Self-protection microcapsule-encapsulated therapeutic cell clusters and preparation method and application thereof

Abstract

The present invention discloses a self-protection microcapsule-encapsulated therapeutic cell cluster, comprising a therapeutic cell cluster and a microcapsule encapsulating the therapeutic cell cluster, wherein the microcapsule also encapsulates spermidine engineered cells. During preparation, the therapeutic cell cluster and the spermidine engineered cells are first obtained; then they are mixed into a microcapsule generating solution to prepare microcapsules, forming a self-protection microcapsule-encapsulated therapeutic cell cluster. The self-protection microcapsule-encapsulated therapeutic cell cluster of the present invention can be used as a cell transplant material in cell transplantation to treat diseases, can effectively prevent the release of inflammatory cytokines or microcapsule fibrosis caused by hypoxia of therapeutic cells, can protect therapeutic cells, reduce inflammation and the fibrosis produced by it, and is conducive to improving the therapeutic effect of the therapeutic cell cluster.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy, and specifically relates to a microcapsule encapsulation product that can be used for clinical treatment of chronic diseases, and a preparation method and application thereof. Background Art

[0002] Cell transplantation has attracted widespread attention from peers in the treatment of chronic diseases such as diabetes and Parkinson's disease because of its ability to deliver target molecules directly to the target area in a long-term and stable manner.

[0003] Treating diabetes by transplanting healthy pancreatic islet cell clusters has the advantages of high safety compared to other organ transplants, long-term blood sugar control, continuous normalization of glycosylated hemoglobin, and avoidance of side effects of subcutaneous insulin injections. Cell transplantation is a method of treating diabetes that has emerged in recent years, but receiving allogeneic pancreatic islet cell transplantation means the need for lifelong use of immunosuppressants, which can lead to a series of immunosuppressive complications such as infection, cancer, and organ system toxicity.

[0004] Parkinson's disease is a disease that is common in the elderly. The prevalence rate in people over 65 years old in my country is about 1.7%. For neurological diseases such as Parkinson's disease, which are characterized by central nervous system failure, "neural stem / precursor cell transplantation" (such as NT2 cells (induced to transform into dopamine-secreting cells)) has become a promising treatment strategy with its endogenous mechanism advantages such as repairing damaged tissues and restoring lost functions. However, directly transplanting neural stem cells into the brain will also produce a series of complications such as low survival rate of transplanted cells, immune rejection, and teratoma. These problems have seriously affected the application and development of the "cell transplantation" program in clinical treatment.

[0005] Microencapsulated cell transplantation is an advanced treatment technology. Although this technology has overcome the problems of transplanted cell damage and low survival rate caused by immune rejection to a certain extent, a large number of studies have shown that microencapsulated cells inevitably cause hypoxia of therapeutic cells in the microcapsules. In a hypoxic environment, therapeutic cells will be stressed and produce a series of cytokines that help recruit and activate inflammatory macrophages (such as monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP), nitric oxide (NO), interleukin 6 (IL-6), etc.), and damaged cells will also secrete damage-related molecular patterns (such as DNA fragments, uric acid, high mobility group protein 1 (HMGB1), heat shock proteins (HSPs), etc.). These molecules will be released into the body through the pores of the microcapsules and recognized by pattern recognition receptors, activating pro-inflammatory pathways and further aggravating the inflammatory response; causing collagen to accumulate around the microcapsules and fibrosis, ultimately leading to transplantation failure. Summary of the invention

[0006] The purpose of the present invention is to overcome the technical problems existing in the prior art, and to propose a self-protective microcapsule-encapsulated therapeutic cell cluster and a preparation method thereof. The product encapsulates spermidine-producing cells and therapeutic cells together in microcapsules, which can reduce inflammation and capsule fibrosis caused by inflammatory macrophage recruitment, activation-related proteins and damage-related molecular patterns released by cells under ischemic and hypoxic environments. At the same time, the self-protective microcapsule-encapsulated therapeutic cell cluster is also provided as a cell transplant material in cell transplantation treatment of diseases, thereby optimizing the therapeutic effect of cell-encapsulated microcapsules in treating chronic diseases.

[0007] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0008] A self-protection microcapsule-encapsulated therapeutic cell cluster comprises a therapeutic cell cluster and a microcapsule encapsulating the therapeutic cell cluster, wherein spermidine engineered cells are also encapsulated in the microcapsule.

[0009] The natural protective factor spermidine has the functions of inhibiting inflammation and regulating oxidative stress, such as inhibiting eosinophils, mast cells, CD4 + / CD8 + Infiltration of inflammatory cells such as T lymphocytes and macrophages. The above technical solution of the present invention creatively introduces spermidine engineered cells to encapsulate them together with therapeutic cell clusters in microcapsules, which greatly improves its application prospects for cell transplantation in vivo. By transforming the cell line, the self-protection microcapsule-encapsulated therapeutic cell cluster of the present invention can obtain cells that continuously autosecrete and produce spermidine, thereby preventing the release of inflammatory cytokines and microcapsule fibrosis caused by cell hypoxia, achieving a one-time treatment and a continuous functional effect.

[0010] The above-mentioned self-protection microcapsule-encapsulated therapeutic cell mass, preferably, the spermidine engineered cell is formed by transferring a spermidine synthase overexpression plasmid into a low oxygen consumption cell line, and particularly preferably, by transferring a spermidine synthase overexpression plasmid into a mesenchymal stem cell. The spermidine engineered cell formed in such a preferred manner can significantly increase the secretion (synthesis) of spermidine in the engineered cell.

[0011] The above self-protection microcapsules encapsulate the therapeutic cell clusters, preferably, the ratio of the number of cells of the therapeutic cell clusters to the number of spermidine engineered cells in the microcapsules is 10:1 to 20:1 (particularly preferably 15:1 to 20:1). The two cells are mixed in the above preferred ratio to meet the requirements of both the secretion of spermidine and the survival rate of the therapeutic cell clusters and the performance of the therapeutic function.

[0012] The above-mentioned self-protection microcapsule-encapsulated therapeutic cell cluster is preferably at least one of a pancreatic islet cell cluster, a neural stem cell cluster or a neural precursor cell cluster. This is mainly based on the potential therapeutic value of the self-protection microcapsule-encapsulated therapeutic cell cluster of the present invention in treating diabetes and neurodegenerative diseases.

[0013] The above-mentioned self-protection microcapsule encapsulates the therapeutic cell cluster, and preferably, the therapeutic cell cluster is an NT2 cell cluster.

[0014] The above-mentioned self-protection microcapsules encapsulate the therapeutic cell clusters. Preferably, the microcapsules are made of alginate-derived polymers, preferably sodium alginate-derived polymers. By using alginate-derived polymers with biological semipermeable membranes and high tissue compatibility to encapsulate the therapeutic cell clusters and spermidine engineered cells, they can be prevented from being recognized by the host immune system and immune rejection, thereby avoiding the side effects of using immunosuppressants, and at the same time, the survival rate of the therapeutic cells in the body can be improved.

[0015] In the above-mentioned self-protection microcapsule-encapsulated therapeutic cell cluster, preferably, the secretion amount of spermidine of the spermidine engineered cells in the microcapsule is 5-10 μmol / L.

[0016] As a general technical concept, the present invention also provides a method for preparing a self-protection microcapsule-encapsulated therapeutic cell cluster, comprising the following steps:

[0017] (1) obtaining therapeutic cell clusters and spermidine engineered cells;

[0018] (2) The therapeutic cell clusters obtained in step (1) and the spermidine engineered cells are mixed into a microencapsulation solution, and microcapsules are prepared by a microencapsulation device to form self-protective microcapsules encapsulating the therapeutic cell clusters.

[0019] In the above-mentioned preparation method, preferably, obtaining the spermidine engineered cells comprises the following steps: constructing a lentivirus overexpressing spermidine synthase plasmid to produce a spermidine synthase overexpressing lentivirus, which is then infecting mesenchymal stem cells, and obtaining stably overexpressed spermidine engineered cells after drug screening.

[0020] In the above preparation method, preferably, the microcapsule generating solution is a sodium alginate solution, and the step (2) specifically refers to: adding 5×10 5 ~5×10 6 The amount of the obtained therapeutic cell clusters is mixed with the obtained therapeutic cell clusters, and the amount of the therapeutic cell clusters added is 5000-10000 IEQ / mL sodium alginate solution (the unit of IEQ is pancreatic islet equivalent, which is approximately 1 IEQ = 1000 cells after conversion) or 5×10-1×10 5The mass concentration of the sodium alginate solution is 3% to 5%.

[0021] As a general technical concept, the present invention also provides a use of the above-mentioned self-protection microcapsule-encapsulated therapeutic cell cluster as a cell transplantation material in cell transplantation to treat diseases.

[0022] In the above applications, preferably, the self-protection microcapsule-encapsulated therapeutic cell cluster is used as a cell transplantation material to prevent the release of inflammatory cytokines or microcapsule fibrosis caused by hypoxia of the therapeutic cells in cell transplantation to treat diseases.

[0023] In the above applications, preferably, the cell transplantation treatment of diseases includes diabetes or Parkinson's disease. Our research found that after the microcapsules are transplanted into the peritoneum, deposition and aggregation will inevitably occur, which will cause hypoxia of the therapeutic cells in the microcapsules. In the hypoxic and low-nutrient environment in the body, the therapeutic cells will be stressed and produce a series of cytokines that help recruit and activate inflammatory macrophages. The damaged pancreatic islets will also secrete damage-related molecular patterns. These molecules will be released into the body through the pores of the microcapsules and recognized by pattern recognition receptors, activating pro-inflammatory pathways; and blood vessels damaged by surgery will release platelets, complement, etc., and recruit neutrophils, which will further induce immune responses; therefore, the use of microencapsulation technology to prevent direct contact with immune cells will inevitably recruit and activate inflammatory pathways, causing collagen to accumulate around the microcapsules and cause fibrosis, ultimately leading to transplant failure. The present invention transforms the cell line so that the self-protective microcapsule-encapsulated therapeutic cell cluster of the present invention can obtain cells that continuously autosecrete and produce spermidine. The secreted spermidine inhibits the secretion of proinflammatory cytokines such as IL-1β, IL-4, IL-6, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and the production of proinflammatory mediators such as nitric oxide (NO), prostaglandin 2 (PEG2), and reactive oxygen species (ROS) by down-regulating the expression of proinflammatory cytokine and mediator synthesis genes, and blocks inflammation-related signaling pathways, including PI3K / Akt signaling pathway, MARKs signaling pathway, NF-κB signaling pathway, etc., by reducing the phosphorylation and nuclear translocation of key proteins, thereby preventing the release of inflammatory cytokines or microcapsule fibrosis caused by hypoxia of therapeutic cells.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: the enzymes related to the synthesis of protective factors are overexpressed in specific auxiliary cells, so that the cells have the ability to continuously secrete protective factors, and are encapsulated in microcapsules together with therapeutic cells. This can reduce the release of inflammatory factors by therapeutic cells in an oxygen-deficient environment, avoid the inflammation-fibrosis phenomenon caused by the activation of inflammatory-related molecular patterns, and thus improve the efficacy of microencapsulated therapeutic cells in treating chronic diseases.

[0025] The product of the present invention not only changes the microenvironment in which the therapeutic cell encapsulated microcapsules are located, but also provides a device for sustainably generating anti-inflammatory substances, thereby improving the survival rate of therapeutic cells in an oxygen-deficient environment; reducing the expression of inflammatory cytokine genes in therapeutic cells, reducing the expression of inflammatory macrophage recruitment / chemotaxis-related genes in therapeutic cells, and reducing the expression of oxidative stress-related genes in therapeutic cells, thereby effectively improving the survival time of therapeutic cells in the microcapsules.

[0026] The product of the present invention can effectively protect therapeutic cells, reduce inflammation and the fibrosis it produces, and is beneficial to improving the therapeutic effect of therapeutic cell clusters. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is a comparison of the amount of spermidine synthesized by the spermidine synthase-overexpressing cells in Example 1 of the present invention.

[0028] Figure 2 This is a comparison of the effect of spermidine engineered cells on the viability of therapeutic pancreatic islet cell clusters in a hypoxic environment in Example 1 of the present invention.

[0029] Figure 3 This is a comparison of the expression of the inflammatory cytokine IL-6 by the spermidine engineered cells and the therapeutic cells in a hypoxic environment in Example 1 of the present invention.

[0030] Figure 4 This is a comparison of the expression of the inflammatory cytokine TNF-α by the spermidine engineered cells and the therapeutic cells in a hypoxic environment in Example 1 of the present invention.

[0031] Figure 5 This is a comparison of the expression of the monocyte chemotaxis-related gene MCP-1 (CCL2) in Example 1 of the present invention between the spermidine engineered cells and the therapeutic cells under a hypoxic environment.

[0032] Figure 6 This is a comparison of the expression of the monocyte chemotaxis-related gene RANTES (CCL5) in Example 1 of the present invention with the therapeutic cells under a hypoxic environment.

[0033] Figure 7 This is a comparison of the expression of the macrophage recruitment / chemotaxis-related gene MIP-1α (CCL3) in Example 1 of the present invention between the spermidine engineered cells and the therapeutic cells under a hypoxic environment.

[0034] Figure 8 This is a comparison of the expression of the oxidative stress-related gene iNOS in the spermidine engineered cells and therapeutic cells in Example 1 of the present invention under a hypoxic environment.

[0035] Fig. 9The results are comparative test results of measuring the spermidine content in the culture medium after spermidine was encapsulated by microcapsules in Example 1 of the present invention.

[0036] Fig.10 The comparative test results of the present invention show that the microcapsule encapsulated spermidine can prevent microcapsule fibrosis. DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present invention, the present invention is described in more comprehensive and detailed manner below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0038] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0039] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0040] The materials and reagents used in the following examples include:

[0041] 1640 culture medium, DMEM high glucose medium, fetal bovine serum, Ficoll, type V collagenase, HBSS buffer, DTZ, sodium alginate, calcium chloride, EGTA, AO / PI dye, 50mL centrifuge tube, ELISA kit

[0042] Embodiment 1:

[0043] A self-protective microcapsule-encapsulated therapeutic cell cluster of the present invention comprises a pancreatic islet cell cluster and a microcapsule encapsulating the pancreatic islet cell cluster, wherein the microcapsule also encapsulates spermidine engineered cells. In this embodiment, the spermidine engineered cells are formed by transferring a spermidine synthase overexpression plasmid into mesenchymal stem cells, and encapsulating the spermidine synthase overexpression plasmid and the islet cells together in a sodium alginate microcapsule. The secretion amount of spermidine by the spermidine engineered cells in the microcapsule of this embodiment is 9.27±0.04μmol / L, and the ratio of the number of cells of the pancreatic islet cell cluster to the number of spermidine engineered cells in the microcapsule of this embodiment is 10000IEQ:5×10 5 , i.e. 20:1. In this embodiment, the microcapsules are made of sodium alginate derived polymer.

[0044] The method for preparing the self-protection microcapsule-encapsulated therapeutic cell cluster in the above embodiment, taking microcapsule-encapsulated pancreatic islet cells and spermidine engineered cells as an example, specifically comprises the following steps:

[0045] 1. Obtaining Pancreatic Islet Cells and Spermidine-engineered Cells

[0046] 1.1 Acquisition of spermidine engineered cells

[0047] In this embodiment, the acquisition of spermidine engineered cells includes the following steps: constructing a lentivirus overexpressing spermidine synthase plasmid, producing spermidine synthase overexpressing lentivirus, infecting mesenchymal stem cells, obtaining a stably overexpressed spermidine engineered cell line after drug screening, and collecting the supernatant of the spermidine engineered cell line culture medium to detect the spermidine content.

[0048] Detection of spermidine secretion by spermidine engineered cells: The spermidine engineered cells obtained in this example were cultured at 5×10 4 ~1×10 6 The number of cells / mL was inoculated into a 6-well plate and cultured at 37°C and 5% CO2 for 48 to 72 hours. The supernatant of the culture medium was collected and the concentration of spermidine in the supernatant was detected by ELISA kit. The test results were as follows: Figure 1 As shown, spermidine engineered cells / non-engineered cells attached to the wall and floating in the 6-well plate were collected for cell number calibration. Figure 1 It can be seen that in the spermidine engineered cells in this embodiment, the spermidine secretion of the engineered cells overexpressing spermidine synthase is 9.27±0.04 μmol / L (the spermidine secretion of the cells transfected with the empty vector is 7.29±0.28 μmol / L, and the cells transfected with the empty vector are cells into which the "blank lentiviral plasmid" has been transferred). After calibration by cell number, the spermidine synthesis secretion is increased by 2.36 times.

[0049] 1.2 Isolation and purification of pancreatic islet cells

[0050] Purified pancreatic islet cell clusters were obtained using the mouse, pig and human islet separation technology established by our team, with a purity greater than 85% and an activity greater than 90%.

[0051] 2. Construction of spermidine engineered cells / islet cell cluster co-encapsulation microcapsules

[0052] The spermidine engineered cells prepared in step 1.1 were cultured at a rate of 5 × 10 5 / mL sodium alginate solution and the pancreatic islet cell clusters separated in step 1.2 are mixed with a sodium alginate solution with a mass concentration of 4% at 10000IEQ / mL sodium alginate solution, and microcapsules are prepared by a microcapsule generating device to finally form spermidine engineered cells and pancreatic islet cell clusters co-encapsulated microcapsules, which are placed in a 120mM calcium chloride solution for equilibrium for 30 minutes to obtain the self-protection microcapsule-encapsulated therapeutic cell clusters of the present invention.

[0053] 3. Performance description of the self-protective microcapsule-encapsulated therapeutic cell clusters in this example

[0054] The spermidine-secreting engineered mesenchymal stem cells constructed by the above method and the pancreatic islet cell clusters were co-encapsulated in microcapsules, placed in a 37°C hypoxic culture environment (5% CO2, 1% O2), and cultured in 1640 complete medium for 48 to 96 hours; after collecting the microcapsules, EGTA was used to dissolve the microcapsules, and then the spermidine-engineered cells and pancreatic islet cell clusters were mixed and precipitated, and the pancreatic islet cell clusters were separated and collected through a 70μm cell sieve, and the activity was detected using the AO / PI staining method. The test results can be seen Figure 2 : The viability of islet cell clusters in co-encapsulated microcapsules was increased by 15.87% compared with that in single-encapsulated microcapsules (93.20±3.04% vs 77.33±2.16%, P<0.05).

[0055] 4. Detection of the expression of inflammatory cytokine genes in the self-protective microcapsule-encapsulated therapeutic cell clusters under hypoxic culture conditions in this example

[0056] The self-protected microcapsule-encapsulated therapeutic cell clusters constructed in this example were cultured in 1640 complete medium at 37°C, 5% CO2, 1% O2 for 48 to 96 hours, the microcapsules were collected and dissolved with EGTA, spermidine engineered cells and islet cell clusters were mixed and precipitated, and the islet cell clusters were separated and collected by a 70 μm cell sieve, RNA extraction, cDNA reverse transcription, and qPCR detection of IL-6 and TNF-α gene expression were performed, and the gene expression results were compared with those of the islet cell clusters encapsulated alone. The comparison results are as follows: Figure 3 , Figure 4 As shown. Figure 3 , Figure 4 It can be seen that the gene expression of inflammatory cytokine IL-6 in islet cells co-encapsulated in microcapsules with spermidine synthase-overexpressing engineered cells and islet cell clusters was reduced by 19.00% (81.00±6.28% vs 100.52±10.23%, P<0.05) and the gene expression of TNF-α was reduced by 57.38% (42.72±1.35% vs 100.13±7.50%, P<0.05) compared with islet cells encapsulated in microcapsules alone.

[0057] 5. Detection of the expression of monocyte chemotaxis-related genes in the self-protective microcapsule-encapsulated therapeutic cell clusters under hypoxic culture conditions in this example

[0058] The self-protected microcapsule-encapsulated therapeutic cell clusters constructed in this example were cultured in 1640 complete medium at 37°C, 5% CO2, 1% O2 for 48 to 96 hours, the microcapsules were collected and dissolved with EGTA, spermidine engineered cells and islet cell clusters were mixed and precipitated, and the islet cell clusters were separated and collected by a 70 μm cell sieve, RNA extraction, cDNA reverse transcription, and qPCR detection of MCP-1 (CCL2) and RANTES (CCL5) gene expression were performed, and the gene expression results were compared with those of the islet cell clusters encapsulated alone. The comparison results are as follows: Figure 5 , Figure 6 As shown. Figure 5 , Figure 6 It can be seen that the expression of monocyte chemotaxis-related gene MCP-1 (CCL2) decreased by 55.07% (44.93±1.34% vs 100.15±7.80%, P<0.05), and the expression of RANTES (CCL5) decreased by 32.36% (67.64±5.84% vs 100.47±9.56%, P<0.05).

[0059] 6. Detection of the expression of macrophage recruitment / chemotaxis-related genes in the self-protection microcapsule-encapsulated therapeutic cell clusters under hypoxic culture conditions in this example

[0060] The self-protected microcapsule-encapsulated therapeutic cell clusters constructed in this example were cultured in 1640 complete medium at 37°C, 5% CO2, 1% O2 for 48 to 96 hours, the microcapsules were collected and dissolved with EGTA, spermidine engineered cells and islet cell clusters were mixed and precipitated, and the islet cell clusters were separated and collected by a 70 μm cell sieve, RNA extraction, cDNA reverse transcription, and qPCR detection of MIP-1α (CCL3) gene expression were performed, and the gene expression results were compared with those of the islet cell clusters encapsulated alone. The comparison results are as follows: Figure 7 As shown. Figure 7 It can be seen that the expression of inflammatory macrophage recruitment / chemotaxis-related gene MIP-1α (CCL3) was reduced by 59.29% (40.71±0.85% vs 100.58±11.05%, P<0.05).

[0061] 7. Detection of the expression of oxidative stress genes in the therapeutic cell clusters encapsulated by the self-protective microcapsules of this example under hypoxic culture conditions

[0062] The self-protected microcapsule-encapsulated therapeutic cell clusters constructed in this example were cultured in 1640 complete medium at 37° C., 5% CO 2 , 1% O 2 for 48 to 96 hours, the microcapsules were collected and dissolved with EGTA, spermidine engineered cells and islet cell clusters were collected and mixed, and the islet cell clusters were separated and collected by a 70 μm cell sieve, RNA extraction, cDNA reverse transcription, and qPCR detection of iNOS gene expression were performed, and the gene expression results were compared with those of the islet cell clusters encapsulated alone. The comparison results are as follows: Figure 8 As shown. Figure 8 It can be seen that the expression of oxidative stress-related gene iNOS was reduced by 78.66% (21.34±0.92% vs 100.04±2.80%, P<0.05).

[0063] Since the therapeutic microcapsules may need to stay in the patient's body for a long time, the technology of embedding spermidine in microcapsules can slowly release spermidine in the body, but it will eventually be exhausted after a period of time, and after exhaustion, it will no longer be able to protect the therapeutic cells from hypoxic damage; the present invention does not directly embed spermidine but uses engineered cells that secrete spermidine, which will ensure that the engineered cells in the microcapsules can continue to secrete spermidine, achieving long-term and continuous protection of the therapeutic cells. Fig. 9 As shown, Fig. 9 In order to measure the result of spermidine content in the culture medium after spermidine was encapsulated in microcapsules, that is, the result of spermidine being released from the microcapsules, we made 5mL of sodium alginate microcapsules with a spermidine content of 10μmol / L. The results showed that after the microcapsules were placed in the culture medium for 15 minutes, the spermidine content in the culture medium had reached 10μmol / L, and spermidine gradually decreased with the passage of time. The analysis showed that this might be due to the degradation of spermidine.

[0064] Obviously, the self-protection microcapsule-encapsulated therapeutic cell clusters described in this embodiment can be used as a new therapeutic cell protection technology in cell transplantation to treat diseases and has obvious application prospects.

[0065] Other application prospects:

[0066] Application of self-protective microcapsules encapsulating therapeutic cell clusters in Parkinson's disease to block the release of inflammatory cytokines caused by hypoxia in therapeutic cells.

[0067] Since our team has reported the use of microcapsule-encapsulated choroid plexus cells to treat Parkinson's syndrome (see Luo Xianming, Wang Wei. Histological observation of intracerebral transplantation of neural cell microcapsules in monkeys with hemiparkinsonism [J]. Life Science Research, 2012, 16(03): 196-200. DOI: 10.16605 / j.cnki.1007-7847.2012.03.005; see Luo Xianming, Liao Jian, Wang Wei. Study on the physical and biochemical properties of choroid plexus cell microcapsules before and after intracerebral transplantation [J]. Life Science Research, 2012, 16(01): 15-19. DOI: 10.16605 / j.cnki.1007-7847.2012.01.001).

[0068] It has been previously reported in the literature (see Dalen, Marit Lunde et al. "Post-hypoxichypothermia is protective in human NT2-N neurons regardless of oxygen concentration during reoxygenation." Brain research vol.1259(2009):80-9.doi:10.1016 / j.brainres.2008.12.055) that the viability of NT2 cells decreased significantly under hypoxic conditions, and the expression of inflammation-related genes and encoded proteins (interleukin-8, IL-8; interferon-induced protein 10, IP-10; monocyte chemoattractant protein-1, MCP-1; basic fibroblast growth factor, bFGF) increased significantly. Some studies have also pointed out (see Elisabeth et al. "Inflammatory receptors and pathways in human NT2-N neurons during hypoxia and reoxygenation. Impact of acidosis." Brain research vol. 1217 (2008): 37-49. doi: 10.1016 / j.brainres.2008.04.038), inflammatory receptors (chemokine receptors CXCR1, CXCR2, CCR2; tumor necrosis factor receptor TNFR1) and apoptosis pathways (Bax expression is significantly upregulated; Bcl-2 and Bcl-xL expression is significantly downregulated) are activated during hypoxia and reoxygenation of NT2-N neurons, which will affect the viability of neuronal cell transplantation and the therapeutic effect, suggesting that there is room for optimization in the use of microencapsulated therapeutic cells to treat Parkinson's syndrome.

[0069] According to the process principle of the present invention, the self-protection microcapsule-encapsulated therapeutic cell cluster of the present invention can be foreseen to be used as a cell transplant material for preventing the release of inflammatory cytokines caused by hypoxia of therapeutic cells in Parkinson's disease.

[0070] Application of self-protective microcapsule-encapsulated therapeutic cell clusters as cell transplantation materials to prevent microcapsule fibrosis in Parkinson's disease.

[0071] Obviously, the above self-protective microcapsule-encapsulated therapeutic cell clusters can be used as cell transplant materials in cell transplantation to treat diseases, especially as cell transplant materials that prevent microcapsule fibrosis in cell transplantation to treat diseases.

[0072] Microcapsules were prepared by mixing sodium alginate with 100 μM exogenous spermidine and encapsulating pancreatic islet cell clusters. The microcapsules were transplanted into C57BL / C mice and the microcapsules were removed 22 and 36 days after transplantation. Fig.10 As shown, it was observed that the microcapsules mixed with 100 μM exogenous spermidine produced only a very small amount of fibrosis, while the microcapsules without exogenous spermidine produced a large amount of fibrosis. It can be fully predicted that the self-protected microcapsule-encapsulated therapeutic cell mass of the present invention contains spermidine produced by spermidine engineered cells, and the continuously accumulated spermidine can significantly reduce the fibrosis of the pancreatic islet encapsulated microcapsules.

Claims

1. A self-protective microcapsule-encapsulated therapeutic cell cluster, comprising a therapeutic cell cluster and a microcapsule encapsulating the therapeutic cell cluster, It is characterized in that The microcapsule also encapsulates spermidine engineered cells, which are formed by transferring spermidine synthase overexpression plasmid into mesenchymal stem cells, and the therapeutic cell cluster is a pancreatic islet cell cluster.

2. The self-protective microcapsule-encapsulated therapeutic cell cluster according to claim 1, It is characterized in that The ratio of the number of therapeutic cell clusters to the number of spermidine engineered cells in the microcapsule is 10:1 to 20:

1.

3. The self-protective microcapsule-encapsulated therapeutic cell cluster according to claim 1, It is characterized in that The microcapsules are made of alginate-derived polymers.

4. The self-protective microcapsule-encapsulated therapeutic cell cluster according to claim 3, It is characterized in that The alginate-derived polymer is a sodium alginate-derived polymer.

5. The self-protective microcapsule-encapsulated therapeutic cell cluster according to claim 1, It is characterized in that The secretion amount of spermidine of the spermidine engineered cells in the microcapsule is 5-10 μmol / L.

6. A method for preparing a self-protective microcapsule-encapsulated therapeutic cell cluster, The following steps are involved: (1) obtaining therapeutic cell clusters and spermidine engineered cells, wherein the therapeutic cell clusters are pancreatic islet cell clusters; (2) mixing the therapeutic cell cluster obtained in step (1) and the spermidine engineered cells into a microencapsulation solution, and preparing microcapsules using a microencapsulation device to form a self-protective microcapsule-encapsulated therapeutic cell cluster; The acquisition of the spermidine engineered cells comprises the following steps: constructing a lentivirus overexpressing spermidine synthase plasmid to produce a spermidine synthase overexpressing lentivirus, which is then infecting mesenchymal stem cells, and obtaining stably overexpressed spermidine engineered cells after drug screening.

7. The preparation method according to claim 6, It is characterized in that The microcapsule generating solution is a sodium alginate solution, and the step (2) specifically refers to: adding the obtained spermidine engineered cells at a concentration of 5×10 5 ~5×10 6 The amount of the therapeutic cell clusters added is 5000 to 10000 IEQ / mL of sodium alginate solution or 5×10 to 1×10 5 The mass concentration of the sodium alginate solution is 3% to 5%.

8. Use of the self-protection microcapsule-encapsulated therapeutic cell cluster according to any one of claims 1 to 5 in the preparation of cell transplantation materials for cell transplantation treatment of diabetes.

9. The use according to claim 8, It is characterized in that The cell transplant material is a cell transplant material that blocks the release of inflammatory cytokines or microcapsule fibrosis caused by therapeutic cell hypoxia.

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