CN107158034A - Stroma cell and candidate stem cell combined therapy senium praecox or the purposes of early ageing disease - Google Patents
Stroma cell and candidate stem cell combined therapy senium praecox or the purposes of early ageing disease Download PDFInfo
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Abstract
The present invention relates to stroma cell and candidate stem cell combined therapy senium praecox or the purposes of early ageing disease.Specifically, the invention belongs to cell therapy technology field, it is related to a kind of method for treating senium praecox or early ageing disease, more particularly to a kind of therapeutic agent for being used to repair the aging of body function and delay organ function to fail, more particularly to a kind of therapeutic agent including GD2+ stroma cells and KDR+ candidate stem cells.Further, the present invention relates to both GD2+ stroma cells and KDR+ candidate stem cells combine prepare be used for treat senium praecox or early ageing disease or for repair the aging of body function and delay organ function decline therapeutic agent in purposes.It has been found that therapeutic agent of the present invention can be efficiently used for treatment senium praecox or early ageing disease, particularly for repairing the aging of body function and delaying organ function to fail, and the inventive method can substantially reduce the usage amount of mesenchymal cell.
Description
Technical field
The invention belongs to cell therapy technology field, it is related to a kind of method for treating senium praecox or early ageing disease, particularly relates to
And a kind of therapeutic agent for being used to repair the aging of body function and delay organ function to fail, it is more particularly to a kind of to include GD2+ bases
The therapeutic agent of cell plastid and KDR+ candidate stem cells.Further, the present invention relates to GD2+ stroma cells and KDR+ Hematopoietic Stems are thin
Both born of the same parents, which combine, to be prepared for treating senium praecox or early ageing disease or for repairing the aging of body function and delaying organ function
Purposes in the therapeutic agent of decline.It has been found that therapeutic agent of the present invention can be efficiently used for treatment senium praecox or early ageing disease, it is special
It is not to be used to repair the aging of body function and delay organ function to fail.
Background technology
Aging can be divided into physiological aging and pathological seaility, physiological aging be body development it is ripe after, with the time
Passage, the degeneration for occurring gradual organism structure and function of organization, and to be disease or abnormal factorses led pathological seaility
The aging of cause accelerates, such as early ageing disease etc..
Early ageing disease (hutchinson-Gilford syndrome) belongs to hereditary disease, and the process of body aging is relatively normal fast 5 to 10 times, and patient's complexion picture is old
People, organ also fails quickly, causes physiological function to decline.Symptom includes diminutive in stature, alopecia and later long teeth.Affected children ranges one
As can only live by 7 to 20 years old, most of all to die from diseases of aging, such as cardiovascular disease does not have effective treatment method now, only leaned on
Medicine is for treatment
1999《science》In the 10 big sciences progress that magazine is chosen, the research work of stem cell is especially made us looking steadily
Mesh.On the one hand disclose many about cell growth and the basic theory problem of development；On the other hand, stem cell is expected to be used
In wound repair, nerve regneration and anti-ageing clinic study of waiting for a long time.British scientist Anastasia is in Nature Journal within 2007
Write articles and point out that adult stem cell is most important to human body self-regeneration and regeneration, it is human senility that adult stem cell, which is reduced,
Main cause.
As the mescenchymal stem cell (mesenchymal stem cells, MSC) of one of adult stem cell from hair
The mesoderm and ectoderm of early stage is educated, the features such as with multi-lineage potential, immunological regulation and self-replacation, people are increasingly subject to
Concern.Mescenchymal stem cell under specific inductive condition, can be divided into fat, bone, cartilage, muscle, flesh in vivo or in vitro
The Various Tissues cell such as tendon, ligament, nerve, liver, cardiac muscle, endothelium, still has multidirectional point after continuous passage culture and freezen protective
Change potential.
Mescenchymal stem cell can increase the expression of VEGF etc., be played an important role in terms of anti-aging.Greatly
Quantity research shows that the change of adult human stem cell's type and functional characteristic has lesion such as the growth at age.Particularly
Oxidative metabolism stress act on the consumption and the consumption in terms of DNA reparations of increase telomere with chronic depression, can cause DNA destruction
It is unstable with gene, cause that aging and death occur in a replication process.Repairing aging can be related to aging for preventing
Disorder, including hematolymphiod is disorderly, heart failure and cardiovascular disease nervous function reduction disease, muscle and intestines problem, artery
The disease such as atherosis and malignant tumour.
It is now recognized that aging is the reduction of cytoactive, the aging of cell, tissue and organ is ultimately resulted in, therefore aging is
It is the aging of cell.Aging and regeneration exist in vivo simultaneously in body, and the process of aging is more than the ability regenerated and is
Can aging.If cell can be immortalized, the process of that tissue aging will be delayed or even block.Stem cell is at present in animal
Experiment is able to effect of the confirmation with anti-aging, and part stem cell has been applied in clinical anti-aging, these stem cells
With raising function of organization, definite curative effect is had been achieved with terms of improvement tissue morphology, it is contemplated that stem cell has antigen recognizing work
With, stem cell lifting body antiradical activities, the functional status of body is regulated and controled on the whole, be it is a kind of it is systemic, systemic,
Essence is promoted longevity.Mescenchymal stem cell is not only safe and reliable, and without immunological rejection, also has no adverse reaction.Can conduct
Preferable seed cell is used for injuries of tissues and organs reparation caused by aging and lesion, and especially treatment aging and histoorgan are damaged
Wound, which is repaired, very big clinical value.
Research group early in Han Zhongchao professors in 2009 finds that the more unsorted mesenchyma of GD2+ mescenchymal stem cells is done
Cell is compared, and GD2+ Derived from Mesenchymal Stem Cells abilities are remarkably reinforced, in addition, the embryonic stem cell in GD2+ mescenchymal stem cells
Mark SSEA-4, Oct-4, Sox-2, Nanog expression are also significantly improved.
Candidate stem cell (is commonly abbreviated as HSC), refers to the class cell with self-renewing and Multidirectional Differentiation ability.It
Fundamental characteristics be with self-renewal capacity, i.e., after a cell cycle events, can produce two with division before
Property identical candidate stem cell, while there is Multidirectional Differentiation ability again, i.e., under certain environmental conditions, candidate stem cell tool
Oriented is respectively the ability of haemocyte differentiation.
In general, candidate stem cell is present in three positions, is marrow, peripheral blood and Cord blood respectively, according to its come
Source is referred to as marrow hemopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell.With medical science and biology
The development of technology, Recent study finds that the mature placenta of the mankind is present and contains what is enriched in substantial amounts of candidate stem cell, placental blood
Various early stage in stage candidate stem cells, its content is about the candidate stem cell in more than ten times of Cord blood, a placenta can be complete
It is full up to be enough to two adult Man's Demands, if with cord blood cell patient can be applied to together, undoubtedly considerably increase Hematopoietic Stem
The content of cell, this makes candidate stem cell to be entirely applied to all applicable crowds.And these placental hematopoietic stem cells exist
It can be separated before and after stored frozen.What the activity of placental hematopoietic stem cell CFU (CFU) was to determine, exempting from
Transplantation experiments in epidemic disease defect mouse potentiality of the verified placental hematopoietic stem cell in transplanting.In addition, placenta Hematopoietic Stem is transplanted
The distribution type requirement of cell need not be very strict, and react relatively light after transplanting and need not use medicine.In addition, being used as placenta hematopoiesis
Source of human stem cell-placenta, wide material sources often turn into discarded object, its collection will not cause mother and neonate after pregnant woman's production
The sensation of any discomfort produces any bad influence.Plurality of advantages makes placental hematopoietic stem cell be expected to replace Bone Marrow Hematopoietic Stem
Cell, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cell are used in HSCT.The strong earth's surface of these results
Bright, the mature placenta of the mankind is likely to become a kind of source of the new candidate stem cell for being used to transplant.
Early in Amy Wagers research groups of stem-cell research institute of Harvard University in 2014, it is found that young decimal blood can
So that old mouse is recovered one's youthful vigour, this achievement in research quilt attracting people's attention《Science》Magazine is chosen as ten big sciences and breaks through it
One.2017《It is natural》Exist in the blood of what magazine was delivered recent studies have shown that young man it is a kind of can be with the albumen of anti-aging
Matter, can reverse senile symptom, include reduction and the skeletal structure of failure of memory, decreased muscle function, metabolism
Lose.These researchs show that candidate stem cell has the function of anti-aging.
0.1%~0.5% expression blood vessel in people's marrow, bleeding of the umbilicus, the CD34+ cells of normal or mobilization derived from peripheral blood
Endothelial growth factor receptor 2 (vascular endothelial growth facror receptor 2, VEGFR 2), it is this
Acceptor is also referred to as KDR or Flk-1.Found in Zieglar research groups in 1999, in HSCT, only
KDR+ candidate stem cells can be successfully implanted into, and CD34+KDR- cells can not be implanted into.Restricted dilution experiment shows：Can be long-term
The cell being persistently implanted into is that to have 20% be HSC for CD34+KDR+ cells in KDR+, marrow.It is sub- that HSC is present in CD34+KDR+
Group, and HPC is present in CD34+KDR- subgroups, thus hematopoietic stem/progenitor can be made a distinction using KDR.
The aging of body function and organ function decline are more special and typically show as senium praecox (people are generally also known as
Early ageing disease).Repair the aging of body function and delay organ function decline to be the necessary means for treating senium praecox.It is unluckily
It is the method that clinically there is no effective treatment senium praecox so far.
Therefore, those skilled in the art urgently expect there is a kind of effective method to treat senium praecox or early ageing disease, so that
Realize the purpose repaired the aging of body function and delay organ function to fail.
The content of the invention
Delay it is an object of the invention to provide a kind of effective method and treat senium praecox or early ageing disease, so as to realize
The purpose repaired the aging of body function and delay organ function to fail.Present inventors have surprisingly discovered that, use mesenchyma
Stem cell can effectively repair the aging of body function with candidate stem cell and delay organ function to fail, so as to reach treatment
Senium praecox or the purpose of early ageing disease.The present invention is accomplished based on this discovery.
Prepared therefore, one aspect of the present invention is combined there is provided mescenchymal stem cell with candidate stem cell for treating senilism
Disease or early ageing disease or for repair the aging of body function and delay organ function fail therapeutic agent in purposes.
Purposes according to a first aspect of the present invention, wherein the mescenchymal stem cell is GD2+ stroma cells.
Purposes according to a first aspect of the present invention, wherein the mescenchymal stem cell is derived from placenta and/or umbilical cord
Mescenchymal stem cell.
Purposes according to a first aspect of the present invention, wherein the mescenchymal stem cell is derived from placenta and/or umbilical cord
GD2+ stroma cells.
Purposes according to a first aspect of the present invention, wherein the mescenchymal stem cell is derived from placenta and/or umbilical cord
GD2+ stroma cells, and GD2 positive expression rates are more than 90%, are greater than 95%.
Purposes according to a first aspect of the present invention, wherein in the cell suspension of the mescenchymal stem cell, added with dimension life
Plain C and EGF.In one embodiment, vitamin C is added with the cell suspension of the mescenchymal stem cell
And EGF, every 106The 0.1 μm of ol vitamin C and 2500IU EGFs of individual cell addition.People's will is gone out
The discovery of material, when adding appropriate vitamin C and EGF in the mescenchymal stem cell in injection, between obvious reduction
The biology effect essentially identical with high mescenchymal stem cell consumption can be obtained in the case of mesenchymal stem cells, this meaning is
Unusual, reason is that the source of mescenchymal stem cell is rare.Said vitamin C and EGF can in advance
It is added in the cell suspension of mescenchymal stem cell, can also be added to before mescenchymal stem cell is expelled in organism thin
In born of the same parents' suspension.EGF (Epidermal Growth Factor, EGF), also known as epithelical cell growth factor, also known as
Oligopeptides -1, is a kind of active small peptide material in human body, the active peptides being made up of 53 amino acid residues, by stimulation epidermis
The tyrosine phosphorylation of growth factor acceptor, reaches repairing hyperplasia skin surface cell, it is stated that to injured, impaired epidermis
Skin possesses excellent curative effect.Its maximum feature is can to promote the Proliferation, Differentiation of cell, so as to replace declining with newborn cell
Old and dead cell.EGF can also stop blooding, and with skin and mucous membrane wound healing, anti-inflammatory analgesic is accelerated, prevent the work(of ulcer
Effect.EGF stability is fabulous, and flowing of scattering is difficult at normal temperatures, can form good coordinating effect with various enzymes in human body.
Initial EGF is mainly employed for medical domain, is primarily to facilitate the reparation and regeneration of impaired epidermis, and such as treatment is burnt, scalded
Wound etc..EGF EGF directly can be bought from commercially available approach, for example, ratify to give birth to through state food pharmaceuticals administration general bureau
The EGF that production and marketing is sold.
Purposes according to a first aspect of the present invention, wherein the derived from hematopoietic precursor cells in：Cord blood, marrow, placental blood,
And/or mobilized peripheral blood.
Purposes according to a first aspect of the present invention, wherein the candidate stem cell is the candidate stem cell of KDR positive expressions.
Purposes according to a first aspect of the present invention, wherein the candidate stem cell is the candidate stem cell of KDR positive expressions,
KDR positive expression rates are more than 85% in the candidate stem cell of the KDR positive expressions, are greater than 90%.
Purposes according to a first aspect of the present invention, wherein CD34 positive expression rates are more than 80% in the candidate stem cell,
It is greater than 85%.
Purposes according to a first aspect of the present invention, wherein the therapeutic agent is in the form of medicine box, the medicine box includes individually
Seal-packed mescenchymal stem cell and independent seal-packed candidate stem cell.
Purposes according to a first aspect of the present invention, wherein between the therapeutic agent is described in when for mammal, such as people
The each consumption of dosage of mesenchymal stem cells is that per kilogram weight in patients consumption is (0.1~20) × 105Individual stroma cell, for example often
Kg patient body weight's consumption is (0.1~10) × 105Individual stroma cell, such as per kilogram weight in patients consumption are (0.2~1.0)
×105Individual stroma cell, such as per kilogram weight in patients consumption are 0.5 × 105Individual stroma cell.
Purposes according to a first aspect of the present invention, wherein the therapeutic agent is made described in when for mammal, such as people
The dosage of hemocytoblast is that per kilogram weight in patients consumption is (1~5) × 10 every time7Individual mononuclearcell, such as per kilogram are suffered from
Person's body weight consumption is (2~4) × 107Individual mononuclearcell, such as per kilogram weight in patients consumption are 3 × 107Individual single core is thin
Born of the same parents.Or, in one embodiment, wherein therapeutic agent candidate stem cell when for mammal, such as people
Dosage be every time per kilogram weight in patients consumption be (2~10) × 105Individual mononuclearcell, such as per kilogram weight in patients are used
Measure as (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 the therapeutic agent is first used when for mammal such as people
Mescenchymal stem cell, candidate stem cell was used after 1 month.
Further, second aspect of the present invention provides a kind of for treating senium praecox or early ageing disease or for repair machine
The aging of body function and the therapeutic agent for delaying organ function to fail, it is in the form of medicine box, and the medicine box includes individually packing
Mescenchymal stem cell and independent seal-packed candidate stem cell.
Therapeutic agent according to a second aspect of the present invention, wherein the mescenchymal stem cell is GD2+ stroma cells.
Therapeutic agent according to a second aspect of the present invention, wherein the mescenchymal stem cell is derived from placenta and/or umbilical cord
Mescenchymal stem cell.
Therapeutic agent according to a second aspect of the present invention, wherein the mescenchymal stem cell is derived from placenta and/or umbilical cord
GD2+ stroma cells.
Therapeutic agent according to a second aspect of the present invention, wherein the mescenchymal stem cell is derived from placenta and/or umbilical cord
GD2+ stroma cells, and GD2 positive expression rates be more than 90%, be greater than 95%.
Therapeutic agent according to a second aspect of the present invention, wherein in the cell suspension of the mescenchymal stem cell, added with dimension
Raw element C and EGF.In one embodiment, vitamin is added with the cell suspension of the mescenchymal stem cell
C and EGF, every 106The 0.1 μm of ol vitamin C and 2500IU EGFs of individual cell addition.
Therapeutic agent according to a second aspect of the present invention, wherein the derived from hematopoietic precursor cells in：Cord blood, marrow, placenta
Blood, and/or mobilized peripheral blood.
Therapeutic agent according to a second aspect of the present invention, wherein the candidate stem cell be KDR positive expressions Hematopoietic Stem it is thin
Born of the same parents.
Therapeutic agent according to a second aspect of the present invention, wherein the candidate stem cell be KDR positive expressions Hematopoietic Stem it is thin
KDR positive expression rates are more than 85% in born of the same parents, the candidate stem cell of the KDR positive expressions, are greater than 90%.
Therapeutic agent according to a second aspect of the present invention, wherein CD34 positive expression rates are more than in the candidate stem cell
80%, it is greater than 85%.
Therapeutic agent according to a second aspect of the present invention, wherein the therapeutic agent is described in when for mammal, such as people
The each consumption of dosage of mescenchymal stem cell is that per kilogram weight in patients consumption is (0.1~20) × 105Individual stroma cell, for example
Per kilogram weight in patients consumption is (0.1~10) × 105Individual stroma cell, such as per kilogram weight in patients consumption for (0.2~
1.0)×105Individual stroma cell, such as per kilogram weight in patients consumption are 0.5 × 105Individual stroma cell.
Therapeutic agent according to a second aspect of the present invention, wherein the therapeutic agent is made described in when for mammal such as people
The dosage of hemocytoblast is that per kilogram weight in patients consumption is (1~5) × 10 every time7Individual mononuclearcell, such as per kilogram are suffered from
Person's body weight consumption is (2~4) × 107Individual mononuclearcell, such as per kilogram weight in patients consumption are 3 × 107Individual single core is thin
Born of the same parents.Or, in one embodiment, wherein therapeutic agent candidate stem cell when for mammal such as people
Dosage is that per kilogram weight in patients consumption is (2~10) × 10 every time5Individual mononuclearcell, such as per kilogram weight in patients consumption
For (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 the therapeutic agent first makes when for mammal such as people
With mescenchymal stem cell, candidate stem cell was used after 1 month.
Mescenchymal stem cell used in the present invention can use methods known in the art to prepare.
For example, for the mescenchymal stem cell obtained by placenta, being referred to Chinese Patent Application No.
Method in 201210292509.9 is obtained, and the GD2 positive expression rates for example obtained by document method are not less than 90%
GD2+ stroma cells.In an example, the method for obtaining the GD2+ stroma cells comprises the following steps：
(1) placenta tissue is cleaned：Placenta tissue is handled in Biohazard Safety Equipment, is used according to placenta size appropriate
PBS is rinsed 2-3 times to placenta tissue, rinses placenta tissue remained on surface blood well, placenta surface is without blood
Liquid grumeleuse；
(2) placenta tissue digestion process：Placental lobules is cut from the placenta tissue that step (1) is obtained using operating scissors,
Leaflet is transferred on culture dish, 25ml PBSs and placental lobules is tried one's best is added and shreds, add the pancreases of 25ml 0.25%
(pancreatin is 1 with PBS volume ratio to enzyme (Gibco):1) and tissue is mixed, culture dish is put into 37 DEG C of constant-temperature table
In digest 20 minutes；
(3) placenta tissue filtration treatment：5ml FBS (Gibco) are added in toward culture dish and are mixed and terminate digestion to reach
Purpose, the placenta tissue fragment after digestion is transferred on 200 mesh metallic filters, placenta tissue fragment is ground
And the liquid filtered is collected using another culture dish, separate the PBS cleaning for adding 10ml toward metallic filter twice
Organize and continue grinding, the filtrate of collection is transferred to 50ml centrifuge tubes, centrifuged 10 minutes with speed 1400rpm, supernatant is removed
Liquid simultaneously adds PBS resuspension cell, is centrifuged 10 minutes with speed 1400rpm, removes supernatant, adds PBS weight
Outstanding cell, extracts a small amount of samples and carries out cell count, and 10 minutes effects to reach cleaning cell are centrifuged with speed 1400rpm；
(4) the full cells frozen storing liquid of placenta is prepared：The white egg of human blood of 15 parts by weight is included in the full cells frozen storing liquid of placenta
In vain, the DMSO (dimethyl sulfoxide (DMSO)) and the DMEM-F12 of 65 parts by weight of 10 parts by weight, the frozen stock solution prepared is placed on 4 DEG C of refrigerator preservations
Until using；
(5) the full cell cryopreservation of placenta：Supernatant is removed after the placenta tissue filtered fluid centrifugation that step (3) is obtained, at 4 DEG C
Low temperature environment under, step (4) obtained full cells frozen storing liquid is added, then with each every milliliter of pipe 4 × 107To 1 × 108's
Cell density is added in cryopreservation tube, and this process need to be carried out under 4 DEG C of cryogenic conditions, and cryopreservation tube is put into program temperature reduction box,
First under 4 DEG C of temperature conditionss deepfreeze 0.5 hour, then under -80 DEG C of temperature conditionss freeze 1 day, then by cryopreservation tube in
Freezed in liquid nitrogen, it is standby；
If necessary, further it can include and the matching used method for resuscitation of cryopreservation methods：
(6) the full cell recovery of placenta is frozen:The full cell of placenta that step (4) is freezed takes out from liquid nitrogen, is placed on constant temperature
Thawed in water-bath to half frozen stock solution and start to melt, (it is for example comprising 15%FBS+1%L- using mescenchymal stem cell culture medium
Glutamine+0.05%Gentamicin+84%DMEM-F12 the drop method cleaning full cell of placenta, the thin of acquisition of thawing) are carried out
The volume ratio of born of the same parents' suspension and mescenchymal stem cell culture medium is 1: 3 (1ml:3ml), it is blended with mescenchymal stem cell culture medium
Cell suspension is transferred to centrifuge tube, centrifuges 10 minutes cleaning DMSO with speed 1400rpm, removes supernatant, add PBS
Cell, and the ratio addition erythrocyte cracked liquid (Roche) with 1 part of cell suspension than 2-3 parts of erythrocyte cracked liquids is resuspended,
Cultivated 10-15 minutes in the environment of 15-25 DEG C, put centrifuge into and centrifuged within 10 minutes with speed 1400rpm centrifugations, after centrifugation
Supernatant is removed, erythrocyte splitting situation is observed, the step of erythrocyte splitting is repeated if needed is cracked, be eventually adding
PBS, which is resuspended cell and extracts a small amount of samples, carries out cell count, puts centrifuge into and is centrifuged 10 minutes with speed 1400rpm
Centrifuged to clean cell, remove supernatant；
If necessary, the method for mescenchymal stem cell separation and amplification after recovery can further be included：
(7) cell culture：Appropriate mescenchymal stem cell culture medium is added in the full cell obtained to step (6) to be resuspended carefully
Born of the same parents, are transferred to T25 blake bottles, then put T25 blake bottles into CO2 concentration to be cultivated in 5% 37 DEG C of incubators, and culture is extremely
T25 blake bottles are taken out from incubator at the 6th day, carry out partly changing liquid for the first time, continues to cultivate, cultivated T25 at the 9th day
Bottle is taken out from incubator, and liquid is partly changed for the second time, the culture medium inside plate is taken away at the 12nd day, is filled between addition 15ml
Matter stem cell media continues to cultivate, and liquid is once changed entirely within every 2 days backward；
(8) passage：Attached cell fusion rate inside T25 blake bottles reaches 80% or so, using digestive ferment
Attached cell is departed from T25 blake bottles bottom by (TrypLE Express), and supernatant is removed after centrifugation, and it is dry thin to add mesenchyma
Born of the same parents' culture medium suspension cell again, is inoculated in T25 Tissue Culture Flasks and is passed on, and carry out amplification cultivation；Hereafter change within every two days
Liquid once until after fusion rate reaches 80%, produce (can replenishing vitamins C and EGF wherein, for biology
Body injection treatment；Or at it after the processing such as passing on, freezing, the supplement dimension life thereto before for organism injection treatment
Plain C and EGF), passed on again if necessary.
If necessary, placenta mesenchyma stem cell obtained by above step (8) can be further directed to, detection following items
At least one of：Cytoactive, cell contamination, hereditary disease, HLA-ABC/DR distribution type；
If necessary, can be further by the placenta mesenchyma stem cell after being passed on obtained by above step (8) in liquid nitrogen
Freeze, it is standby.
This placenta mesenchyma for being not less than 90% GD2+ stroma cells in the GD2 positive expression rates of above-mentioned acquisition is dry thin
During born of the same parents, GD2 positive expression rate detections are carried out to the cell obtained, GD2 positive expression rates is chosen and is not less than 90% for example
It is not less than the mescenchymal stem cell that 95% GD2+ stroma cells are used as the present invention.
In another example, for the mescenchymal stem cell obtained by umbilical cord, it is referred to Chinese Patent Application No.
Method in 201210159916.2 is obtained, and the GD2 positive expression rates for example obtained by document method are not less than 90%
GD2+ stroma cells.In an example, the method for obtaining the GD2+ stroma cells comprises the following steps：
(1) umbilical cord tissue frozen stock solution is prepared：In the umbilical cord tissue frozen stock solution comprising 80 parts by weight human serum albumin and
The DMSO (dimethyl sulfoxide (DMSO), dimethyl sulfoxide) of 10 parts by weight, the cold liquid storage prepared be placed on 4 DEG C of refrigerators preserve until
Use；
(2) sterilize and clean：In Biohazard Safety Equipment, umbilical cord tissue surface is carried out disinfection with alcohol, by umbilical cord therefrom
Between cut off, be laid on sterile 10cm cell culture plates, using PBS tissue, to reduce the red blood cell above tissue；
(3) umbilical cord tissue is handled：The umbilical cord tissue that step (2) is obtained is transferred to another 10cm cell culture plate
In, umbilical cord tissue is cut into size 1cm3 square shape；
(4) umbilical cord tissue is cold deposits：Under 4 DEG C of low temperature environment, tissue block and frozen stock solution are added in cryopreservation tube, then will
Cryopreservation tube is put into program temperature reduction box, first deepfreeze 0.5 hour under 4 DEG C of temperature conditionss, then under -80 DEG C of temperature conditionss it is cold
Freeze 1 day, then freeze cryopreservation tube in liquid nitrogen, it is standby；
If necessary, further it can include and the matching used method for resuscitation of cryopreservation methods：
(5) freezing and storing umbilical tissue is recovered：The umbilical cord tissue that step (4) is freezed takes out from liquid nitrogen, is placed on water bath with thermostatic control
In thaw to half frozen stock solution start melt, using mescenchymal stem cell culture medium, (it is for example comprising 15%FBS+1%L-
Glutamine+0.05%Gentamicin+84%DMEM-F12 drop method cleaning umbilical cord tissue) is carried out, is filtered using 100um
Device removes waste liquid；
If necessary, the method for mescenchymal stem cell separation and amplification after recovery can further be included：
(6) the adherent processing of umbilical cord tissue：The freezing and storing umbilical tissue of recovery is laid in another 10cm cell culture plate
In, the tissue number of blocks in each plate maintains 10-15 blocks, tissue block is air-dried 10-15 minutes until tissue is attached to plate
On；
(7) umbilical cord tissue culture：Being slowly added into mescenchymal stem cell culture medium along plate edge, (it is for example comprising 15%
FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) flooded to tissue；Plate is placed in CO2
Concentration is cultivated for 5% 37 DEG C of incubators, and culture adds 5ml mesenchymas to taking out plate from incubator at the 5th day
Stem cell media；At the tenth day by the media transfer in plate, the fresh mescenchymal stem cell culture mediums of 15ml are added；
Remove within 12nd day all umbilical cord tissue blocks and continue culture, liquid is once changed entirely within hereafter every two days；
(8) passage：Attached cell fusion rate inside plate reaches 60% or so, using digestive ferment
Attached cell is departed from plate bottom by (TrypLE Express), supernatant is removed after centrifugation, and add mescenchymal stem cell training
Foster base suspension cell again, is inoculated in T25 Tissue Culture Flasks and is passed on, and carry out amplification cultivation；Hereafter liquid one is changed within every two days
It is secondary until fusion rate reaches 80% after, produce (can replenishing vitamins C and EGF wherein, noted for organism
Penetrate treatment；Or at it after the processing such as passing on, freezing, the replenishing vitamins C thereto before for organism injection treatment
And EGF), passed on again if necessary；
If necessary, umbilical cord mesenchymal stem cells obtained by above step (8) can be further directed to, detection following items
At least one of：Cytoactive, cell contamination, hereditary disease, HLA-ABC/DR distribution type；
If necessary, can be further by the umbilical cord mesenchymal stem cells after being passed on obtained by above step (8) in liquid nitrogen
Freeze, it is standby.
This umbilical cord mesenchyma for being not less than 90% GD2+ stroma cells in the GD2 positive expression rates of above-mentioned acquisition is dry thin
During born of the same parents, GD2 positive expression rate detections are carried out to the cell obtained, GD2 positive expression rates is chosen and is not less than 90% for example
It is not less than the mescenchymal stem cell that 95% GD2+ stroma cells are used as the present invention.
Candidate stem cell used in the present invention can use methods known in the art to prepare.
For example, for the candidate stem cell obtained by placenta, being referred to Chinese Patent Application No. 2014104050724
In method obtain, such as CD34 positive expression rates are more than 80% in the candidate stem cell obtained by document method, for example
More than 85%；Also, KDR positive expression rates are more than 85% in the candidate stem cell obtained by document method, are greater than
90%.In an example, the method for obtaining the cell of the KDR positive expressions comprises the following steps：The collection of placenta, placenta
Initial survey, the pre-sterilization of placenta, placenta and female blood examination survey, the lavation of placental hematopoietic stem cell, the concentrating and purifying of candidate stem cell.Respectively
Step is specially：
(1) collection of placenta：Placenta and female blood are collected under operating room gnotobasis, sterile placenta accumulating box is deposited in
In, label, bar code are posted, the temperature of placenta accumulating box, which is maintained in 4-10 DEG C, 24 hours, is sent to laboratory；
(2) initial survey of placenta：Check temperature in placenta accumulating box box, label, the date of service, accumulating box whether have seepage,
Whether female blood blood sample is had；
(3) pre-sterilization of placenta：The fetus face expansion of placenta is positioned over the bottom that placenta rinses box, rushed with physiological saline
Placenta surface is washed, the osculum that placenta rinses cassette bottom face is opened, removes the physiological saline of flushing, check placenta Surface calcification feelings
Condition；
(4) placenta and female blood examination are surveyed：The project of detection, which includes hepatitis, to be detected to the placenta and female blood sample taken
Poison detection, AIDS virus detection, venereal disease detection, tissue matching (HLA) detection, hematopoietic stem/progenitor qualitative detection (CFU-GM)；
(5) lavation of placental hematopoietic stem cell：Placenta is aseptically washed on placenta with sterile saline
Blood clot and hematocele, after being sterilized using disinfectant, by irrigating solution syringe needle insert placenta arteria umbilicalis in, placental perfusion returnable bottle pin
In head insertion umbilical vein, haemostatic clamp is clamped, and slowly opens constant flow pump, and irrigating solution is moved through sebific duct, switch, peristaltic pump, syringe needle, placenta
Arteries and veins, placenta, placental vein, placental perfusion returnable bottle syringe needle, finally receive irrigating solution in irrigating solution returnable bottle；
(6) concentrating and purifying of candidate stem cell：The irrigating solution that lavation is come out, in centrifuge with 1500-2000rpm from
Heart 15-20 minutes, removes supernatant, collects the liquid of precipitation and lower floor, by the precipitation being collected into and the liquid and physiology of lower floor
Salt solution presses 2：1-1：2 ratio mixes to obtain mixed liquor, and mixed liquor and lymphocyte separation medium then are pressed into 2：1-1：2 ratio point
It is not added in centrifuge tube, the order of addition is slow added into mixed liquor first to add lymphocyte separation medium in centrifuge tube,
Note keeping the liquid level of lymphocyte separation medium smooth in adition process, centrifuged 20-25 minutes with 2200-2500rpm after adding,
Slow acceleration is slow during centrifugation slows down, after centrifugation terminates, and middle tunica albuginea layer is collected into new centrifuge tube, with 2：1-1：2 ratio adds
Physiological saline is mixed, and 1200-1500rpm is centrifuged 10-15 minutes；Supernatant is removed in centrifugation after terminating, add 10-20mL physiology salts
Water piping and druming precipitation, then 1200-1500rpm are centrifuged 10-15 minutes, after centrifugation terminates, are removed supernatant, are resuspended with DMEM culture mediums
Precipitation, collects candidate stem cell group, the cell mass of resuspension is carried out into cell and mycotic culture, candidate stem cell is quantitatively detected
(CD34), candidate stem cell Activity determination (Trypan Blue), candidate stem cell qualitative detection (CFU-GM).
More than 80% 85% is greater than in the CD34 positive expression rates of above-mentioned acquisition, and KDR positive expression rates are more than
85% is greater than during 90% candidate stem cell, and CD34 positive expression rate detections are carried out to the candidate stem cell obtained
With KDR positive expression rates, choose CD34 positive expression rates and 85% and KDR positive expression rates are greater than more than 80% more than 85%
The candidate stem cell for being greater than 90% crosses the candidate stem cell used as the present invention.
Or, in one embodiment, the candidate stem cell used in the present invention can be obtained according to following method：Take Hematopoietic Stem
Cell derived tissue (includes but is not limited to Cord blood, marrow, placental blood, mobilized peripheral blood), by 8：1~1：8 ratios add hydroxyl
Hydroxyethyl starch, after being well mixed, is transferred in the supporting processing consumptive material of AXP candidate stem cell piece-rate systems, loads AXP Hematopoietic Stems thin
Born of the same parents' piece-rate system, is put into centrifuge, centrifuges 10~30min with eccentricity (RCF) 1200~1600, then centrifuge with RCF50~200
Taken out after 5~15min, be automatically separated and obtain mononuclearcell, export AXP data obtain the separation quantity of candidate stem cell.Make
CD34 the and KDR contents of the cell arrived separated with flow cytomery.Then isolated cell is pressed 8：1~1：8
Ratio adds DMSO, and program is cooled to after -90 DEG C to be put under liquid nitrogen environment and frozen.Recovered before use, being placed in 37 degree of water-baths.Make
The CD34 positive expression rates obtained in aforementioned manners are greater than 85% more than 80%, and KDR positive expression rates are more than 85%
Such as larger than 90% candidate stem cell.
Further, can be that GD2+ stroma cells and KDR+ make by mescenchymal stem cell of the present invention before freezing
Hemocytoblast is sub-packed in bottle independently of one another, and then two bottles are placed in medicine box of the present invention, the present invention is obtained
For the therapeutic agent repaired the aging of body function and delay organ function to fail, then the 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 being used for people
Particularly there is the patient of senium praecox feature to repair the aging of body function and delay organ function to fail.As a result show, this hair
Excellent result is not only presented in bright method in animal experiment, and encouraging results are presented in human body.Although CN
106074604 A also disclose a kind of method for treating early ageing disease or senium praecox, but present invention discover that the inventive method is than it
Clinical value is had more in the method described in the A of CN 106074604.
Any technical characteristic that any embodiment of either side or the either side of the present invention has is equally applicable
Any embodiment of other any embodiments or other either sides, as long as they will not be conflicting, certainly mutual
Between where applicable, if necessary can individual features be made with appropriate modification.Make to various aspects of the present invention with feature into one below
The description of step.
All documents recited in the present invention, their full content is incorporated herein by reference, and if these are literary
Offer expressed implication with it is of the invention inconsistent when, be defined by the statement of the present invention.In addition, the various terms that use of the present invention and
Phrase has well known to a person skilled in the art general sense, nonetheless, the present invention remain desirable at this to these terms and
Phrase is described in more detail and explained that the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute's table of the present invention
The implication stated is defined.
Embodiment
The present invention can be further described by the following examples, however, the scope of the present invention is not limited
In following embodiments.One of skill in the art, can be with it is understood that on the premise of without departing substantially from the spirit and scope of the present invention
Various change and modification are carried out to the present invention.The present invention carries out general to the material and test method that are arrived used in experiment
And/or specific description.Although for realize many materials used in the object of the invention and operating method be it is known in the art that
But the present invention is still described in detail as far as possible herein.
The present invention specific experiment in, using to placenta mesenchyma stem cell be GD2+ stroma cells be with reference to China
What the method for the embodiment 1,6,7 of number of patent application 201210292509.9 was obtained, and GD2 positive expression rates are more than 95%.
The present invention specific experiment in, using to umbilical cord mesenchymal stem cells be GD2+ stroma cells be with reference to China
What the method for the embodiment 1,6,7 of number of patent application 201210159916.2 was obtained, and GD2 positive expression rates are more than 95%.
In the specific experiment of the present invention, using to placental hematopoietic stem cell be with reference to Chinese Patent Application No.
What the method for 2014104050724 embodiment 1 was obtained, and CD34 positive markers expression rate is more than 85%, KDR marks
Positive expression rate is more than 90%.
In the specific experiment of the present invention, its CD34 positive markers expression rate of the umbilical cord blood hematopoietic stem cell used is big
Be more than 90% in 85%, KDR/2 positive markers expression rate, and prepared with reference to following method：By 8：1~1：8 ratios
HES is added, after being well mixed, is transferred in the supporting processing consumptive material of AXP candidate stem cell piece-rate systems, loads AXP and make
Hemocytoblast piece-rate system, is put into centrifuge, with eccentricity (RCF) 1200~1600 centrifuge 10~30min, then with RCF50~
Taken out after 200 5~15min of centrifugation, be automatically separated and obtain mononuclearcell, export AXP data obtain the separation of candidate stem cell
Quantity.CD34 the and KDR contents of the cell arrived separated using flow cytomery.Then isolated cell is pressed 8：
1~1：8 ratios add DMSO, and program is cooled to after -90 DEG C to be put under liquid nitrogen environment and frozen.Before use, being placed in 37 degree of water-baths
Recovery.In the experiment that vitamin C and EGF are with the addition of in candidate stem cell, each candidate stem cell adds before the injection
Plus vitamin C and EGF, with every 106Individual cytometer adds 0.1 μm of ol vitamin C and 2500IU epidermal growth factors
Son.
Embodiment 1：The use of placenta mesenchyma stem cell is that GD2+ stroma cells are treated with Cord blood KDR+ candidate stem cells
Mouse aging
First, the selection and packet of animal
1st, the selection of animal：Model mice is health C57BL/6J mouse, and female, 8 week old weigh 18~22g, 60.
2nd, animal packet：It is randomly divided into control group, model group, three groups for the treatment of group, every group 20 merely.
2nd, mice aging model is set up
5% D- galactolipins are subcutaneously injected in the daily nape part of model group and treatment group, and injection volume is 0.25ml/10g, continuously
Administration sets up aged animal model in 8-16 weeks.Control group then gives the physiological saline of same volume.
3rd, it is transfused
Aging model was built up after two weeks, treatment group injection GD2+ placenta MSC and KDR+ Cord bloods HSC.It is green using expressing
The slow virus carrier mark GD2+MSC and Cord blood KDR+HSC of color fluorescin, to determine GD2+MSC and Cord blood KDR+HSC
In the implantation situation of Organs of Mice.Aging marker before and after detection treatment：Including mouse weight, thymus gland, spleen, serum,
Hepatic tissue, lungs, brain tissue catalase (CAT), superoxide dismutase (SOD) vigor and MDA (MDA) content
Change.
First course for the treatment of of treatment group：Through mouse tail vein infusion people source GD2+ placenta MSC single cell suspensions 2 × 106/
Only, it is a course for the treatment of that weekly administration once, is altogether administered 4 times to 0.2mL/.Second course for the treatment of：People is transfused through mouse tail vein within first week
Source GD2+ placenta MSC single cell suspensions 2 × 106Only, second week is transfused people source KDR+ Cord blood HSC single cell suspensions 2 to/0.2mL/
×106/ 0.2mL/ only, is transfused GD2+ placentas MSC per the course for the treatment of later；Control group and aging model group give the thin of Isodose
Born of the same parents' nutrient solution.
4th, method
L, with tail vein injection frame fix mouse.
2nd, tail is wiped with cotton ball soaked in alcohol to distend the blood vessels；Injection state is turned white for tail, and the coccyx both sides against white are clear
Clear visible two red veins.
3rd, left-handed forefinger, middle finger, nameless and thumb fixes mousetail.Hold before 1ml syringes
At 0.1CM.Right hand little finger of toe is ridden at the left hand thumb for dragging rat-tail, by this hand inserting needle.
4th, inject：Left hand pulls tail during injection, tail is close to desktop, and tail is close to turning with table side for inserting needle position,
It is typically chosen the inserting needle at tail point 1/4 or l/3.Syringe needle enter after skin at once syringe needle slightly up, parallel inserting needle, when pin is penetrated
There is the sense that falls through, slowly inject.
5th, result：
1. treatment group is colonized in Recipient mice, treatment group's mouse thymus, spleen, serum, hepatic tissue, lungs, brain tissue
MDA and CAT contents are significantly lower than control group, SOD vigor is apparently higher than control group and there is significant difference (P≤0.05),
Compared with model group, then there is pole significant difference (P≤0.01), it is statistically significant.
2. the tissue pathological slice of each group mouse is shown, each anatomical feature heavy damage of model group mouse, and cell is controlled
Each visceral organ injury for the treatment of group mouse has obvious reparation.As a result visceral organ injuries of the visible MSC to mouse aging has repair, so that
Play its anti-aging effects.
6th, conclusion：It is small that GD2+MSC and KDR+HSC therapeutic alliances are obviously improved D- galactolipin induced subacute aging models
The Aging marker of mouse, show both GD2+MSC and KDR+HSC be applied in combination with it is significant repair the aging of body function and
The effect of organ function decline.
Complementary testing 1a：With reference to above example 1, but in treatment using only GD2+MSC and using only KDR+HSC
Treatment, it has been found that any therapeutic action is not presented.
The present inventor attempts to treat the trouble of patient's senium praecox on the basis of sufficient biological test, using the inventive method
Person, as a result shows that the inventive method has the therapeutic action excellent to senium praecox, this therapeutic action is also embodied in it and can repaiied
Answer a pager's call and the aging of body function and delay organ function to fail.
Complementary testing 1b：The method of reference implementation example 1, is GD2+ stroma cells and KDR+ using placenta mesenchyma stem cell
Placental hematopoietic stem cell treats mouse aging, and essentially identical therapeutic effect is presented with embodiment 1 in as a result display.
Complementary testing 1c：The method of reference implementation example 1, is GD2+ stroma cells and KDR+ using umbilical cord mesenchymal stem cells
Placental hematopoietic stem cell treats mouse aging, and essentially identical therapeutic effect is presented with embodiment 1 in as a result display.
Complementary testing 1d：The method of reference implementation example 1, is GD2+ stroma cells and KDR+ using umbilical cord mesenchymal stem cells
Umbilical cord blood hematopoietic stem cell treats mouse aging, and essentially identical therapeutic effect is presented with embodiment 1 in as a result display.
Embodiment 2：The use of placenta mesenchyma stem cell is that GD2+ stroma cells are treated with Cord blood KDR+ candidate stem cells
Mouse aging
In the present embodiment 2 and its complementary testing, if not otherwise indicated, dimension is with the addition of in mesenchyma stem cell suspension used
Raw element C and EGF.
First, the selection and packet of animal
1st, the selection of animal：Model mice is health C57BL/6J mouse, and female, 8 week old weigh 18~22g, 60.
2nd, animal packet：It is randomly divided into control group, model group, three groups for the treatment of group, every group 20 merely.
2nd, mice aging model is set up
5% D- galactolipins are subcutaneously injected in the daily nape part of model group and treatment group, and injection volume is 0.25ml/10g, continuously
Administration sets up aged animal model in 8-16 weeks.Control group then gives the physiological saline of same volume.
3rd, it is transfused
Aging model was built up after two weeks, treatment group injection GD2+ placenta MSC and KDR+ Cord bloods HSC.It is green using expressing
The slow virus carrier mark GD2+MSC and Cord blood KDR+HSC of color fluorescin, to determine GD2+MSC and Cord blood KDR+HSC
In the implantation situation of Organs of Mice.Aging marker before and after detection treatment：Including mouse weight, thymus gland, spleen, serum,
Hepatic tissue, lungs, brain tissue catalase (CAT), superoxide dismutase (SOD) vigor and MDA (MDA) content
Change.
First course for the treatment of of treatment group：Through mouse tail vein infusion people source GD2+ placenta MSC single cell suspensions 1 × 104/
Only, it is a course for the treatment of that weekly administration once, is altogether administered 4 times to 0.2mL/.Second course for the treatment of：People is transfused through mouse tail vein within first week
Source GD2+ placenta MSC single cell suspensions 1 × 104Only, second week is transfused people source KDR+ Cord blood HSC single cell suspensions 2 to/0.2mL/
×106/ 0.2mL/ only, is transfused GD2+ placentas MSC per the course for the treatment of later；Control group and aging model group give the thin of Isodose
Born of the same parents' nutrient solution.It is above-mentioned to be transfused people source GD2+ placenta MSC single cell suspensions 1 × 10 through mouse tail vein4/ 0.2mL/ only, is converted to
During dosage (mouse dose is typically 10 times of people's dosage) of people, about 0.5 × 105/ kg body weight.
4th, method
L, with tail vein injection frame fix mouse.
2nd, tail is wiped with cotton ball soaked in alcohol to distend the blood vessels；Injection state is turned white for tail, and the coccyx both sides against white are clear
Clear visible two red veins.
3rd, left-handed forefinger, middle finger, nameless and thumb fixes mousetail.Hold before 1ml syringes
At 0.1CM.Right hand little finger of toe is ridden at the left hand thumb for dragging rat-tail, by this hand inserting needle.
4th, inject：Left hand pulls tail during injection, tail is close to desktop, and tail is close to turning with table side for inserting needle position,
It is typically chosen the inserting needle at tail point 1/4 or l/3.Syringe needle enter after skin at once syringe needle slightly up, parallel inserting needle, when pin is penetrated
There is the sense that falls through, slowly inject.
5th, result：
1. treatment group is colonized in Recipient mice, treatment group's mouse thymus, spleen, serum, hepatic tissue, lungs, brain tissue
MDA and CAT contents are significantly lower than control group, SOD vigor is apparently higher than control group and there is significant difference (P≤0.05),
Compared with model group, then there is pole significant difference (P≤0.01), it is statistically significant；In addition, for the above results, this reality
Apply the result of example 2 and the same parameters of embodiment 1 result is essentially identical and no difference of science of statistics.
2. the tissue pathological slice of each group mouse is shown, each anatomical feature heavy damage of model group mouse, and cell is controlled
Each visceral organ injury for the treatment of group mouse has obvious reparation.As a result visceral organ injuries of the visible MSC to mouse aging has repair, so that
Play its anti-aging effects；In addition, for the above results, the result of the result of the present embodiment 2 and the same index of embodiment 1 substantially without
Difference.
6th, conclusion：It is small that GD2+MSC and KDR+HSC therapeutic alliances are obviously improved D- galactolipin induced subacute aging models
The Aging marker of mouse, show both GD2+MSC and KDR+HSC be applied in combination with it is significant repair the aging of body function and
The effect of organ function decline；In addition, be only in cell supplement addition mcg vitamin C and EGF and it is other
In the case that experimental condition is constant, the present embodiment 2 can just be obtained and the institute of embodiment 1 using notable lesser amount of mescenchymal stem cell
With biological effect as cell quantity.
Complementary testing 2a：With reference to above example 2, but in treatment using only GD2+MSC and using only KDR+HSC
Treatment, it has been found that any therapeutic action is not presented.
The present inventor attempts to treat the trouble of patient's senium praecox on the basis of sufficient biological test, using the inventive method
Person, as a result shows that the inventive method has the therapeutic action excellent to senium praecox, this therapeutic action is also embodied in it and can repaiied
Answer a pager's call and the aging of body function and delay organ function to fail.
Complementary testing 2b：The method of reference implementation example 2, is GD2+ stroma cells and KDR+ using placenta mesenchyma stem cell
Placental hematopoietic stem cell treats mouse aging, and essentially identical therapeutic effect is presented with embodiment 2 in as a result display.
Complementary testing 2c：The method of reference implementation example 2, is GD2+ stroma cells and KDR+ using umbilical cord mesenchymal stem cells
Placental hematopoietic stem cell treats mouse aging, and essentially identical therapeutic effect is presented with embodiment 2 in as a result display.
Complementary testing 2d：The method of reference implementation example 2, is GD2+ stroma cells and KDR+ using umbilical cord mesenchymal stem cells
Umbilical cord blood hematopoietic stem cell treats mouse aging, and essentially identical therapeutic effect is presented with embodiment 2 in as a result display.
Complementary testing 2e：The method of reference implementation example 2, different is only that dimension life is not added with mesenchyma stem cell suspension
Plain C (but still EGF of addition respective amount), as a result in MSC single cell suspensions with 3 × 106The dosage of/0.2mL/ only
When can be only achieved the effect of embodiment 1, this show in mesenchyma stem cell suspension only addition EGF and without
Vitamin C can not obtain the effect for reducing mescenchymal stem cell consumption.
Complementary testing 2f：The method of reference implementation example 2, different is only that epidermis is not added with mesenchyma stem cell suspension
Growth factor (but still vitamin C of addition respective amount), as a result in MSC single cell suspensions with 2.5 × 106The agent of/0.2mL/ only
The effect of embodiment 1 is can be only achieved during amount, this show in mesenchyma stem cell suspension only addition vitamin C and without epidermis
Growth factor can not obtain the effect for reducing mescenchymal stem cell consumption.
Embodiment 3：MSC+HSC treats the Mechanism Study of mouse aging
To the gained biological sample of embodiment 1, related beta-galactosidase (SA- β-Gal) Activity determination of aging is carried out.
1st, cell specimen is collected takes ld, 3d, 5d, 7d, 14d, 28d MSC after control group and treatment group's treatment respectively, with 3%
Neutral formalin room temperature fixes 5min poststainings.
2nd, SA- β-Gal are dyed
X-Gal is first dissolved in into dimethylformamide with 20/L concentration and is made into X-Gal storing liquids, 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 ferrocyanides+
The 5mmol/L potassium ferricyanide+150mmol/L NaCl+2mmol/L MgCl2。
Dyeing：Sample is immersed into fresh dye liquor room temperature and dyes 12~16h, distilled water rinsing, dimethyl diaminophenazine chloride or Giemsa dye liquors
Redye, light Microscopic observation.
3rd, SA- β-Gal colour developings result observation
SA- β-Gal staining positive cells ratios are with the positive stained cells of random selected 5 visual field/slides under microscope
Mean is as the positive rate of every slide, and positive rate is with the mean ± standard deviation calculating of 3 slides.
4th, related beta-galactosidase (SA- β-Gal) Activity determination result of aging
Control group SA- β-Gal stained positives rate is 3.03 ± 0.66%；And GD2+MSC and KDR+HSC treatment after 1d, 3d,
After 7d, 14d, 28d MSC SA- β-Gal stained positive rates be respectively 2.92 ± 0.58%, 3.17 ± 0.76%, 3.13 ±
0.76%, 2.37 ± 0.76%, 2.61 ± 0.76%；They are compared with control group respectively, without significant difference (p>0.05).It is real
Test without significant difference between group and control group, illustrate that GD2+MSC and KDR+HSC can not cause MSC agings.
5. Identifying cellular senescence-associated genes p16Ink4a, p53, p21cipl/wafl relative expression
(1)p16：1d, 3d, 7d, 14d MSC p16 have no significantly raised after GD2+MSC and KDR++HSC treatments, and right
According between group without significant difference (p>0.05).Difference after GD2+MSC and KDR+HSC treatments between 28d p16 and control group, which has, extremely to be shown
Write meaning (p<0.001), hence it is evident that reduction.
(2)p21：Ld, 7d, 14d, 28d MSC p2l have no significantly raised after GD2+MSC and KDR+HSC treatments, and right
According between group without significant difference (p>0.05).Difference after GD2+MSC and KDR+HSC treatments between 3d p21 and control group has significantly
Meaning (p<0.05), hence it is evident that reduction.
(3)p53：GD2+MSC ld, 3d, 7d, 14d, 28d MSC after KDR+HSC is treated p53 is bright compared with control group
Aobvious reduction, group difference significance (p<0.05).
Result above shows that GD2+MSC and KDR+HSC combined therapies have significant therapeutic effect for mouse aging.
In addition, in the supplement experiment 3a carried out with reference to above-described embodiment 3, to the gained biological sample of embodiment 2, entering
Row aging correlation beta-galactosidase (SA- β-Gal) Activity determination, as a result shows every detection parameter and index and above-mentioned implementation
The result of example 3 is essentially identical, no significant difference.
Industrial applicability
The invention belongs to cell therapy technology field, it is related to a kind of for repairing the aging of body function and delaying organ function
The therapeutic agent of decline, more particularly to a kind of therapeutic agent including GD2+ stroma cells and KDR+ candidate stem cells.Further,
Combined the present invention relates to both GD2+ stroma cells and KDR+ candidate stem cells is used to repair the aging of body function and prolongs preparing
Purposes in the therapeutic agent of slow organ function decline.Therapeutic agent of the present invention can be efficiently used for repairing the aging of body function and prolong
Slow organ function decline.
Claims (10)
- Prepared 1. mescenchymal stem cell is combined with candidate stem cell for treating senium praecox or early ageing disease or for repair machine The aging of body function and delay organ function fail therapeutic agent in purposes.
- 2. purposes according to claim 1, wherein the mescenchymal stem cell is GD2+ stroma cells.
- 3. purposes according to claim 1, wherein：The mescenchymal stem cell is derived from the mescenchymal stem cell of placenta and/or umbilical cord；The mescenchymal stem cell is derived from the GD2+ stroma cells of placenta and/or umbilical cord；The mescenchymal stem cell is derived from the GD2+ stroma cells of placenta and/or umbilical cord, and GD2 positive expression rates are more than 90%, it is greater than 95%；With or, the cell suspension of the mescenchymal stem cell in, added with vitamin C and epidermal growth factor Son.
- 4. purposes according to claim 1, wherein：The derived from hematopoietic precursor cells in：Cord blood, marrow, placental blood, and/or mobilized peripheral blood；The candidate stem cell is the candidate stem cell of KDR positive expressions；The candidate stem cell is KDR sun in the candidate stem cell of KDR positive expressions, the candidate stem cell of the KDR positive expressions Property expression rate be more than 85%, be greater than 90%；And/orCD34 positive expression rates are more than 80% in the candidate stem cell, are greater than 85%.
- 5. purposes according to claim 1, wherein：The therapeutic agent is in the form of medicine box, and the medicine box includes independent seal-packed mescenchymal stem cell and independent sealed bundle The candidate stem cell of dress；The therapeutic agent each consumption of dosage of mescenchymal stem cell when for mammal, such as people is that per kilogram is suffered from Person's body weight consumption is (0.1~20) × 105Individual stroma cell, such as per kilogram weight in patients consumption are (0.1~10) × 105It is individual Stroma cell, such as per kilogram weight in patients consumption are (0.2~1.0) × 105Individual stroma cell, such as per kilogram weight in patients Consumption is 0.5 × 105Individual stroma cell；Therapeutic agent dosage of candidate stem cell when for mammal, such as people is per kilogram weight in patients every time Consumption is (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 the treatment Agent dosage of candidate stem cell when for mammal, such as people be per kilogram weight in patients consumption every time for (2~ 10)×105Individual mononuclearcell, such as per kilogram weight in patients consumption are (2~5) × 105Individual mononuclearcell, such as per public Jin body weight consumption is (2~4) × 105Individual mononuclearcell；The therapeutic agent is when for mammal such as people, first using mescenchymal stem cell, and Hematopoietic Stem was used after 1 month Cell.
- 6. the treatment for treating senium praecox or early ageing disease or for repairing the aging of body function and delaying organ function to fail Agent, it is in the form of medicine box, and the medicine box includes independent seal-packed mescenchymal stem cell and independent seal-packed hematopoiesis Stem cell.
- 7. therapeutic agent according to claim 6, wherein the mescenchymal stem cell is GD2+ stroma cells.
- 8. therapeutic agent according to claim 6, wherein：The mescenchymal stem cell is derived from the mescenchymal stem cell of placenta and/or umbilical cord；The mescenchymal stem cell is derived from the GD2+ stroma cells of placenta and/or umbilical cord；The mescenchymal stem cell is derived from the GD2+ stroma cells of placenta and/or umbilical cord, and GD2 positive expression rates are more than 90%, it is greater than 95%；And/or, in the cell suspension of the mescenchymal stem cell, added with vitamin C and epidermal growth The factor.
- 9. therapeutic agent according to claim 6, wherein：The derived from hematopoietic precursor cells in：Cord blood, marrow, placental blood, and/or mobilized peripheral blood；The candidate stem cell is the candidate stem cell of KDR positive expressions；The candidate stem cell is KDR sun in the candidate stem cell of KDR positive expressions, the candidate stem cell of the KDR positive expressions Property expression rate be more than 85%, be greater than 90%；And/orCD34 positive expression rates are more than 80% in the candidate stem cell, are greater than 85%.
- 10. therapeutic agent according to claim 6, wherein：The therapeutic agent each consumption of dosage of mescenchymal stem cell when for mammal, such as people is that per kilogram is suffered from Person's body weight consumption is (0.1~20) × 105Individual stroma cell, such as per kilogram weight in patients consumption are (0.1~10) × 105It is individual Stroma cell, such as per kilogram weight in patients consumption are (0.2~1.0) × 105Individual stroma cell, such as per kilogram weight in patients Consumption is 0.5 × 105Individual stroma cell；Therapeutic agent dosage of candidate stem cell when for mammal such as people is per kilogram weight in patients every time Consumption is (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 the treatment Agent dosage of candidate stem cell when for mammal such as people is that per kilogram weight in patients consumption is (2~10) every time ×105Individual mononuclearcell, such as per kilogram weight in patients consumption are (2~5) × 105Individual mononuclearcell, such as per kilogram are suffered from Person's body weight consumption is (2~4) × 105Individual mononuclearcell；And/orThe therapeutic agent is when for mammal such as people, first using mescenchymal stem cell, and Hematopoietic Stem was used after 1 month Cell.
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