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WO2018010588A1 - Principe thérapeutique indiqué pour lutter contre le vieillissement fonctionnel et retarder les défaillances organiques - Google Patents

Principe thérapeutique indiqué pour lutter contre le vieillissement fonctionnel et retarder les défaillances organiques Download PDF

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Publication number
WO2018010588A1
WO2018010588A1 PCT/CN2017/091926 CN2017091926W WO2018010588A1 WO 2018010588 A1 WO2018010588 A1 WO 2018010588A1 CN 2017091926 W CN2017091926 W CN 2017091926W WO 2018010588 A1 WO2018010588 A1 WO 2018010588A1
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stem cells
cells
hematopoietic stem
therapeutic agent
patient
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Chinese (zh)
Inventor
许晓椿
肖海蓉
刘冰
程霞
周丹
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BOYALIFE Inc
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BOYALIFE Inc
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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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic 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
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention belongs to the technical field of cell therapy, and relates to a therapeutic agent for repairing functional aging of a body and delaying the decline of organ function, in particular to a therapeutic agent comprising GD2+ stromal cells and KDR2+ hematopoietic stem cells. Further, the present invention relates to the use of a combination of both GD2+ stromal cells and KDR2+ hematopoietic stem cells for the preparation of a therapeutic agent for repairing functional aging of the body and delaying the decline of organ function. It has been found that the therapeutic agents of the present invention can be effectively used to repair the aging of the body and delay the decline of organ function.
  • Mesenchymal stem cells are derived from mesoderm and ectoderm in early development, and have multi-directional differentiation potential, immune regulation and self-replication, and have attracted more and more attention.
  • Mesenchymal stem cells can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, heart muscle, endothelium, etc. under in vivo or in vitro specific induction conditions, and continue to be subcultured and cryopreserved. It has multi-directional differentiation potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and lesions, especially for the treatment of aging and tissue damage repair.
  • MSCs are abundant in the bone marrow, but as the age ages, the number of stem cells in the bone marrow is also significantly reduced, and the ability to proliferate and differentiate is also greatly degraded.
  • bone marrow MSC transplantation may cause an immune response, and the damage of the stem cell process to the patient and other problems encountered during collection directly affect the clinical application of bone marrow MSC, making it possible to find alternatives other than bone marrow.
  • the source of mesenchymal stem cells has become an important issue.
  • umbilical cord tissue and placental tissue also contain mesenchymal stem cells and can be successfully isolated.
  • This tissue-derived mesenchymal stem cell not only maintains the biological characteristics of mesenchymal stem cells, but also isolates stem cells more primitive and has a stronger ability to proliferate and differentiate.
  • Its immune cells have low functional activity, greatly reducing the risk of triggering an immune response and causing graft-versus-host disease.
  • the risk of infection and transmission of latent viruses and microorganisms is relatively low.
  • the collection process is simple and there is no harm or damage to the mother and newborn. The above reasons are sufficient to make umbilical cord mesenchymal stem cells an ideal substitute for bone marrow mesenchymal stem cells.
  • Hematopoietic stem cells (often abbreviated HSC) refer to a class of cells that have the ability to self-renew and multi-differentiate. It The basic characteristic is self-renewal ability, that is, after a cell cycle activity, it can produce two hematopoietic stem cells with the same properties as before the division, and at the same time have the ability of multi-directional differentiation, that is, under certain environmental conditions, hematopoietic stem cells have a direction The ability of each line to differentiate blood cells.
  • Hematopoietic stem cell transplantation is currently widely used in hematological malignancies (such as acute leukemia, chronic myeloid leukemia, etc.), non-malignant refractory blood diseases (such as aplastic anemia, myelodysplastic syndrome, etc.) hereditary diseases (innate immunity) Defective diseases, thalassemia, etc.) and treatment of certain solid tumors.
  • Hematopoietic stem cell transplantation refers to the transplantation of normal donor or autologous hematopoietic stem cells into a patient after systemic irradiation, chemotherapy and immunosuppressive preconditioning, so as to restore normal hematopoiesis and immune function.
  • hematopoietic stem cells exist in three parts, namely bone marrow, peripheral blood, and cord blood, and are called bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, and cord blood hematopoietic stem cells according to their sources.
  • the placenta contains a large number of hematopoietic stem cells.
  • the number of hematopoietic stem cells contained in the placenta is high, and the type of placental hematopoietic stem cells is transplanted.
  • placental hematopoietic stem cells a source of placental hematopoietic stem cells - placenta
  • placental hematopoietic stem cells a wide range of sources, pregnant women often become waste after production, the collection will not cause any discomfort or any adverse effects of the mother and newborn.
  • placental hematopoietic stem cells to replace bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, and cord blood hematopoietic stem cells for hematopoietic stem cell transplantation.
  • the main methods of hematopoietic stem cell transplantation in the treatment of international hematological diseases can be divided into three categories according to the source of the cells: bone marrow hematopoietic stem cell transplantation (BMT), mobilization of peripheral blood stem cell transplantation (MPST), and cord blood stem cell transplantation. (UCBT).
  • BMT bone marrow hematopoietic stem cell transplantation
  • MPST mobilization of peripheral blood stem cell transplantation
  • UCBT cord blood stem cell transplantation.
  • the former two stem cells are rich in source, generally have a number of nuclear cells up to 5-10 ⁇ 10 8 /Kg, and CD34+ cells (a surface marker of hematopoietic stem/progenitor cells) can reach 1-5 ⁇ 10 6 /Kg, but
  • the HLA is strictly consistent between the donor and the recipient to ensure the success of the transplant.
  • graft-versus-host disease graft-versus-host disease
  • Umbilical cord blood comes from the placenta and is usually discarded after delivery. It is now found that cord blood is rich in hematopoietic stem cells. The concentration of CD34+ cells is similar to that of bone marrow, accounting for about 0.1-0.5% of total cells, and earlier hematopoietic stem cells. CD34- is also higher than bone marrow. As a source of hematopoietic cells, umbilical cord blood transplantation is now increasing. Compared with BMT, UCBT has the advantage of reducing severe graft-versus-host response.
  • cord blood transplantation is the limited number of hematopoietic stem cells in cord blood. This restriction has led to the need to use twice the dose of cord blood in the clinic and to transplant adult recipients with larger body weight; another method is to perform hematopoiesis in vitro. Stem cell expansion and culture, but in vitro expansion takes time and cost, and more importantly, hematopoietic stem cells also differentiate while amplifying. The clinical application results show that there is no significant difference in transplantation between cord blood hematopoietic stem cells before and after amplification.
  • placenta There are a large number of hematopoietic stem cells in the human term placenta, and there are more hematopoietic stem cells than cord blood, and these placental hematopoietic stem cells can be isolated before and after cryopreservation.
  • the activity of placental hematopoietic stem cell colony forming units (CFU) is well established, and transplantation experiments in immunodeficient mice have demonstrated the potential of placental hematopoietic stem cells in transplantation. These results strongly suggest that human term placenta may be a new source of hematopoietic stem cells for transplantation.
  • placenta There are a large number of hematopoietic stem cells in the placenta.
  • the placental hematopoietic stem cells are relatively early stem cells, which can be differentiated into various cells in the body.
  • the placental blood is rich in various stages of early hematopoietic stem cells, and its content is about ten times that of cord blood.
  • Hematopoietic stem cells in a placenta can be fully satisfied by the needs of two adults. If used together with cord blood cells, it will undoubtedly increase the content of hematopoietic stem cells, which makes hematopoietic stem cells fully applicable to all applicable populations.
  • the aging of the body and the decline of organ function are more specific and typically manifest as premature aging (also commonly referred to as premature aging). Repairing the aging of the body and delaying the decline of organ function is a necessary means to treat premature aging. Regrettably, there has been no clinically effective method for treating premature aging.
  • the object of the present invention is to provide an effective method for delaying and treating aging, thereby achieving the purpose of repairing the aging of the body and delaying the decline of organ function.
  • the present inventors have surprisingly found that the use of mesenchymal stem cells and hematopoietic stem cells can effectively repair the aging of the body function and delay the decline of organ function, thereby achieving the purpose of treating premature aging.
  • the present invention has been completed based on this finding.
  • the present invention provides, in one aspect, the use of mesenchymal stem cells in combination with hematopoietic stem cells for the preparation of a therapeutic agent for repairing functional aging of the body and delaying the decline of organ function.
  • mesenchymal stem cells are GD2+ stromal cells.
  • mesenchymal stem cell is a charge derived from a placenta and/or an umbilical cord Stem cells.
  • mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord.
  • mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord, and the GD2 positive expression rate is greater than 90%, such as greater than 95%.
  • hematopoietic stem cells are derived from: cord blood, bone marrow, placental blood, and/or mobilized peripheral blood.
  • hematopoietic stem cell is a KDR2-positively expressed hematopoietic stem cell.
  • the hematopoietic stem cell is a KDR2-positively expressed hematopoietic stem cell
  • the positive expression rate of KDR2 in the KDR2-positive hematopoietic stem cell is greater than 85%, such as greater than 90%.
  • the positive expression rate of CD34 in the hematopoietic stem cells is greater than 80%, such as greater than 85%.
  • the therapeutic agent is in the form of a kit comprising mesenchymal stem cells individually sealed and individually packaged and individually packaged hematopoietic stem cells.
  • the therapeutic agent is used in a mammal, for example, a human
  • the dose of the mesenchymal stem cells is (0.1 to 10) ⁇ 10 7 stromal cells per kg of body weight.
  • weight amounts per kilogram of patient 0.5 ⁇ 5)
  • ⁇ 10 7 th stromal cells such as weight amounts per kilogram of patient (0.75 ⁇ 1.5
  • ⁇ 10 7 th stromal cells such as weight amounts per kilogram of patient 10 7 stromal cells .
  • the therapeutic agent is used in a mammal, such as a human, in a dose of (1 to 5) x 10 7 mononuclear cells per kilogram of patient body weight, for example
  • the amount of body weight per kilogram of patient is (2 ⁇ 4) ⁇ 10 7 mononuclear cells, for example, 3 ⁇ 10 7 mononuclear cells per kilogram of patient weight.
  • the hematopoietic stem cell is administered in an amount of (2 to 10) x 10 5 mononuclear cells per kilogram of patient body weight, for example per The body weight of kilogram patients is (2 ⁇ 5) ⁇ 10 5 mononuclear cells, for example, (2 ⁇ 4) ⁇ 10 5 mononuclear cells per kilogram of body weight.
  • the therapeutic agent is used in a mammal such as a human, first using mesenchymal stem cells, and after one month using hematopoietic stem cells.
  • a second aspect of the present invention provides a therapeutic agent for repairing aging of a body function and delaying degeneration of an organ function, which is in the form of a kit comprising a mesenchymal stem cell separately sealed and separately Sealed packaging of hematopoietic stem cells.
  • the therapeutic agent according to the second aspect of the invention wherein the mesenchymal stem cell is a GD2+ stromal cell.
  • mesenchymal stem cells are mesenchymal stem cells derived from the placenta and/or the umbilical cord.
  • the therapeutic agent according to the second aspect of the invention wherein the mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord.
  • the therapeutic agent according to the second aspect of the invention wherein the mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord, and the GD2 positive expression rate is greater than 90%, such as greater than 95%.
  • the therapeutic agent according to the second aspect of the present invention wherein the hematopoietic stem cell is derived from: cord blood, bone marrow, placental blood, and/or mobilized peripheral blood.
  • hematopoietic stem cell is a hematopoietic stem cell positively expressed by KDR2.
  • the hematopoietic stem cell is a KDR2-positively expressed hematopoietic stem cell
  • the positive expression rate of KDR2 in the KDR2-positive hematopoietic stem cell is greater than 85%, for example, greater than 90%.
  • the therapeutic agent according to the second aspect of the present invention wherein the hematopoietic stem cell has a positive expression rate of CD34 of more than 80%, for example, more than 85%.
  • the therapeutic agent according to the second aspect of the present invention wherein the therapeutic agent is used in a mammal such as a human, and the dose of the mesenchymal stem cells is (0.1 to 10) ⁇ 10 7 stromal cells per kg of body weight.
  • the dose of the mesenchymal stem cells is (0.1 to 10) ⁇ 10 7 stromal cells per kg of body weight.
  • weight amounts per kilogram of patient 0.5 ⁇ 5)
  • ⁇ 10 7 th stromal cells such as weight amounts per kilogram of patient (0.75 ⁇ 1.5
  • ⁇ 10 7 th stromal cells such as weight amounts per kilogram of patient 10 7 stromal cells .
  • the therapeutic agent according to the second aspect of the present invention wherein the therapeutic agent is used in a mammal such as a human, and the dose of the hematopoietic stem cells is (1 to 5) ⁇ 10 7 mononuclear cells per kg of patient body weight, for example The amount of body weight per kilogram of patient is (2 ⁇ 4) ⁇ 10 7 mononuclear cells, for example, 3 ⁇ 10 7 mononuclear cells per kilogram of patient weight.
  • the hematopoietic stem cell is administered in an amount of (2 to 10) x 10 5 mononuclear cells per kilogram of patient body weight, for example, per kilogram.
  • the patient's body weight is (2 ⁇ 5) ⁇ 10 5 mononuclear cells, for example, the body weight of each patient is (2 ⁇ 4) ⁇ 10 5 mononuclear cells.
  • the therapeutic agent according to the second aspect of the present invention wherein the therapeutic agent is used for a mammal such as a human, first using mesenchymal stem cells, and after one month using hematopoietic stem cells.
  • the mesenchymal stem cells used in the present invention can be prepared using methods known in the art.
  • the method of obtaining, for example, the GD2 positive expression rate obtained by the literature method is not less than 90% of GD2+ stromal cells.
  • the method of obtaining the GD2+ stromal cells comprises the steps of:
  • Placental tissue cleaning The placental tissue is processed in the biosafety cabinet. The placental tissue is washed 2-3 times according to the size of the placenta, and the residual blood on the surface of the placenta tissue is rinsed clean. There is no blood coagulation on the surface of the placenta. Piece;
  • Placental tissue digestion cut the placenta leaflets from the placenta tissue obtained in step (1) using a surgical scissors, transfer the leaflets to the culture dish, add 25 ml PBS buffer and cut the placenta leaflets as much as possible, add 25 ml 0.25 % trypsin (Gibco) (the ratio of trypsin to PBS buffer is 1:1) and mix the tissue, and put the culture dish into a constant temperature shaker at 37 ° C for 20 minutes;
  • Placental tissue filtration treatment Add 5ml FBS (Gibco) to the culture dish and mix to achieve the purpose of terminating digestion. Transfer the digested placental tissue fragments to a 200 mesh metal filter to grind the placental tissue fragments. The collected liquid was collected by another culture dish, and the tissue was washed twice by adding 10 ml of PBS buffer to the metal filter and grinding was continued. The collected filtrate was transferred to a 50 ml centrifuge tube, centrifuged at a speed of 1400 rpm for 10 minutes, and removed.
  • FBS Gibco
  • the supernatant was resuspended in PBS buffer, centrifuged at 1400 rpm for 10 minutes, the supernatant was removed, the cells were resuspended in PBS buffer, and a small sample was taken for cell counting, and centrifuged at 1400 rpm for 10 minutes to achieve cell washing. effect;
  • the placental whole cell cryopreservation solution comprises 15 parts by weight of human serum albumin, 10 parts by weight of DMSO (dimethyl sulfoxide) and 65 parts by weight of DMEM-F12 Store the frozen stock solution in a refrigerator at 4 °C until use;
  • Placental whole cell cryopreservation centrifuge the placental tissue filtrate obtained in step (3), remove the supernatant, and add the whole cell cryopreservation solution obtained in step (4) at a low temperature of 4 ° C, and then Add 4 ⁇ 10 7 to 1 ⁇ 10 8 cell density per ml to the cryotube.
  • This process should be carried out at a low temperature of 4 ° C. Place the cryotube into the programmed cooling box, first at 4 ° C. Under the temperature condition, the temperature was refrigerated for 0.5 hour, and then frozen at a temperature of -80 ° C for 1 day, and then the frozen tube was frozen in liquid nitrogen for use;
  • cryopreservation method used in conjunction with the cryopreservation method may be further included:
  • red blood cell lysate (Roche), incubate for 10-15 minutes in the environment of 15-25 ° C, centrifuge in a centrifuge at 1400 rpm for 10 minutes, centrifuge, remove the supernatant, observe the red blood cell lysis, if necessary The procedure of red blood cell lysis is repeated, and finally, the cells are resuspended by adding PBS buffer, and a small sample is taken for cell counting, and centrifuged at a speed of 1400 rpm for 10 minutes to perform centrifugation to wash the cells, and the supernatant is removed;
  • Cell culture Resuspend the cells in the appropriate amount of mesenchymal stem cell medium in the whole cells obtained in the step (6), transfer to the T25 flask, and then place the T25 flask into a CO2 concentration of 5%.
  • the culture was carried out in a °C incubator. When the culture was carried out until the 6th day, the T25 flask was taken out from the incubator, and the first half of the flask was changed, and the culture was continued. On the 9th day, the T25 flask was taken out of the incubator for the second time. The second half of the change, the medium in the plate was removed on the 12th day, 15ml of mesenchymal stem cell culture medium was added to continue the culture, and the whole liquid exchange was performed every 2 days;
  • the placental mesenchymal stem cells obtained in the above step (8) may be further tested for at least one of the following items: cell activity, cell contamination, genetic disease, HLA-ABC/DR matching;
  • the placental mesenchymal stem cells obtained after passage of the above step (8) can be further frozen in liquid nitrogen for use.
  • the GD2 positive expression rate of the obtained cells was detected, and the positive expression rate of GD2 was not less than 90%.
  • the mesenchymal stem cells used in the present invention not less than 95% of GD2+ stromal cells are used as the mesenchymal stem cells used in the present invention.
  • the mesenchymal stem cells obtained by the umbilical cord can be obtained by the method of Chinese Patent Application No. 201210159916.2, for example, the GD2 positive expression rate of GD2 positive expression rate obtained by the literature method is not less than 90%.
  • the method of obtaining the GD2+ stromal cells comprises the steps of:
  • the umbilical cord tissue cryopreservation solution comprises 80 parts by weight of human serum albumin and 10 parts by weight of DMSO (dimethyl sulfoxide), and a cold storage solution Store in a refrigerator at 4 ° C until use;
  • Umbilical cord tissue treatment the umbilical cord tissue obtained in the step (2) was transferred to another 10 cm cell culture plate, and the umbilical cord tissue was cut into a square shape of 1 cm 3 in size;
  • umbilical cord tissue cold storage in a low temperature environment of 4 ° C, the tissue block and cryopreservation solution were added to the cryotube, and then the cryotube was placed in the program cooling box, first frozen at a temperature of 4 ° C After 0.5 hours, freeze at a temperature of -80 ° C for 1 day, then freeze the frozen tube in liquid nitrogen for use;
  • cryopreservation method used in conjunction with the cryopreservation method may be further included:
  • cryopreservation of umbilical cord tissue recovery the umbilical cord tissue frozen in step (4) is taken out from liquid nitrogen, thawed in a constant temperature water bath until half of the frozen storage solution begins to melt, and the mesenchymal stem cell culture medium (which contains, for example, 15 %FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) The umbilical cord tissue was cleaned by spotting, and the waste liquid was removed by a 100um filter;
  • Umbilical cord tissue culture slowly add mesenchymal stem cell culture medium (for example, containing 15% FBS + 1% L-Glutamine + 0.05% Gentamicin + 84% DMEM-F12) along the edge of the plate until the tissue is submerged; The plate was placed in a 37 ° C incubator with a CO 2 concentration of 5%. The plate was removed from the incubator on the fifth day, and 5 ml of mesenchymal stem cell medium was added; on the tenth day, the medium in the plate was transferred. Add 15 ml of fresh mesenchymal stem cell culture medium; remove all umbilical cord tissue blocks on the twelfth day and continue the culture, and then perform a full liquid change every two days;
  • mesenchymal stem cell culture medium for example, containing 15% FBS + 1% L-Glutamine + 0.05% Gentamicin + 84% DMEM-F12
  • the umbilical cord mesenchymal stem cells obtained in the above step (8) may be further tested for at least one of the following items: cell activity, cell contamination, genetic disease, HLA-ABC/DR matching;
  • the passaged umbilical cord mesenchymal stem cells obtained in the above step (8) may be further frozen in liquid nitrogen for use.
  • the GD2 positive expression rate of the obtained cells was detected, and the positive expression rate of GD2 was not less than 90%.
  • the mesenchymal stem cells used in the present invention not less than 95% of GD2+ stromal cells are used as the mesenchymal stem cells used in the present invention.
  • the hematopoietic stem cells used in the present invention can be prepared using methods known in the art.
  • the positive expression rate of CD34 in hematopoietic stem cells obtained by the literature method is greater than 80%, for example, greater than 85%; and, by the literature
  • the positive expression rate of KDR2 in the hematopoietic stem cells obtained by the method is greater than 85%, for example greater than 90%.
  • the method for obtaining KDR2-positively expressed cells comprises the steps of: collection of placenta, initial examination of placenta, pre-sterilization of placenta, detection of placenta and maternal blood, lavage of placental stem cells, and purification of hematopoietic stem cells .
  • Each step is specifically as follows:
  • Placenta and maternal blood test The placenta and maternal blood samples taken are tested. The items tested include hepatitis virus detection, HIV detection, sexually transmitted diseases detection, tissue matching (HLA) detection, and hematopoietic stem/progenitor cell characterization. Detection (CFU-GM);
  • lavage of placental hematopoietic stem cells the placenta is washed under sterile conditions with sterile saline to remove blood clots and hemorrhage on the placenta. After disinfection with a disinfectant, the lavage needle is inserted into the placenta umbilical artery. The placenta perfusion bottle is inserted into the umbilical vein, the hemostat is clamped, and the constant flow pump is slowly opened. The lavage fluid is filled into the recovery bottle needle through the hose, switch, peristaltic pump, needle, placental artery, placenta, placenta vein, placenta, and finally Receiving a lavage fluid in a lavage recovery bottle;
  • the lavage lavage fluid is centrifuged at 1500-2000 rpm for 15-20 minutes in a centrifuge, the supernatant is removed, and the precipitate and the underlying liquid are collected, and the collected precipitate and
  • the lower layer of liquid and physiological saline are mixed in a ratio of 2:1 to 1:2 to obtain a mixed solution, and then the mixed solution and the lymphocyte separation liquid are separately added to the centrifuge tube in a ratio of 2:1 to 1:2, and added.
  • the order is to first add the lymphocyte separation solution to the centrifuge tube, and then slowly add the mixture. During the addition process, keep the liquid level of the lymphocyte separation solution flat.
  • centrifuge at 20-2500 rpm for 20-25 minutes After the addition, centrifuge at 20-2500 rpm for 20-25 minutes, and slowly accelerate during centrifugation. Slow deceleration, after the end of centrifugation, collect the intermediate white membrane layer into a new centrifuge tube, Mix with physiological saline at a ratio of 2:1-1:2, centrifuge at 1200-1500 rpm for 10-15 minutes; remove the supernatant after centrifugation, add 10-20 mL of normal saline to blow the pellet, and centrifuge at 1200-1500 rpm for 10-15. Minutes, after centrifugation, the supernatant was removed, the pellet was resuspended in DMEM medium, and the hematopoietic stem cell population was collected.
  • the resuspended cell population was cultured in cells and mold, and hematopoietic stem cell quantitative assay (CD34), hematopoietic stem cell activity assay Pan blue staining), qualitative detection of hematopoietic stem cells (CFU-GM).
  • CD34 hematopoietic stem cell quantitative assay
  • Pan blue staining hematopoietic stem cell activity assay Pan blue staining
  • CFU-GM qualitative detection of hematopoietic stem cells
  • the obtained hematopoietic stem cells are detected for CD34 positive expression rate and KDR2 positive expression.
  • the hematopoietic stem cells used in the present invention can be obtained by taking hematopoietic stem cell-derived tissues (including but not limited to cord blood, bone marrow, placental blood, mobilizing peripheral blood) at a ratio of 8:1 to 1: Add hydroxyethyl starch to a ratio of 8 and mix it evenly. Transfer it to the AXP hematopoietic stem cell separation system to process the consumables. Load it into the AXP hematopoietic stem cell isolation system, place it in a centrifuge, and centrifuge at a centrifuge rate (RCF) of 1200-1600 for 10 to 30 minutes.
  • RCF centrifuge rate
  • the CD34 and KDR2 levels of the isolated cells were detected using a flow cytometer. Then, the isolated cells were added to DMSO at a ratio of 8:1 to 1:8, and the procedure was cooled to -90 ° C and then stored in a liquid nitrogen atmosphere. Resuscitate in a 37 degree water bath before use.
  • the CD34 positive expression rate obtained using the above method is greater than 80%, such as greater than 85%, and the KDR2 positive expression rate is greater than 85%, such as greater than 90% of hematopoietic stem cells.
  • the mesenchymal stem cells of the present invention namely GD2+ stromal cells and KDR2+ hematopoietic stem cells
  • the mesenchymal stem cells of the present invention can be separately dispensed into a bottle, and then two bottles are placed in the kit of the present invention.
  • the therapeutic agent of the present invention for repairing functional aging of the body and delaying the decline of organ function is obtained, and then the therapeutic agent is stored under cryopreservation conditions.
  • the inventors attempted to apply the method of the present invention to a person, particularly a patient having the characteristics of premature aging, to repair the aging of the body function and delay the deterioration of organ function.
  • the results show that the method of the present invention not only exhibits excellent results in animal experiments, but also presents encouraging results in humans.
  • the placental mesenchymal stem cells i.e., GD2+ stromal cells, which were used were obtained by the methods of Examples 1, 6, and 7 of Chinese Patent Application No. 201210292509.9, and the GD2 positive expression rate was more than 95%.
  • the umbilical cord mesenchymal stem cells i.e., GD2+ stromal cells
  • the positive expression rate of GD2 was more than 95%.
  • the placental hematopoietic stem cells used are obtained by the method of Example 1 of Chinese Patent Application No. 2014104050724, and the positive expression rate of CD34 markers is greater than 85%, and the positive expression rate of KDR2 markers is greater than 90%. .
  • the cord blood hematopoietic stem cells used have a positive expression rate of CD34 markers greater than 85% and a positive expression rate of KDR/2 markers greater than 90%, and are prepared by the following method: 8:1 ⁇ 1:8 ratio of hydroxyethyl starch, mixed evenly, transferred to AXP hematopoietic stem cell separation system supporting treatment materials, loaded into AXP hematopoietic stem cell separation system, placed in a centrifuge, centrifuged at a centrifuge rate (RCF) 1200 ⁇ 1600 After 10 to 30 minutes, the cells were removed by centrifugation for 5 to 15 minutes with RCF 50 to 200, and single nuclear cells were automatically isolated, and AXP data were derived to obtain the number of hematopoietic stem cells isolated.
  • RCF centrifuge rate
  • the CD34 and KDR2 levels of the isolated cells were detected using a flow cytometer. Then, the isolated cells were added to DMSO at a ratio of 8:1 to 1:8, and the procedure was cooled to -90 ° C and then stored in a liquid nitrogen atmosphere. Resuscitate in a 37 degree water bath before use.
  • Example 1 Treatment of placental mesenchymal stem cells, GD2+ stromal cells, and cord blood KDR2+ hematopoietic stem cells Aging mice
  • mice were healthy C57BL/6J mice, female, 8 weeks old, weighing 18-22 g, 60 rats.
  • Animal grouping They were randomly divided into control group, model group and treatment group, with 20 groups in each group.
  • the model group and the treatment group were injected subcutaneously with 5% D-galactose daily at a dose of 0.25 ml/10 g, and a aging animal model was established by continuous administration for 8-16 weeks.
  • the control group was given the same volume of physiological saline.
  • GD2+MSC and umbilical cord blood KDR2+HSC were labeled with lentiviral vector expressing green fluorescent protein to determine the implantation of GD2+MSC and cord blood KDR2+HSC in various organs of mice.
  • Detection of aging related indicators before and after treatment including mouse body weight, thymus, spleen, serum, liver tissue, lung, brain tissue catalase (CAT), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content The change.
  • the first course of treatment the human GD2+ placenta MSC single cell suspension was injected into the tail vein of the mice 2 ⁇ 10 6 /0.2 mL / only once a week, a total of 4 times for a course of treatment.
  • the second course of treatment the first week of infusion of human GD2+ placenta MSC single cell suspension 2 ⁇ 10 6 /0.2mL / only, the second week of infusion of human KDR 2+ cord blood HSC single cell suspension 2 ⁇ 10 6 /0.2 mL/only, GD2+ placenta MSC was infused every course of treatment; the control group and the aging model group were given the same dose of cell culture medium.
  • mice were fixed with a tail vein injection frame.
  • the treatment group was colonized in the recipient mice.
  • the contents of MDA and CAT in the thymus, spleen, serum, liver tissue, lung and brain of the treatment group were significantly lower than those of the control group, and the SOD activity was significantly higher than that of the control group.
  • mice in each group showed that the organs of the model group were severely damaged, and the damage of various organs in the cells of the cell treatment group was significantly repaired.
  • MSC can repair the visceral damage of aging mice, thus exerting its anti-aging effect.
  • the cell specimens were collected from the control group and the treatment group, and MSCs were treated with 3%, 3d, 5d, 7d, 14d, and 28d after treatment for 5%, and then stained with 3% neutral formaldehyde for 5 minutes at room temperature.
  • X-Gal was first dissolved in dimethylformamide at a concentration of 20/L to form an X-Gal stock solution, which was stored at room temperature for use.
  • Preparation of fresh dye solution 1mg/mL X-Gal+40mmol/L citrate buffer (pH 6.0)+5mmol/L potassium ferrocyanide+5mmol/L potassium ferricyanide+150mmol/L NaCl+2mmol/L MgCl 2 .
  • Dyeing The specimen was immersed in fresh dyeing solution for 12 to 16 hours at room temperature, rinsed with distilled water, neutralized with neutral red or Giemsa dyeing solution, and observed under light microscope.
  • the ratio of SA- ⁇ -Gal staining positive cells was determined by the number of positive staining cells of 5 fields/slides randomly selected under the microscope as the positive rate of each slide.
  • the positive rate was calculated by the mean ⁇ standard deviation of 3 slides. .
  • the positive rate of SA- ⁇ -Gal staining in the control group was 3.03 ⁇ 0.66%.
  • the positive rate of SA- ⁇ -Gal staining in MSCs after 1.31, 3d, 7d, 14d and 28d after GD2+MSC and KDR2+HSC treatment was 2.92 ⁇ . 0.58%, 3.17 ⁇ 0.76%, 3.13 ⁇ 0.76%, 2.37 ⁇ 0.76%, 2.61 ⁇ 0.76%; they were not significantly different from the control group (p>0.05). There was no significant difference between the experimental group and the control group, indicating that GD2+MSC and KDR2+HSC could not cause MSC senescence.
  • p16 There was no significant increase in p16 of MSCs at 1d, 3d, 7d and 14d after treatment with GD2+MSC and KDR2++HSC, and there was no significant difference between the two groups (p>0.05). The difference between p16 and control group at 28d after GD2+MSC and KDR2+HSC treatment was extremely significant (p ⁇ 0.001), which was significantly reduced.
  • the present inventors have attempted to treat patients with premature aging by using the method of the present invention on the basis of sufficient biological tests, and the results show that the method of the present invention has an excellent therapeutic effect on premature aging, and the therapeutic effect is also reflected in its ability to repair the body function. Aging and delaying the decline of organ function.
  • mice were treated with umbilical cord mesenchymal stem cells, GD2+ stromal cells and KDR2+ placental hematopoietic stem cells, according to the method of Example 1, and the results showed substantially the same therapeutic effects as in Example 1.
  • the invention belongs to the technical field of cell therapy, and relates to a therapeutic agent for repairing functional aging of a body and delaying the decline of organ function, in particular to a therapeutic agent comprising GD2+ stromal cells and KDR2+ hematopoietic stem cells. Further, the present invention relates to the use of a combination of both GD2+ stromal cells and KDR2+ hematopoietic stem cells for the preparation of a therapeutic agent for repairing functional aging of the body and delaying the decline of organ function.
  • the therapeutic agent of the invention can be effectively used for repairing the aging of the body function and delaying the decline of organ function.

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Abstract

L'invention concerne un principe thérapeutique indiqué pour lutter contre le vieillissement fonctionnel et retarder les défaillances organiques et, plus particulièrement, un principe thérapeutique comprenant des cellules souches mésenchymes, et plus spécifiquement, des cellules stomatiques d'expression positive GD2 et des cellules souches hématopoïétiques d'expression positive KDR2. Les cellules souches mésenchymes, et plus spécifiquement, les cellules stomatiques d'expression positive GD2 et les cellules souches hématopoïétiques d'expression positive KDR2 trouvent une application thérapeutique dans la préparation du principe thérapeutique de la présente invention pour lutter contre le vieillissement fonctionnel et retarder les défaillances organiques. Le principe thérapeutique et la méthode thérapeutique permettent de lutter efficacement contre le vieillissement fonctionnel et de retarder les défaillances organiques et sont efficaces dans le traitement du syndrome de Hutchinson-Gilford et des démences préséniles.
PCT/CN2017/091926 2016-07-13 2017-07-06 Principe thérapeutique indiqué pour lutter contre le vieillissement fonctionnel et retarder les défaillances organiques Ceased WO2018010588A1 (fr)

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CN112494519A (zh) * 2020-12-16 2021-03-16 广州杜德生物科技有限公司 一种基于卵巢靶向的干细胞组合物的干细胞制剂

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CN107158034A (zh) * 2017-05-17 2017-09-15 博雅干细胞科技有限公司 基质细胞和造血干细胞组合治疗早老症或早衰症的用途
CN107412265B (zh) * 2017-05-17 2019-09-20 博雅干细胞科技有限公司 治疗早老症或早衰症的方法
CN111420034A (zh) * 2019-01-08 2020-07-17 上海莱馥医疗科技有限公司 一种用于治疗银屑病的混合干细胞制剂及其制备方法

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