WO2018010588A1 - Therapeutic agent for restoring body function aging and delaying organ function decline - Google Patents

Abstract

A therapeutic agent for restoring body function aging and delaying organ function decline, and particularly a therapeutic agent comprising mesenchymal stem cells, especially stromal cells having positive expression of GD2 and hematopoietic stem cells having positive expression of KDR2. Applications of the mesenchymal stem cells and particularly applications of a combination the stromal cells having positive expression of GD2 and the hematopoietic stem cells having positive expression of KDR2 in the preparation of the therapeutic agent for restoring body function aging and delaying organ function decline. The therapeutic agent and the therapeutic method can be effectively used for restoring body function aging and delaying organ function decline and can be effectively applied to patients having progeria and senilism.

Description

A therapeutic agent for repairing aging of the body and delaying the decline of organ function Technical field

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.

Background technique

Mesenchymal stem cells (MSCs) 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. In addition, 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.

Recent studies have shown that 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.

Generally, 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. With the development of medicine and biotechnology, it has been found in recent years that the placenta contains a large number of hematopoietic stem cells. Compared with the above three types 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. The requirements do not need to be very strict, and the response after transplantation is lighter and does not require the use of drugs. In addition, as a source of placental hematopoietic stem cells - placenta, 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. Many advantages make it possible for 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.

At present, 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). 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. Otherwise, the donor hematopoietic stem cells are not easily incubated in the patient, and even more severe graft-versus-host disease (GVHD) will occur. In a family of children, there is only a 1/4 HLA match probability, and in non-related unrelated donors, this probability is only one percent, which greatly limits the clinical application of BMT or MPST. .

The successful clinical application of cord blood hematopoietic stem cell transplantation in the past ten years has promoted the development of cord blood hematopoietic stem cell research. 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. The effect is expanded for those who lack the proper human leukocyte antigen-matching family or unrelated donor transplants. The main limitation of 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.

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. 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.

Therefore, those skilled in the art are eager to have an effective method for treating premature aging, thereby achieving the purpose of repairing the aging of the body and delaying the decline of organ function.

Summary of the invention

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.

To this end, 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.

The use according to the first aspect of the invention, wherein the mesenchymal stem cells are GD2+ stromal cells.

The use according to the first aspect of the invention, wherein the mesenchymal stem cell is a charge derived from a placenta and/or an umbilical cord Stem cells.

The use according to the first aspect of the invention, wherein the mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord.

The use according to the first 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 use according to the first aspect of the invention, wherein the hematopoietic stem cells are derived from: cord blood, bone marrow, placental blood, and/or mobilized peripheral blood.

The use according to the first aspect of the invention, wherein the hematopoietic stem cell is a KDR2-positively expressed hematopoietic stem cell.

The use according to the first aspect of the invention, wherein the hematopoietic stem cell is a KDR2-positively expressed hematopoietic stem cell, and the positive expression rate of KDR2 in the KDR2-positive hematopoietic stem cell is greater than 85%, such as greater than 90%.

The use according to the first aspect of the invention, wherein the positive expression rate of CD34 in the hematopoietic stem cells is greater than 80%, such as greater than 85%.

The use according to the first aspect of the invention, wherein 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 use according to the first aspect of the present invention, wherein the therapeutic agent is used in a mammal, for example, a human, and the dose of the mesenchymal stem cells is (0.1 to 10) × 10 ⁷ stromal cells per kg of body weight. For example 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 use according to the first aspect of the invention, wherein 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. Alternatively, in one embodiment, wherein the therapeutic agent is used in a mammal, such as a human, 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 use according to the first aspect of the invention, wherein 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.

Further, 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.

The therapeutic agent according to the second aspect of the invention, wherein the 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.

The therapeutic agent according to the second aspect of the invention, wherein the hematopoietic stem cell is a hematopoietic stem cell positively expressed by KDR2.

The therapeutic agent according to the second aspect of the present invention, wherein the hematopoietic stem cell is a KDR2-positively expressed hematopoietic stem cell, and 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. For example 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. Alternatively, in one embodiment, wherein the therapeutic agent is used in a mammal, such as a human, 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.

For example, for mesenchymal stem cells obtained through the placenta, reference can be made to Chinese Patent Application No. 201210292509.9 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. In one example, the method of obtaining the GD2+ stromal cells comprises the steps of:

(1) 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;

(2) 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;

(3) 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. 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;

(4) Formulating a placental whole cell cryopreservation solution: 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;

(5) 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;

If necessary, the resuscitation method used in conjunction with the cryopreservation method may be further included:

(6) Cryopreservation of placental whole cell resuscitation: the whole cell of the placenta frozen in step (4) is taken out from liquid nitrogen, thawed in a constant temperature water bath until half of the frozen solution begins to melt, using mesenchymal stem cell culture medium (for example Containing 15% FBS + 1% L-Glutamine + 0.05% Gentamicin + 84% DMEM-F12) The whole placenta cells were washed by spotting, and the volume ratio of the cell suspension obtained by thawing to mesenchymal stem cell medium was 1:3 ( 1ml: 3ml), mixed with mesenchymal stem cell culture The cell suspension of the nutrient was transferred to a centrifuge tube, and the DMSO was washed by centrifugation at 1400 rpm for 10 minutes, the supernatant was removed, and the cells were resuspended in PBS buffer, and the ratio of 1 part of the cell suspension to 2-3 parts of the erythrocyte lysate was used. Add 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;

If necessary, methods for isolation and expansion of mesenchymal stem cells after resuscitation may be further included:

(7) 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;

(8) Cell passage: When the adherence rate of adherent cells in the T25 flask reaches 80%, the adherent cells can be removed from the bottom of the T25 flask by digestive enzymes (TrypLE Express), the supernatant is removed after centrifugation, and added. The mesenchymal stem cell culture medium was resuspended, inoculated into a T25 cell culture flask for passage, and subjected to expansion culture; after that, the liquid was changed every two days until the fusion rate reached 80%, and then, if necessary, passage was carried out.

If necessary, 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;

If necessary, the placental mesenchymal stem cells obtained after passage of the above step (8) can be further frozen in liquid nitrogen for use.

In the process of obtaining the placental mesenchymal stem cells of GD2+ stromal cells with a GD2 positive expression rate of not less than 90%, the GD2 positive expression rate of the obtained cells was detected, and the positive expression rate of GD2 was not less than 90%. For example, not less than 95% of GD2+ stromal cells are used as the mesenchymal stem cells used in the present invention.

For another example, 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%. In one example, the method of obtaining the GD2+ stromal cells comprises the steps of:

(1) preparing a umbilical cord tissue cryopreservation solution: 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;

(2) Disinfection and cleaning: In the biological safety cabinet, the surface of the umbilical cord tissue is disinfected with alcohol, the umbilical cord is cut from the middle, and laid flat on a sterile 10 cm cell culture plate, and the tissue is washed with PBS to reduce the tissue. Red blood cells

(3) 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;

(4) 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;

If necessary, the resuscitation method used in conjunction with the cryopreservation method may be further included:

(5) 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;

If necessary, methods for isolation and expansion of mesenchymal stem cells after resuscitation may be further included:

(6) Attachment treatment of umbilical cord tissue: The resuscitation frozen umbilical cord tissue was plated in another 10 cm cell culture plate, and the number of tissue blocks in each plate was maintained at 10-15 pieces, and the tissue block was air-dried for 10-15 minutes. Until the tissue is attached to the plate;

(7) 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;

(8) Cell passage: When the adherence rate of adherent cells in the plate reaches 60%, the adherent cells can be removed from the bottom of the plate by using TrypLE Express, the supernatant is removed after centrifugation, and mesenchymal stem cells are added. The medium is resuspended, inoculated into a T25 cell culture flask for passage, and subjected to expansion culture; after that, the liquid is changed every two days until the fusion rate reaches 80%, and then, if necessary, passage is performed;

If necessary, 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;

If necessary, the passaged umbilical cord mesenchymal stem cells obtained in the above step (8) may be further frozen in liquid nitrogen for use.

In the umbilical mesenchymal stem cells of GD2+ stromal cells with a GD2 positive expression rate of not less than 90%, the GD2 positive expression rate of the obtained cells was detected, and the positive expression rate of GD2 was not less than 90%. For example, 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.

For example, for hematopoietic stem cells obtained through the placenta, it can be obtained by the method of Chinese Patent Application No. 2014104050724, for example, 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%. In one example, 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:

(1) Collection of placenta: Collect placenta and maternal blood in the aseptic environment of the operating room, store in a sterile placenta storage and transportation box, stick the label and barcode, and keep the temperature of the placenta storage box at 4-10 °C. Delivered to the laboratory within 24 hours;

(2) Initial examination of placenta: check the temperature, label, delivery date, storage and transportation box of the placenta storage and transportation box, whether there is leakage of blood, and whether there is blood sample of maternal blood;

(3) Pre-disinfection of placenta: Place the fetal face of the placenta on the bottom of the placenta washing box, rinse the surface of the placenta with physiological saline, open the drainage hole on the bottom of the placenta washing box, remove the washed saline, and check the calcification of the surface of the placenta;

(4) 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);

(5) 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;

(6) Concentration and purification of hematopoietic stem cells: 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. 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).

In the hematopoietic stem cell process in which the CD34 positive expression rate obtained above is greater than 80%, for example, greater than 85%, and the KDR2 positive expression rate is greater than 85%, for example, greater than 90%, the obtained hematopoietic stem cells are detected for CD34 positive expression rate and KDR2 positive expression. The hematopoietic stem cells having a CD34 positive expression rate greater than 80%, for example greater than 85% and a KDR2 positive expression rate greater than 85%, for example greater than 90%, were selected as hematopoietic stem cells for use in the present invention.

Alternatively, in one embodiment, 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. Then, it was taken out by centrifugation at RCF50-200 for 5-15 minutes, and mononuclear cells were automatically isolated, and AXP data were derived to obtain the number of hematopoietic stem cells isolated. 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.

Further, before the cryopreservation, the mesenchymal stem cells of the present invention, namely GD2+ stromal cells and KDR2+ hematopoietic stem cells, 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.

According to the results obtained in the animal test described in the present invention, 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.

Any of the technical features of any of the aspects of the invention or any one of the aspects of the invention are equally applicable to any one of any other embodiment or any other aspect, as long as they do not contradict each other, of course When applicable, the corresponding features may be appropriately modified as necessary. Various aspects and features of the present invention are further described below.

All documents cited in the present invention are hereby incorporated by reference in their entirety, and if the meanings expressed by these documents are inconsistent with the present invention, the expression of the present invention shall prevail. Moreover, the various terms and phrases used in the present invention have the ordinary meanings well known to those skilled in the art, and even though the present invention is intended to provide a more detailed description and explanation of the terms and phrases herein, such terms and phrases are Inconsistent with the well-known meaning, the meaning expressed in the present invention shall prevail.

detailed description

The invention is further described by the following examples, however, the scope of the invention is not limited to the embodiments described below. A person skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope of the invention. The present invention provides a general and/or specific description of the materials and test methods used in the tests. While many of the materials and methods of operation used to accomplish the objectives of the present invention are well known in the art, the present invention is still described in detail herein.

In the specific experiment of the present invention, 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%.

In the specific experiment of the present invention, the umbilical cord mesenchymal stem cells, i.e., GD2+ stromal cells, were obtained by the method of Examples 1, 6, and 7 of Chinese Patent Application No. 201210159916.2, and the positive expression rate of GD2 was more than 95%.

In the specific experiment of the present invention, 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%. .

In the specific experiment of the present invention, 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. 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

I. Selection and grouping of animals

1. Animal selection: The model mice were healthy C57BL/6J mice, female, 8 weeks old, weighing 18-22 g, 60 rats.

2. Animal grouping: They were randomly divided into control group, model group and treatment group, with 20 groups in each group.

Second, establish a mouse aging model

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.

Third, the infusion

Two weeks after the aging model was built, the treatment group was injected with GD2+ placental MSC and KDR2+ cord blood HSC. 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.

Fourth, the method

l. The mice were fixed with a tail vein injection frame.

2, rubbing the tail with an alcohol cotton ball to expand the blood vessels; the injection state is white tail, two red veins are clearly visible on both sides of the white tailbone.

3. Fix the mouse tail with the index finger of the left hand, the middle finger, the ring finger and the thumb. Hold 0.1 ml in front of the 1 ml syringe. The little finger of the right hand is placed on the thumb of the left hand holding the tail of the mouse, and the needle is inserted into the hand.

4. Injection: When the injection is done, the left hand is tailed, so that the tail is close to the table, and the tail and the edge of the table are close to the turning point for the needle insertion. Generally, the needle is inserted at 1/4 or l/3 from the tail tip. Immediately after the needle enters the skin, the needle is slightly upward, and the needle is inserted in parallel. When the needle is inserted, there is a sense of falling, and the bolus is slowly pushed.

V. Results:

1. 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. The difference (P ≤ 0.05), compared with the model group, there was a significant difference (P ≤ 0.01), which was statistically significant.

2. Histopathological sections of 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. The results showed that MSC can repair the visceral damage of aging mice, thus exerting its anti-aging effect.

Conclusion: The combination of GD2+MSC and KDR2+HSC significantly improved the aging-related indicators of D-galactose-induced subacute aging model mice, indicating that the combination of GD2+MSC and KDR2+HSC has significant repairing function. And the role of organ function decline.

Supplemental test: Refer to Example 1 above, but only treatment with GD2+MSC and only KDR2+HSC at the time of treatment, but found no therapeutic effect.

Example 2: Mechanism of MSC+HSC in the treatment of aging mice

Senescence-associated B-galactosidase (SA-β-Gal) activity assay

1. 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.

2. SA-β-Gal staining

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.

3, SA-β-Gal coloration results observation

The ratio of SA-β-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. .

4, aging-related B-galactosidase (SA-β-Gal) activity test results

The positive rate of SA-β-Gal staining in the control group was 3.03±0.66%. The positive rate of SA-β-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.

5. Relative expression of cell senescence-associated genes p16Ink4a, p53, p21cipl/wafl

(1) 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.

(2) p21: GD2+MSCs and KDR2+HSC treatment ld, 7d, 14d, 28d MSC p2l did not increase significantly, and there was no significant difference between the control group (p>0.05). The difference between p21 and control group 3d after GD2+MSC and KDR2+HSC treatment was significant (p<0.05), which was significantly lower.

(3) p53: GD2+MSCs and KDR2+HSC treatment ld, 3d, 7d, 14d, 28d MSC p53 was significantly lower than the control group, the difference between the groups was significant (p<0.05).

The above results show that the combination therapy of GD2+MSC and KDR2+HSC has a significant therapeutic effect on aging mice.

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.

Supplementary test: The senile mice were treated with placental mesenchymal stem cells, i.e., 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.

Supplementary test: The senile 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.

Supplementary test: The senile mice were treated with umbilical cord mesenchymal stem cells, GD2+ stromal cells and KDR2+ cord blood hematopoietic stem cells, according to the method of Example 1, and the results showed substantially the same therapeutic effects as in Example 1.

Industrial applicability

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.

Claims (10)

1. The use of mesenchymal stem cells in combination with hematopoietic stem cells in the preparation of a therapeutic agent for repairing functional aging of the body and delaying the decline of organ function.
2. The use according to claim 1, wherein said mesenchymal stem cells are GD2+ stromal cells.
3. The use according to claim 1, characterized in that:

   Wherein the mesenchymal stem cells are mesenchymal stem cells derived from the placenta and/or the umbilical cord;

   Wherein the mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord; and/or

   Wherein the mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord, and the positive expression rate of GD2 is greater than 90%, such as greater than 95%.
4. The use according to claim 1, characterized in that:

   Wherein the hematopoietic stem cells are derived from: cord blood, bone marrow, placental blood, and/or mobilized peripheral blood;

   Wherein the hematopoietic stem cell is a hematopoietic stem cell positively expressed by KDR2;

   Wherein the hematopoietic stem cells are KDR2-positive hematopoietic stem cells, and the positive expression rate of KDR2 in the KDR2-positive hematopoietic stem cells is greater than 85%, such as greater than 90%; and/or

   The CD34 positive expression rate in the hematopoietic stem cells is greater than 80%, for example greater than 85%.
5. The use according to claim 1, wherein said therapeutic agent is in the form of a kit comprising mesenchymal stem cells individually sealed and individually packaged and individually packaged hematopoietic stem cells.
6. The use according to claim 5, characterized in that:

   When the therapeutic agent is used in a mammal such as a human, the dose of the mesenchymal stem cells is (0.1 to 10) × 10 ⁷ stromal cells per kilogram of the patient's body weight, for example, the body weight per kilogram of the patient is (0.5 to 5). ) × ¹⁰⁷ stromal cells, such as an amount per kg body weight of the patient (0.75 ~ 1.5) × 10 ⁷ th stromal cells, e.g. kilogram of patient body weight per an amount of 10 ⁷ stromal cells;

   When the therapeutic agent is used in a mammal such as a human, the dose of the hematopoietic stem cells is (1 to 5) × 10 ⁷ mononuclear cells per kilogram of the patient's body weight, for example, the body weight per kilogram of the patient is (2 to 4). ×10 ⁷ mononuclear cells, for example, 3 × 10 ⁷ mononuclear cells per kg of patient weight; or, in one embodiment, wherein the therapeutic agent is used for a hematopoietic stem cell in a mammal such as a human The dose is (2 ~ 10) × 10 ⁵ mononuclear cells per kilogram of patient weight, for example, the amount of body weight per kilogram of patients is (2 ~ 5) × 10 ⁵ mononuclear cells, for example, the body weight per kg patient is (2 ~4) x 10 ⁵ mononuclear cells; and/or

   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.
7. A therapeutic agent for repairing aging of the body and delaying the decline of organ function, in the form of a kit comprising mesenchymal stem cells individually sealed and individually sealed and packaged hematopoietic stem cells.
8. The therapeutic agent according to claim 7 wherein:

   The mesenchymal stem cells are GD2+ stromal cells;

   The mesenchymal stem cells are mesenchymal stem cells derived from the placenta and/or the umbilical cord;

   The mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or the umbilical cord;

   The mesenchymal stem cells are GD2+ stromal cells derived from the placenta and/or umbilical cord, and the GD2 positive expression rate is greater than 90%, such as greater than 95%.
9. The therapeutic agent according to claim 7 wherein:

   The hematopoietic stem cells are derived from: cord blood, bone marrow, placental blood, and/or mobilized peripheral blood;

   The hematopoietic stem cell is a hematopoietic stem cell positively expressed by KDR2;

   The hematopoietic stem cell is a KDR2-positive hematopoietic stem cell, and the positive expression rate of KDR2 in the KDR2-positive hematopoietic stem cell is greater than 85%, such as greater than 90%; and/or

   The CD34 positive expression rate in the hematopoietic stem cells is greater than 80%, such as greater than 85%.
10. The therapeutic agent according to claim 7 wherein:

    When the therapeutic agent is used in a mammal such as a human, the dose of the mesenchymal stem cells is (0.1 to 10) × 10 ⁷ stromal cells per kilogram of the patient's body weight, for example, the body weight per kilogram of the patient is (0.5 to 5). ) × ¹⁰⁷ stromal cells, such as an amount per kg body weight of the patient (0.75 ~ 1.5) × 10 ⁷ th stromal cells, e.g. kilogram of patient body weight per an amount of 10 ⁷ stromal cells;

    When the therapeutic agent is used in a mammal such as a human, the dose of the hematopoietic stem cells is (1 to 5) × 10 ⁷ mononuclear cells per kilogram of the patient's body weight, for example, the body weight per kilogram of the patient is (2 to 4). ×10 ⁷ mononuclear cells, for example, 3 × 10 ⁷ mononuclear cells per kg of patient weight; or, in one embodiment, wherein the therapeutic agent is used for a hematopoietic stem cell in a mammal such as a human The dose is (2 ~ 10) × 10 ⁵ mononuclear cells per kilogram of patient weight, for example, the amount of body weight per kilogram of patients is (2 ~ 5) × 10 ⁵ mononuclear cells, for example, the body weight per kg patient is (2 ~4) x 10 ⁵ mononuclear cells; and/or

    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.