24, and the result is shown in figure 42, at 3 weeks after the operation, the depth of the PBS group defect is deepest, and safranine staining of filling tissues is negative; the M groups of defect parts are filled with fibrous tissues, the tissue trend is irregular, and the surface is uneven; the defect part of the MCP group is filled with a small amount of cells, the safranin staining is negative, and the surface of the filled tissue is uneven; the MNP tissue surface filling tissue has uniform thickness and smoother surface, but the safranin staining result of the injured part is negative; the MENP group filling tissue has the thickness close to that of the natural cartilage tissue, the surface is smooth, the cells are filled compactly, and a small amount of safranin positive tissue is filled in the damaged part. At week 6 after surgery, there was still significant cartilage defect in the PBS group; the tissue fibrosis of the M groups of defect parts is serious, the surface defect is obvious, and obvious gaps exist among tissues; the defect parts of the MCP group still have obvious depressions, and large gaps exist among cells at the defect parts; the MNP group defect part is more compact in filling tissue, has a part of safranine positive tissue, but is thicker than the natural cartilage tissue in filling thickness; the new tissue formed at the defect part of the MENP group is similar to the thickness of the natural cartilage tissue, has safranin positive tissue and is uniform in surface. This indicates that there was more cartilage tissue in the filling tissue at the defect site of the MENP group and the repair was better 6 weeks after the operation.
Example 27 study of hE/N-cadherin-Fc in combination with modified P/C-h microsphere-mediated hMSCs aggregates for treatment of rat articular subchondral bone defects
The femur of SD rat 3 and 6 weeks after the operation in example 26 was removed and subjected to MicroCT (SCANCO MEDICAL vivac 80, Switerland) analysis, and the results are shown in fig. 43, and as can be seen from fig. 43Aa and 43Ab, the defect depth of subchondral bone in MENP group was significantly reduced and the defect site had different degrees of osteogenic filling compared to PBS group at 3 weeks after the operation; the subchondral bones of the M group, the MCP group and the MNP group still have obvious subchondral bone defect traces, and the defect depth is not effectively repaired compared with that of the PBS group. As can be seen from FIG. 43Ac and FIG. 43Ad, the BV/TV values were significantly higher in the MNP group and the MENP group than in the M group; Tb/Th values were also significantly higher in both MNP and MENP groups than in M group. This indicates that the MENP group has a better overall degree of repair for subchondral bone than the MNP group, and more than the PBS group, M group and MCP group at 3 weeks after the operation.
As can be seen from fig. 43Ba and 43Bb, at week 6 after the operation, the defect depth of subchondral bone of the MENP group became significantly shallower compared to the PBS group and the M group, and the depression trace of the defect site was almost filled with newly-generated bone tissue, and no more significant depression trace was observed; the subchondral bones of the M group, the MCP group and the MNP group still have obvious subchondral bone defect traces, the defect depth is not effectively repaired compared with that of the PBS group, and meanwhile, the hyperplasia of the defect part of the M group is more serious compared with that of other groups. As can be seen from FIGS. 43Bc and 43Bd, the BV/TV values of MNP group and MENP group are significantly higher than those of M group, and the BV/TV values of MNP group are also significantly higher than those of MNP group; Tb/Th values were also significantly higher in both MNP and MENP groups than in M group. This indicates that the MENP group had better repair of subchondral bone than the MNP group, more significantly better than the PBS group, M group and MCP group at 6 weeks after surgery.
Example 28 hE/N-cadherin-Fc combination modification of P/C-h microsphere-mediated repair mechanism of hMSCs aggregates on rat articular cartilage defects
Cartilage samples at 3 and 6 weeks were fixed and decalcified as in example 26, embedded and sectioned as in example 6. Immunostaining for Ku80 protein and type ii collagen was then performed. 0.4% pepsin solution is prepared with 0.1M hydrochloric acid, and is dripped on the cartilage sample, and incubated for 30min at 37 ℃ for antigen retrieval. Blocking was performed according to the method of example 25, incubation of Ku80 antibody (PBS buffer 1: 500 dilution, Abcam) and collagen type 2 antibody (PBS buffer 1: 500 dilution, Abcam), washing, incubation of FITC labeled goat anti-mouse IgG antibody and Rhodamine labeled goat anti-rabbit secondary antibody, followed by addition of 20 μ L of DAPI-containing anti-fluorescence quencher, and photographing with a laser confocal microscope, as a result shown in fig. 44, after 3 weeks of in vivo injection treatment, staining of the MENP group Ku80 more showed more positive human cells and more expression of the surrounding collagen type 2, compared to M, MCP and MNP groups, indicating that the MENP group aggregates could be better localized to the cartilage defect site, survived longer in vivo, could be more differentiated into mature chondrocytes, expressed collagen type 2, and filled in situ to the cartilage defect site.
Different compositions of hMSCs aggregates were prepared and cultured in alginate hydrogel in 3D according to the method of example 22. IL-1. beta. was added at a concentration of 10ng/mL to a cartilage differentiation medium (group M: DF12+ 5% FBS +10ng/mL TGF. beta.1 +200nM 2-P-asc +100nM Dex, group of aggregates containing P/C-h 2: 1-TGF. beta.1 composite microspheres: DF12+ 5% FBS +200nM 2-P-asc +100nM Dex) to simulate an arthritic microenvironment in vitro, the solution was changed every 24 hours, after 48 hours of induction, the aggregates were recovered according to the method of example 6, RNA was extracted according to the method of example 7, and RT-PCR was performed to detect the gene expression of TGF. beta., IL-10 and IL-4, using the primer sequences shown in the following Table.
The result is shown in figure 45, after 48 hours of in vitro arthritis microenvironment induction, the expression quantity of the genes of the IL-4, IL-10 and TGF beta of the MENP group aggregate is obviously higher than that of the M group, and the IL-10 gene expression in the MENP group is obviously different from that of the MNP group, which indicates that the MENP aggregate has stronger gene expression of the anti-inflammatory factors IL-4, IL-10 and TGF beta in the arthritis microenvironment. When the articular cartilage defect model is repaired, the MENP aggregate can respond to inflammatory reaction of an injury part at an early stage, secrete more anti-inflammatory factors and weaken the inflammatory reaction of the injury part.
Claims (14)
1. A modified substrate comprising an epithelial cadherin-Fc fusion protein and a neuronal cadherin-Fc fusion protein, and said substrate is a hydrophobic microsphere.
2. The modified matrix according to claim 1, wherein said epithelial cadherin is human epithelial cadherin and the Fc is that of an IgG (preferably IgG 1); the neural cell cadherin is human neural cell cadherin, and Fc is Fc of IgG (preferably IgG 1);
preferably, the sequence of the epithelial cell cadherin is represented by SEQ ID NO. 2, the sequence of the neural cell cadherin is represented by SEQ ID NO. 5,
more preferably, the Fc of the epithelial cell cadherin-Fc fusion protein and the Fc of the neural cell cadherin-Fc fusion protein are identical or different, preferably identical, more preferably the sequences shown in SEQ ID NO 3,
more preferably, the epithelial cell cadherin-Fc fusion protein is preferably the sequence shown in SEQ ID NO 1; the nerve cell cadherin-Fc fusion protein is preferably a sequence shown in SEQ ID NO. 4.
3. The modified matrix according to claim 1, wherein said matrix is a PLGA microsphere, preferably a PLGA/chitosan-heparin core-shell structure composite microsphere.
4.A method of preparing a modified substrate comprising modifying a substrate by mixing an epithelial cell cadherin-Fc fusion protein and the neuronal cell cadherin-Fc fusion protein with the substrate.
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 endocyst-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 3:1 to 10:1, preferably 3:1 or 5: 1.
9. Use of an epithelial cadherin-Fc fusion protein and a neuronal cadherin-Fc 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 for promoting a cellular immunoregulatory function and/or for promoting cartilage repair, preferably the epithelial cadherin-Fc fusion protein and the neuronal cadherin-Fc fusion protein are immobilized on the same substrate or on different substrates, preferably the substrates are hydrophobic microspheres (preferably PLGA microspheres, more preferably PLGA composite microspheres modified with chitosan and heparin).
10. Use of an epithelial cell cadherin-Fc fusion protein and a neuronal cell cadherin-Fc 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 the manufacture of a medicament for the treatment of: acute and chronic inflammation of colitis, arthritis and soft tissue, and acute and chronic articular cartilage defect.
11. Use according to claim 9, wherein, when used to promote cellular immune regulatory function, the epithelial cell cadherin-Fc fusion protein and the neuronal cell cadherin-Fc fusion protein are in a ratio of from 1:5 to 5:1, preferably 1: 3.
12. The use of claim 11, wherein the promotion of immune regulation comprises enhancement of the ability of paracrine-related factors (e.g., VEGF, TGF- β, IL-6, EGF, HGF, FGF-2, etc.), enhancement of the expression of anti-inflammatory factor (e.g., IL-1, IL-10, TGF β, etc.) genes, enhancement of the regulatory ability on macrophages.
13. The use of claim 9, wherein the ratio of the epithelial cadherin-Fc fusion protein to the neuronal cadherin-Fc fusion protein is from 1:1 to 1:3, preferably 1:2, when used to promote cartilage repair.
14. The use of claim 13, wherein the promotion of cartilage repair comprises promotion of chondrocyte differentiation, inhibition of mature chondrocyte calcification, joint defect repair, subchondral bone repair, and the like.
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