阅读说明：本技术 一种间充质干细胞制剂及其应用 (Mesenchymal stem cell preparation and application thereof ) 是由 沈萍萍 孙璐琛 杨南飞 郑薇 左诗曼 于 2021-04-08 设计创作，主要内容包括：本发明属于医药生物技术领域,具体涉及一种间充质干细胞制剂的制备方法与应用。本发明对人源间充质干细胞(MSCs),经过由特定的多种生物活性分子和化合物组成的活化剂处理得到活化的间充质干细胞(Active MSCs)。所述Active MSCs特异性大量分泌白介素6,(IL6),吲哚胺2,3加双氧酶(IDO),骨形成蛋白6(BMP6),中性粒细胞弹性蛋白酶,组织蛋白酶G,超氧化物歧化酶,组织金属蛋白酶抑制因子3(TIMP3),蛋白质赖氨酸6氧化酶(LOX),IGFL3,TNFAIP6,CYR61,TGFβ等多种生物活性物质,能够治疗自身免疫性疾病、提高机体的抗肿瘤能力、预防衰老,增加机体免疫力以及促进机体组织再生能力。本发明采用间充质干细胞活化剂对间充质干细胞进行诱导活化得到的Active MSCs无药物残留,并能高表达具有免疫调节和组织再生能力的生物活性物质。相较于化疗,Active MSCs移植的毒副作用更低,且具备低免疫原性,在肿瘤和免疫性疾病的治疗以及预防衰老方面具有重大的意义。(The invention belongs to the technical field of medical biology, and particularly relates to a preparation method and application of a mesenchymal stem cell preparation. The invention obtains activated mesenchymal stem cells (Active MSCs) by treating human Mesenchymal Stem Cells (MSCs) with an activating agent consisting of specific various bioactive molecules and compounds. The Active MSCs specifically secrete a large amount of interleukin 6, (IL6), indoleamine 2, 3 dioxygenase (IDO), bone morphogenetic protein 6(BMP6), neutrophil elastase, cathepsin G, superoxide dismutase, tissue metalloproteinase inhibitory factor 3(TIMP3), protein lysine 6 oxidase (LOX), IGFL3, TNFAIP6, CYR61, TGF beta and other bioactive substances, and can treat autoimmune diseases, improve the anti-tumor capacity of organisms, prevent aging, increase the immunity of the organisms and promote the regeneration capacity of tissues of the organisms. The Active MSCs obtained by inducing and activating the mesenchymal stem cells by using the mesenchymal stem cell activator have no drug residue and can highly express bioactive substances with immunoregulation and tissue regeneration capabilities. Compared with chemotherapy, the Active MSCs has lower toxic and side effects in transplantation, low immunogenicity and great significance in treating tumors and immune diseases and preventing aging.)

1. The components and compatibility of the mesenchymal stem cell activator are as follows:

alpha-MEM medium of 10% fetal bovine serum, 0.5-10. mu.M calcitriol (1, 25-dihydrouvitamin D3, 1, 25D3), 1-10. mu.M asiaticoside, 0.1-1. mu.M rosiglitazone (rosiglitazone), 2-10mM metformin, 0.5-2mM vitamin C and 0.5-1.5. mu.M sodium pyruvate. In some embodiments, the pharmaceutical excipients may include: phosphate Buffered Saline (PBS), 0.9% aqueous sodium chloride, Tween-20 (Tween-20), dimethyl sulfoxide (DMSO).

2. A mesenchymal stem cell preparation: and (3) incubating the extracted mesenchymal stem cells by using a mesenchymal stem cell activator to prepare induced activated mesenchymal stem cells (Active MSCs)3. the application of the mesenchymal stem cell preparation in immune system diseases.

3. An application of the mesenchymal stem cell preparation in treating autoimmune diseases, acute myelogenous leukemia, preventing senility, improving immunity and promoting tissue regeneration is disclosed.

4. The mesenchymal stem cell activator according to claim 1, wherein the Active MSCs can be prepared using any one or more of the activator components. The components of the mesenchymal stem cell activator are compatible, wherein any component can be replaced by other homologous or similar effector compounds, and the mesenchymal stem cell activator specifically comprises the following components:

(1)1, 25D3 is replaceable as: alfacalcidol (25(OH)₂D3) Paricalcitol, doxercalciferol, fluorocalcitol, maxacalcitol and the like.

(2) Rosiglitazone can be replaced by: thiazolidinedione drugs (TZDs) such as troglitazone (troglitazone), pioglitazone (pioglitazone), and ciglitazone (ciglitazone).

(3) Metformin can be replaced by: allantoin and ginsenoside

(4) The CDDO may be replaced with: oleanolic acid, CDDO-Me, CDDO-imidazolide, CDDO ethyl ester.

5. The mesenchymal stem cell preparation according to claim 2, wherein the Mesenchymal Stem Cells (MSCs) are any one or more of human umbilical cord-derived mesenchymal stem cells (UC-MSCs), human bone marrow-derived mesenchymal stem cells (BM-MSCs), or human adipose tissue-derived mesenchymal stem cells (ADSCs).

6. The mesenchymal stem cell preparation of claim 2, wherein the acellular component comprises an activator component and a pharmaceuticauy acceptable adjuvant component; the activator component is any one or any combination of the components of the activator component of claim 1. The preparation of the preparation can comprise the following three types:

(1) the stem cell preparation prepared by treating MSCs with the activator of claim 1 to obtain Active MSCs and mixing with pharmaceutical excipients.

(2) Treating MSCs with the activator of claim 1 to produce Active MSCs; mixing the obtained Active MSCs with an activator and pharmaceutic adjuvants to prepare the stem cell preparation.

(3) MSCs are directly mixed with the activator of claim 1 and mixed with a pharmaceutically acceptable excipient to produce a stem cell preparation.

7. The use of claim 3, wherein the Active MSCs meet the following criteria:

(1) cell surface markers: CD14, CD34, CD45 negative expression; CD73, CD90, CD105 positive expression.

(2) Differentiation characteristics of three lineages (osteogenic, chondrogenic, adipogenic)

(3) Secretion of factors: interleukin 6, (IL6), indoleamine 2, 3 dioxygenase (IDO), bone morphogenetic protein 6(BMP6), neutrophil elastase, cathepsin G, superoxide dismutase, tissue metalloproteinase inhibitor 3(TIMP3), protein lysine 6 oxidase (LOX), IGFL3, TNFAIP6, CYR61, TGF β.

8. According to the criteria of claim 7, the secretion factor is identified as a functional active factor of the mesenchymal stem cell preparation for disease treatment and prevention. The functionally active factor should be any one or combination of more of claims 11. In some embodiments, a means of promoting upregulated expression or release of a secreted factor as described herein by using gene editing or drug-induced means in an mesenchymal stem cell is considered to be within the scope of the present invention.

9. Use according to claim 3, wherein the immune system disorders comprise: autoimmune hepatitis, pulmonary fibrosis, liver fibrosis; acute myeloid leukemia includes: acute monocytic leukemia, acute granulo-monocytic leukemia; the application of preventing aging, enhancing immunity and promoting regeneration of organism tissues comprises the following steps: skin injury repair (burns, rubbing injuries), diabetic foot, allergic dermatitis.

10. Use of a disease according to claims 13-15, wherein the stem cell preparation is administered by: peripheral intravenous infusion at effective dose of 5 × 10⁶/kg-10×10⁷/kg。

Technical Field

The invention belongs to the field of stem cell treatment, and particularly relates to a preparation method and application of a mesenchymal stem cell preparation.

Background

Mesenchymal Stem Cells (MSCs) are multipotent adult stem cells that are widely distributed in Mesenchymal tissues, such as fat, bone marrow, etc. Mesenchymal stem cells not only have the same strong self-renewal and multipotentiality as other stem cells, but also have the following three characteristics: (1) the source is wide, the separation, the culture and the purification are easy, and the stem cells still have the characteristics after multiple passages; (2) MSCs exert their immunomodulatory functions by interacting with immune cells in the natural and acquired immune system by direct contact or paracrine means; (3) the MSCs have low immunogenicity, so the allotype requirement is not strict during allografting and the rejection reaction is light; (4) MSCs have the property of "homing" to inflammatory and tumor sites. Due to the biological characteristics of the mesenchymal stem cells, the mesenchymal stem cells have wide clinical application prospects in the treatment aspects of autoimmune diseases, inflammations, cancers and the like. And can also be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathological changes.

Bone marrow derived mesenchymal stem cells (BM-MSCs) are the most commonly used clinically, mainly used for autologous transplantation and can be used for treating various chronic diseases. But gradually find many disadvantages in the application process of the autologous mesenchymal stem cells. (1) The amplification capacity is greatly different among individuals; (2) the culture time is long, and the requirement of the state of an illness cannot be met in time; (3) it has been reported that BM-MSCs derived from cancer patients can also protect tumor cells from the killing effect of chemotherapeutic drugs. Therefore, autologous transplanted BM-MSCs fail to achieve a cancer course alleviating effect and may even exacerbate tumor growth. These problems severely restrict the clinical application of autologous mesenchymal stem cells.

Umbilical cord mesenchymal stem cells (UC-MSCs) are mesenchymal stem cells isolated from Umbilical cord tissue of a newborn. Compared with other adult mesenchymal stem cells, the UC-MSCs have some unique advantages, such as rapid growth, strong cell secretion capacity, lower immunogenicity and the like. Evidence suggests that allogeneic UC-MSCs from healthy newborns have greater immunomodulatory activity and have been successfully used to treat a variety of autoimmune diseases. Meanwhile, some reports indicate that UC-MSCs have certain antitumor activity. Based on the above, we propose a method for activating mesenchymal stem cells, so that the mesenchymal stem cells can specifically express bioactive molecules and enhance the anti-tumor and immunoregulatory capabilities of the mesenchymal stem cells.

Adipose-derived stem cells (ADSCs) are stem cells with multipotential differentiation potential isolated from Adipose tissue. The adipose-derived stem cells can secrete various cytokines and growth factor factors, and act on the microenvironment around the damaged tissue through paracrine to interfere the proliferation and apoptosis of cells and participate in the growth, regeneration and reconstruction of tissue cells. For example: the fibroblast growth factor secreted by the ADSC promotes the long fiber cells to secrete collagen I and III and fibronectin, promotes the synthesis of collagen, and plays roles of anti-inflammation, antioxidation, injury repair and the like. In addition, the ADSCs have the function of immunoregulation, influence the differentiation and activation of immune cells through the direct action or the paracrine action of the ADSCs and the immune cells, and reestablish the immune balance of the body. Based on the above, we propose a method for activating mesenchymal stem cells, so that the mesenchymal stem cells can specifically express bioactive molecules, and the tissue repair and immunoregulation capabilities of the mesenchymal stem cells are enhanced.

Disclosure of Invention

The first purpose of the invention is to provide a compatibility method of the activating agent of the mesenchymal stem cells.

The second purpose of the invention is to provide a preparation method of the mesenchymal stem cell preparation.

The third purpose of the invention is to provide the application of the mesenchymal stem cell preparation in immune system diseases.

The fourth purpose of the invention is to provide the application of the mesenchymal stem cell preparation in the treatment of acute myeloid leukemia.

The fifth purpose of the invention is to provide the application of the mesenchymal stem cell preparation in the aspects of preventing aging, enhancing the immunity of the organism and promoting the regeneration of the organism tissue.

The activating agent component of the mesenchymal stem cell comprises: 10% fetal bovine serum α -MEM medium, 0.01-10 μ M calcitriol (1, 25-dihydroyvitamin D3, 1, 25D3), 0.1-50 μ M bardoxolone (CDDO), 0.1-10 μ M asiaticoside, 0.1-1 μ M rosiglitazone (rosiglitazone), 0.2-10mM metformin, 0.1-2mM vitamin C and 0.5-1.5 μ M sodium pyruvate.

In some embodiments, the components of the activator may be substituted with other homologous or homologous effector compounds:

(1)1, 25D3 is replaceable as: alfacalcidol (25(OH)₂D3) Paricalcitol, doxercalciferol, fluorocalcitol, maxacalcitol and the like.

(2) Rosiglitazone can be replaced by: thiazolidinedione drugs (TZDs) such as troglitazone (troglitazone), pioglitazone (pioglitazone), and ciglitazone (ciglitazone).

(3) Metformin can be replaced by: allantoin and ginsenoside

(4) The CDDO may be replaced with: oleanolic acid, CDDO-Me, CDDO-imidazolide, CDDO ethyl ester

The preparation method of the mesenchymal stem cell preparation comprises two parts: extraction of mesenchymal stem cells, activation of mesenchymal stem cells and preparation of preparations.

The mesenchymal stem cells are obtained by separating and extracting umbilical cord of newborn, subcutaneous fat of adult or bone marrow of adult respectively by enzyme digestion method.

The activation of mesenchymal stem cells is carried out by incubating the extracted mesenchymal stem cells (12-72 hours) with the mesenchymal stem cell activator of [0014], and preparing induced activated mesenchymal stem cells (Active MSCs)

The preparation of the preparation can be divided into two types:

(1) and (3) mixing the Active MSCs prepared in the step (0015) with pharmaceutic adjuvants to prepare the stem cell preparation.

(2) And (3) mixing the Active MSCs obtained in the step (0015) with the mesenchymal stem cell activator of the step (0011) and pharmaceutic adjuvants to prepare a stem cell preparation.

(3) And (3) mixing the mesenchymal stem cells obtained in the step (0014), the mesenchymal stem cell activator of the step (0011) and pharmaceutic adjuvants to prepare a stem cell preparation.

In some embodiments, the pharmaceutical excipients may include: disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, albumin and glycerol.

The Active MSCs of the invention play the Active ingredients for preventing or treating diseases, and comprise: interleukin 6, (IL6), indoleamine 2, 3 dioxygenase (IDO), bone morphogenetic protein 6(BMP6), neutrophil elastase, cathepsin G, superoxide dismutase, tissue metalloproteinase inhibitor 3(TIMP3), protein lysine 6 oxidase (LOX), IGFL3, TNFAIP6, CYR61, TGF β.

In some embodiments, Active MSCs can be prepared using any of the activator components. In some embodiments, the mesenchymal stem cell preparation may be prepared using any of the activator components and preparing Active MSCs and mixing with a pharmaceutical excipient.

The Active MSCs prepared by the method have the following characteristics:

(1) surface markers: CD14, CD34, CD45 negative expression; CD73, CD90, CD105 positive expression;

(2) can induce differentiation into adipocytes, osteoblasts and chondrocytes, respectively;

(3) one or more active ingredients in the high-expression active ingredients are combined.

The secretion factor in [0018] is identified as a functional active factor of the mesenchymal stem cell preparation for treating and preventing diseases. In some embodiments, a means of promoting upregulated expression or release of a secreted factor as described herein by using gene editing or drug-induced means in an mesenchymal stem cell is considered to be within the scope of the present invention.

The mesenchymal stem cell preparation is applied to immune system diseases. Immune system disorders include: autoimmune hepatitis, idiopathic pulmonary fibrosis, diabetic dermatosis. The prepared Active MSCs can inhibit M1 type polarization of macrophages in vitro, reduce expression of macrophage TNF-alpha, and promote proliferation and migration of endothelial cells and fibroblasts. The prepared mesenchymal stem cell preparation containing Active MSCs can effectively slow down inflammatory reaction and Liver injury in an Autoimmune Liver disease (AIH) model induced by sword bean protein; effectively relieve the level of pulmonary fibrosis in a pulmonary fibrosis model. Meanwhile, the mesenchymal stem cell preparation does not cause obvious toxic and side effects on main organs.

The mesenchymal stem cell preparation is applied to the treatment of Acute Myelogenous Leukemia (AML). The prepared Active MSCs can remarkably promote the apoptosis of AML cells in vitro, promote the cell cycle arrest of leukemia cells, inhibit proliferation and promote the differentiation of the leukemia cells to mature monocytes. The prepared mesenchymal stem cell preparation containing Active MSCs can effectively delay the process of leukemia in an AML mouse model and improve the survival rate of AML mice.

The mesenchymal stem cell preparation is applied to the aspects of preventing aging, improving the immunity of the organism and promoting the regeneration of organism tissues. The prepared Active MSCs can obviously improve the proliferation capacity and the migration capacity of endothelial cells and fibroblasts in vitro. The prepared mesenchymal stem cell preparation containing Active MSCs can obviously promote wound healing and hair regeneration of skin in a skin injury model.

The quality control method of the Active MSCs comprises the following steps:

quality control of induced molecular residue content in culture supernatant of Active MSCs.

And (5) detecting the cell viability of the Active MSCs.

Evaluation of Active MSCs phenotype and multipotentiality.

Evaluation of biological efficacy of Active MSCs.

Drawings

FIG. 1 shows the residue of the formulation components in Active MSCs (B) as measured by High Performance Liquid Chromatography (HPLC) compared to untreated MSCs (A) of the same batch.

FIG. 2 is a CCK8 test for the effect of induced activation of culture on the proliferation of MSCs.

FIG. 3 shows the flow assay of Active MSCs for the expression of markers CD73, CD90, CD105, CD14, CD34 and CD 45.

FIG. 4 shows the differentiation-inducing identification of osteogenic (A), adipogenic (B) and chondrogenic (C) Active MSCs

FIG. 5 shows Active MSCs secreted histone GO clustering analysis

FIG. 6 shows the expression levels of IL-6 and IDO in Active MSCs.

Figure 7 shows that Active MSCs improve macrophage inflammatory function. Detecting the expression of macrophage-related inflammatory factors by flow cytometry (A) Q-pcr, (B) implanting a mesenchymal stem cell preparation containing Active MSCs into an AIH mouse, and analyzing liver morphology and liver pathological sections after 1 week, and (C) analyzing the enzyme activity of ALT/AST in the serum of the mouse treated with liver injury by the mesenchymal stem cell preparation containing Active MSCs.

FIG. 8 shows that Active MSCs alleviate pulmonary fibrosis. Masson staining examined the level of collagen deposition in mice with pulmonary fibrosis.

FIG. 9 is the in vitro apoptosis induction of Active MSCs on U937(A) and THP-1(B) cells.

FIG. 10 is a graph of the ability of Active MSCs to induce differentiation of acute monocytic leukemia cells. Active MSCs treatment of leukemia patients primary samples were flow cytometrically tested for expression of CD14 and CD11b on the cell surface.

FIG. 11 shows the activity of Active MSCs in delaying senescence. 12-month-old mice (A) liver lipid accumulation, (B) steatosis, and (C) the number of leukocytes in peripheral blood.

FIG. 12 is a test of the ability of Active MSCs to promote skin lesion repair. (A) Endothelial cell migration capacity, (B) mesenchymal stem cell preparation containing Active MSCs in mouse skin lesions.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Separation and subculture of umbilical cord mesenchymal stem cells

Preparing tissue digestive enzyme (type II collagenase 250U/ml, neutral protease 100U/ml, hyaluronidase 10U/ml), dissolving in alpha-MEM culture medium at 37 deg.C, and filtering with 0.22 μm filter to remove bacteria;

collecting umbilical cord tissue of newborn infant, cleaning umbilical cord in culture dish with PBS containing 0.1% penicillin-streptomycin double antibody, placing in alpha-MEM culture medium, peeling off three blood vessels, and cutting umbilical cord into pieces of 1-2mm³The tissue mass of (a);

mixing the cut tissue blocks and the prepared tissue digestive enzyme solution in a volume ratio of 1: 1 in a 50ml centrifuge tube, digesting for 3 hours at 37 ℃ and 200rpm until the tissue blocks are basically completely digested;

the digested tissue fluid was centrifuged at 300g for 5min at 4 ℃ and the supernatant was discarded. PBS was resuspended in 50ml of α -MEM medium, centrifuged at 300g for 5min at 4 ℃ and the supernatant was discarded. PBS washing twice, suspending the precipitate in 10% fetal bovine serum, 1% double antibody alpha-MEM medium, inoculating in 10cm diameter cell culture dish, placing at 37 deg.C, 5% CO₂Standing in an incubator with 80% of saturated humidity for adherent culture;

after 3 days, half the volume was changed. Thereafter, every two days, the MSCs grew along adherent tissue blocks or adherent cells;

when the cells grow to 80% abundance, digesting the lower cells by using cell digestive juice containing 0.25% of pancreatin and 0.02% of EDTA; resuspending the cells, centrifuging at 1000rpm for 5min, discarding the supernatant, washing with PBS, centrifuging, discarding the supernatant, resuspending the obtained cell pellet with fresh culture medium, inoculating to a new culture dish, and passaging the cells. After the cells grow to 90% confluence, the next subculture is performed.

The quality control method and standard of the Active MSCs are as follows:

quality control of the induction activation culture solution residues in the culture supernatant of Active MSCs: washing Active MSCs twice with PBS, culturing in α -MEM complete culture medium containing 10% FBS at 37 deg.C and 5% CO2 incubator for 2 days, collecting culture supernatant, and detecting content of inducing molecule in the supernatant by High Performance Liquid Chromatography (HPLC), wherein the content of supernatant should be below 0.1 nM.

Quality control of cell viability of Active MSCs: CFSE tested the cell proliferation capacity of Active MSCs as a control of untreated MSCs from the same batch. The proliferative capacity should be unchanged compared to before treatment.

Quality control of Active MSCs phenotype: the expression of the Active MSCs cell surface markers CD14, CD34, CD45, CD73, CD90, CD105 was identified by flow cytometry as controls on untreated MSCs from the same batch. This indicates that marker expression should not be significantly altered compared to before treatment.

Active MSCs multipotency evaluation: using untreated MSCs of the same batch as a control, and simultaneously carrying out adipogenic, osteogenic and chondrogenic induced differentiation and identification on Active MSCs: adopting MSCs osteogenic and adipogenic differentiation induction culture medium and induction scheme of Guangzhou Seisakuchen organisms to induce adipogenic and osteogenic differentiation; inducing chondrogenic differentiation by adopting MSCs chondrogenic differentiation induction culture medium and induction scheme of Stem Cell. The strength change of the differentiation capacity is judged by morphological staining and observation. The differentiation capacity of the three lineages should not be significantly changed compared to that before induction.

The performance evaluation method and standard of Active MSCs are as follows: the expression of IL-6, IDO, BMP6, neutrophil elastase, cathepsin G, superoxide dismutase, tissue metalloproteinase inhibitor 3 and insulin-like growth factor binding protein 3 in Active MSCs is detected by real-time quantitative PCR (RT-qPCR) by using untreated MSCs of the same batch as a control, and is increased by 3 times or more.

The foregoing examples further illustrate the present invention but are not to be construed as limiting thereof. It will be apparent to those skilled in the art that modifications and substitutions to methods, steps or conditions of the invention can be made without departing from the spirit and substance of the invention.