CN106074604A - For repairing the therapeutic agent that body function is aging and delays organ function to fail - Google Patents
For repairing the therapeutic agent that body function is aging and delays organ function to fail Download PDFInfo
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- CN106074604A CN106074604A CN201610551770.4A CN201610551770A CN106074604A CN 106074604 A CN106074604 A CN 106074604A CN 201610551770 A CN201610551770 A CN 201610551770A CN 106074604 A CN106074604 A CN 106074604A
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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
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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
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
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- 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
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- 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
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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
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
Abstract
The present invention relates to for repairing the therapeutic agent that body function is aging and delays organ function to fail.Specifically, the present invention relates to belong to cell therapy technology field, relate to that a kind of body function is aging and the therapeutic agent that delays organ function fail for repairing, particularly relate to a kind of therapeutic agent including mescenchymal stem cell particularly GD2 expresses the stromal cell being positive and KDR2 expression is positive hematopoietic stem cell.Further, the present invention relates to mescenchymal stem cell particularly GD2 express the stromal cell that is positive and KDR2 express both the hematopoietic stem cell being positive combine preparation for repair body function aging and delay organ function to fail therapeutic agent in purposes.It is aging and delay organ function to fail that therapeutic agent of the present invention and Therapeutic Method can be efficiently used for repairing body function, and is effectively applied to progeria and senilism patient.
Description
Technical field
The invention belongs to cell therapy technology field, relate to a kind of aging and delay organ function for repairing body function
The therapeutic agent of decline, particularly relates to a kind of therapeutic agent including GD2+ stromal cell and KDR2+ hematopoietic stem cell.Further
, the present invention relates to both GD2+ stromal cell and KDR2+ hematopoietic stem cell and combine in preparation aging for repairing body function
With the purposes in the therapeutic agent delaying organ function to fail.Have been found that therapeutic agent of the present invention can be efficiently used for repair machine
Body function is aging and delays organ function to fail.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) derives from grows mesoderm in early days and outer embryo
Layer, has the features such as multi-lineage potential, immunomodulating and self replication, is increasingly subject to the concern of people.Mescenchymal stem cell
In vivo or under external specific inductive condition, fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, the heart can be divided into
The Various Tissues cell such as flesh, endothelium, still has multi-lineage potential after continuous passage cultivation and freezen protective, can be as preferably
The injuries of tissues and organs reparation that seed cell causes for old and feeble and pathological changes, especially old and feeble to treatment and injuries of tissues and organs reparation
There is the biggest clinical value.
MSC contains abundant in bone marrow, but aging along with the age, the stem cell population in bone marrow also can significantly reduce,
Proliferation and differentiation ability the most significantly fails.It addition, bone marrow MSC transplants may cause immunoreation to allosome, and extract stem cell
Process to the damaging of patient and when gathering the other problems that runs into, all directly affects the clinical practice of bone marrow MSC so that
Find other alternative source for mesenchymal stem cells beyond bone marrow and become an important problem.
Recent study shows, also contains mescenchymal stem cell and can be successfully separated in umbilical cord tissue and placenta tissue.
This tissue-derived mescenchymal stem cell not only maintains the biological characteristics of mescenchymal stem cell, and separate dry
Cell is more original, has higher proliferation and differentiation ability.The functional activity of its immunocyte is low, is greatly reduced triggering immunoreation
And cause the risk of graft versus host disease.Latent virus and the infection of microorganism and to propagate probability ratio relatively low.Gatherer process
Simply, to puerpera and neonate without any harm and damage.Above reason be enough to make umbilical cord mesenchymal stem cells become between bone marrow
The ideal substitute of mesenchymal stem cells.
Hematopoietic stem cell (is commonly abbreviated as HSC), refers to a class cell with self renewal and Multidirectional Differentiation ability.It
Fundamental characteristics be that there is self-renewal capacity, i.e. after a cell cycle events, can produce two with division before
The hematopoietic stem cell that character is identical, has again Multidirectional Differentiation ability simultaneously, and the most under certain environmental conditions, hematopoietic stem cell has
The oriented ability being respectively hemocyte and breaking up.
Hematopoietic stem cell transplantation is now widely used for malignant hematologic disease (such as acute leukemia, chronic myelocytic leukemia
Deng), non-malignant intractable hematopathy (such as aplastic anemia, myelodysplastic syndrome etc.) hereditary (congenital
Property immunodeficiency thalassemia etc.) and some treatment of solid tumors.Hematopoietic stem cell transplantation refers to patient is carried out whole body photograph
Penetrate, after chemotherapy and immunosuppressant pretreatment, by Normal donor or autologous hematopoietic stem cell intravascular infusion to patient, make reconstruction
Normal hemopoietic and immunologic function.
In general, hematopoietic stem cell is present in three positions, is bone marrow, peripheral blood and Cord blood respectively, comes according to it
Source is referred to as marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell.Along with medical science and biology
The development of technology, containing substantial amounts of hematopoietic stem cell in discovered in recent years Placenta Hominis, with the hematopoietic stem cell phase in above-mentioned three kinds of sources
Ratio, in Placenta Hominis, the quantity of contained hematopoietic stem cell is the highest, and the distribution type requirement transplanting placental hematopoietic stem cell need not the tightest
React relatively light after lattice, and transplanting and need not use medicine.Additionally, as placental hematopoietic stem cell source-Placenta Hominis, source is wide
General, anemia of pregnant woman often becomes garbage after producing, and it gathers and mother and the sensation of neonate any discomfort or generation will not be caused to appoint
The best impact.Plurality of advantages makes placental hematopoietic stem cell be expected to replace marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells
With umbilical cord blood hematopoietic stem cell in hematopoietic stem cell transplantation.
The method of the hematopoietic stem cell transplantation mainly taked in the treatment of international disease in the blood system at present, according to cell
Source, can be divided three classes: marrow hemopoietic stem cells is transplanted (BMT), mobilized Peripheral blood stem cell transplantation (MPST) and Cord blood
Stem cell transplantation (UCBT).The above two source of human stem cell enrich, and general number of nucleated cells is up to 5-10 × 108/ Kg, CD34+ are thin
Born of the same parents' (surface markers of a kind of hematopoietic stem/progenitor) are up to 1-5 × 106/ Kg, but owing to needing HLA tight between donor and receptor
Lattice are consistent, the success that guarantee is transplanted, and otherwise donor hematopoietic stem cell is difficult to live in patient body's interplantation, also can send out even if surviving
Raw more serious graft versus host disease (GVHD).In the children of one family, the probability that only 1/4HLA is consistent, and non-
In the unrelated donor of blood relationship, this probability only has one of percentage, significantly limit BMT or MPST clinically extensive
Application.
Umbilical cord blood stem cell transplantation the most successfully applies that to promote umbilical cord blood hematopoietic dry thin in the recent decade
The development of born of the same parents' research.Cord blood comes from Placenta Hominis, generally discarded after Delivery, has now found that in Cord blood and makes containing abundant
Hemocytoblast, the concentration of its CD34+ cell is similar with bone marrow, accounts for the 0.1-0.5% of total cell number, and Hematopoietic Stem more in early days
Cell CD34-the most relatively bone marrow concentration is higher.As the source of a kind of hematopoietic cell, now with Umbilical Cord Blood Transplantation operation by
Cumulative add.Compared with BMT, the advantage of UCBT resides in reduced serious graft versus host disease, effectively expand for
Those lack suitable human leukocyte antigen matching family or the probability of irrelevant donor transplanting.Umbilical Cord Blood Transplantation is main
The hematopoietic stem cell limited amount being limited in umbilical blood, this restriction promotes the umbilicus needing to use 2 multiple dose units in clinic
Blood, transplants bigger the growing up of body weight and is help others；Another kind of method is by Hematopoiesis in Vitro expansion of stem cells and cultivates, but amplification in vitro
Needing the cost time, expense is the highest, it is often more important that while amplification, and hematopoietic stem cell also breaks up, the knot of clinical practice
Fruit show umbilical hemopoietic stem cell amplification before and after to transplant without much difference.
There is substantial amounts of hematopoietic stem cell in the mature Placenta Hominis of the mankind, has a more hematopoietic stem cell than Cord blood, and these
Placental hematopoietic stem cell can be separated before and after stored frozen.Placental hematopoietic stem cell colony-forming units (CFU)
Activity determines that, the transplantation experiments in Immune deficient mice has turned out placental hematopoietic stem cell potentiality in transplanting.This
A little results strongly suggest that, the mature Placenta Hominis of the mankind is likely to become the source of a kind of novel hematopoietic stem cell for transplanting.
Having substantial amounts of hematopoietic stem cell in Placenta Hominis, placental hematopoietic stem cell is stem cell more in early days, can be divided into various in vivo
Cell, placental blood is contained within various stages of enriching hematopoietic stem cell in early days, and its content is about tens times of Cord blood, one
Hematopoietic stem cell in Placenta Hominis can fully meet requirements of two adult Man's Demands, if disease can be applied to together with cord blood cell
People, considerably increases the content of hematopoietic stem cell undoubtedly, and this makes hematopoietic stem cell can be entirely applied to all of applicable crowd.
Body function is aging and organ function decline is the most special and typically show as progeria (people are generally also known as
Senilism disease).Repair body function aging and delay organ function decline be treat progeric necessary means.Unluckily
It is to there is no the progeric method of effective treatment the most clinically.
Therefore, those skilled in the art urgently expect there is a kind of effective method to treat progeria, thus realize repairing
Body function is aging and the purpose that delays organ function to fail.
Summary of the invention
It is an object of the invention to provide a kind of effective method and delay and treat progeria, thus realize repairing body
Function is aging and the purpose that delays organ function to fail.Present inventors have surprisingly discovered that, use mescenchymal stem cell with
It is aging and delay organ function to fail that hematopoietic stem cell can repair body function effectively, thus reaches to treat progeric
Purpose.The present invention finds based on this and is accomplished.
Preparing for repairing body to this end, one aspect of the present invention provides mescenchymal stem cell with hematopoietic stem cell combination
Function aging and delay organ function to fail therapeutic agent in purposes.
Purposes according to a first aspect of the present invention, wherein said mescenchymal stem cell is GD2+ stromal cell.
Purposes according to a first aspect of the present invention, wherein said mescenchymal stem cell is derived from Placenta Hominis and/or umbilical cord
Mescenchymal stem cell.
Purposes according to a first aspect of the present invention, wherein said mescenchymal stem cell is derived from Placenta Hominis and/or umbilical cord
GD2+ stromal cell.
Purposes according to a first aspect of the present invention, wherein said mescenchymal stem cell is derived from Placenta Hominis and/or umbilical cord
GD2+ stromal cell, and GD2 positive expression rate is more than 90%, is greater than 95%.
Purposes according to a first aspect of the present invention, wherein said derived from hematopoietic precursor cells in: Cord blood, bone marrow, placental blood,
And/or mobilized peripheral blood.
Purposes according to a first aspect of the present invention, wherein said hematopoietic stem cell is that the Hematopoietic Stem of KDR2 positive expression is thin
Born of the same parents.
Purposes according to a first aspect of the present invention, wherein said hematopoietic stem cell is that the Hematopoietic Stem of KDR2 positive expression is thin
Born of the same parents, in the hematopoietic stem cell of this KDR2 positive expression, KDR2 positive expression rate is more than 85%, is greater than 90%.
Purposes according to a first aspect of the present invention, in wherein said hematopoietic stem cell, CD34 positive expression rate is more than 80%,
It is greater than 85%.
Purposes according to a first aspect of the present invention, wherein said therapeutic agent is the form of medicine box, and this medicine box includes individually
Seal-packed mescenchymal stem cell and independent seal-packed hematopoietic stem cell.
Purposes according to a first aspect of the present invention, wherein said therapeutic agent for mammal, such as described in during people between
The dosage of mesenchymal stem cells is per kilogram weight in patients consumption for (0.1～10) × 107Individual stromal cell, such as per kilogram patient
Body weight consumption is (0.5～5) × 107Individual stromal cell, such as per kilogram weight in patients consumption are (0.75～1.5) × 107Individual base
Cell plastid, such as per kilogram weight in patients consumption are 107Individual stromal cell.
Purposes according to a first aspect of the present invention, wherein said therapeutic agent for mammal, is such as made described in during people
The dosage of hemocytoblast is per kilogram weight in patients consumption for (1～5) × 107Individual mononuclearcell, such as per kilogram patient body
The amount of reusing is (2～4) × 107Individual mononuclearcell, such as per kilogram weight in patients consumption are 3 × 107Individual mononuclearcell.Or
Person, in one embodiment, wherein said therapeutic agent for mammal, the such as dosage of described hematopoietic stem cell during people
It is per kilogram weight in patients consumption for (2～10) × 105Individual mononuclearcell, such as per kilogram weight in patients consumption are (2～5)
×105Individual mononuclearcell, such as per kilogram of body weight consumption are (2～4) × 105Individual mononuclearcell.
Purposes according to a first aspect of the present invention, wherein said therapeutic agent, when for mammal such as people, first uses
Mescenchymal stem cell, used hematopoietic stem cell after 1 month.
Further, second aspect present invention provides a kind of aging and delay organ function to decline for repairing body function
The therapeutic agent moved back, it is the form of medicine box, and this medicine box includes independent seal-packed mescenchymal stem cell and independent sealed bundle
The hematopoietic stem cell of dress.
Therapeutic agent according to a second aspect of the present invention, wherein said mescenchymal stem cell is GD2+ stromal cell.
Therapeutic agent according to a second aspect of the present invention, wherein said mescenchymal stem cell is derived from Placenta Hominis and/or umbilical cord
Mescenchymal stem cell.
Therapeutic agent according to a second aspect of the present invention, wherein said mescenchymal stem cell is derived from Placenta Hominis and/or umbilical cord
GD2+ stromal cell.
Therapeutic agent according to a second aspect of the present invention, wherein said mescenchymal stem cell is derived from Placenta Hominis and/or umbilical cord
GD2+ stromal cell, and GD2 positive expression rate is more than 90%, is greater than 95%.
Therapeutic agent according to a second aspect of the present invention, wherein said derived from hematopoietic precursor cells is in Cord blood, bone marrow, Placenta Hominis
Blood and/or mobilized peripheral blood.
Therapeutic agent according to a second aspect of the present invention, wherein said hematopoietic stem cell is that the Hematopoietic Stem of KDR2 positive expression is thin
Born of the same parents.
Therapeutic agent according to a second aspect of the present invention, wherein said hematopoietic stem cell is that the Hematopoietic Stem of KDR2 positive expression is thin
Born of the same parents, in the hematopoietic stem cell of this KDR2 positive expression, KDR2 positive expression rate is more than 85%, is greater than 90%.
Therapeutic agent according to a second aspect of the present invention, in wherein said hematopoietic stem cell, CD34 positive expression rate is more than
80%, it is greater than 85%.
Therapeutic agent according to a second aspect of the present invention, between wherein said therapeutic agent is described in when for mammal such as people
The dosage of mesenchymal stem cells is per kilogram weight in patients consumption for (0.1～10) × 107Individual stromal cell, such as per kilogram patient
Body weight consumption is (0.5～5) × 107Individual stromal cell, such as per kilogram weight in patients consumption are (0.75～1.5) × 107Individual base
Cell plastid, such as per kilogram weight in patients consumption are 107Individual stromal cell.
Therapeutic agent according to a second aspect of the present invention, wherein said therapeutic agent is made described in when for mammal such as people
The dosage of hemocytoblast is per kilogram weight in patients consumption for (1～5) × 107Individual mononuclearcell, such as per kilogram patient body
The amount of reusing is (2～4) × 107Individual mononuclearcell, such as per kilogram weight in patients consumption are 3 × 107Individual mononuclearcell.Or
Person, in one embodiment, wherein said therapeutic agent is the dosage of described hematopoietic stem cell when for mammal such as people
It is per kilogram weight in patients consumption for (2～10) × 105Individual mononuclearcell, such as per kilogram weight in patients consumption are (2～5)
×105Individual mononuclearcell, such as per kilogram weight in patients consumption are (2～4) × 105Individual mononuclearcell.
Therapeutic agent according to a second aspect of the present invention, wherein said therapeutic agent, when for mammal such as people, first makes
With mescenchymal stem cell, after 1 month, use hematopoietic stem cell.
Mescenchymal stem cell used by the present invention can use methods known in the art to prepare.
Such as, for the mescenchymal stem cell obtained by Placenta Hominis, it is referred to Chinese Patent Application No.
Method in 201210292509.9 obtains, and the GD2 positive expression rate such as obtained by document method is not less than 90%
GD2+ stromal cell.In an example, it is thus achieved that the method for this GD2+ stromal cell comprises the following steps:
(1) placenta tissue cleans: placenta tissue is to process in Biohazard Safety Equipment, uses appropriate according to Placenta Hominis size
Placenta tissue is rinsed 2-3 time by PBS, makes placenta tissue remained on surface blood rinse well, Placenta Hominis surface depletion of blood
Liquid grumeleuse；
(2) placenta tissue digestion process: use operating scissors to cut placental lobules from the placenta tissue that step (1) obtains,
Lobule is transferred on culture dish, adds 25ml PBS and placental lobules is shredded as far as possible, add 25ml 0.25% pancreas
Enzyme (Gibco) (pancreatin is 1:1 with PBS volume ratio) also mixes tissue, culture dish is put into the constant-temperature table of 37 DEG C
In digest 20 minutes；
(3) placenta tissue filtration treatment: add 5ml FBS (Gibco) in culture dish and mix to reach to terminate digestion
Purpose, digestion after placenta tissue fragment transfer to, on 200 mesh metallic filters, placenta tissue fragment is ground
And utilize another culture dish to collect the liquid filtered, the PBS cleaning that separately twice adds 10ml toward metallic filter
Organize and continue to grind, the filtrate of collection being transferred to 50ml centrifuge tube, is centrifuged 10 minutes with speed 1400rpm, remove supernatant
Liquid also adds PBS re-suspended cell, is centrifuged 10 minutes with speed 1400rpm, removes supernatant, adds PBS weight
Outstanding cell, extraction sample in a small amount carries out cell counting, is centrifuged 10 minutes with the effect reaching to clean cell with speed 1400rpm；
(4) the preparation full cells frozen storing liquid of Placenta Hominis: comprise the white egg of human blood of 15 weight portions in the full cells frozen storing liquid of described Placenta Hominis
In vain, the DMSO (dimethyl sulfoxide) and the DMEM-F12 of 65 weight portions of 10 weight portions, the frozen stock solution prepared is placed on 4 DEG C of Refrigerator stores
Until using；
(5) the full cell cryopreservation of Placenta Hominis: remove supernatant, at 4 DEG C after placenta tissue filtrate step (3) obtained is centrifugal
Low temperature environment under, add step (4) the full cells frozen storing liquid that obtains, then with each pipe every milliliter 4 × 107To 1 × 108's
Cell density adds in cryopreservation tube, and this process need to be carried out under the cryogenic conditions of 4 DEG C, is put into by cryopreservation tube in program temperature reduction box,
First deepfreeze 0.5 hour under the temperature conditions of 4 DEG C, then under the temperature conditions of-80 DEG C freezing 1 day, then by cryopreservation tube in
In liquid nitrogen, freezing, standby；
If desired, method for resuscitation matching used with cryopreservation methods can be included further:
(6) the full cell recovery of frozen Placenta Hominis: the full cell of Placenta Hominis that step (4) is freezing is taken out from liquid nitrogen, is placed on constant temperature
Thawing in water-bath to half frozen stock solution and start to melt, (it such as comprises 15%FBS+1%L-to utilize mescenchymal stem cell culture medium
Glutamine+0.05%Gentamicin+84%DMEM-F12) carry out drop method and clean the full cell of Placenta Hominis, the thin of acquisition of thawing
Born of the same parents' suspension is 1:3 (1ml:3ml) with the volume ratio of mescenchymal stem cell culture medium, is blended with mescenchymal stem cell culture medium
Cell suspension transfers to centrifuge tube, is centrifuged 10 minutes with speed 1400rpm and cleans DMSO, removes supernatant, add PBS
Re-suspended cell, and add erythrocyte cracked liquid (Roche) with 1 part of cell suspension than the ratio of 2-3 part erythrocyte cracked liquid,
Cultivate 10-15 minute in the environment of 15-25 DEG C, put centrifuge into and be centrifuged 10 minutes with speed 1400rpm and be centrifuged, centrifugal after
Removing supernatant, observe erythrocyte splitting situation, the step if needed repeating erythrocyte splitting cracks, and is eventually adding
PBS re-suspended cell also extracts in a small amount sample and carries out cell counting, puts centrifuge into and is centrifuged 10 minutes with speed 1400rpm
It is centrifuged cleaning cell, removes supernatant；
If desired, after can including recovery further, mescenchymal stem cell separates and the method for amplification:
(7) cell is cultivated: add appropriate mescenchymal stem cell culture medium in the full cell that step (6) obtains resuspended carefully
Born of the same parents, transfer to T25 culture bottle, then are put into by T25 culture bottle in 37 DEG C of incubators that CO2 concentration is 5% and cultivate, and cultivate extremely
When the 6th day, T25 culture bottle is taken out from incubator, carry out partly changing for the first time liquid, continue to cultivate, when the 9th day, T25 is cultivated
Bottle takes out from incubator, carries out second time and partly changes liquid, the 12nd day plate inside culture medium take away, add between 15ml and fill
Matter stem cell media continues to cultivate, and within the most every 2 days, the most entirely changes liquid；
(8) passage: the attached cell fusion rate inside T25 culture bottle reaches about 80%, available digestive enzyme
Attached cell is departed from bottom T25 culture bottle by (TrypLE Express), removes supernatant, and it is dry thin to add mesenchyme after being centrifuged
Born of the same parents' culture medium Eddy diffusion cell, is inoculated in T25 Tissue Culture Flask and passes on, and carry out amplification cultivation；Within the most every two days, change
Liquid is once until after fusion rate reaches 80%, to obtain final product, to pass on.
If desired, following items can be detected further for above step (8) gained placenta mesenchyma stem cell
At least one: cytoactive, cell contamination, heredopathia, HLA-ABC/DR distribution type；
If desired, the placenta mesenchyma stem cell after can being passed on by above step (8) gained further is in liquid nitrogen
Frozen, standby.
This placenta mesenchyma that GD2 positive expression rate in above-mentioned acquisition is not less than the GD2+ stromal cell of 90% is dry thin
During born of the same parents, the cell obtained is carried out GD2 positive expression rate detection, choose GD2 positive expression rate and be not less than 90% such as
It is not less than the mescenchymal stem cell that the GD2+ stromal cell of 95% uses as the present invention.
The most such as, for the mescenchymal stem cell obtained by umbilical cord, it is referred to Chinese Patent Application No.
Method in 201210159916.2 obtains, and the GD2 positive expression rate such as obtained by document method is not less than 90%
GD2+ stromal cell.In an example, it is thus achieved that the method for this GD2+ stromal cell comprises the following steps:
(1) preparation umbilical cord tissue frozen stock solution: described umbilical cord tissue frozen stock solution comprises 80 weight portions human albumin and
The DMSO (dimethyl sulfoxide, dimethyl sulfoxide) of 10 weight portions, the cold liquid storage prepared be placed on 4 DEG C of Refrigerator stores until
Use；
(2) sterilize and clean: in Biohazard Safety Equipment, with ethanol, umbilical cord tissue surface being carried out disinfection, by umbilical cord therefrom
Between cut off, be laid on aseptic 10cm cell culture plate, utilize PBS tissue, to reduce the above erythrocyte of tissue；
(3) umbilical cord tissue processes: the umbilical cord tissue that step (2) obtains is transferred to another 10cm cell culture plate
In, umbilical cord tissue is cut into the square shape of size 1cm3；
(4) umbilical cord tissue is cold deposits: under the low temperature environment of 4 DEG C, adds in cryopreservation tube by piece of tissue and frozen stock solution, then will
Cryopreservation tube puts into program temperature reduction box, first deepfreeze 0.5 hour under the temperature conditions of 4 DEG C, colder under the temperature conditions of-80 DEG C
Freeze 1 day, then by cryopreservation tube freezing in liquid nitrogen, standby；
If desired, method for resuscitation matching used with cryopreservation methods can be included further:
(5) freezing and storing umbilical tissue recovery: the umbilical cord tissue that step (4) is freezing is taken out from liquid nitrogen, is placed on water bath with thermostatic control
In thaw to half frozen stock solution start melt, (it such as comprises 15%FBS+1%L-to utilize mescenchymal stem cell culture medium
Glutamine+0.05%Gentamicin+84%DMEM-F12) carry out drop method and clean umbilical cord tissue, utilize 100um to filter
Waste liquid is removed by device；
If desired, after can including recovery further, mescenchymal stem cell separates and the method for amplification:
(6) the adherent process of umbilical cord tissue: the freezing and storing umbilical tissue of recovery is laid in another 10cm cell culture plate
In, the piece of tissue quantity in each plate maintains 10-15 block, makes piece of tissue air-dry 10-15 minute until organizing and being attached to plate
On；
(7) umbilical cord tissue is cultivated: (it such as comprises 15% to be slowly added into mescenchymal stem cell culture medium along plate edge
FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) extremely tissue floods；Plate is placed in CO2
Concentration be 5% 37 DEG C of incubators cultivate, cultivate to when the 5th day by plate from incubator take out, add 5ml mesenchyme
Stem cell media；At the tenth day by the media transfer in plate, add the fresh mescenchymal stem cell culture medium of 15ml；?
Within 12nd day, remove all umbilical cord tissue blocks and continue to cultivate, within the most every two days, the most entirely changing liquid；
(8) passage: the attached cell fusion rate inside plate reaches about 60%, available digestive enzyme
Attached cell is departed from bottom plate by (TrypLE Express), removes supernatant, and add mescenchymal stem cell training after being centrifuged
Support the new suspension cell of basic weight, be inoculated in T25 Tissue Culture Flask and pass on, and carry out amplification cultivation；Within the most every two days, change liquid one
Secondary until after fusion rate reaches 80%, to obtain final product, to pass on the most again；
If desired, following items can be detected further for above step (8) gained umbilical cord mesenchymal stem cells
At least one: cytoactive, cell contamination, heredopathia, HLA-ABC/DR distribution type；
If desired, the umbilical cord mesenchymal stem cells after can being passed on by above step (8) gained further is in liquid nitrogen
Frozen, standby.
This umbilical cord mesenchyma that GD2 positive expression rate in above-mentioned acquisition is not less than the GD2+ stromal cell of 90% is dry thin
During born of the same parents, the cell obtained is carried out GD2 positive expression rate detection, choose GD2 positive expression rate and be not less than 90% such as
It is not less than the mescenchymal stem cell that the GD2+ stromal cell of 95% uses as the present invention.
Hematopoietic stem cell used by the present invention can use methods known in the art to prepare.
Such as, for the hematopoietic stem cell obtained by Placenta Hominis, it is referred to Chinese Patent Application No. 2014104050724
In method obtain, such as in the hematopoietic stem cell obtained by document method, CD34 positive expression rate is more than 80%, such as
More than 85%；Further, by KDR2 positive expression rate in the hematopoietic stem cell that document method obtains more than 85%, it is greater than
90%.In an example, it is thus achieved that the method for the cell of this KDR2 positive expression comprises the following steps: the collection of Placenta Hominis, Placenta Hominis
Initial survey, the pre-sterilization of Placenta Hominis, Placenta Hominis and female blood examination survey, the lavation of placental hematopoietic stem cell, the concentrating and purifying of hematopoietic stem cell.
Each step particularly as follows:
(1) collection of Placenta Hominis: collect Placenta Hominis and female blood under operating room gnotobasis, deposit in aseptic Placenta Hominis accumulating box
In, posting label, bar code, the temperature of Placenta Hominis accumulating box is maintained at 4-10 DEG C, sends to laboratory in 24 hours；
(2) initial survey of Placenta Hominis: check temperature in Placenta Hominis accumulating box box, label, the date of service, accumulating box whether have seepage,
Whether there is female blood blood sample；
(3) pre-sterilization of Placenta Hominis: the fetus face of Placenta Hominis is launched is positioned over Placenta Hominis and rinses the bottom of box, rush with normal saline
Wash Placenta Hominis surface, open Placenta Hominis and rinse the osculum in cassette bottom face, remove the normal saline of flushing, check Placenta Hominis Surface calcification feelings
Condition；
(4) Placenta Hominis and female blood examination are surveyed: detecting the Placenta Hominis taken and female blood sample, the project of detection includes hepatitis
Poison detection, HIV (human immunodeficiency virus) detection, sexually transmitted disease (STD) detection, tissue matching (HLA) detection, hematopoietic stem/progenitor qualitative detection (CFU-GM)；
(5) lavation of placental hematopoietic stem cell: Placenta Hominis is aseptically washed on Placenta Hominis with physiological saline solution
Blood clot and hematocele, after using disinfectant sterilization, irrigating solution syringe needle is inserted in Placenta Hominis umbilical artery, placental perfusion returnable bottle pin
Head inserts in umbilical vein, and mosquito forceps clamps, and slowly opens constant flow pump, and irrigating solution moves through sebific duct, switch, peristaltic pump, syringe needle, Placenta Hominis
Arteries and veins, Placenta Hominis, placental vein, placental perfusion returnable bottle syringe needle, finally receive irrigating solution in irrigating solution returnable bottle；
(6) concentrating and purifying of hematopoietic stem cell: by lavation irrigating solution out, in centrifuge with 1500-2000rpm from
Heart 15-20 minute, removes supernatant, collects the liquid of precipitation and lower floor, precipitates the liquid with lower floor and physiology by collect
Saline mixes to obtain mixed liquor in the ratio of 2:1-1:2, then mixed liquor and lymphocyte separation medium is divided in the ratio of 2:1-1:2
Not joining in centrifuge tube, the order of interpolation is first to add lymphocyte separation medium in centrifuge tube, is slow added into mixed liquor,
Adition process noting, the liquid level keeping lymphocyte separation medium is smooth, be centrifuged 20-25 minute with 2200-2500rpm after adding,
Slowly accelerate time centrifugal to slow down slowly, after centrifugal end, in the middle tunica albuginea layer of collection to new centrifuge tube, add with the ratio of 2:1-1:2
Normal saline mixes, and 1200-1500rpm is centrifuged 10-15 minute；Remove supernatant after centrifugal end, add 10-20mL physiology salt
Water piping and druming precipitates, then 1200-1500rpm is centrifuged 10-15 minute, after centrifugal end, removes supernatant, resuspended by DMEM culture medium
Precipitation, collects hematopoietic stem cell group, resuspended cell mass carries out cell and mycotic culture, hematopoietic stem cell detection by quantitative
(CD34), hematopoietic stem cell Activity determination (Trypan Blue), hematopoietic stem cell qualitative detection (CFU-GM).
CD34 positive expression rate in above-mentioned acquisition is greater than 85% more than 80%, and KDR2 positive expression rate is more than
During 85% is greater than the hematopoietic stem cell of 90%, the hematopoietic stem cell obtained is carried out CD34 positive expression rate detection
With KDR2 positive expression rate, choose CD34 positive expression rate and be greater than 85% and KDR2 positive expression rate be more than more than 80%
85% is greater than the hematopoietic stem cell of 90% crosses the hematopoietic stem cell used as the present invention.
Or, in one embodiment, the hematopoietic stem cell used by the present invention can obtain according to following method: takes Hematopoietic Stem
Cell derived tissue (includes but not limited to Cord blood, bone marrow, placental blood, mobilized peripheral blood), adds hydroxyl in 8:1～1:8 ratio
Hydroxyethyl starch, after mix homogeneously, transfers in AXP hematopoietic stem cell piece-rate system supporting process consumptive material, loads AXP Hematopoietic Stem thin
Born of the same parents' piece-rate system, puts into centrifuge, is centrifuged 10～30min with eccentricity (RCF) 1200～1600, then is centrifuged with RCF50～200
Take out after 5～15min, be automatically separated and obtain mononuclearcell, derive AXP data and obtain the separation quantity of hematopoietic stem cell.Make
With CD34 and the KDR2 content of the cell arrived that flow cytomery separates.Then the cell of isolated is pressed 8:1～1:8
Ratio adds DMSO, puts under liquid nitrogen environment frozen after programmed cooling extremely-90 DEG C.Before using, it is placed in 37 degree of water-baths recovery.Make
The CD34 positive expression rate obtained in aforementioned manners is greater than 85% more than 80%, and KDR2 positive expression rate is more than 85% example
The hematopoietic stem cell of such as larger than 90%.
Further, before frozen, mescenchymal stem cell of the present invention i.e. GD2+ stromal cell and KDR2+ can be made
Hemocytoblast is sub-packed in bottle independently of one another, is then placed in medicine box of the present invention by two bottles, obtains the present invention
For repairing the therapeutic agent that body function is aging and delays organ function to fail, then this therapeutic agent is protected under conditions of cryopreservation
Deposit.
The result obtained in animal experiment recorded according to the present invention, the present inventor attempts the inventive method is used for people
Particularly there is the patient of progeria feature aging and delay organ function to fail to repair body function.Result shows, this
Bright method not only presents excellent result in animal experiment, and presents encouraging results in human body.
Arbitrary technical characteristic that any embodiment of either side of the present invention or this either side is had is equally applicable
Other any embodiment or any embodiment of other either side, as long as they will not be conflicting, certainly mutually
Between where applicable, if necessary can make individual features suitably to modify.Make into one with feature the most to various aspects of the present invention
The description of step.
All documents recited in the present invention, their full content is incorporated herein by, and if these literary compositions
Offer expressed implication and the present invention inconsistent time, be as the criterion with the statement of the present invention.Additionally, the present invention use various terms and
Phrase has and well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this these terms and
Phrase is described in more detail and explains, the term mentioned and phrase are if any inconsistent with common art-recognized meanings, with institute of the present invention table
The implication stated is as the criterion.
Detailed description of the invention
The present invention can be conducted further description by the following examples, but, the scope of the present invention does not limit
In following embodiment.One of skill in the art is it is understood that on the premise of without departing substantially from the spirit and scope of the present invention, permissible
The present invention is carried out various change and modification.The present invention to test used in material and test method carry out generality
And/or concrete description.Although being to it is known in the art that by realizing many materials that the object of the invention used and operational approach
But the present invention still describes in detail as far as possible at this.
In the specific experiment of the present invention, the placenta mesenchyma stem cell i.e. GD2+ stromal cell used is with reference to China
The method of the embodiment 1,6,7 of number of patent application 201210292509.9 obtains, and GD2 positive expression rate is more than 95%.
In the specific experiment of the present invention, the umbilical cord mesenchymal stem cells i.e. GD2+ stromal cell used is with reference to China
The method of the embodiment 1,6,7 of number of patent application 201210159916.2 obtains, and GD2 positive expression rate is more than 95%.
In the specific experiment of the present invention, the placental hematopoietic stem cell used is with reference to Chinese Patent Application No.
The method of the embodiment 1 of 2014104050724 obtains, and CD34 positive markers expression rate more than 85%, KDR2 mark
Thing positive expression rate is more than 90%.
In the specific experiment of the present invention, use its CD34 positive markers expression rate of umbilical cord blood hematopoietic stem cell arrived big
In 85%, KDR/2 positive markers expression rate is more than 90%, and prepares with reference to following method: in 8:1～1:8 ratio
Add hetastarch, after mix homogeneously, transfer in AXP hematopoietic stem cell piece-rate system supporting process consumptive material, load AXP and make
Hemocytoblast piece-rate system, puts into centrifuge, is centrifuged 10～30min with eccentricity (RCF) 1200～1600, then with RCF50～
200 be centrifuged 5～15min after take out, be automatically separated and obtain mononuclearcell, derive AXP data and obtain the separation of hematopoietic stem cell
Quantity.Use CD34 and the KDR2 content of the cell arrived of flow cytomery separation.Then the cell of isolated is pressed
8:1～1:8 ratio adds DMSO, puts under liquid nitrogen environment frozen after programmed cooling extremely-90 DEG C.Before using, it is placed on 37 degree of water-baths
Middle recovery.
Embodiment 1: use placenta mesenchyma stem cell i.e. GD2+ stromal cell to treat with Cord blood KDR2+ hematopoietic stem cell
Mouse aging
One, the selection of animal and packet
1, the selection of animal: model mice is healthy C57BL/6J mice, female, 8 week old, weighs 18～22g, 60.
2, animal packet: be randomly divided into merely matched group, model group, treatment group three groups, often group 20.
Two, mice aging model is set up
Model group and the D-galactose for the treatment of group neck every day dorsal sc injection 5%, injection volume is 0.25ml/10g, continuously
It is administered and 8-16 week sets up aged animal model.Matched group then gives the normal saline of same volume.
Three, infusion
After aging model builds up two weeks, treatment group injection GD2+ Placenta Hominis MSC and KDR2+ Cord blood HSC.Use expression green
The slow virus carrier labelling GD2+MSC of color fluorescin and Cord blood KDR2+HSC, to determine GD2+MSC and Cord blood KDR2+
HSC is in the implantation situation of Organs of Mice.Aging marker before and after detection treatment: include Mouse Weight, thymus, spleen, blood
Clearly, hepatic tissue, lungs, cerebral tissue catalase (CAT), superoxide dismutase (SOD) vigor and malonaldehyde (MDA) content
Change.
First course for the treatment of of treatment group: through mouse tail vein infusion people source GD2+ Placenta Hominis MSC single cell suspension 2 × 106/
Only, once, be administered altogether is for 4 times a course for the treatment of to Per-Hop behavior to 0.2mL/.Second course for the treatment of: first week through mouse tail vein infusion people
Source GD2+ Placenta Hominis MSC single cell suspension 2 × 106/ 0.2mL/, second week infusion people source KDR2+ Cord blood HSC single cell suspension
2×106/ 0.2mL/, the most per equal infusion GD2+ Placenta Hominis MSC course for the treatment of；Matched group and aging model group give Isodose
Cell culture fluid.
Four, method
L, fix mice with tail vein injection frame.
2, wipe tail with cotton ball soaked in alcohol to distend the blood vessels；Injection state is that tail turns white, clear against the coccyx both sides of white
Clear visible two red veins.
3, left-handed forefinger, middle finger, mousetail is fixed by nameless and thumb.Hold before 1ml syringe
At 0.1CM.Right hand little finger of toe rides over to be dragged at the left hand thumb of rat-tail, by this hand inserting needle.
4, injection: during injection, left hand pulls tail, makes tail be close to desktop, and it is inserting needle position that turning is close on tail and table limit,
It is typically chosen inserting needle at distance tail point 1/4 or 1/3.Syringe needle enters after skin syringe needle the most up, and parallel inserting needle, when pin penetrates
There is the sense that falls through, slowly inject.
Five, result:
1. treatment group is field planting in Recipient mice, treatment group mouse thymus, spleen, serum, hepatic tissue, lungs, cerebral tissue
MDA and CAT content is significantly lower than matched group, SOD vigor is apparently higher than matched group and there is significant difference (P≤0.05),
Compared with model group, then there is pole significant difference (P≤0.01), statistically significant.
2. the tissue pathological slice of each group mice shows, each anatomical feature heavy damage of model group mice, and cell is controlled
Each visceral organ injury for the treatment of group mice has substantially reparation.MSC seen from result has repair to the visceral organ injury of mouse aging, thus
Play its anti-aging effects.
Six, conclusion: it is little that GD2+MSC with KDR2+HSC therapeutic alliance is obviously improved D-galactose induced subacute aging model
The Aging marker of Mus, shows that both GD2+MSC and KDR2+HSC are applied in combination and have that significantly to repair body function aging
Effect with organ function decline.
Complementary testing: with reference to above example 1, but only use GD2+MSC when treatment and only use KDR2+HSC
Treatment, it has been found that all do not present any therapeutical effect.
The Mechanism Study of embodiment 2:MSC+HSC treatment mouse aging
Old and feeble relevant beta-galactosidase (SA-β-Gal) Activity determination
1, cell specimen is collected and is taken ld, 3d, 5d, 7d, 14d, 28d MSC after matched group and the treatment for the treatment of group respectively, with 3%
Neutral formalin room temperature fixes 5min poststaining.
2, SA-β-Gal dyeing
First with the concentration of 20/L, X-Gal being dissolved in dimethylformamide and be made into X-Gal storage liquid, normal temperature storage is standby.Newly
The preparation of fresh dye liquor: 1mg/mL X-Gal+40mmol/L citrate buffer solution (PH 6.0)+5mmol/L potassium ferrocyanide+
The 5mmol/L potassium ferricyanide+150mmol/L NaCl+2mmol/L MgCl2。
Dyeing: specimen immerses fresh dye liquor room temperature dyeing 12～16h, and distilled water rinses, dimethyl diaminophenazine chloride or Giemsa dye liquor
Redye, light Microscopic observation.
3, SA-β-Gal colour developing result is observed
SA-β-Gal staining positive cells ratio is with the positive stained cells of 5 visual field/slides the most selected under microscope
Mean calculates with the mean ± standard deviation of 3 slides as the positive rate of every slide, positive rate.
4, old and feeble relevant beta-galactosidase (SA-β-Gal) Activity determination result
Matched group SA-β-Gal stained positive rate is 3.03 ± 0.66%；And GD2+MSC and KDR2+HSC treatment after 1d,
After 3d, 7d, 14d, 28d, the SA-β-Gal stained positive rate of MSC is respectively 2.92 ± 0.58%, 3.17 ± 0.76%, 3.13 ±
0.76%, 2.37 ± 0.76%, 2.61 ± 0.76%；They compare with matched group respectively, all without significant difference (p > 0.05).Real
Test without significant difference between group and matched group, illustrate that GD2+MSC Yu KDR2+HSC can not cause MSC old and feeble.
5. the relative expression of Identifying cellular senescence-associated genes p16Ink4a, p53, p21cipl/wafl
(1) after p16:GD2+MSC with KDR2++HSC treatment, the p16 of the MSC of 1d, 3d, 7d, 14d has no significantly raised, with
Without significant difference (p > 0.05) between matched group.After GD2+MSC and KDR2+HSC treatment, the difference between the p16 of 28d and matched group has
Extremely notable meaning (p < 0.001), hence it is evident that reduce.
(2) after p21:GD2+MSC with KDR2+HSC treatment, the p21 of the MSC of ld, 7d, 14d, 28d has no significantly raised, with
Without significant difference (p > 0.05) between matched group.After GD2+MSC and KDR2+HSC treatment, the difference between the p21 of 3d and matched group has aobvious
Write meaning (p < 0.05), hence it is evident that reduce.
(3) after p53:GD2+MSC with KDR2+HSC treats, the p53 of the MSC of ld, 3d, 7d, 14d, 28d is compared with matched group
Substantially reduce, group difference significance (p < 0.05).
Result above display GD2+MSC with KDR2+HSC combined therapy has significant therapeutic effect for mouse aging.
The present inventor, on the basis of sufficient biological test, uses the inventive method to attempt the treatment progeric trouble of patient
Person, result shows that the inventive method has the therapeutical effect excellent to progeria, and this therapeutical effect is also embodied in it and can repair
Body function of answering a pager's call is aging and delays organ function to fail.
Complementary testing: the method for reference example 1, uses placenta mesenchyma stem cell i.e. GD2+ stromal cell and KDR2+
Placental hematopoietic stem cell treatment mouse aging, result shows and embodiment 1 presents essentially identical therapeutic effect.
Complementary testing: the method for reference example 1, uses umbilical cord mesenchymal stem cells i.e. GD2+ stromal cell and KDR2+
Placental hematopoietic stem cell treatment mouse aging, result shows and embodiment 1 presents essentially identical therapeutic effect.
Complementary testing: the method for reference example 1, uses umbilical cord mesenchymal stem cells i.e. GD2+ stromal cell and KDR2+
Umbilical cord blood hematopoietic stem cell treatment mouse aging, result shows and embodiment 1 presents essentially identical therapeutic effect.
Industrial applicability
The invention belongs to cell therapy technology field, relate to a kind of aging and delay organ function for repairing body function
The therapeutic agent of decline, particularly relates to a kind of therapeutic agent including GD2+ stromal cell and KDR2+ hematopoietic stem cell.Further
, the present invention relates to both GD2+ stromal cell and KDR2+ hematopoietic stem cell and combine in preparation aging for repairing body function
With the purposes in the therapeutic agent delaying organ function to fail.It is aging that therapeutic agent of the present invention can be efficiently used for repairing body function
Fail with delaying organ function.
Claims (10)
1. to be used for repairing body function in preparation aging and delay organ function to decline for mescenchymal stem cell and hematopoietic stem cell combination
Purposes in the therapeutic agent moved back.
Purposes the most according to claim 1, wherein said mescenchymal stem cell is GD2+ stromal cell.
Purposes the most according to claim 1, it is characterised in that:
Wherein said mescenchymal stem cell is derived from the mescenchymal stem cell of Placenta Hominis and/or umbilical cord；
Wherein said mescenchymal stem cell is derived from the GD2+ stromal cell of Placenta Hominis and/or umbilical cord；And/or
Wherein said mescenchymal stem cell is derived from the GD2+ stromal cell of Placenta Hominis and/or umbilical cord, and GD2 positive expression rate is big
In 90%, it is greater than 95%.
Purposes the most according to claim 1, it is characterised in that:
Wherein said derived from hematopoietic precursor cells is in Cord blood, bone marrow, placental blood and/or mobilized peripheral blood；
Wherein said hematopoietic stem cell is the hematopoietic stem cell of KDR2 positive expression；
Wherein said hematopoietic stem cell is the hematopoietic stem cell of KDR2 positive expression, in the hematopoietic stem cell of this KDR2 positive expression
KDR2 positive expression rate is more than 85%, is greater than 90%；And/or
In wherein said hematopoietic stem cell, CD34 positive expression rate is more than 80%, is greater than 85%.
Purposes the most according to claim 1, wherein said therapeutic agent is the form of medicine box, and this medicine box includes individually packing
Mescenchymal stem cell and independent seal-packed hematopoietic stem cell.
Purposes the most according to claim 5, it is characterised in that:
Described therapeutic agent dosage of described mescenchymal stem cell when for mammal such as people is that per kilogram weight in patients is used
Amount is (0.1～10) × 107Individual stromal cell, such as per kilogram weight in patients consumption are (0.5～5) × 107Individual stromal cell,
Such as per kilogram weight in patients consumption is (0.75～1.5) × 107Individual stromal cell, such as per kilogram weight in patients consumption are 107
Individual stromal cell；
Described therapeutic agent dosage of described hematopoietic stem cell when for mammal such as people is per kilogram weight in patients consumption
For (1～5) × 107Individual mononuclearcell, such as per kilogram weight in patients consumption are (2～4) × 107Individual mononuclearcell, such as
Per kilogram weight in patients consumption is 3 × 107Individual mononuclearcell；Or, in one embodiment, wherein said therapeutic agent exists
When mammal such as people, the dosage of described hematopoietic stem cell is per kilogram weight in patients consumption for (2～10) × 105Individual
Mononuclearcell, such as per kilogram weight in patients consumption are (2～5) × 105Individual mononuclearcell, such as per kilogram weight in patients
Consumption is (2～4) × 105Individual mononuclearcell；And/or
Described therapeutic agent, when for mammal such as people, first uses mescenchymal stem cell, uses Hematopoietic Stem after 1 month
Cell.
7., for repairing the therapeutic agent that body function is aging and delays organ function to fail, it is the form of medicine box, in this medicine box
Including independent seal-packed mescenchymal stem cell and independent seal-packed hematopoietic stem cell.
Therapeutic agent the most according to claim 7, it is characterised in that:
Described mescenchymal stem cell is GD2+ stromal cell；
Described mescenchymal stem cell is derived from the mescenchymal stem cell of Placenta Hominis and/or umbilical cord；
Described mescenchymal stem cell is derived from the GD2+ stromal cell of Placenta Hominis and/or umbilical cord；
Described mescenchymal stem cell is derived from the GD2+ stromal cell of Placenta Hominis and/or umbilical cord, and GD2 positive expression rate is more than
90%, it is greater than 95%.
Therapeutic agent the most according to claim 7, it is characterised in that:
Described derived from hematopoietic precursor cells is in Cord blood, bone marrow, placental blood and/or mobilized peripheral blood；
Described hematopoietic stem cell is the hematopoietic stem cell of KDR2 positive expression；
Described hematopoietic stem cell is the hematopoietic stem cell of KDR2 positive expression, KDR2 in the hematopoietic stem cell of this KDR2 positive expression
Positive expression rate is more than 85%, is greater than 90%；And/or
In described hematopoietic stem cell, CD34 positive expression rate is more than 80%, is greater than 85%.
Therapeutic agent the most according to claim 7, it is characterised in that:
Described therapeutic agent dosage of described mescenchymal stem cell when for mammal such as people is that per kilogram weight in patients is used
Amount is (0.1～10) × 107Individual stromal cell, such as per kilogram weight in patients consumption are (0.5～5) × 107Individual stromal cell,
Such as per kilogram weight in patients consumption is (0.75～1.5) × 107Individual stromal cell, such as per kilogram weight in patients consumption are 107
Individual stromal cell；
Described therapeutic agent dosage of described hematopoietic stem cell when for mammal such as people is per kilogram weight in patients consumption
For (1～5) × 107Individual mononuclearcell, such as per kilogram weight in patients consumption are (2～4) × 107Individual mononuclearcell, such as
Per kilogram weight in patients consumption is 3 × 107Individual mononuclearcell；Or, in one embodiment, wherein said therapeutic agent exists
When mammal such as people, the dosage of described hematopoietic stem cell is per kilogram weight in patients consumption for (2～10) × 105Individual
Mononuclearcell, such as per kilogram weight in patients consumption are (2～5) × 105Individual mononuclearcell, such as per kilogram weight in patients
Consumption is (2～4) × 105Individual mononuclearcell；And/or
Described therapeutic agent, when for mammal such as people, first uses mescenchymal stem cell, uses Hematopoietic Stem after 1 month
Cell.
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| CN111420034A (en) * | 2019-01-08 | 2020-07-17 | 上海莱馥医疗科技有限公司 | Mixed stem cell preparation for treating psoriasis and preparation method thereof |
| CN112494519A (en) * | 2020-12-16 | 2021-03-16 | 广州杜德生物科技有限公司 | Stem cell preparation based on ovary-targeted stem cell composition |
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