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WO2014090111A1 - Utilisation de cellules de fraction vasculaire stromale et de cellules progénitrices mésenchymateuses pour la prévention ou le traitement de la polyarthrite rhumatoïde - Google Patents

Utilisation de cellules de fraction vasculaire stromale et de cellules progénitrices mésenchymateuses pour la prévention ou le traitement de la polyarthrite rhumatoïde Download PDF

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Publication number
WO2014090111A1
WO2014090111A1 PCT/CN2013/088683 CN2013088683W WO2014090111A1 WO 2014090111 A1 WO2014090111 A1 WO 2014090111A1 CN 2013088683 W CN2013088683 W CN 2013088683W WO 2014090111 A1 WO2014090111 A1 WO 2014090111A1
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Prior art keywords
cells
surface antigen
mesenchymal
mesenchymal progenitor
progenitor cells
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English (en)
Chinese (zh)
Inventor
曹卫
张丽
周玉洁
曾晓聆
赵光宇
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • Interstitial vascular layer cells and mesenchymal progenitor cells in prevention or treatment are interstitial vascular layer cells and mesenchymal progenitor cells in prevention or treatment
  • the invention belongs to the field of stem cells and biomedicine.
  • 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
  • RA Rheumatoid arthritis
  • 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.
  • cytokine involvement mediates the entire pathological process.
  • CD4+ T cell-mediated autoimmune responses and B cells macrophages that infiltrate the synovium play a key role in the pathogenesis of RA.
  • TNF- ⁇ 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.
  • granulocyte macrophage colony-stimulating factors affect the occurrence of RA.
  • the treatment of rheumatoid arthritis includes medical treatment, surgical treatment and psychological rehabilitation treatment, and the treatment should be individualized.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs anti-rheumatic drugs
  • glucocorticoids glucocorticoids
  • botanicals glucocorticoids
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARDs anti-rheumatic drugs
  • None of the above drugs can completely control joint destruction, but only relieve pain, reduce or delay the development of inflammation.
  • 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.
  • surgery does not cure rheumatoid arthritis, so medical treatment is still needed after surgery.
  • mesenchymal vascular layer cells and mesenchymal progenitor cells for the prevention or treatment of rheumatoid arthritis.
  • 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.
  • the mesenchymal vascular layer cells are mesenchymal vascular layer cell populations.
  • the mesenchymal progenitor cell is a mesenchymal progenitor cell population.
  • the mesenchymal vascular layer cells have any one or more of the following characteristics selected from the group consisting of:
  • more than 35% of the cells have the surface antigen CD29.
  • more than 55% of the cells have the surface antigen CD73.
  • more than 90% of the cells have the surface antigen CD49d.
  • more than 60% of the cells have the surface antigen CD90.
  • the mesenchymal vascular layer cells have any one or more of the following characteristics selected from the group consisting of:
  • less than 80% of the cells have the surface antigen CD34.
  • less than 12% of the cells have the surface antigen CD45.
  • 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.
  • HGF stem cell growth factor
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived factor
  • TGF-P human transforming growth factor P(TGF-P)
  • GM-CSF macrophage colony-stimulating factor
  • IL-2 interleukin-2
  • IL-10 interleukin-10
  • the mesenchymal vascular layer cells are mesenchymal vascular layer cell populations.
  • ng/ml 0.5 ng/ml, preferably 0.8 ng/ml.
  • the concentration of vascular endothelial growth factor (VEGF) secreted by the mesenchymal vascular layer cells is 35 pg/ml, preferably 40 pg/ml.
  • 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.
  • 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.
  • 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.
  • IL-IO interleukin-IO
  • the mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:
  • more than 98% of the cells have the surface antigen CD90.
  • more than 98% of the cells have the surface antigen CD73.
  • more than 98% of the cells have the surface antigen CD29.
  • more than 98% of the cells have the surface antigen CD49d.
  • 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;
  • less than 1% of the cells have the surface antigen HLA-DR.
  • less than 1% of the cells have a surface antigen Actin.
  • less than 1% of the cells have the surface antigen CD34.
  • less than 1% of the cells have the surface antigen CD45.
  • less than 1% of the cells have the surface antigen CD14.
  • 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 ⁇ - ⁇ , 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).
  • VEGF vascular endothelial growth factor
  • aCTGF-o human transforming growth factor ⁇ - ⁇
  • GM-CSF Granulocyte colony-stimulating biological factors
  • HGF hepatocyte growth factor
  • PDGF platelet-derived factor
  • IL-2 interleukin-2
  • IL-4 interleukin-4
  • IL-10 interleukin-10
  • the concentration of vascular endothelial growth factor (VEGF) secreted by the mesenchymal progenitor cells is 10 pg/ml, preferably 15 pg/ml.
  • 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.
  • the concentration of granulocyte colony-stimulating biological factor (GM-CSF) secreted by mesenchymal progenitor cells is 3011 ⁇ /1111, preferably 40 ng/ml.
  • the concentration of hepatocyte growth factor (HGF) secreted by mesenchymal progenitor cells 0.4 ng/ml, preferably 0.5 ng/ml.
  • the concentration of platelet-derived factor (PDGF) secreted by mesenchymal progenitor cells is PDGF.
  • the mesenchymal progenitor cells secrete interleukin-2 (IL-2) at a concentration of 25 pg/ml, preferably
  • the mesenchymal progenitor cells secrete an interleukin-IO (IL-IO) concentration of 30 pg/ml, preferably 40 pg/ml.
  • IL-IO interleukin-IO
  • a pharmaceutical composition for preventing and/or treating rheumatoid arthritis comprising: an effective amount of interstitial vascular layer cells (SVF) and Proliferating progenitor cells (haMPCs), and a pharmaceutically acceptable carrier.
  • SVF interstitial vascular layer cells
  • haMPCs Proliferating progenitor cells
  • the pharmaceutical composition is an intravenous injection, and/or an intra-articular injection of the agent.
  • the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the concentration of the mesenchymal vascular layer cells is ⁇ . ⁇ -lOOx lO 4 /ml, preferably 1-lOx lO 4 /ml, more preferably 2 ⁇ 10 5 / Ml.
  • the mesenchymal progenitor cell concentration is ⁇ . ⁇ - lOOx lO 4 / ml, preferably 1-lOx lO 4 / ml, more preferably 2 ⁇ 10 5 / ml .
  • a method of 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 a pharmaceutical composition comprising mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs).
  • SVF interstitial vascular layer cells
  • haMPCs mesenchymal progenitor cells
  • a pharmaceutical composition comprising mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs).
  • the subject is a human or non-human mammal, preferably a human.
  • the method includes the steps of:
  • the site of administration is the vein of the subject, and/or within the joint cavity.
  • the interval between step (1) and step (2) is 1 month or longer, and/or 3 months or longer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 cells 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • fat-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.
  • 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;
  • SVF stromal vasvular fraction cells
  • 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;
  • Cell culture adjust the inoculation density according to the amount of counted cells, inoculate the culture flask to the CO 2 incubator;
  • 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
  • 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.
  • haMPCs for operations such as use, handling, administration, and the like using conventional methods.
  • 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.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of mesenchymal vascular layer cells and mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • 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.
  • the concentration of the interstitial vascular layer cells of the subcutaneous injection agent is ⁇ . ⁇ - lOOx lO 4 / ml, preferably 1-lOx lO 4 / ml, more preferably 2 ⁇
  • 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 ⁇ 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:
  • 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).
  • 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 SVF and haMPCs of the present invention are safe for use in vivo;
  • haMPCs 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. ;
  • 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;
  • haMPCs can be converted into chondrocytes under specific induction conditions, and are used for preventing or treating rheumatoid arthritis.
  • 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 Example 1
  • 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.
  • 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.
  • 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.
  • 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.
  • CD90 99.89 Actin ⁇ 0% negative
  • 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.
  • Fig. 3A 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.
  • 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 ⁇ 10 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne l'utilisation de cellules de fraction vasculaire stromale et de cellules progénitrices mésenchymateuses dans la fabrication d'un médicament destiné à la prévention et/ou au traitement de la polyarthrite rhumatoïde. Les cellules progénitrices mésenchymateuses ont une capacité élevée de sécrétion de cytokines, et peuvent réparer les lésions corporelles in vivo. Les cellules de fraction vasculaire stromale et les cellules progénitrices mésenchymateuses ont la capacité de se différencier en tissu cartilagineux et osseux. La présente invention concerne également une composition pharmaceutique comprenant des cellules de fraction vasculaire stromale et des cellules progénitrices mésenchymateuses, et une méthode pour la prévention et/ou le traitement de la polyarthrite rhumatoïde.
PCT/CN2013/088683 2012-12-11 2013-12-05 Utilisation de cellules de fraction vasculaire stromale et de cellules progénitrices mésenchymateuses pour la prévention ou le traitement de la polyarthrite rhumatoïde Ceased WO2014090111A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011008277A2 (fr) * 2009-07-14 2011-01-20 Massachusetts Institute Of Technology Cellules progénitrices stromales hématopoïétiques et utilisations afférentes
WO2012091911A1 (fr) * 2010-12-27 2012-07-05 Intellicell Biosciences, Inc. Extraits vasculaires de stroma et de mésenchyme dérivés par cavitation ultrasonique et cellules dérivées de ceux-ci obtenues à partir de tissu adipeux et utilisation de ceux-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011008277A2 (fr) * 2009-07-14 2011-01-20 Massachusetts Institute Of Technology Cellules progénitrices stromales hématopoïétiques et utilisations afférentes
WO2012091911A1 (fr) * 2010-12-27 2012-07-05 Intellicell Biosciences, Inc. Extraits vasculaires de stroma et de mésenchyme dérivés par cavitation ultrasonique et cellules dérivées de ceux-ci obtenues à partir de tissu adipeux et utilisation de ceux-ci

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, DONGXU ET AL.: "Mesenchymal stem cells in the treatment of rheumatoid arthritis", JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH. 03, vol. 15, no. 49, 3 December 2011 (2011-12-03), pages 9267 - 9270 *
RODRIGUEZ, J.P . ET AL.: "Autologous stromal vascular fraction therapy for theumatoid arthritis: rationale and clinical safety.", INTERNATIONAL ARCHIVES OF MEDICINE, vol. 5, 8 February 2012 (2012-02-08), pages 1 - 9 *

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