WO2014075634A1

WO2014075634A1 - Use of stromal vascular layer cell and mesenchymal progenitor cell for preventing or treating osteoarthritis

Info

Publication number: WO2014075634A1
Application number: PCT/CN2013/087284
Authority: WO
Inventors: 曹卫; 张丽; 赵光宇; 关战军; 王新华
Original assignee: 西比曼生物科技（上海）有限公司; 西比曼生物科技（无锡）有限公司
Priority date: 2012-11-16
Filing date: 2013-11-16
Publication date: 2014-05-22
Also published as: CN103816183A

Abstract

Disclosed are uses of stromal vascular layer cells and mesenchymal progenitor cells for preventing and treating osteoarthritis. Also provided are a pharmaceutical composition of stromal vascular layer cells and mesenchymal progenitor cells, and a method for preventing and treating osteoarthritis.

Description

Application of interstitial vascular layer cells and mesenchymal progenitor cells in preventing or treating osteoarthritis

The invention belongs to the field of stem cells and biomedicine. In particular, the invention relates to the use of mesenchymal vascular layer cells and mesenchymal progenitor cells for the prevention or treatment of osteoarthritis. Background technique

Osteoarthritis (OA) is a chronic joint disease characterized by degeneration, destruction and bone hyperplasia of articular cartilage. OA has a prevalence of 50% in people over 60 years of age and 80% in people over 75 years of age. The disability rate of the disease can be as high as 53% (Chinese Society of Orthopaedic Branch, 2007 Revision of the Guide to Osteoarthritis). OA occurs in joints that are heavy and active, such as the knee joint. Therefore, it is called knee osteoarthritis (KOA). Knee osteoarthritis is a serious health hazard to human health and is one of the major diseases that cause dysfunction in the elderly.

At present, the overall treatment principle of knee osteoarthritis (KOA) is to combine medical and non-pharmacological treatments, if necessary, to perform surgery to reduce or eliminate pain, correct deformity, improve or restore joint function, and improve quality of life. .

Drug treatment mainly includes analgesics such as non-organic anti-inflammatory drugs (KSAIDs), intra-articular injection of hyaluronic acid (HA) or glucocorticoids, improvement of disease-like drugs and cartilage protective agents. These drugs can delay the course of the disease to a certain extent, improve the symptoms of patients, but the curative effect is limited, can not reverse the pathological process, or repair cartilage damage, and most of the drugs have obvious side effects.

When the conventional therapy is ineffective, if there is joint deformity and joint dysfunction, surgical treatment must be performed. The main surgical approach is through arthroscopy (open endoscopy) and open surgery. Although it can temporarily relieve pain, the long-term effect is not ideal.

In addition, tissue engineering efforts, such as autologous chondrocyte transplantation or matrix-induced autologous soft bone cell transplantation, offer long-term solutions to the bioremediation or regeneration of degenerative joint tissue. However, a major limitation of autologous chondrocyte transplantation or matrix-induced autologous chondrocyte transplantation is the inability to treat larger cartilage defects, its effects on donor site destruction, dedifferentiation, and limited survival time of chondrocytes, Not for KOA patients.

It is precisely because the immune rejection of allogeneic chondrocyte transplantation is very strong, and there is currently no method for preventing or treating knee osteoarthritis in the field, so it is urgent to find novel treatment and repair strategies. Summary of the invention It is an object of the present invention to provide the use of mesenchymal vascular layer cells and mesenchymal progenitor cells for the prevention or treatment of knee osteoarthritis. In a first aspect of the invention, there is provided the use of a mesenchymal vascular layer cell (SVF) and mesenchymal progenitor cells (haMPCs) for the preparation of a pharmaceutical composition for the prevention and/or treatment of osteoarthritis .

In another preferred embodiment, the osteoarthritis is selected from the group consisting of knee osteoarthritis, spinal osteoarthritis, hip osteoarthritis, or a combination thereof.

In another preferred embodiment, the osteoarthritis is knee osteoarthritis.

In another preferred embodiment, the mesenchymal vascular layer cells are mesenchymal vascular layer cell populations.

In another preferred embodiment, the mesenchymal progenitor cell is a mesenchymal progenitor cell population.

In another preferred embodiment, the mesenchymal vascular layer cells have any one or more of the following characteristics selected from the group consisting of:

(i) more than 30% of cells have a surface antigen CD29;

(ii) more than 50% of the cells have a surface antigen CD73;

(iii) more than 85% of the cells have the surface antigen CD49d;

(iv) More than 55% of the cells have the surface antigen CD90.

In another preferred embodiment, more than 35% of the cells have the surface antigen CD29.

In another preferred embodiment, more than 55% of the cells have the surface antigen CD73.

In another preferred embodiment, more than 90% of the cells have the surface antigen CD49d.

In another preferred embodiment, more than 60% of the cells have the surface antigen CD90.

(V) 85% or less of cells have surface antigen CD34;

(vi) 15% or less of the cells have the surface antigen CD45.

In another preferred embodiment, less than 80% of the cells have the surface antigen CD34.

In another preferred embodiment, less than 12% of the cells have the surface antigen CD45.

In another preferred embodiment, the mesenchymal vascular layer cells secrete a cytokine selected from the group consisting of stem cell growth factor (HGF), vascular endothelial growth factor (VEGF), platelet-derived factor (PDGF), human transforming growth factor P(TGF-P), macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-10 (IL-10), or a combination thereof.

In another preferred embodiment, the concentration of stem cell growth factor (HGF) secreted by the mesenchymal vascular layer cells

0.5 ng/ml, preferably 0.8 ng/ml.

In another preferred embodiment, the concentration of vascular endothelial growth factor (VEGF) secreted by the mesenchymal vascular layer cells 35 pg/ml, preferably 40 pg/ml.

In another preferred embodiment, the concentration of human transforming growth factor P (TGF-P) secreted by the mesenchymal vascular layer cells is 150 pg/ml, preferably 180 pg/ml.

In another preferred embodiment, the interstitial vascular layer cells secrete interleukin-2 (IL-2) at a concentration of S3⁄4 15 pg/ml, preferably 20 pg/ml, more preferably 30 pg/ml.

In another preferred embodiment, the interstitial vascular layer cells secrete an interleukin-IO (IL-IO) concentration of S? 15 pg/ml, preferably 20 pg/ml, more preferably 30 pg/ml, optimally 40 pg/ml. .

In another preferred embodiment, the mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:

(i) more than 95% of cells have a surface antigen CD90;

(ii) more than 95% of the cells have a surface antigen CD73;

(iii) more than 95% of the cells have a surface antigen CD29;

(iv) More than 95% of cells have the surface antigen CD49d.

In another preferred embodiment, more than 98% of the cells have the surface antigen CD90.

In another preferred embodiment, more than 98% of the cells have the surface antigen CD73.

In another preferred embodiment, more than 98% of the cells have the surface antigen CD29.

In another preferred embodiment, more than 98% of the cells have the surface antigen CD49d.

(v) 2% or less of the cells have a surface antigen HLA-DR;

(vi) 2% or less of the cells have a surface antigen Actin;

(vii) 2% or less of the cells have the surface antigen CD34;

(viii) 2% or less of the cells have the surface antigen CD45;

(viiii) 2% or less of the cells have the surface antigen CD 14.

In another preferred embodiment, less than 1% of the cells have the surface antigen HLA-DR.

In another preferred embodiment, less than 1% of the cells have a surface antigen Actin.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD34.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD45.

In another preferred embodiment, less than 1% of the cells have the surface antigen CD14.

In another preferred embodiment, the mesenchymal progenitor cells secrete a cytokine selected from the group consisting of vascular endothelial growth factor (VEGF), human transforming growth factor aCTGF-a, and human transforming growth factor P (TGF-P). ), granulocyte colony-stimulating biological factors (GM-CSF), hepatocyte growth factor (HGF), platelet-derived factor (PDGF), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10).

In another preferred embodiment, the concentration of vascular endothelial growth factor (VEGF) secreted by mesenchymal progenitor cells lOpg/ml, preferably 15 pg/ml.

In another preferred embodiment, the concentration of human transforming growth factor P (TGF-P) secreted by mesenchymal progenitor cells

300 pg/ml, preferably 400 pg/ml.

In another preferred embodiment, the granulocyte colony stimulating biological factor (GM-CSF) secreted by the mesenchymal progenitor cells has a concentration of 30 ng/ml, preferably 40 ng/ml.

In another preferred embodiment, the concentration of hepatocyte growth factor (HGF) secreted by mesenchymal progenitor cells

0.4 ng/ml, preferably 0.5 ng/ml.

In another preferred embodiment, the concentration of platelet-derived factor (PDGF) secreted by mesenchymal progenitor cells

0.008 ng/ml, preferably 550.01 ng/ml.

In another preferred embodiment, the concentration of interleukin-2 (IL-2) secreted by the mesenchymal progenitor cells is 25 pg/ml, preferably

In another preferred embodiment, the mesenchymal progenitor cells secrete an interleukin-IO (IL-IO) concentration of 30 pg/ml, preferably 3⁄4 40 pg/ml.

In a second aspect of the invention, there is provided a pharmaceutical composition for preventing and/or treating osteoarthritis, the pharmaceutical composition comprising: an effective amount of interstitial vascular layer cells (SVF) and a charge Progenitor cells (haMPCs), and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a subcutaneous injection agent.

In another preferred embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.

In another preferred embodiment, the concentration of the mesenchymal vascular layer cells is Ο.Ι-lOOx lO ⁴ cells/ml, preferably

1-lOx lO ⁴ / ml, more preferably 2 x l 0 ⁵ / ml.

In another preferred embodiment, the mesenchymal progenitor cell concentration is from 0.1 to 100 x 10 ⁴ / ml, preferably from 1 to 10 x 10 / ⁴ , more preferably 2 x 10 ⁵ / ml.

In a third aspect of the invention, a method for preventing and/or treating osteoarthritis is provided, comprising the steps of: administering to a subject in need of interstitial vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs), Or a pharmaceutical composition comprising mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs).

In another preferred embodiment, the subject is a human or non-human mammal, preferably a human.

In another preferred embodiment, the method includes the steps of:

(1) administering interstitial vascular layer cells to a subject in need;

(2) Applying mesenchymal progenitor cells to the desired subject.

In another preferred embodiment, the site of application is within the joint cavity of the subject.

In another preferred embodiment, the interval between step (1) and step (2) is 1 month or more, and/or 3 months. On. It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described in the following (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS

The following drawings are used to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the appended claims.

Figure 1 shows the combination of SVF and haMPCs for the treatment of KOA.

Figure 2 shows the results of SVF surface antigen detection. Figures 2A–21 show the results of CD34, CD29, CD73, CD49d, CD90, CD 14, CD45, Actin, and HLA-DR antigens, respectively.

Figure 3 shows the change in VEGF secretion of haMPCs; Figure 3A shows the change in VEGF secretion of haMPCs after 24 h of LPS stimulation; Figure 3B shows the effect of hypoxia stimulation on VEGF secretion of haMPCs.

Figure 4 shows the results of chondrogenic induction experiments of haMPCs. detailed description

The inventors have extensively and intensively studied, and for the first time, unexpectedly found that interstitial vascular layer cells and mesenchymal progenitor cells have extremely excellent effects in preventing or treating osteoarthritis. Specifically, the autologous adipose-derived mesenchymal vascular layer cells and mesenchymal progenitor cells of the present invention are administered to a subject in need thereof, or a pharmaceutical composition containing autologous adipose-derived mesenchymal vascular layer cells and mesenchymal progenitor cells is administered, It has a significant preventive or therapeutic effect on osteoarthritis. Mesenchymal progenitor cells have high cytokine secretion ability and can repair the damage of the body under suitable conditions in vivo: Interstitial vascular layer cells and mesenchymal progenitor cells have the ability to differentiate into cartilage and osteogenesis. The present invention also provides a method for preventing and treating osteoarthritis and a pharmaceutical composition comprising interstitial vascular layer cells and mesenchymal progenitor cells. The present invention has been completed on this basis. the term

As used herein, the terms "above" and "below" include the number, for example "95% or more" means S3⁄495%, and "0.2% or less" means 0.2%. Osteoarthritis

As used herein, the term "osteoarthritis" is used interchangeably with "osteoarthritis" or 'ΌΑ' antigen. Osteoarthritis is a chronic joint disease. Its main changes are degenerative changes in the articular cartilage surface and secondary bone hyperplasia, which are manifested in joint pain and inflexibility. X-ray shows narrowing of joint space, cartilage The lower bone is dense, the trabecular bone is broken, there is hardening and cystic change; the edge of the joint has lip-like hyperplasia; the end of the bone is deformed, the joint surface is uneven; the cartilage in the joint is spalled, the bone is broken into the joint, and the joint is free. body.

In the present invention, the osteoarthritis may be osteoarthritis selected from the group consisting of knee osteoarthritis, spinal osteoarthritis, hip osteoarthritis, or a combination thereof. The osteoarthritis according to the present invention is preferably knee osteoarthritis. Fat

Autologous fat is an excellent source of plastic and anti-aging treatments. Adipose tissue materials can be derived from the waist, hips, abdomen, thighs, upper arms and more. Those skilled in the art can obtain autologous lipid tissue using a general technical method including, but not limited to, aspiration, surgical separation, and the like.

In the present invention, the adipose tissue or the fat raw material is not particularly limited, and may be an adipose tissue derived from any part of an animal or a human, preferably a human adipose tissue. Preferably, the adipose tissue may be tissue of the waist, buttocks, abdomen, thighs, upper arms, and the like. Interstitial vascular layer cell

As used herein, the terms "interstitial vascular layer cells", "SVF", or "interstitial vascular fragments" are used interchangeably. The interstitial vascular layer cells are stem cells with multi-directional differentiation potential isolated from adipose tissue.

SVF can stably proliferate in vitro and has a low mortality rate. It is easy to obtain, has a large amount of reserves in the body, is suitable for large-scale cultivation, has little damage to the body, has a wide range of sources, and is suitable for autologous transplantation. SVF is the most important component of stem cell-assisted fat transplantation. The cell cluster formed by a mixture of various cells separated from adipose tissue by collagenase digestion is called interstitial blood vessel debris. Interstitial vascular debris is rich in mesenchymal cells, which can differentiate into multiple lineage cells. It is the ideal seed cell for regenerative medicine and tissue engineering.

Those skilled in the art will be able to perform the separation and purification of SVF using a general method.

As used herein, the terms "separation method", "separation method of SVF" are used interchangeably and refer to methods and processes for obtaining isolated SVF from the original adipose tissue. For the obtained SVF fat cell material, the first washing is performed to remove blood cells; the fat is broken and digested; the undigested tissue is removed, and the SVF-containing filtrate is obtained; and the SVF is obtained by centrifugation. The resulting SVF can be used for further passage, culture or cryopreservation.

In a preferred embodiment, the separation of the SVF may include a step (but not limited to): the liposuction fat is repeatedly washed twice with PBS, and then digested with collagenase at 37 ° C for 30 min, after centrifugation at 1200 g for 10 min. That is, high-density SVF fragments are obtained, which mainly include mesenchymal cells, vascular endothelial cells, and parietal cells. Another In addition, SVF also includes some blood vessel-derived cells, such as white blood cells and red blood cells, which have synergistic effects between various cells. Antigen detection of interstitial vascular layer cells

The SVF used in the present invention has a high purity and is substantially free of other types of cells or stem cells. This can be verified by detection of cell surface antigens.

SVF has a variety of specific antigens and receptors, mainly CD3, CD13, D29, CD34, CD45,

CD49e, CD59, CD73, CD90, CD105, HLA-ABC, etc.

CD34 antigen is a highly glycosylated type I transmembrane protein that is selectively expressed on human hematopoietic stem (HSC), progenitor (PC) and vascular endothelial (EC) surfaces, adipose tissue progenitor cells with CD34 The proportion of total stem cells is preferably ≤ 0.2%, more preferably ≤ 0.2%.

CD45 is present on the surface of all hematopoietic cells, including hematopoietic stem cells and osteoclasts. The proportion of adipose tissue progenitor cells bearing CD45 in total stem cells is preferably ≤ 0.1%.

CD29, CD73, CD49d, CD90, etc. are mainly present on the surface of adipose mesenchymal progenitor cells.

The proportion of SVF with CD29 in total stem cells is preferably ≥ 30%, more preferably ≥ 32%, optimally ≥ 35%. The proportion of SVF with CD73 in total stem cells is preferably ≥ 50%, more preferably ≥ 60%, optimally ≥ 70%. The proportion of SVF with CD49d in total stem cells is preferably ≥ 85%, more preferably ≥ 90%, optimally ≥ 95%. The proportion of SVF with CD90 in total stem cells is preferably ≥ 55%, more preferably ≥ 60%, optimally ≥ 65%. One skilled in the art can use a general method to detect the purity and degree of differentiation of SVF, such as flow cytometry. At the time of detection, different specific and targeted specific antibodies are added, and the antibody may be a complete monoclonal or polyclonal antibody, or may be an immunologically active antibody fragment, such as a Fab' or (Fab) 2 fragment; an antibody heavy chain; Antibody light chain; genetically engineered single chain Fv molecule (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibody, such as an antibody having murine antibody binding specificity but still retaining antibody portions from humans. The antibody is added to the cell surface antigen for a certain period of time, and the cells are automatically analyzed and sorted by flow cytometry. Adipose-derived mesenchymal progenitor cells

As used herein, the terms "fat-derived mesenchymal progenitor cells", "haMPCs" or "adipose tissue-derived mesenchymal progenitor cells" have the same meaning and are used interchangeably.

The adipose-derived mesenchymal progenitor cells used in the present invention are preferably human-derived adipose-derived mesenchymal progenitor cells; more preferably human autologous adipose-derived mesenchymal progenitor cells.

One of ordinary skill in the art can perform the separation and culture of haMPCs using conventional methods. In a preferred embodiment, the steps are as follows: a. Dispensing fat: The extracted fat is packed into a centrifuge tube. The extracted fat is divided into two parts. Part of the fat is digested and washed with collagenase, and then used to prepare SVF cell suspension, which is directly returned to the site; the other part is obtained by SVF. Continue to culture to obtain fat progenitor cells;

b. Decomposition of fat: Add the enzyme to the centrifuge tube, place it in a constant temperature oscillator, and perform fat digestion. After the digestion is completed, discard the fat from the upper layer after centrifugation, collect the precipitate and wash it;

c Filtration: Add cell washing solution, mix well, filter with cell strainer, count the cells, centrifuge, and wash the pellet. The filtered cells are SVF (stromal vasvular fraction Cells). SVF is a mixture of various cells isolated from adipose tissue. It is a group of cells composed of various allogeneic cells, including MPCs-like cells, endothelial (progenitor) cells, vascular smooth muscle cells, and other parts of blood-derived cells. Including granulocytes, monocytes, lymphocytes, etc.;

d. Preparation of SVF suspension: The filtered SVF cells were formulated into 5 ml of cell suspension, and the suspension was inhaled with an injection, and the suspension was poured into a 100 ml saline bag;

e. Cell culture: adjust the inoculation density according to the amount of counted cells, inoculate into the culture flask and set up C0 ₂ incubator culture; f. Passage: a small amount of adherent mesenchymal progenitor cells appear in the inoculation for about 3 days, and culture the adherent cells for 5-7 days. In the case of colonies, digestion and passage, collecting and counting cells, primary adherence 1 : 1-2 ratio subculture, subculture for 2 to 3 weeks, collecting cells (autologous fat derived progenitor cells);

g. Preparation of adipose-derived progenitor cell suspension: After centrifugation of the collected adipose-derived progenitor cells, physiological saline is injected to prepare a cell suspension. Antigen detection of adipose-derived mesenchymal progenitor cells

The adipose-derived mesenchymal progenitor cells used in the present invention have extremely high purity and activity.

One of ordinary skill in the art can detect mesenchymal progenitor cell surface antigens using conventional methods, such as flow cytometry.

Adipose-derived mesenchymal progenitor cells have a variety of specific antigens and receptors, mainly including: CD29, CD73, CD90, CD49d and the like.

The proportion of mesenchymal progenitor cells bearing CD73 antigen in total mesenchymal progenitor cells is ≥ 95%, more preferably ≥ 98%, more preferably ≥ 99%, most preferably 100%.

The proportion of mesenchymal progenitor cells bearing CD90 antigen in total mesenchymal progenitor cells is ≥ 95%, more preferably ≥ 98%, more preferably ≥ 99%, most preferably 100%.

The proportion of mesenchymal progenitor cells with CD29 antigen in total mesenchymal progenitor cells is ≥95%, more preferably ≥98

%, more preferably ≥99%, optimally 100%.

The proportion of mesenchymal progenitor cells with CD49d antigen in total mesenchymal progenitor cells is ≥95%, more preferably ≥98 %, more preferably ≥99%, optimally 100%.

Negative indicators of adipose-derived mesenchymal progenitor cells include: HLA-DR, Actin, CD34, CD45, CD14, and the like.

The proportion of mesenchymal progenitor cells with HLA-DR antigen in total mesenchymal progenitor cells is ≤ 2%, more preferably <1%, more preferably ≤ 0.5%, and optimally no HLA-DR antigen.

The proportion of mesenchymal progenitor cells with Actin antigen in total mesenchymal progenitor cells is ≤ 2%, more preferably ≤ 1%, more preferably ≤ 0.5%, optimally free of Actin antigen.

The proportion of mesenchymal progenitor cells bearing CD34 in total mesenchymal progenitor cells is < 2%, more preferably < 1%, more preferably < 0.5%, optimally free of CD34.

The proportion of mesenchymal progenitor cells bearing CD45 in total mesenchymal progenitor cells is < 2%, more preferably < 1%, more preferably < 0.5%, optimally without CD45.

The proportion of mesenchymal progenitor cells bearing CD14 in total mesenchymal progenitor cells is < 2%, more preferably < 1%, more preferably < 0.5%, optimally free of CD14.

The haMPCs used in the present invention are preferably human-derived haMPCs capable of secreting a large amount of VEGF, TGF-a, TGF-β, GM-CSF, HGF, PDGF, IL-2, IL-4, IL-10 and other gene factors. Strong colony forming ability and extremely low immunogenicity.

One of ordinary skill in the art can use, process, apply, etc. the haMPCs using conventional methods. For example, each batch of haMPCs must be tested for sterility, endotoxin and mycoplasma, and DNA identification before delivery or use. The cells should be in compliance with cell viability ≥95% and cell purity (positive index >95%, negative index <2%). The haMPCs were all acute and allergic to the test results, and there was a corresponding test report. Pharmaceutical composition and application thereof

The invention also provides a pharmaceutical composition comprising an effective amount of mesenchymal vascular layer cells and mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.

Generally, interstitial vascular layer cells and mesenchymal progenitor cells can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is usually about 5-8, preferably. Ground, pH is about 7-8.

As used herein, the term "effective amount" or "effective amount" refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, a substance having a reasonable benefit/risk ratio . Term "medicine "Study acceptable carrier" means a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical compositions of the present invention comprise, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of the active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.

The effective amount of the mesenchymal vascular layer cells and mesenchymal progenitor cells of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

The pharmaceutical composition of the present invention is preferably a subcutaneous injection agent. In another preferred embodiment, the concentration of the interstitial vascular layer cells of the subcutaneous injection agent is 0.1-100×10 ⁴ /ml, preferably 1-lOx10 ⁴ /ml, more preferably 2χ10 ⁵ / The concentration of the mouth and/or mesenchymal progenitor cells is 0.1-100×10 ⁴ /ml, preferably 1-10 χ 10 ⁴ /ml, more preferably 2 χ 10 ⁵ /ml.

The invention also provides a method of using the pharmaceutical composition of the invention, in a specific embodiment, comprising the steps of:

(1) administering a mesenchymal vascular layer cell to a subject in need thereof, the preferred site of administration being within the joint cavity of the subject;

(2) The mesenchymal progenitor cells are administered to a subject in need thereof, and the preferred site of administration is within the joint cavity of the subject; the preferred period of use is 1 month, and/or 2 months after step (1).

The application procedure described in the present invention is shown in Figure 1. The main advantages of the present invention are as follows - (1) The SVF and haMPCs of the present invention are safe for use in vivo;

(2) After the stimulation of LPS, hypoxia, etc., the expression levels of various cytokines are significantly increased in haMPCs used in the present invention, haMPCs have high cytokine secretion ability, and can repair body damage under suitable conditions in vivo. ;

(3) The haMPCs of the present invention have typical mesenchymal stem cell characteristics and have potential differentiation ability under suitable conditions in vivo, thereby satisfying the relative needs of the body;

(4) In the mixed therapy of SVF and haMPCs of the present invention, haMPCs can be converted into chondrocytes under specific induction conditions, and are used for preventing or treating osteoarthritis. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Example 1

Treatment of knee osteoarthritis (KOA) with SVF and haMPCs

example 1 :

Female patient, 45 years old, bilateral knee pain for more than 3 years, morning stiffness, limited mobility, difficulty in getting up and down stairs, pain in flat walking, X-ray examination showed obvious osteophytes, narrow joint space, sclerosing change. The WOMAC score is 92 points. Diagnosis: Moderate knee osteoarthritis.

About 30 ml of fat was taken from the lower abdomen of KOA patients, wherein the vascular matrix fraction was separated from 10 ml of freshly obtained adipose tissue, and the mature adipocytes were removed by collagenase digestion, filtration, and centrifugation to obtain SVF, and the SVF was subjected to surface antigen identification. The results of the identification are shown in Figure 2.

SVF of 5*10 ⁶ cells/2 ml per side was injected into the bilateral joint cavity of the patient. After the separation and purification of the remaining adipose tissue, the P2-P5 generation was cultured to obtain haMPCs. The haMPCs were cryopreserved and cultured again after thawing and resuscitation. The bilateral joint cavities were received once in the first and third months after the first injection, and the amount of each side was 1*10 ⁷ / 2 / 2 ml.

RESULTS: The patient's symptoms improved significantly, the pain was relieved, the pain on the ground was not obvious, no morning stiffness appeared, and the activity ability was significantly improved. X-ray examination showed that the articular surface was smoother than before; WOMAC score was 55 points. Control example:

Patient male, 62 years old, knee pain for more than 3 years, morning stiffness, limited mobility, can only do easy housework, difficulty getting up and down stairs; X-ray: moderate amount of callus formation, narrow joint space, hardening change; WOMAC Score 98 points. Diagnosis: Moderate knee osteoarthritis.

About 30 ml of fat was taken from the lower abdomen of KOA patients, and the vascular matrix fraction was isolated. After collagenase digestion, filtration, and centrifugation to remove mature adipocytes, SVF was obtained, and bilateral intra-articular injection was performed. The dose per side was 5*10 ⁶ / 2 ml. . The above procedure was repeated for the first and third months after the first injection, and bilateral intra-articular injection was again performed at a dose of 5*10 ⁶ / 2 ml per side.

RESULTS: The patient's symptoms were not improved obviously, the pain was slightly relieved, there was still morning stiffness, and the mobility was slightly improved; the X-ray examination was almost the same as before; the WOMAC score was 86 points. The results showed that the combined use of SVF and haMPCs had significant effects in the treatment of knee osteoarthritis (KOA). Example 2 Identification of SVF and haMPCs

1. Flow detection

The cells were collected into a centrifuge tube by enzymatic digestion. The cell suspension was adjusted to a density of lx l0 ⁵ /mL, 800 r/min (120 g), centrifuged for 5 min, the supernatant was discarded, and washed with cold D-Hanks at 4 °C. The cells were resuspended, and the cell suspension was again centrifuged at 800 r/mm for 5 min, after which the supernatant was discarded. The cells were then resuspended to 1 mL with D-Hanks, 5 to 10 L of antibody was added, protected from light, and placed on ice for 30 min. Rinse with D-Hanks, centrifuge, discard the supernatant, repeat the rinsing process 2~3 times, ensure that the unbound antibody is removed, and add about 200 to 300 L of D-Hanks to make a suspension, using flow cytometry. Detection (Figure 2).

The flow detection results of SVF are shown in Table 1.

Table 1

The expression of cell surface antigen marker expression by SVF was analyzed by flow cytometry. The results showed that the ratio of SVF in freshly isolated progenitor cells was 60%, and the content of hematopoietic progenitor cells was 70%, and there were more mixed cells.

The flow detection results of haMPCs are shown in Table 2.

Table 2

2. Detection of cell secreting factors

SVF and haMPCs were cultured in the culture chamber for 48 hours, and the supernatant was taken to detect the cell secretory factor. The umbilical cord stem cells were used as the control group, and the test results are shown in Table 3. table 3

The results showed that both SVF and haMPCs have a good function of secreting cytokines. Among them, the TGF-β secreted by haMPCs is as high as 401.00 pg/ml, which is much higher than that of SVF, which is equivalent to umbilical cord stem cells. Example 3

haMPCs stimulation test

(1) Fresh and frozen haMPCs were separately cultured in complete medium and 5% FBS medium, and VEGF secreted by haMPCs was detected.

The results showed (Fig. 3A) that the concentration of VEGF decreased with the increase of LPS concentration in fresh haMPCs cultured in complete medium; the concentration of VEGF in the fresh haMPCs group cultured in 5% FBS medium was basically the same as that in the control group at 200 ng/ml. 100ng/ml and 300ng/ml were decreased respectively. The concentration of VEGF in the cryopreserved haMPCs group in complete medium culture did not change much with the increase of LPS concentration. Overall, serum culture is higher than VEGF in complete medium.

(2) To detect the secretion of VEGF in haMPCs under hypoxic stimulation, it was found that the secretion of VEGF under hypoxia was 2-3 times that in normal culture, and the secretion in 48 hours was twice that in 24 hours. ,

The increase in VEGF is directly proportional to the increase in cell concentration (Fig. 3B). Example 3

haMPCs differentiation test

The chondrogenic differentiation medium was prepared using the GIBCO STEMPRO into a chondrogenic differentiation kit, and the p3 generation cells were obtained by digesting the haMPCs by centrifugation, and a cell suspension of 1.6 X 10 ^Λ 7 viable cells/mL was prepared using standard medium. A 5 L cell suspension (cell volume 8 x l0 ⁴ ) was pipetted with a sample gun and inoculated into the center of the well of a multi-well plate to be placed. Pre-culture for 2 hours in a 0 ₂ incubator.

After the pre-culture, add 1 mL of 37 ° C water bath to pre-heat the chondrogenic differentiation complete culture Base, add 1 mL of 37 ° C water bath to pre-heat standard medium in the control well, and place the porous culture plate in a C0 ₂ incubator at 37 ° C, 5% CO ₂ concentration. During the cultivation process, the corresponding plate holes were exchanged every 2-3 days. After sufficient incubation time (7, 14 days), 4% formalin solution (lmL 40% formaldehyde dissolved in 9mL water) and 1% Alcian Blue dye solution (O.lg Alcian Blue dissolved in lOmL 0.1 N) In HCL). Each well was fixed and stained. The medium was aspirated, washed once with DPBS, and the cells were fixed with a 4% formalin solution for 30 minutes. After fixation, it was washed with DPBS, and then stained with a 1% Alcian Blue solution (dissolved in 0.1 N of HC1) for 30 minutes. The cells were washed three times with 0.1 N of HC1, added to distilled water and acidic, and then observed under light microscope for each experimental group and control group, and the pictures were saved. The experimental group that successfully differentiated into chondrocytes stained with Alcian Blue and became blue due to the proteoglycan synthesized by chondrocytes.

Experimental results: The cell growth was observed on the 7th day of the experiment, and the Al, A3, and C1 wells were selected for photo analysis. It can be seen that the cells in the well A1 (Fig. 4.1) are concentrated in the central region of the inoculation. The morphology of the cells is circular, close to the chondrocytes, and the adherence of the cells is generally tight, but the adherence state can be maintained after several fluid changes; The cells in well A3 (Fig. 4.2) form a spherical highly aggregated cell mass structure with a milky white globular structure visible under the naked eye, which remains after several fluid changes and eliminates the possibility of contamination; CK Figure 4.3) As a control group, standard medium was added, and the cell morphology was consistent with standard haMPCs adherent cells. Therefore, from the morphological point of view, the haMPCs cultured in the differentiation medium have a significant difference compared with the control group.

Alcian Blue staining on day 14 of the experiment showed that there was no obvious blue staining in the C1 well (Fig. 4.6); in contrast, A1 (Fig. 4.4) showed obvious blue staining, indicating the presence of chondrocyte synthesis. The mucopolysaccharide; while the A3 (Fig. 4.5) pore may be too blue in color, resulting in a blue-black stain. Therefore, haMPCs cultured in a differentiation medium exhibited characteristics of differentiation into chondrocytes under classical staining conditions.

In summary, this example shows that the haMPCs of the present invention exhibit chondrocyte characteristics in cell morphology and classical chemical staining under the culture conditions of the differentiation medium, indicating that haMPCs have the differentiation potential to the chondrocytes. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entirety in the the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art after the above-described teachings of the present invention, which are also within the scope defined by the appended claims.

Claims

Rights request

A use of mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs), characterized in that they are used for the preparation of a pharmaceutical composition for preventing and/or treating osteoarthritis.

2. The use according to claim 1, wherein the mesenchymal vascular layer cells have any one or more of the following characteristics selected from the group consisting of:

(i) more than 30% of cells have a surface antigen CD29;

(ii) More than 50% of the cells have the surface antigen CD73;

(iii) more than 85% of the cells have the surface antigen CD49d;

(iv) More than 55% of the cells have the surface antigen CD90.

3. The use according to claim 1, wherein the mesenchymal vascular layer cells have any one or more of the following characteristics selected from the group consisting of:

(V) 85% or less of cells have surface antigen CD34;

(vi) 15% or less of the cells have the surface antigen CD45.

The use according to claim 1, wherein the mesenchymal vascular layer cells secrete a cytokine selected from the group consisting of stem cell growth factor (HGF), vascular endothelial growth factor (VEGF), and platelet-derived factor. (PDGF), human transforming growth factor P (TGF-P), macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-10 (IL-10), or a combination thereof.

5. The use according to claim 1, wherein said mesenchymal progenitor cells have one or more characteristics selected from the group consisting of:

(i) more than 95% of cells have a surface antigen CD90;

(ii) More than 95% of the cells have the surface antigen CD73;

(iii) more than 95% of the cells have a surface antigen CD29;

(iv) More than 95% of cells have the surface antigen CD49d.

6. The use according to claim 1, wherein said mesenchymal progenitor cells have any one or more of the following characteristics selected from the group consisting of:

(V) 2% or less of cells have a surface antigen HLA-DR;

(vi) 2% or less of the cells have a surface antigen Actin;

(vii) 2% or less of the cells have the surface antigen CD34;

(viii) 2% or less of the cells have the surface antigen CD45; (viiii) 2% or less of the cells have the surface antigen CD 14 .

7. The use according to claim 1, wherein the mesenchymal progenitor cells secrete a cytokine selected from the group consisting of vascular endothelial growth factor (VEGF) and human transforming growth factor _a (TGF-a). , human transforming growth factor P (TGF), granulocyte colony-stimulating biological factor (GM-CSF), hepatocyte growth factor (HGF), platelet-derived factor (PDGF), interleukin-2 (IL-2), interleukin-4 ( IL-4), interleukin-10 (IL-10).

A pharmaceutical composition for preventing and/or treating osteoarthritis, characterized in that the pharmaceutical composition comprises: an effective amount of mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells ( haMPCs), and a pharmaceutically acceptable carrier.

A method for preventing and/or treating osteoarthritis, which comprises the steps of: administering to a subject in need of interstitial vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs), or administering A pharmaceutical composition of mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs).

10. The method of claim 9, comprising:

(1) administering interstitial vascular layer cells to a subject in need;

(2) Applying mesenchymal progenitor cells to the desired subject.