WO2014090111A1

WO2014090111A1 - Use of stromal vascular fraction cells and mesenchymal progenitor cells for prevention or treatment of rheumatoid arthritis

Info

Publication number: WO2014090111A1
Application number: PCT/CN2013/088683
Authority: WO
Inventors: 曹卫; 张丽; 周玉洁; 曾晓聆; 赵光宇
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: 2012-12-11
Filing date: 2013-12-05
Publication date: 2014-06-19
Also published as: CN103860593A

Abstract

The present invention provides the use of stromal vascular fraction cells and mesenchymal progenitor cells in manufacture of medicine for prevention and/or treatment of rheumatoid arthritis. The mesenchymal progenitor cells have a high ability to secrete cytokines, and can repair the body injuries in vivo. The stromal vascular fraction cells and mesenchymal progenitor cells have the ability to differentiate to cartilage and ossify. The present invention also provides a pharmaceutical composition comprising stromal vascular fraction cells and mesenchymal progenitor cells, and a method for prevention and/or treatment of rheumatoid arthritis.

Description

Interstitial vascular layer cells and mesenchymal progenitor cells in prevention or treatment

Application in rheumatoid arthritis

Technical field

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 rheumatoid arthritis. Background technique

Rheumatoid arthritis (RA) is a chronic inflammation of the joint that is common in the world by the immune system caused by autoimmune disorders. Its pathological features are chronic inflammatory hyperplasia of the synovial membrane and vasospasm formation. , cartilage and subchondral bone destruction, eventually leading to joint deformity and rigidity. Its prevalence rate accounts for about 1% of the total adult population in the world, and China is about 0.32% to 0.36%. The average life expectancy of patients is shortened by 5 to 10 years, and at least 50% of patients lose their ability to work after 10 years of onset. Clinical manifestations of chronic, progressive, symmetrical, erosive polyarthritis, with the most common finger, finger, wrist, elbow and bipedal toe, ankle, and knee joint involvement, resulting in articular cartilage, bone and joints The capsule is destroyed, eventually leading to joint deformity and loss of function. Patients may have symptoms such as fever, joint pain and morning stiffness. Disease progression also systematically affects other extra-articular tissues, including skin, blood vessels, heart, lungs, and muscles.

The current pathogenesis of rheumatoid arthritis is not well understood. The main point is that cytokine involvement mediates the entire pathological process. There is increasing evidence that CD4+ T cell-mediated autoimmune responses and B cells, macrophages that infiltrate the synovium play a key role in the pathogenesis of RA. TNF-α secreted by monocytes/macrophages and fibroblasts also plays an important role in RA by inducing some cytokines including IL-1, IL-6, IL-15 and IL-18. And granulocyte macrophage colony-stimulating factors affect the occurrence of RA.

At present, the treatment of rheumatoid arthritis includes medical treatment, surgical treatment and psychological rehabilitation treatment, and the treatment should be individualized. Choose the appropriate treatment plan based on the patient's own condition, such as age, gender, weight, self-risk factors, lesion location and degree.

Current drugs for the treatment of rheumatoid arthritis are divided into four categories: non-steroidal anti-inflammatory drugs (NSAIDs), anti-rheumatic drugs (DMARDs) that improve the condition, glucocorticoids, and botanicals. However, none of the above drugs can completely control joint destruction, but only relieve pain, reduce or delay the development of inflammation. After active and regular medical treatment, patients who are still unable to control the disease can be treated for surgery to correct deformity and improve quality of life. Commonly used operations include synovectomy, arthroplasty, soft tissue release or repair surgery, and arthrodesis. However, surgery does not cure rheumatoid arthritis, so medical treatment is still needed after surgery.

Therefore, traditional treatment options are far from being able to control the progression of the disease or completely cure rheumatoid arthritis, thus forcing people to seek new treatments.

Therefore, there is currently no method for preventing or treating rheumatoid arthritis in the field, and there is an urgent need to explore new ways. Ying's treatment and repair strategy. 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 rheumatoid arthritis. 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 combination of drugs for the prevention and/or treatment of rheumatoid arthritis Things.

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 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.

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 a surface antigen CD34;

(vi) 15% or less of 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 is 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 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 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 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.

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.

In another preferred embodiment, the mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of: (V) 2% or less of cells having 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;

(iv) 2% or less of cells have the surface antigen CD14.

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-o, human transforming growth factor βΟ^-β, 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 the mesenchymal progenitor cells is 10 pg/ml, preferably 15 pg/ml.

In another preferred embodiment, the human transforming growth factor P (TGF-P) secreted by the mesenchymal progenitor cells has a concentration of 300 pg/ml, preferably 400 pg/ml.

In another preferred embodiment, the concentration of granulocyte colony-stimulating biological factor (GM-CSF) secreted by mesenchymal progenitor cells is 3011 _§ /1111, 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 0.01 ng/ml.

In another preferred embodiment, the mesenchymal progenitor cells secrete interleukin-2 (IL-2) at a concentration of 25 pg/ml, preferably

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

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

In another preferred embodiment, the pharmaceutical composition is an intravenous injection, and/or an intra-articular injection of the 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 /ml, preferably 1-lOx lO ⁴ /ml, more preferably 2 χ 10 ⁵ / Ml.

In another preferred embodiment, the mesenchymal progenitor cell concentration is Ο. Ι - lOOx lO ⁴ / ml, preferably 1-lOx lO ⁴ / ml, more preferably 2 χ 10 ⁵ / ml .

In a third aspect of the invention, a method of preventing and/or treating rheumatoid arthritis 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 administration is the vein of the subject, and/or within the joint cavity.

In another preferred embodiment, the interval between step (1) and step (2) is 1 month or longer, and/or 3 months or longer. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (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 figures are used to illustrate specific embodiments of the invention and are not intended to be limited by the scope of the claims The scope of the invention is intended.

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

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

Figure 3 shows changes in VEGF secretion of haMPCs; Figure 3A shows changes 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.

Figure 5 shows the results of osteogenic 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 rheumatoid arthritis. 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 rheumatoid arthritis.

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 rheumatoid arthritis 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"

^ 95%, "0. 2%以下" means 0.2%. Rheumatoid arthritis

As used herein, the term "rheumatoid arthritis" is used interchangeably with "rhematodid arthritis" or "RA". Rheumatoid arthritis is a chronic symmetrical multi-joint disease, especially a systemic autoimmune disease; its pathological features are chronic inflammatory hyperplasia of the synovial membrane, vasospasm formation, cartilage and subchondral bone destruction, Eventually lead to joint deformity and rigidity. Clinical manifestations of chronic, progressive, symmetrical, erosive polyarthritis, with the most common finger, finger, wrist, elbow and bipedal toe, ankle, and knee joint involvement, resulting in articular cartilage, bone and joints The capsule is destroyed, eventually leading to joint deformity and loss of function. Disease progression can also systematically affect other extra-articular tissues, including skin, blood vessels, heart, lungs, and muscles.

In particular, rheumatoid arthritis is different from common osteoarthritis (OA). Osteoarthritis is not an autoimmune disease, and lesioned joints are more common in weight-bearing joints, such as knee joints. Joints, etc. 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 the like. A person skilled in the art can obtain autonomous adipose 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 a tissue of a part of the waist, the buttocks, the abdomen, the thigh, the upper arm, 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 multipotential 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 culture, 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 progenitor 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 be differentiated into cells of various lineages. 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, washing is first performed to remove blood cells; fat is broken, digested; undigested tissue is removed, and SVF-containing filtrate is obtained; and 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. High-density SVF fragments are obtained, which mainly include interstitial cells, vascular endothelial cells, and parietal cells. 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 and so on. 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%, most preferably ≥ 35%.

The proportion of SVF with CD73 in total stem cells is preferably ≥ 50%, more preferably ≥ 60%, most preferably ≥ 70%.

The proportion of SVF with CD49d in total stem cells is preferably ≥ 85%, more preferably ≥ 90%, most preferably ≥ 95%.

The proportion of SVF with CD90 in total stem cells is preferably ≥ 55%, more preferably ≥ 60%, most preferably ≥ 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., US patent)

No. 4,946,778); or a chimeric antibody, such as an antibody having murine antibody binding specificity but still retaining an antibody portion derived from human. The antibody is added to the antigen on the cell surface 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 stromal vasvular fraction cells (SVF). 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 the culture flask to the CO ₂ incubator;

f. Passage: A small amount of adherent mesenchymal progenitor cells appeared in the vaccination for about 3 days. When the adherent cells were colonized for 5-7 days, they were digested and collected, and the cells were collected and counted. The original adherence condition 1: 1-2 ratio Passage, subculture for 2 to 3 weeks, collection of cells (autologous fat-derived progenitor cells);

g. Preparation of adipose-derived progenitor cell suspension: The collected adipose-derived progenitor cells are washed by centrifugation, and then injected with physiological saline 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 with 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%, most preferably 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 without Actin antigen.

The proportion of mesenchymal progenitor cells with CD34 in total mesenchymal progenitor cells is ≤ 2%, more preferably ≤ 1%, more Good land ≤ 0.5%, optimally no CD34.

The proportion of mesenchymal progenitor cells bearing CD45 in total mesenchymal progenitor cells is ≤ 2%, more preferably ≤ 1%, more preferably ≤ 0.5%, and optimally no CD45.

The proportion of mesenchymal progenitor cells bearing CD14 in total mesenchymal progenitor cells is ≤ 2%, more preferably ≤ 1%, more preferably ≤ 0.5%, and most preferably no CD14.

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

One of ordinary skill in the art can use haMPCs for operations such as use, handling, administration, and the like 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 results of acute and allergic tests of haMPCs were negative, 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 (e.g., toxicity, irritation, and allergies), i.e., materials having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to 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. In general, 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 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 used in combination with a subcutaneous injection reagent and an intravenous injection reagent. In another preferred embodiment, the concentration of the interstitial vascular layer cells of the subcutaneous injection agent is Ο. Ι - lOOx lO ⁴ / ml, preferably 1-lOx lO ⁴ / ml, more preferably 2 χ The concentration of 10 ⁵ /ml; and / or mesenchymal progenitor cells is 0.1 - lOOx lO ⁴ / ml, preferably 1-lOx lO 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 interstitial vascular layer cells to a subject in need, and

(2) The mesenchymal progenitor cells are administered to a subject in need thereof, and the preferred use time is one month, and/or three months after the step (1).

In the present invention, an interstitial vascular layer mesenchymal progenitor cell is administered to a subject in need thereof, and a preferred site of administration is the joint cavity of the subject, and/or intravenous, the mechanism of which is to achieve systemic immunomodulation by intravenous injection. The purpose of the intra-articular injection is to achieve local regulation, and to stimulate the ossification and cartilage of the progenitor cells to repair the lesion.

The application procedure described in the present invention is shown in Figure 1.

The main advantages of the invention are as follows:

(1) The SVF and haMPCs of the present invention are safe for use in vivo;

(2) After stimulation with 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 progenitor cell characteristics and have potential differentiation ability under suitable conditions in vivo, thereby satisfying the relative needs of the organism;

(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 rheumatoid arthritis.

(5) In the mixed therapy of SVF and haMPCs of the present invention, haMPCs can be converted into bone cells under specific induction conditions, and are used for preventing or treating rheumatoid arthritis. 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

SVF and haMPCs for rheumatoid arthritis (RA)

Patient female, 45 years old, repeated swelling and pain for 7 years, test RF 320 RU/ml, anti-CCP 569.5 RU/ml, diagnosed as rheumatoid arthritis.

Oral anti-inflammatory drugs, total sputum and leflunomide, etc., relieved symptoms, but increased liver enzymes and elevated bilirubin. Switch to immunosuppressive therapy, patients can not tolerate. SVF and haMPCs were treated with the patient's consent.

About 35 ml of fat was taken from the lower abdomen of RA 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 centrifuged to obtain SVF, and the surface antigen was identified by the SVF. The results of the identification are shown in Figure 2.

An intravenous infusion of 1*10 ⁷ cells/100 ml of SVF was administered for a duration of approximately 30 minutes, and a dose of 1*10 ⁷ cells/2 ml of SVF was injected into the affected 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 first and third months after the first injection, the same method of intravenous infusion plus intra-articular injection was performed, and the dose was 1*10 ⁷ /piece.

RESULTS: After 4 weeks of treatment, the patient's symptoms were significantly improved. After 3 months, serum antibodies were significantly lower than before treatment. The RF is 40RU/ml and the anti-CCP is 98.5 RU/ml.

Example 2

Identification of SVF and haMPCs

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/min for 5 min, after which the supernatant was discarded. The cells were then resuspended to 1 mL with D-Hanks, and 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 D-Hanks to make a suspension, which is detected by flow cytometry. (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 Antibody ratio (%) Antibody ratio (%)

CD90 99.89 Actin ~0% negative

CD29 99.96 DR ~0% negative

CD73 99.98 CD34 ~0% negative

CD49d 99.91 CD14 ~0% negative

CD45 ~0% negative

CD80 0.01

Detection of CD86 0.01 cell secreting factor

SVF and haMPCs were cultured for 48 hours in the culture chamber, 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 secretion of TGF-β 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 in the fresh haMPCs group with complete medium culture, and the concentration of VEGF decreased in the fresh haMPCs group cultured at 5% FBS medium 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 was higher than VEGF in complete medium.

(2) Detecting the secretion of VEGF in haMPCs under hypoxic stimulation, and 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 4

HaMPCs chondrogenic differentiation test

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

After the pre-culture, add 1 mL of the chondrocyte differentiation complete medium preheated in a 37 ° C water bath pot to the test well, add 1 mL of 37 ° C water bath pot to pre-heat the standard medium in the control well, and place the multi-well culture plate The culture was carried out in a CO ₂ incubator at 37 ° C in a 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) 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 Al, A3,

C1 hole photo analysis. It can be seen that the cells in the well A1 (Fig. 4A) are concentrated in the central region of the inoculation. The cell morphology is circular, close to the chondrocytes, and the cell adhesion is generally tight, but the adherence state can be maintained after several fluid changes; The cells in well A3 (Fig. 4B) form a globular 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; C1 (Fig. 4C) was added as a control medium to the standard medium, 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 significant differences 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. 4F); in contrast, A1 (Fig. 4D) showed obvious blue staining, indicating the presence of chondrocyte synthesis. The mucopolysaccharide; and the A3 (Fig. 4E) pore may be blue-black due to the cell being too dense. Therefore, haMPCs cultured in a differentiation medium exhibited characteristics of differentiation into chondrocytes under classical staining conditions.

In summary, this example demonstrates 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 chondrocytes. Example 5

HaMPCs osteogenic differentiation test

The osteogenic differentiation medium was prepared by using the GIBCO STEMPRO osteogenic differentiation kit, and the haMPCs were digested and centrifuged to obtain P3 cells, and the cell suspension was prepared using an appropriate amount of growth medium for use. HaMPCs were inoculated to a multi-well plate at a density of 5*10 ³ cells/cm ² and pre-incubated for 2 hours in a CO ₂ incubator.

After the pre-culture, add 1 mL of the osteogenic differentiation complete medium preheated in a 37 ° C water bath, and add 1 mL of a 37 ° C water bath to pre-heat the standard medium in the control well, and place the multi-well plate The culture was carried out in a CO ₂ incubator at 37 ° C, 5% CO ₂ concentration. During the cultivation process, the corresponding plate holes were exchanged every 3-4 days. After sufficient incubation time (>21 days), 4% formalin solution and 2% Alizarin Red S dye solution were placed on the same day, and the wells were 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 twice with distilled water, and then stained with 2% Alizarin Red S dye solution (pH 4.2) for 2-3 minutes. Wash three times with distilled water, and then observe each experimental group and control group under light microscope to save the picture.

Experimental results: Cell growth was observed on the 25th day of the experiment: The experimental group showed that the nodular deposit was light brown, confirming that the nodular deposit was calcium salt deposition (Fig. 5A); the control group showed negative staining (Fig. 5A) Figure 5B).

In summary, this example demonstrates that the haMPCs of the present invention exhibit osteocyte characteristics in cell morphology and classical chemical staining under the conditions of osteogenic differentiation medium, indicating that haMPCs have differentiation potential to osteoblasts. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

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

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 a surface antigen CD34;

(vi) 15% or less of 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 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;

(iv) 2% or less of cells have the surface antigen CD14.

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), human transforming growth factor _a (TGF- _a ) , human transforming growth factor P (TGF-P), 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 rheumatoid arthritis, 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 rheumatoid arthritis, comprising 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);

Preferably, the desired subject is administered by intravenous injection, and/or local intra-articular injection.

10. The method according to claim 9, comprising the steps of:

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

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