CN104450608B

CN104450608B - Promote the adjuvant of Human Meta's stem cell proliferation, expand Human Meta's stem cell methods, medical composition, growth factor and application thereof

Info

Publication number: CN104450608B
Application number: CN201310414614.XA
Authority: CN
Inventors: 孙立易; 冯清荣; 郑敬枫; 李典锟
Original assignee: BUDDHIST TCBC MEDICAL FOUNDATION
Current assignee: BUDDHIST TCBC MEDICAL FOUNDATION
Priority date: 2013-09-12
Filing date: 2013-09-12
Publication date: 2017-10-24
Anticipated expiration: 2033-09-12
Also published as: CN104450608A

Abstract

The present invention is on a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant, to solve the problem of cell amplification efficiency is not good during known Human Meta stem cell culture.The Human Meta stem cell culture addition adjuvant of the present invention includes at least one antioxidant, a Second-Type fiber mother cell growth factor（FGF‑2）.Whereby, foregoing adjuvant is added in the culture medium comprising Human Meta stem cell, in normal oxygen environment（About 21% partial pressure of oxygen）After culture, occur cell quickly divide, the cell cycle synthesis phase（S phase）Ratio improves, reduces aging and improve the phenomenons such as differentiation potential, the present invention not only can be rapidly expanded Human Meta stem cell and is obtained growth factor, and can still possess the feature of stem cell multifunctional, culture and the purposes of amplification as Human Meta stem cell.

Description

Promote the adjuvant, amplification Human Meta stem cell methods, doctor of Human Meta's stem cell proliferation Drug composition, growth factor and application thereof

Technical field

The present invention is related to a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant, it is espespecially a kind of comprising antioxidant with The adjuvant of growth factor to make an addition in the culture medium of Human Meta stem cell, to make mankind's interstital stem cell in primary or after It is commissioned to train and supports and rapid multiplication and growth factor can be obtained.

Background technology

Press, current scientific circles have descended some basic definitions with being with characteristic for stem cell：Referring to a group has cell certainly I updates（Self-renewal）, hyperplasia（Proliferation）Ability, while can maintain for a long time undifferentiated （Undifferentiation）The cell of state, and can be divided into the thin of different pedigree after being stimulated by appropriate induction The multi-differentiation of the tissue of born of the same parents group and specific function（multi-differentation）Characteristic.

According to the difference of source of human stem cell and its differentiation potential, it can be classified as：

1st, totipotency stem cell（Totipotent stem cell）：It, which has, can develop into complete independent biochemical body Ability, such as embryonated egg（Zygote）Or embryonic development is to the cell mass of about eight cell stages.

2nd, multi-functional stem cell（Pluripotent stem cell）：Embryonated egg（Zygote）After fertilization about 4 days, entirely Feature stem cell starts differentiation and enters the blastaea body phase, and blastaea body can be divided into two parts：Outer trophocyte（outer layer of cells）With inner cell mass（inner cell mass）, in embryo development procedure, outer trophocyte can form placenta The supporting tissue needed for uterus is attached to fetus, and inner cell mass can then form ectoderm, mesoderm and entoderm, each Germinal layer is down divided into a variety of systems and organ again.Although the ability of each part of the tangible adult body of inner cell mass, if Inner cell mass is individually put into the intrauterine of human mature's women's health, in default of outer trophocyte, it is impossible to form placenta And the external support System and Surroundings needed for fetal growth, so a complete individuals can not be developed into, therefore its multifunctionality is still deposited Have what part was limited.

3rd, multipotency stem cell（Multipotent stem cell）：It is the stem cell of current most study, from above-mentioned Multi-functional stem cell down breaks up again, can specialization into the stem cell of particular organization, such as candidate stem cell（hematopoietic stem cell）, mesenchymal stem cell（mesenchymal stem cells）Deng, wherein, candidate stem cell come from peripheral blood, Cord blood, marrow etc., can be divided into various blood cells and lymph, and mesenchymal stem cell may be from fat, periosteum （periosteum）, membrana articulata（synovial membrane）, marrow and some internal organs interstitial tissue（mesenchymal tissue）, such as placenta

4th, single-minded/single type stem cell（Unipotent stem cell）：Refer to and have the ability to be divided into specific a certain kind The stem cell of tissue or referred to as precursor cell（progenitor cell）, this kind of cell is prevalent in the group of each several part Knit, be the stem cell for being easiest to be found, such as liver precursor cell, neural precursor cell.

Mesenchymal stem cell is academicly being defined as being formed fine micro- mother cell of settlement unit for the first time（colony- forming unit of fibroblast- CFU-Fs）.During culture, meeting individual layer is attached to plastic culture dish table Fusiform is presented in face, the similar fine micro- mother cell of form, in vitro can rapid multiplication formation settlement, and possess that to be divided into bone female thin Born of the same parents（osteoblast）, fat cell（adipocyte）, cartilage cell（chondrocyte）Potential.Also there is research in recent years Point out that liver cell, cardiac muscle cell, nerve cell, islet cells etc. can be divided into（Minguell et al., 2001）.

The source of mesenchymal stem cell can be separated from many different tissues of human body, for example by operation directly cut off or via The adipose tissue that liposuction is obtained is the abundant source of a stem cell, may separate out fat stem cell（Adipose tissue-derived stem cells, ADSCs）, it has had the advantage that：Acquisition mode is invasive low, to human injury Amount that is small and once obtaining is more, can be in external hyperplasia culture etc.；Also possess plus fat stem cell can be divided into os osseum, it is soft The potentiality of bone, muscle and fat cell（Zuk et al., 2002）, therefore, fat stem cell is considered as that most research is sent out Open up one of stem cell of potentiality.

The research and application of stem cell, no matter on basic medical research or on clinical treatment, are required for enough thin Born of the same parents' number, and must cultivate in the environment of one is appropriate, stimulated comprising suitable microenvironment, growth factor etc., to avoid stem cell In culture, hyperplastic process premature aging, lose activity or be divided into other cells.However, due to cell separation technology, life The difference of long culture medium and condition of culture causes stem cell dramatically different with having on differentiation capability in hyperplasia（Pittenger et al.,2008）.In addition, many reported in literature all unanimously notice that aging tendency of the interstital stem cell in culture hyperplastic process is asked Topic（Bonab et al., 2006；Shibata et al., 2007；Wagner et al., 2008）.Therefore, stem cell increases Difference encountered in raw process in cell performance and aging, may urgently hinder the clinical application of interstital stem cell.Therefore, such as The stem cell that acquirement effectively and quickly can be difficult by what is cultivated and amplifies number, while can keep undifferentiated state and reduction again The phenomenon of aging, and still there is multi-functional characteristic, it is always that scientists are strongly pursued.

And in recent years, research report is also noted that, bone marrow interstital stem cell was cultivated（BMMSC）Conditioned medium in, quilt It was found that with the presence of can largely promote the paracrine factor of wound healing, such as：VEGF（VEGF）, para-insulin Growth factor 1（IGF-I）, EGF（EGF）, keratinocyte growth factor（KGF）, angiogenin （angiopoietin-1）, stromal-derived factor（stromal-derived factor-1）, macrophage inflammatory albumen （macrophage inflammatory protein-1α、macrophage inflammatory protein-1 β）And promote Erythropoietin(EPO)（erythropoietin）Deng（Martin et al., 1997）.And adipose stromal stem cell（ADSC） Through being proved and bone marrow interstital stem cell, Cord blood interstital stem cell, Periosteal Mesenchymal Stem Cells, synovial membrane interstital stem cell and muscle Interstital stem cell has no significant difference in gene performance and phenotype（Sheehy et al., 2012；Hung et al., 2012；Hung et al., 2007）.More preferably separated in view of adipose stromal stem cell has compared to bone marrow interstital stem cell And external hyperplasia condition, it is very hopeful at present to be applied to wound reparation and regeneration.It is known that in conditioned medium, fat Interstital stem cell has secreted following growth factor, such as：Second-Type fibroblast growth factor（bFGF）, horn cell（KGF）、 Make the transition growth factor（TGF-β）, HGF（HGF）, VEGF（VEGF）Deng, and these growth because Son all may be relevant with wound healing.Therefore how to make interstital stem cell rapid multiplication and produce the raised growth factor, actually one It is worth the problem for making great efforts research.

Just like No. 201331366 one kind of TaiWan, China patent " external serum-free adult stem cell amplification culture technique ", carry For a kind of method of external serum-free adult stem cell amplification culture, its method utilizes and adds autologous growth factor（PRGF）Yu Wu The Initial culture and squamous subculture of human adult stem cell, the mankind cultivated in this approach are carried out in serum stem cell medium Adult stem cell is after multiple squamous subculture, and human adult stem cell remains at the undifferentiated state of essence.Only, through it The stem cell population that cultural method is obtained is in squamous subculture to the third generation（P3）Preceding about 55,000 cell numbers/every square of just reaching Centimetre, to obtain more stem cell populations, must may improve the algebraically of cultivated days and squamous subculture to meet demand, Relative, it is also necessary to undertake the risk that multiple squamous subculture is potentially contaminated.

Just like No. 201118172 one kind of TaiWan, China patent, " low-density merges hypoxemia culture amplification interstital stem cell again Method ", this method can quickly be done carefully not influenceing cell to breed with the lower of stem cell properties with efficiently amplification Human Meta Born of the same parents, are included in external or internal increase propagation, reduce aging and increase differentiation potential.Only because most of research unit is used for training Support cell incubator mostly only provide carbon dioxide partial pressure and humidity adjustment function, if need under low-oxygen environment cultivate, Zero type is needed to purchase the incubator of tool partial pressure of oxygen adjustment function；Actually one born greatly for the research unit of reasearch funds not very abundance Load.

For another TaiWan, China patent 201231087 " a kind of skin care products processing procedure containing a variety of growth factors ", take first Go out healthy fat；The solvent of a predetermined volumes is mixed into adipose tissue again, to be cleaned to adipose tissue；By predetermined close Reagent adds the adipose tissue of predetermined volumes, and is centrifuged；The adipose tissue produced after separation is then mixed into predetermined agent The ferment of amount shakes in a centrifuge tube, and to the centrifuge tube；The sediment of generation is subjected to cell culture thin to produce body Born of the same parents；And obtain after the body cell, it is rich in the growth factor that cell is secreted through the cell culture fluid acquired by culture aforesaid way （Such as：VEGF、HGF、b-FGF、TGF、IGF）, to complete efficient skin care products raw material.Precisely because the cultural method disclosed is by body cell Insert one and include percent 10 hyclones（10%FBS）Basal medium DMEM（Dulbecco`s Modified Eagle Medium）Middle culture, the body cell proliferation rate is slow, and the growth factor secreted by it is relatively fewer, to obtain substantial amounts of life The long factor, must just improve the algebraically of cultivated days and squamous subculture to reach, therefore, it may have because multiple squamous subculture can The risk that can be contaminated is present.

Summary, in order that external Human Meta stem cell can rapid multiplication, and can still possess stem cell multifunctional Feature, and can largely obtain the growth factor of Human Meta's stem cell secretion simultaneously, we, which need badly, wants a Human Meta dry thin Born of the same parents cultivate adjuvant and cultural method, and efficiently amplifying cells number and the raised growth factor is obtained again with quick.

The content of the invention

The present inventor is in view of existing for the culture of Human Meta stem cell is with amplifying cells number and obtains Human Meta The missing of the growth factor of stem cell secretion studied again there is provided a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant and Its cultural method and a kind of method of external rapid amplifying Human Meta stem cell to obtain growth factor and application thereof, to up to To more rapidly and efficiently amplification Human Meta stem cell and obtain the purpose of growth factor.

For up to above-mentioned purpose, solution of the invention is：

The present invention provides a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant, it include at least one antioxidant with And a Second-Type fiber mother cell growth factor（Basic fibroblast growth factor, FGF-2）.

Further, aforementioned human's interstital stem cell body is selected from：Between adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord Matter stem cell constitutes one of group.

Further, foregoing antioxidant includes a long-acting type ascorbic acid derivates（Long-acting ascorbic acid derivative）And an ACETYLCYSTEINE（N-acetyl-L-cysteine, NAC）Combination.

Further, foregoing long-acting type ascorbic acid derivates are an ascoltin -2- phosphate（L-ascorbic acid-2 -phosphate, AsA2P）.

Further, foregoing Second-Type fiber mother cell growth factor concentration is 1 ng/ml（ng/mL）To 20 Ng/ml（ng/mL）.

Further, it suppresses by the cell within a cell cyclin-dependent kinase for suppressing aforementioned human's interstital stem cell Thing：The performance of p21 and p27 albumen is to improve cell cycle protein dependent kinase -2（CDK-2）, cyclin dependant Kinases -4（CDK-4）And cell division cycle protein（CDC2）Performance.

The present invention provides a kind of method of external rapid amplifying Human Meta stem cell, including foregoing adjuvant is added into one In culture medium comprising Human Meta stem cell, to expand Human Meta stem cell, and obtain between the undifferentiated mankind of essence Matter stem cell.

The present invention provides a kind of method of external rapid amplifying Human Meta stem cell again, including foregoing adjuvant is added In a culture medium comprising a serum additive and a Human Meta stem cell, to expand aforementioned human's interstital stem cell, and obtain Take the undifferentiated Human Meta stem cell of essence.

Further, foregoing serum additive is human serum or hyclone, and used concentration expressed in percentage by volume is percentage 2 to percent 10.

Further, the method for foregoing in vitro rapid amplifying Human Meta stem cell further includes the foregoing amplification of a freezen protective Human Meta's stem cell step, for further using.

Further, using the Human Meta stem cell of the amplification of preceding method institute freezen protective to set up a cell bank.

Further, the method for foregoing in vitro rapid amplifying Human Meta stem cell includes performing an extraction step obtaining one The cell extraction thing of Human Meta stem cell.

Further, the method for foregoing in vitro rapid amplifying Human Meta stem cell further includes execution one and induces differentiation step, To obtain a cell broken up from aforementioned human's interstital stem cell.

Further, the foregoing cell broken up from Human Meta stem cell, including osteocyte, fat cell or cartilage cell One of.

The present invention separately provides a kind of medical composition, and it is included by the side of foregoing in vitro rapid amplifying Human Meta stem cell The undifferentiated Human Meta stem cell of essence or a cell broken up from aforementioned human's interstital stem cell acquired in method.

Further, foregoing medical composition combines a biocompatible material with applied to regenerative medicine or organizational project.

The present invention provides a kind of method of external rapid amplifying Human Meta stem cell to obtain growth factor again, through foregoing After the method culture of external rapid amplifying Human Meta stem cell, to obtain a growth factor in prior culture media.

Further, aforementioned growth factors include：FGF-2、EGF、FGF-4、FGF-6、FGF-7、HB-EGF、HGF、IGFBP- 1、IGFBP-2、IGFBP-3、IGFPB-4、IGFBP-6、IGF-I、IGF-I SR、IGF-II、M-CSF、M-CSF R、PDGF R α、PDGF-Rβ、PDAF-AA、PDGF-AB、PDGF-BB、PIGF、SCF、TGF-β3、VEGF、VEGF R2。

The present invention provides a kind of growth factor, according to foregoing in vitro rapid amplifying Human Meta stem cell with obtain growth because The method of son is obtained.

The present invention provides a kind of medical composition, includes foregoing in vitro rapid amplifying Human Meta stem cell to obtain life The growth factor that the method for the long factor is obtained.

The present invention provides a kind of purposes included just like aforementioned growth factors in the medicine for preparing wound healing promoting.

The present invention provides a kind of include just like purposes of the aforementioned growth factors as skin care product.

Effect of the present invention is：

First, cell rapid multiplication：By Human Meta's stem cell in vitro rapid multiplication adjuvant of the addition present invention in culture In base, mankind's interstital stem cell body is set to occur synthesis phase cell cycle in primary or squamous subculture（S phase）Ratio is improved, And promote the phenomenon of the quick division and proliferation of cell, and can still possess the potential of stem cell multifunctional differentiation.

2nd, cell senescence is reduced：By Human Meta's stem cell in vitro rapid multiplication adjuvant of the addition present invention in culture In base, aforementioned human's interstital stem cell body telomerase reverse transcription ferment can be made（Telomerase Reverse Transcriptase, TERT）Improve, cell telomere can be extended（Telomere）The time of shortening, to slow down cell senescence And extend the life-span of cell.

3rd, the quick obtaining raised growth factor：By Human Meta's stem cell in vitro rapid multiplication assistant of the addition present invention Agent more conventional can be incubated in a culture medium comprising concentration expressed in percentage by volume for percent 10% hyclones, obtain in culture medium Take at least more than 2 to 3 times of growth factor [such as IGF-1（IGF-1）, HGF（HGF）And EGF（EGF）] content.

4th, reduction pathogeny pollution：Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention is in collocation human serum When being cultivated, it can avoid there are allosome or the interaction of xenogeneic pathogeny because being cultivated using the serum of allosome or xenogenesis The risk of pollution.

Brief description of the drawings

Figure 1A-Fig. 1 C are Human Meta stem cell in the present invention in the number of days under different condition of culture and cell density relation Figure；

Fig. 2A is the impact analysis figure of fat stem cell cell cycle after different condition culture in the present invention；

Fat stem cell is after different condition culture in Fig. 2 B explanation present invention, the albumen for being responsible for cell cycle regulation Performance influence；

Fig. 3 for the present invention in fat stem cell after different condition culture, for the influence of cell relative telomere length；

Fig. 4 for the present invention in fat stem cell after different condition culture, the graph of a relation of cultivated days and TCS；

Fig. 4 B are fat stem cell in the present invention after the different condition culture, the albumen for being responsible for cell cycle regulation Performance influence；

Fig. 5 A are fat stem cell in the present invention after different condition culture, cell quantity and kenel schematic diagram；

Fig. 5 B- Fig. 5 D for the present invention in fat stem cell after different condition culture, cell culture number of days and cell quantity Graph of a relation；

Fig. 6 A for the present invention in fat stem cell after different condition culture, its Cell surface antigen analysis result figure；

Fig. 6 B for the present invention in fat stem cell after different condition culture, its stem cell gene relative performance's analysis chart；

Fig. 7 A- Fig. 7 B for the present invention in fat stem cell after different condition culture and being induced to differentiate into os osseum cell, warp Chemical staining result figure；

Fig. 7 C for the present invention in fat stem cell after different condition culture and being induced to differentiate into os osseum cell, its os osseum is thin Extracellular molecule mark relative performance's analysis chart；

Fig. 8 A for the present invention in fat stem cell after different condition culture and being induced to differentiate into cartilage cell, through chemistry dye Color result figure；

Fig. 8 B for the present invention in fat stem cell after different condition culture and being induced to differentiate into cartilage cell, its cartilage is thin Extracellular molecule mark relative performance's analysis chart；

Fig. 8 C for the present invention in fat stem cell after different condition culture and being induced to differentiate into fat cell, through chemistry dye Color result figure；

Fig. 8 D are fat stem cell in the present invention after different condition culture and being induced to differentiate into fat cell, and its fat is thin Extracellular molecule mark relative performance's analysis chart；

Fig. 9 A for the present invention in fat stem cell after different condition culture, cell factor array analysis chart；

Fig. 9 B compare figure for the cell factor array analysis result of Fig. 9 A in the present invention；

Fig. 9 C- Fig. 9 D are relative performance's quantitative analysis figure of cell factor in Fig. 9 B；

Fig. 9 E are fat stem cell in the present invention after different condition culture, the relative performance of cell factor point in its cell Analysis figure；

Fig. 9 F for the present invention in fat stem cell after different condition culture, cell factor array analysis chart；

Fig. 9 G compare figure for the cell factor array analysis result of Fig. 9 F in the present invention；

Figure 10 A are incubated at the light microscope striograph of a microcarrier for adipose stromal stem cell in the present invention；

The fluoroscopic image figure that Figure 10 B are Figure 10 A in the present invention.

Embodiment

In order to which above and other purpose, feature and the advantage of the present invention can be become apparent, preferable implementation cited below particularly Example, and coordinate appended diagram, it is described in detail below：

The present invention provides a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant, it include at least one antioxidant with And a Second-Type fiber mother cell growth factor（FGF-2）；Wherein foregoing antioxidant includes a long-acting type ascorbic acid and derived Thing and an ACETYLCYSTEINE（NAC）Combination.It is preferred that the long-acting type ascorbic acid derivates are anti-for a L- Bad hematic acid -2- phosphate（Asc 2-P）, and Second-Type fiber mother cell growth factor concentration is 1 to 20 nanogram/milli Rise.

It is preferred that aforementioned human's interstital stem cell body is selected from：Adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord Interstital stem cell constitutes one of group.

It is preferred that Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention is done by aforementioned human's interstitial is suppressed The cell within a cell cyclin-dependent kinase mortifier of cell：The performance of p21 and p27 albumen is to improve cyclin Dependant kinase -2（CDK-2）, cell cycle protein dependent kinase -4（CDK-4）And cell division cycle protein（CDC2）'s Performance, and then promote the cell cycle to enter the synthesis phase（S phase）Ratio improve so that the quick division and proliferation of cell.

Add present invention simultaneously provides a kind of method of external rapid amplifying Human Meta stem cell, including by foregoing adjuvant Enter and cultivated in a culture medium comprising Human Meta stem cell, do not divided with expanding Human Meta stem cell, and obtaining essence The Human Meta stem cell changed.

It is preferred that foregoing serum additive is human serum or hyclone, used concentration expressed in percentage by volume is hundred / 2 to percent 10.

It is preferred that the method for foregoing in vitro rapid amplifying Human Meta stem cell further comprises performing an extraction step To obtain the cell extraction thing of a Human Meta stem cell.

It is preferred that the method for foregoing in vitro rapid amplifying Human Meta stem cell further includes the foregoing amplification of a freezen protective Human Meta's stem cell step, for further using.

It is preferred that " freezen protective " one word as used herein typically refers to cell adding cryoprotector such as diformazan Sulfoxide（DMSO）Or the method that subzero temperature is preserved is cooled to after glycerine, for example, minus 80 DEG C or minus 196 DEG C（Liquid nitrogen Boiling point）.Method and flow that freezen protective can be carried out such as those skilled in the art, only non-invention demand emphasis is therefore It will not go into details（Second edition Pollard, J.W. and Walker, base written by J.M.. are published refering to HummaPress companies in 1997 Plinth cell culture flow；Wiley-Liss companies in 2000 publish the 4th edition Freshney, R.I., written by zooblast training Support）.

The present invention provides a kind of cell bank, and it includes cold by the method institute of foregoing in vitro rapid amplifying Human Meta stem cell Freeze the Human Meta stem cell of the amplification preserved.

It is preferred that the method for foregoing in vitro rapid amplifying Human Meta stem cell further includes the induction differentiation step of execution one Suddenly, to obtain a cell broken up from aforementioned human's interstital stem cell.

It is preferred that the foregoing cell broken up from Human Meta stem cell, including osteocyte, fat cell or cartilage are thin One of born of the same parents.

The present invention provides a kind of medical composition again, and it is included by the side of foregoing in vitro rapid amplifying Human Meta stem cell The undifferentiated Human Meta stem cell of essence or a cell broken up from aforementioned human's interstital stem cell acquired in method.

It is preferred that the medical composition is secreted comprising aforementioned human's interstital stem cell, from its cell, cell for being broken up One or a combination set of thing or cell extraction thing and the upper acceptable carrier/excipient of suitable treatment.Wherein, cell is secreted Thing is obtained after can purifying and concentrate in cell culture medium in certain embodiments.

The present invention provides a kind of method of external rapid amplifying Human Meta stem cell to obtain growth factor again, via preceding After the method culture for stating external rapid amplifying Human Meta stem cell, cell also secretes various kinds of cell while rapid multiplication Growth factor can obtain aforementioned growth factors in prior culture media whereby in culture medium.Wherein, aforementioned growth factors bag Include：FGF-2、EGF、FGF-4、FGF-6、FGF-7、HB-EGF、HGF、IGFBP-1、IGFBP-2、IGFBP-3、IGFPB-4、 IGFBP-6、IGF-I、IGF-I SR、IGF-II、M-CSF、M-CSF R、PDGF Rα、PDGF-Rβ、PDAF-AA、PDGF-AB、 PDGF-BB、PIGF、SCF、TGF-β3、VEGF、VEGF R2。

The present invention provides a kind of medical composition, includes foregoing in vitro rapid amplifying Human Meta stem cell to obtain Take the method for growth factor to obtain growth factor, can be used for but do not limit treatment skin trauma.

The present invention provides a kind of skin care product for including aforementioned growth factors, to repair skin sunburn or delay skin Skin cell senescence.

" Human Meta stem cell " word alleged by the present invention refers to any cell for being obtained from Human Meta's tissue and had The unlimited ability voluntarily updated and various kinds of cell or organizational form can be divided into, such as, but not limited to adipose stromal is dry thin Born of the same parents, bone marrow interstital stem cell or umbilical cord mesenchymal stem cells, Cord blood interstital stem cell, Periosteal Mesenchymal Stem Cells, synovial membrane interstitial are done Cell and muscle interstital stem cell constitute one of group.Embodiments of the invention are with the embodiment of human adipose's interstital stem cell Give demonstration to illustrate, but the present invention is not limited by following embodiments.

Experiment one：The adjuvant of the present invention is cultivated to cell for different mankind's interstital stem cells under normal oxygen and low-oxygen environment The influence of growth.

1. separation and the in vitro culture of Human Meta stem cell

This experiment by Buddhism Tzu Chi general hospital inside examination board（IRB100-102）Approval is carried out, respectively with people Class adipose tissue, umbilical cord and myeloid tissue are that stem cell obtains source, and only the separation method of Human Meta stem cell is habit herein Knowing technology and non-this case demand emphasis, therefore it will not go into details, and main purpose is all obtaining Human Meta stem cell be just commissioned to train Support；The cell culture condition of this experiment is divided into three groups：Basal medium is plus FGF-2 in normal oxygen environment culture group, basis culture Base adds the adjuvant of the present invention in normal oxygen environment culture group plus FGF-2 in low-oxygen environment culture group and basal medium；Its In alleged often oxygen environment refer under about percent 21 partials pressure of oxygen, and low-oxygen environment refers under 5 partials pressure of oxygen of about percentage；The basal medium For IMDM（Iscove`s modified Dulbecco`s medium, GIBCO-Invitrogen）Add 10% tire ox blood Clearly（FBS, MSC-Qualified, GIBCO-Invitrogen）And 2 millimolar concentrations（mM）Left-handed bran amic acid（L- glutamine, GIBCO-Invitrogen）Constituted；And concentration used in aforementioned growth factors FGF-2 is 10 ng/mL （R&D Systems）, and foregoing adjuvant of the invention includes 2 millimolar concentrations（mM）ACETYLCYSTEINE（NAC, Sigma）And 0.2 millimolar concentration（mM）Ascoltin -2- phosphate（AsA2P, Sigma）.Every group of stem cell with Every square centimeter of 3000 cells（3000 cells/cm²）Cell density be incubated in 6 hole cell cultures（6-well plates, Becton Dickinson）, and all cells are all incubated at 37 degree Celsius, percent 5 carbon dioxide partial pressures and hundred Incubator under/95 humidity environments（Forma Series II Model 3110, Thermo）, and change once for every 3 days Culture medium；The culture experiment of another hypoxemia culture environment is then in another incubator（MCO-18M, Sanyo）It is middle to carry out.See whereby Different mankind's interstital stem cells are examined in 7 days under different condition of culture, the influence to hyperplasia.

1.1 cell densities are analyzed

By each group stem cell with phosphate buffer solution（PBS）After cleaning once, with trypsin-EDTA （Trypsin-EDTA）Solution is carefully removed with cell spatula again after being acted on 5 minutes in 37 degree Celsius not to be applied completely carefully Born of the same parents, add the ferment action activity with trypsase in culture medium of the equal proportion containing hyclone.All cell number mesh is all with thin Born of the same parents' calculator（Vi-CELL AS, Beckman Coulter）Calculate.The cell of survival is with 0.4% trypan blue（Trypan-blue, GIBCO-Invitrogen）To be distinguished with dead cell, judge during calculating work interstital stem cell parameter setting as：100 10-30 microns of images, Size, 75% live brightness, 5% field regions.As a result presentation is measured with every group of experimental data Three are repeated, and it is represented with average value ± standard deviation.

1.2 experimental result

Above experimental data is tested through Microsoft Excel Software t（t-test）Statistical analysis, with p ＜ 0.05 for notable water It is flat, and result is quantified as statistical chart.First, it is fat stem cell in the life under above-mentioned 3 kinds different condition of culture referring to Figure 1A Long situation, wherein basal medium add FGF-2 in low-oxygen environment plus FGF-2 in normal oxygen environment culture group and basal medium Two groups of culture group compares, it is found that stem cell hyperplasia under low-oxygen environment is more quick, especially there were significant differences at the 3rd day, Start within 4th day even more substantially, the proliferation rate of so display adipose stromal stem cell gets off in more normal oxygen environment under low-oxygen environment Must get well；Another basal medium adds the adjuvant of the present invention in normal oxygen plus FGF-2 in low-oxygen environment culture group and basal medium Two groups of environment culture group compares, and finds to add the adjuvant culture of the present invention under normal oxygen environment, can reach with being trained under low-oxygen environment Close proliferation rate is supported, or even is to surmount the proliferation rate cultivated under low-oxygen environment, it follows that of the invention from the 5th day Adjuvant addition, can make adipose stromal stem cell under normal oxygen environment culture, reach the effect of rapid multiplication.Join again Figure 1B and Fig. 1 C, identical result is also found in the culture experiment of bone marrow interstital stem cell or umbilical cord mesenchymal stem cells；By this experiment Understand, the addition of adjuvant of the invention is for adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord mesenchymal stem cells normal Under oxygen environment culture, the effect of rapid multiplication all can reach.

2. cell cycle analysis

Each experimental group that will be incubated at by above-mentioned adipose stromal stem cell under different condition of culture, is only included along with one The basal medium of 10% hyclone as a control group, with flow cytometer and its software（Phoenix Flow Systems） The change situation of its cell cycle is analyzed in detecting, and each group culture is sampled after three days, and every group of experiment three is repeated, after alcohol fixation, Use propidium iodide（propidium iodide, Sigma）Stain contaminates the DNA of cell to analyze the change of cell cycle.

2.1 experimental result

Join Fig. 2A, be control group under above-mentioned three groups of different condition of culture, to the cell cycle shadow of adipose stromal stem cell Ring, wherein * P< 0.05, ** P < 0.01, *** P < 0.005.Found by experimental result, basal medium is added FGF-2 in low-oxygen environment culture group and basal medium plus the present invention adjuvant in two groups of normal oxygen environment culture group etc., its fat Fat interstital stem cell is in the synthesis phase in the cell cycle（S phase）Percentage compare the control group and the basal medium Plus FGF-2 in normal oxygen environment culture group, hence it is evident that improve many, and basal medium adds FGF-2 in low-oxygen environment culture group With basal medium plus the present invention adjuvant in the G0/G1 phases of two groups of normal oxygen environment culture group etc.（Stop division stage/growth Phase）Percentage come much lower in normal oxygen environment culture group plus FGF-2 with the basal medium compared with the control group, thus may be used Know, under low-oxygen environment or addition the present invention adjuvant under normal oxygen environment cultivate, can all make the cell cycle be in the synthesis phase, promote Enter cell DNA constantly to synthesize, and then make the quick division and proliferation of cell.Wherein, the adjuvant of the present invention is added in training under normal oxygen environment Foster adipose stromal stem cell, the percentage that its cell cycle is in the synthesis phase is even more to exceed culture group under low-oxygen environment, tool There were significant differences；Therefore, the adjuvant of the present invention is added under normal oxygen environment culture, can not only reach rapid multiplication effect, and very To that can surmount the effect of hyperplasia speed can be improved in culture under low-oxygen environment.

3. the performance situation of the GAP-associated protein GAP of cell cycle regulation is analyzed with western blot

Known western blot is the characteristic combined using antibody, antigenic specificity, coordinates SDS-PAGE colloid electrophoresis The quantification and qualification of specific protein performance is detected, only this method is the technology widely known, its details is just no longer gone to live in the household of one's in-laws on getting married herein State.This experiment is for by reported in literature for the relevant albumen of cell cycle regulation, such as：Cyclin A2、Cyclin D1、 The albumen such as Cyclin D3, CDK2, CDK4, CDK6, CDC2, p21 and p27, utilize the selectivity between above-mentioned each albumen and its antibody With reference to coming through above-mentioned western blot in adipose stromal stem cell of the analysis and observation via different condition of culture cultures, its is right In the influence of the relevant albumen performance of cell cycle regulation；Wherein using a β-actin albumen performance amounts as control, as above-mentioned each Albumen performance is quantified as the benchmark foundation of the standardization of data；Because β-actin albumen is a kind of house-keeping gene （Housekeeping gene）The protein transcribe, translated, maintains normal physiological phenomena necessary by cell, and not There is too big change because of various experiment conditions, therefore be suitable as the benchmark foundation of quantized data standardization.

3.1 experimental result

Continuous ginseng Fig. 2 B, are above-mentioned tetra- groups of experimental groups of Fig. 2A, the GAP-associated protein GAP performance analysis of its cell cycle regulation, in figure Performance situation of each protein in different experiments group, finds to be considered to suppress the cell of cell cycle progress by reported in literature Cyclin-dependent kinase mortifier：The performance amount of p21 and p27 albumen, adds compared to above-mentioned control group and basal medium Upper FGF-2 adds plus FGF-2 in normal oxygen environment culture group, the basal medium in low-oxygen environment culture group with the basal medium Upper adjuvant of the invention is in normal oxygen environment culture group, hence it is evident that in down state, or even the basal medium is plus the assistant of the present invention Agent presents close or adds FGF-2 less than the basal medium in normal oxygen environment culture group, the performance amount of itself p21 and p27 albumen In the situation of low-oxygen environment culture group；Another cell cycle protein dependent kinase -2（CDK-2）, cyclin dependent kinase Enzyme -4（CDK-4）And cell division cycle protein（CDC2）Performance amount, added compared to above-mentioned control group and basal medium FGF-2 is added plus FGF-2 in normal oxygen environment culture group, the basal medium in low-oxygen environment culture group with the basal medium The adjuvant of the present invention is in normal oxygen environment culture group, hence it is evident that in the state of raising, or even the basal medium is plus the adjuvant of the present invention In normal oxygen environment culture group, the performance amount of its CDK-2, CDK-4 and CDC2 albumen is presented close or added higher than the basal medium Upper FGF-2 is in the situation of low-oxygen environment culture group.It follows that the adjuvant of the addition present invention is between fat is cultivated under normal oxygen environment Matter stem cell is to reach the effect of rapid multiplication, by suppression Cyclin dependent kinase inhibitor：P21 and p27 eggs White performance, to improve the performance amount of CDK-2, CDK-4 and CDC2 albumen, and then promotes cell quick copy, division, with up to fast The effect of fast hyperplasia.

4. cell is with respect to telomere（Telomere）Length analysis

In the cell, chromosome structure is into and telomere is the repetitive dna sequence of end of chromosome, effect by DNA tissue It is the integrality for protecting chromosome.Chromosome can be replicated first before fissional, and DNA is replicated every time, and telomere will shorten one Point, when telomere shortens to a certain extent, is just unable to maintain that the stabilization of chromosome, and cell is eventually deathward, it is possible to The age of cell is predicted according to the length of telomere.Foregoing three groups different condition of culture and above-mentioned control group will be included（Comprising The basal medium of 10% hyclone）Four groups of adipose stromal stem cells cultivate 14 days after, using can be used to by reported in literature Measure cell relative telomere length（T/S ratio）Technical method（Cawthon, 2002）, to observe different condition of culture Under, for the influence of the telomere of adipose stromal stem cell.

4.1 experimental result

It is shown in Figure 3, via above-mentioned known measurement cell relative telomere length（T/S ratio）Method, with obtain In under foregoing four groups of experimental group condition of culture, the T/S ratio of its cell telomere, wherein * P< 0.05.Found by result, with When on the basis of the control group, the basal medium adds this hair plus FGF-2 in low-oxygen environment culture group and the basal medium Bright adjuvant is more improved compared to the basal medium in normal oxygen environment culture group plus FGF-2 in normal oxygen environment culture group, And tool significant difference；It follows that all may be used in the adjuvant that the present invention is cultivated or added under low-oxygen environment in being cultivated under normal oxygen environment The ratio of relative telomere length is improved, and then reaches the aging effect for delaying cell.

5. adipose stromal stem cell is in the influence cultivated under normal oxygen or oxygen environment

Condition of culture is divided into four groups by this experiment, and it is respectively：Basal medium is plus FGF-2 in normal oxygen environment culture Group, basal medium are trained in oxygen environment culture group, basal medium plus FGF-2 plus the adjuvant of the present invention in normal oxygen environment Group and basal medium are supported plus the adjuvant of the present invention in oxygen environment culture group etc., wherein oxygen environment refers to 37.5 about percent Under partial pressure of oxygen, and other condition of culture parameters（Such as basal medium, incubator and cell culture density）All with previous experiments one 1. contents it is identical.

The cell density analysis of 5.1 adipose stromal stem cells

By cell with phosphate buffer solution（PBS）After cleaning once, with trypsin-EDTA （Trypsin-EDTA）Solution is carefully removed with cell spatula again after being acted on 5 minutes in 37 degree Celsius not to be applied completely carefully Born of the same parents, add the ferment action activity with trypsase in culture medium of the equal proportion containing hyclone.All cell number mesh is all with thin Born of the same parents' calculator（Vi-CELL AS, Beckman Coulter）Calculate.The cell of survival is with 0.4% trypan blue（Trypan-blue, GIBCO-Invitrogen）To be distinguished with dead cell, judge during calculating work interstital stem cell parameter setting as：100 10-30 microns of images, Size, 75% live brightness, 5% field regions.As a result presentation is measured with every group of experimental data Three are repeated, and it is represented with average value ± standard deviation.

5.1.1 experimental result

Join shown in Fig. 4 A, be adipose stromal stem cell under different condition of culture, the relation of cultivated days and total cell number, Found by result, as cultivated days increase, the basal medium is plus adjuvant of the invention in the thin of normal oxygen environment culture group Born of the same parents' number just rapidly rises from after second day, and 1.5 × 10 have been reached in the 5th day⁶Quantity above, compares the basal medium Significantly improved plus FGF-2 in normal oxygen environment culture group and quickly；And the basal medium adds FGF-2 in oxygen environment culture Histocyte number, which is then presented, slowly to be increased, or even the TCS of the 7th day is not yet up to 2.5 × 10⁵Quantity, it is known that Oxygen environment has detrimental effect for the hyperplasia of cell；And the basal medium adds the adjuvant of the present invention in oxygen environment Cultured tissue TCS in compared after second day the basal medium plus FGF-2 in oxygen environment culture group present quickly carry High trend, during to the 6th day, its TCS has reached 1.0 × 10⁶Quantity above, compares the basal medium and adds FGF-2 is significantly improved and quickly in oxygen environment cultured tissue TCS.It follows that the adjuvant of the addition present invention is in normal oxygen Culture can relatively be not added with the adjuvant person of the present invention under environment, and its hyperplasia speed is more quick and number is more, and the basis is trained Base is supported plus the adjuvant of the present invention in oxygen environment culture group, though being cultivated under oxygen environment, has unfavorable for hyperplasia Influence, but the present invention adjuvant can slow down really because oxygen environment is to the adverse effect caused by hyperplasia, so that portion Cell is divided to continue to the phenomenon of hyperplasia.

5.2 adipose stromal stem cells are in the influence showed under different condition of culture p21 albumen and CDK2 albumen

The condition of culture of adipose stromal stem cell with being divided into four groups described in foregoing 5., and using foregoing western blot come In adipose stromal stem cell of the analysis and observation via different condition of culture cultures, it is for the relevant albumen of cell cycle regulation （Such as p21, CDK2）The influence of performance, equally using β-actin albumen performance amounts as control, is quantified as above-mentioned each albumen performance The benchmark foundation of the standardization of data.

5.2.1 experimental result

Join Fig. 4 B, as a result can find, the basal medium is plus FGF-2 in normal oxygen environment culture group and the basal medium There is the phenomenon of attenuating in the p21 albumen performance amounts of normal oxygen environment culture group plus the adjuvant of the present invention, and the table of the CDK2 albumen Now amount has increased phenomenon；And the basal medium adds FGF-2 in oxygen environment culture group then because oxygen environment makes its p21 egg White performance amount significantly improves many, so as to curb the performance of its CDK2 albumen, has a negative impact to hyperplasia；It is worth note Meaning, the basal medium, in oxygen environment culture group, is cultivated plus adjuvant of the invention though being under oxygen environment, by In the adjuvant that with the addition of the present invention, so adding FGF-2 in oxygen environment compared to the basal medium to p21 albumen performance amounts The effect that culture group has substantially been reduced, so that the performance of its CDK2 albumen is also significantly improved.Therefore, reconfirm addition originally , all can more no added adjuvant person of the invention, hence it is evident that between raising no matter the adjuvant of invention is under normal oxygen environment or under oxygen environment The proliferation rate of matter stem cell.

Experiment two：Adipose stromal stem cell is in the influence under different serum additive cultures to hyperplasia

The cell culture condition of this experiment is divided into four groups, and it is respectively：Basal medium adds 10% hyclone group, base Basal culture medium is plus 10% human serum group, basal medium plus 10% human serum plus the adjuvant group of the present invention and basis training Support base and four groups of adjuvant group of the present invention etc. is added plus 2% human serum；The basal medium is IMDM（Iscove`s modified Dulbecco`s medium, GIBCO-Invitrogen）Culture medium adds 2 millimolar concentrations（mM）It is left-handed Bran amic acid（L-glutamine, GIBCO-Invitrogen）Constituted, and foregoing adjuvant of the invention includes 2 mMs Concentration（mM）ACETYLCYSTEINE（NAC, Sigma）And 0.2 millimolar concentration（mM）Ascoltin- 2- phosphate（AsA2P, Sigma）.Every group of stem cell is with every square centimeter of 3000 cells（3000 cells/cm²）Cell Density is incubated in 6 hole cell cultures（6-well plates, Becton Dickinson）, and all cells are all incubated at Incubator under 37 degree Celsius, percent 5 carbon dioxide partial pressures and percent 95 humidity environments（Forma Series II Model 3110, Thermo）In, cultivate one week and every 3 days and change a subculture；Different adipose stromals in 7 days are observed whereby Stem cell is under different condition of culture, the influence to hyperplasia.

1. cell density is analyzed

By each group stem cell with phosphate buffer solution（PBS）After cleaning once, with trypsin-EDTA （Trypsin-EDTA）Solution is carefully removed with cell spatula again after being acted on 5 minutes in 37 degree Celsius not to be applied completely carefully Born of the same parents, add the ferment action activity with trypsase in culture medium of the equal proportion containing hyclone.All cell number mesh is all with thin Born of the same parents' calculator（Vi-CELL AS, Beckman Coulter）Calculate.The cell of survival is with 0.4% trypan blue（Trypan-blue, GIBCO-Invitrogen）To be distinguished with dead cell, judge during calculating work interstital stem cell parameter setting as：100 10-30 microns of images, Size, percent 75 live brightness, percent 5 field regions.As a result presentation is real with every group The repetition of DATA REASONING three is tested, it is represented with average value ± standard deviation.

1.1 experimental result

Above experimental data is tested through Microsoft Excel Software t（t-test）Statistical analysis, with p ＜ 0.05 for notable water It is flat, and result is quantified as statistical chart, wherein * P< 0.05, ** P < 0.01, *** P < 0.005.First, ginseng figure 5A is adipose stromal stem cell under different condition of culture, and in knitting cell kenel under microscope, engineer's scale is 500 microns（μ m）.Found in observation, each histiocytic type state is rather similar, the cell kenel all with fusiform.Referring to Fig. 5 B, trained for the basis Base is supported plus 10% hyclone group and cultivated days and cell density relation of the basal medium plus 10% human serum group Figure, the basal medium was added compared with the basal medium plus 10% human serum histocyte density from after culture four days 10% hyclone group significantly improves about percent 28 to percent 74 cell number, and with significant difference；It follows that Serum additive during with human serum as cell culture, more can make adipose stromal stem cell quick compared with addition hyclone Hyperplasia.Referring to Fig. 5 C, add for the basal medium plus 10% human serum group with the basal medium plus 10% human serum The cell density of upper adjuvant group of the invention compares figure, is found by result, and the basal medium is plus 10% human serum plus this Cell density of the adjuvant group of invention in culture one day after is significantly improved about compared with the basal medium plus 10% human serum group Percent 155 to percent 324 cell number, and with significant difference；It follows that in the adjuvant culture of the addition present invention Under, the speed of adipose stromal stem cell proliferation can be more improved, under identical cultivated days, to obtain more substantial amounts of cell number Mesh.Join Fig. 5 D again, this is added plus 2% human serum with the basal medium plus 10% human serum group for the basal medium The comparison of the adjuvant group of invention, is found by result, though the basal medium is plus adjuvant group of 2% human serum plus the present invention Only 2% human serum, but by after the adjuvant culture for adding the present invention, can still make adipose stromal stem cell in the training of first five day Under supporting, the speed of hyperplasia is still close plus 10% human serum group with the basal medium；It follows that the present invention The addition of adjuvant, can not only reduce the usage amount of human serum, while can also possess promotion adipose stromal stem cell rapid multiplication Effect.

2. the Cell surface antigen analysis of typical mesenchymal stem cell

This experiment is with flow cytometer（FACSCalibur, Becton Dickinson）Carry out the survey of cell surface antigen It is fixed.Cleaned the adipose stromal stem cell attachment removal being incubated at respectively under foregoing four groups different condition of culture and with phosphate buffer Afterwards, back dissolving is contaminated different antigen with corresponding immunofluorescence Primary antibodies respectively in appropriate phosphate buffer Color, including CD13, CD34, CD44, CD73, CD90, CD105, β2-microglobulin（B2M）And the antibody such as HLA-DR（Becton Dickinson）.Lucifuge was dyed after 15 minutes at room temperature, upper machine analysis after appropriate phosphate buffer was added, through flow cytometer Collect after data, with flow cytometry analysis software（FACSCalibur, Becton Dickinson）Analyzed.Wherein bear Control group is to omit the staining procedure of above-mentioned Primary antibodies.

2.1 experimental result

Referring to Fig. 6 A, by result it can be found that adipose stromal stem cell under foregoing four groups different condition of culture, its cell Group be CD13+, CD34-, CD44+, CD73+, CD90+, CD105+, be the cell race of similar interstital stem cell Group, that is, represent to say, the adipose stromal stem cell under foregoing four groups different condition of culture still possesses the table of similar interstital stem cell Face antigen property.Wherein, there are three groups of experiments of addition human serum, its CD44 and CD73 performance amount are higher than the basal medium Plus 10% hyclone group, this result also illustrates three groups of experiments for having addition human serum culture, and it is dry thin that it possesses similar interstitial The effect of the surface antigen feature of born of the same parents is come more preferably compared with the effect of addition hyclone culture.

3. stem cell gene performance analysis

This experiment will be incubated at the adipose stromal stem cell of gained under foregoing four groups different condition of culture respectively, to gather in real time Synthase chain reaction instrument（Real-Time PCR System）Come the performance of the related gene of analyzing undifferentiated stem cell.Will culture After the cell gone out is cleaned with phosphate buffer, collect in 1.5 ml eppendorf, add 1 ml TriZol（10296- 010,Invitrogen）Reagent, is placed five minutes in room temperature, adds 100 μ l BCP（BP. 151,MRC）Solution, with Vortex Mix into after pink solution, room temperature is placed 15 minutes, then 15 minutes are centrifuged at 4 DEG C with 15,000 g.After having centrifuged, Three layers can be divided in eppendorf, lower floor is red color layer, middle a thin layer of white layer, upper strata is hyaline layer, and upper strata is suctioned out It is put into 1.5 milliliters of new centrifuge tubes（eppendorf）In, it is careful to be drawn onto other two layers in suction process.In it is new from 0.5 milliliter of isopropanol is added in heart pipe（isopropanol）, after shaking up, room temperature is placed 30 minutes, afterwards again with 15,000 g Supernatant is extracted out at 4 DEG C, should not be drawn onto agglomerate by centrifugation for 10 minutes（pellet）, add 1 milliliter of 75% alcohol （ethanol）Cleaning, then 10 minutes are centrifuged at 4 DEG C with 15,000 g, take out after alcohol, be air-dried 10 minutes, to contain suppression RNase processed（DEPC）Water back dissolving after i.e. complete RNA extraction.Draw about 10 micrograms（μg）RNA, adds Reverse Transcription group （RT-for-PCR kit, Clontech）, with polymerase chain reaction device（PCR machine）Complete to add after reverse transcription and gather Synthase（GoTaq Green Master Mix, M7122, Promega）Carry out Polymerase Chain Reaction, the setting of its reaction of P Condition is because of different introductions（Primer）Different Tm values and slightly adjust.Analysis related undifferentiated related gene with stem cell, Seem Nanog, SOX2, CXCR4, TERT etc., this experimental analysis Nanog, SOX2, CXCR4, TERT and control group β- Actin genes.Analyze introduction used in each gene as shown in the following chart：

3.1 experimental result

Referring to Fig. 6 B, via result it was found that there is three groups of experiments of addition human serum（10% human serum, 10% people Class serum+adjuvant, 2% human serum+adjuvant）, its stem cell gene（Nanog, SOX2, CXCR4, TERT）Performance it is all bright Show higher than the basal medium plus 10% hyclone group, and it has also been observed that, the basal medium adds 2% mankind's blood Though resetting and adding upper adjuvant group of the invention only 2% human serum, after the adjuvant culture by the addition present invention, it still can such as add Plus 10% as human serum culture, keep the performance of above-mentioned stem cell gene, thus, it can be known that the adjuvant of the addition present invention carry out it is thin Born of the same parents are cultivated, and the usage amount of human serum can be reduced really.

4. the differentiation of adipose stromal stem cell

Reported in literature points out that adipose stromal stem cell, which has, is divided into mesoblastemic ability, seems fat and bone Cell.The same such as experiment two of this experiment after culture under four groups of different condition of culture six days, is induced adipose stromal stem cell Os osseum cell, cartilage cell and fat cell are divided into, to confirm after culture under above-mentioned four groups different condition of culture six days, is It is no that still there is stem cell multifunctional differentiation capability.Induction Analytical Chemical Experiment in the present invention according to generally being made in known document Stem cell induces Differentiation System（Kanda et al., 2011；Song et al., 2010）, because of non-this case demand emphasis Therefore it will not go into details, and its main purpose is to confirm in the present invention after being cultivated under above-mentioned four groups different condition of culture, if still With stem cell multifunctional differentiation capability.

The chemical staining and molecular marker analysis of 4.1 cells

The os osseum cell of differentiation is with alkaline phosphatase（Alkaline phosphatase, ALP）Dyed, the alkalescence Phosphatase is the important indicator of ripe osteoblast differentiation, and its colouring method is carried out according to known staining technique （Yoshimura et al., 2011）, will not be repeated here；It is another to carry out a known Von-kossa dyeing again, to confirm to have The presence of calcium phosphate.The cartilage cell of differentiation is with the blue decoration methods of A Erxin（Alcian blue）To confirm to possess in cartilaginous tissue Proteoglycan（Proteoglycan）Presence（Song et al., 2010）.The fat cell of differentiation is with oil red O stain （Oil red O staining）, to be confirmed whether to have lipid vacuole（Lipid vacuoles）Presence（Kanda et al., 2011）.This another experiment is directed to molecular labeling [the core binding factor that os osseum cell is possessed（cbfa1）, osteocalcin （Osteocalcin, OC）And first collagen type（COL IA1）Deng gene], the molecular labeling that is possessed of cartilage cell it is [soft The poly- glucoprotein of bone（ACAN）, the second collagen type（COL IIA1）Deng gene] and fat cell is fat synthesis related Because of [peroxisome proliferators activated receptor γ（PPARγ）, fatty associated proteins（aP2）] performance, and with β-actin Gene is control group, carries out real time aggregation enzyme chain reaction（Real-Time PCR）To analyze its relative performance amount.Analyze each base Because of used introduction as shown in the following chart：

4.2 experimental result

It is alkaline phosphatase staining result referring to Fig. 7 A, engineer's scale is 500 microns（μm）, adipose stromal stem cell is in preceding State under four groups of different condition of culture, through being induced to differentiate into os osseum cell, after alkaline phosphatase staining, all there is presentation black crystalline Part, that is, represent the presence of alkaline phosphatase, further illustrate this four groups, all adipose stromal stem cell can be made to possess stem cell The ability of differentiation；Continue referring to Fig. 7 B, be Feng Kusa（Von-kossa）Coloration result, engineer's scale is 500 microns（μm）, it is possible to find Foregoing four groups all present black or the calcium phosphate crystal of dark brown, again illustrate this four groups, can all make adipose stromal dry thin Born of the same parents possess the ability of stem cell differentiation；Fig. 7 C, are the molecular marker performance analysis result of os osseum cell, it is possible to find in bone calcium egg again In vain（OC）In relative performance's amount, adjuvant group of the basal medium plus 10% human serum plus the present invention is cultivated with the basis Base adds two groups of adjuvant group of the present invention etc. plus 2% human serum, and the molecular marker performance of its os osseum cell is all trained than the basis Support base to significantly improve plus 10% human serum group with the basal medium plus 10% hyclone group, wherein * P< 0.05, ** P < 0.01, *** P < 0.005；Especially the basal medium adds the adjuvant group of the present invention plus 2% human serum, Its cbfa1, OC and COL IA1 three relative performance amount is even more to add 10% hyclone group apparently higher than the basal medium.By This understands that the adjuvant of the addition present invention carries out adipose stromal stem cell culture plus human serum, can possess stem cell original Differentiation capability, and through being induced to differentiate into the better of os osseum cell.

Fig. 8 A are referred to, are the blue coloration results of A Erxin, engineer's scale is 500 microns（μm）, it is possible to find foregoing four groups are all in Reveal the proteoglycan coloration result of blueness, the especially basal medium plus adjuvant group of 2% human serum plus the present invention Blue protein polysaccharide pigmented section exceed well over other three groups.Continuous ginseng Fig. 8 B, are the molecular labeling performance analysis of cartilage cell, can It was found that adjuvant group and the basal medium of the basal medium plus 10% human serum plus the present invention add 2% human serum Plus adjuvant group of the invention in the isogenic relative performance's amounts of ACAN and COL IIA1, all cultivated apparently higher than the basis Base adds 10% human serum group, wherein * P plus 10% hyclone group with the basal medium< 0.05, ** P < 0.01, *** P < 0.005；The especially basal medium is plus adjuvant group of 2% human serum plus the present invention, and its cartilage is thin The molecular marker performance of born of the same parents is even more to significantly improve many.It follows that the adjuvant of the addition present invention carries out fat plus human serum Fat interstital stem cell culture, can possess the original differentiation capability of stem cell, and through being induced to differentiate into the better of cartilage cell. Join Fig. 8 C again, be oil red O stain result, engineer's scale is 500 microns（μm）, it is possible to find foregoing four groups all show red fat Matter vacuole coloration result.Continuous is that fat synthesis related gene PPAR γ and aP2 relative performance measure referring to Fig. 8 D, wherein * P< 0.05, ** P < 0.01.The basal medium can be found plus adjuvant group of 10% human serum plus the present invention, its PPAR γ and aP2 relative performance's amount adds 10% mankind's blood plus 10% hyclone group compared with the basal medium with the basal medium Clear group is significantly improved.It follows that the adjuvant of the addition present invention carries out adipose stromal stem cell culture plus human serum, it can protect There is the differentiation capability that stem cell is original, and the effect through being induced to differentiate into fat cell is preferable.

5. cell factor array is analyzed to show with Relative gene and analyzed

Three kinds of states in adipose stromal stem cell are tested in this analysis（Before 10% human serum culture, 10% human serum training Support and 10% human serum adds adjuvant culture）Under, take respectively its medium supernatant using it is commercially available include 41 kinds of human cells because Subnumber group analysis set group（Cat #AAH-GF-1, RayBiotech, Norcross, GA）Analyzed, to observe between fat Matter stem cell is under foregoing three kinds of states, the influence to its secrete cytokines and growth factor；Foregoing array analysis set group Therefore it will not go into details for the non-this case demand emphasis of analysis method, and its detailed content can join RayBiotech companies and provide Cat # AAH-GF-1 specification.This another experiment also by the above-mentioned adipose stromal stem cell after culture under four groups of different condition of culture with 3. analysis method for such as testing two is directed to particular growth factor such as insulin-like growth factor（IGF-1）, hepatocyte growth factor Son（HGF）And EGF（EGF）Deng gene, using β-actin genes as control group, real time aggregation enzyme chain lock is carried out anti- Should（Real-Time PCR）To analyze its relative performance amount.Analyze introduction used in each gene as shown in the following chart：

In addition, this experiment also for adipose stromal stem cell in following three condition of culture such as：Basal medium adds 10% Hyclone group, basal medium add 10% hyclone（The low-oxygen environment of 5% partial pressure of oxygen）Group and basal medium are added Under 10% hyclone is supported plus three tissue cultures such as adjuvant group of the present invention, take respectively its medium supernatant carry out human cell because Subnumber group analysis（Cat #AAH-GF-1, RayBiotech, Norcross, GA）, to observe to adipose stromal stem cell point Secrete the influence of cell factor and growth factor.

5.1 experimental result

Ginseng Fig. 9 A figures and Fig. 9 B in the lump, are that cell factor array analysis result is compared with cell factor array analysis result and shown It is intended to, wherein POS represents positive control group, and NEG represents negative control group, * P< 0.05, ** P < 0.01, *** P < 0.005.It can be found by results contrast, adipose stromal stem cell is in after the adjuvant culture of the addition present invention, in conditioned medium Secretion has following cell factor and growth factor：FGF-2、EGF、FGF-4、FGF-6、FGF-7、HB-EGF、HGF、IGFBP-1、 IGFBP-2、IGFBP-3、IGFPB-4、IGFBP-6、IGF-I、IGF-I SR、IGF-II、M-CSF、M-CSF R、PDGF Rα、 PDGF-Rβ、PDAF-AA、PDGF-AB、PDGF-BB、PIGF、SCF、TGF-β3、VEGF、VEGF R2.Wherein 10% shown in Fig. 9 B Before human serum culture with the comparison figure of 10% human serum culture in oblique line to left down block such as：Second-Type fibrocyte Growth factor（bFGF）, EGF（EGF）, platelet derived growth factor（PDGF-AA、AB、BB）And blood vessel endothelium Growth factor（VEGF R3）Deng being that the situation that is absorbed by cell is presented；And in descending the block of oblique line to the right such as in figure：Liver Porcine HGF（HGF）, binding protein of insulin-like growth factor（IGFBP-1、IGFBP-4、IGFBP-6）, para-insulin Growth factor 1（IGF-1）, VEGF（VEGF）Deng, be present the situation into culture medium is secreted by cell.Again Join Fig. 9 C and Fig. 9 D, be the quantization signal that foregoing 10% human serum culture and 10% human serum compare plus adjuvant cultivation results Figure, it is possible to find platelet derived growth factor（PDGF-AA、AB、BB）It is the lasting situation for presenting and being absorbed by cell, and liver Porcine HGF（HGF）, binding protein of insulin-like growth factor（IGFBP-1）And VEGF（VEGF）Then It is not only lasting present and the situation into culture medium is secreted by cell, and through result of 10% human serum plus adjuvant culture Significantly improve.It follows that carrying out adipose stromal stem cell culture plus human serum by the adjuvant of the addition present invention, have The secretion for improving some growth factors such as HGF, IGFBP-1 and VEGF tells on.

Continuous ginseng Fig. 9 E, be adipose stromal stem cell after being cultivated under four groups of different condition of culture, its growth factor I GF-1, HGF and EGF relative performance's amount result, wherein * P< 0.05, ** P < 0.01, *** P < 0.005.Obtained by result Know there are three groups of addition human serum culture（10% human serum, 10% human serum+adjuvant, 2% human serum+adjuvant）Its IGF-1, HGF and EGF relative performance's amount are all significantly improved compared with the basal medium plus 10% hyclone group, especially with this Basal medium is plus relative performance amount highest of 2% human serum plus the adjuvant group of the present invention.It follows that adding this hair Bright adjuvant is carried out after adipose stromal stem cell culture plus human serum, can improve growth factor such as IGF-1, HGF and EGF Relative performance measures.

It is cell factor array analysis result figure and the analysis of above-mentioned cell factor array again also referring to Fig. 9 F and Fig. 9 G In results contrast figure, wherein Fig. 9 G, if showing central clear pie chart sample person in each cell factor block, basis culture is represented Base has this cell factor of secretion plus the adipose stromal stem cell of 10% hyclone group；Similarly, if showing completely black pie chart sample Person, represents the basal medium plus 10% hyclone（The low-oxygen environment of 5% partial pressure of oxygen）The adipose stromal stem cell of group has point Secrete this cell factor；And oblique line pie chart sample person is shown, the basal medium is represented plus 10% hyclone plus the present invention's The adipose stromal stem cell of adjuvant group has this cell factor of secretion or growth factor.It can be found by Fig. 9 G, adipose stromal stem cell Under the conditions of being incubated at, i.e., it can secrete bFGF, EGF, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-6, IGF-II, M- Cell factor or the growth factors such as CSF, M-CSF R, NT-4, PDGF R β, PIGF, TGF-β 3, VEGF.Though it is worth noting that, The basal medium adds 10% hyclone（The low-oxygen environment of 5% partial pressure of oxygen）Group and basal medium add 10% tire ox blood The cytohormone species of adipose stromal stem cell secretion can be increased more by resetting and adding upper adjuvant group of the invention, but the basis is trained Support base plus 10% hyclone plus the increased species of adjuvant group institute of the present invention and measure it is all more, such as β-NGF, FGF-4, FGF-6、FGF-7、IGFBP-3、IGFBP-4、IGF-I、IGF-I SR、GCSF、GDNF、GM-CSF、PDGF-AA、PDGF-AB、 Cell factor or the growth factors such as PDGF-BB, SCF, VEGF R2, VEGF R3, VEGF-D.It follows that the addition present invention The culture that adjuvant carries out adipose stromal stem cell can more increase secreted by adipose stromal stem cell compared with low-oxygen environment culture The species and amount of cell factor or growth factor.

Summary result illustrates that adipose stromal stem cell is whether incubated at the adjuvant of the addition present invention plus the mankind The basal medium of serum or only add the present invention adjuvant under the basal medium containing 10% hyclone, not only all may be used The effect of rapid multiplication is reached, while the growth factor largely secreted during the culture of adipose stromal stem cell can be also obtained, its In again be incubated at addition the present invention adjuvant plus human serum basal medium under the conditions of for optimal culture condition；Whereby Using as being subsequently used for preparing wound healing promoting medicine and purposes for preparing wound healing promoting medicine, or as preparing skin Skin skin care products and the purposes for preparing skin care product.

Experiment three：Adipose stromal stem cell microcarrier culture

The Human Meta stem cell cultivated in vitro is more with sticking type（Anchorage-dependent cells）Culture Mode is carried out, so the cell that a large amount of and uniform quality is produced using a kind of culture systems for meeting sticking type is cured for regeneration The application learned with organizational project is above desirable.This experiment utilizes stirring-type microcarrier cultivation reactor（Spinner Flasks, Bellco Glass, Inc., Vineland, NJ, USA ）Carry out adipose stromal stem cell culture.Inoculating cell enters reaction Before device, first by foregoing cultivation reactor interior surface with silica gel（Sigmacote, Sigma, St. Louis, MO, USA）Place Managed；And used microcarrier（CultiSpher-G；HyClone, Logan, UT, USA）Then according on operation manual Step sequentially weighing, add hydration, and using Sterilization Kettle 15 minute 121 DEG C handle；In be mixed into before cell first remove it is unnecessary Phosphate buffer solution, adds the balance about 24 hours that the nutrient solution of cell to be cultivated makes.By adipose stromal stem cell Add containing in pre-balance nutrient solution and microcarrier altogether about 50 milliliters of stirring-type cultivation reactor, initially with the side of batch (-type) Formula opens external-added electromagnetic stirring system, and it is small to rotate 2 with the frequency that 25 r.p.m rests 10 to 20 minutes after 30 minutes every time When；In after foregoing 2 hours with 25 r.p.m rotating speed start culture, and every 3 days change once accompany nutrient solution, percentage of more exchanging treaties every time 50 to 70 nutrient solution, and first stop stir about 5 minutes before changing, cell and microcarrier is dropped down onto reactor bottom Portion.Wherein microcarrier incubation is cultivated 7 days in the environment of 37 DEG C, the carbon dioxide partial pressure of humidity 95% and 5%, and foregoing culture Base adds 10% human serum, 2 millimolar concentrations comprising IMDM（mM）The adjuvant institute group of left-handed bran amic acid and the present invention Into；And foregoing adjuvant includes 10 ng/mL FGF-2,2 millimolar concentration ACETYLCYSTEINEs and 0.2 mM Concentration ascoltin -2- phosphate.

It is to observe cell in grown on microcarriers and distribution scenario, 1 milliliter of cell liquid containing microcarrier is taken out daily, and Centrifugation is removed culture medium and cleaned once with phosphate buffer solution；It is continuous to fix 10 in room temperature with 10 formalin fixers After minute, remove fixer and cleaned with phosphate buffer solution；Again with 5 mg/ml green fluorescence sodium Diacetates（FDA）With 2 Mg/ml propidium iodide（PI）Dye living cells and dead cell, in after room temperature lucifuge 5 minutes, stain are removed and with phosphorus respectively Acid buffering solution is cleaned three times, then cell and microcarrier are evenly distributed on slide, uses fluorescence microscope.

Experimental result

Figure 10 A and Figure 10 B are referred to, is that adipose stromal stem cell is incubated at microcarrier 7 days, in image under light microscope And the image under fluorescence microscope, wherein green fluorescence represents living cells, and red fluorescence represents dead cell.By Figure 10 B shadow As observation learns that adipose stromal stem cell is easier to be attached at microcarrier, and forms micro-assembly robot state.Understand whereby, by between fat Matter stem cell is incubated at a biocompatible material, such as microcarrier, can form micro-assembly robot state, to be used as subsequent regeneration medical science Or the purposes of organizational project.

Summary experimental result is understood, Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention is added and included In the culture medium of Human Meta stem cell, in normal oxygen environment（About 21% partial pressure of oxygen）, can be as indicated in low-oxygen environment after culture（About 5% Partial pressure of oxygen）Occur as lower culture cell quickly divide, the cell cycle synthesis phase（S phase）Ratio improves, reduces aging and carry The phenomenons such as differentiated potential, and arrange in pairs or groups using human serum substitution hyclone cultivated when, its hyperplasia speed more preferably, And the growth factor amount of cell secretion is improved, species increase, the present invention not only can quickly and be efficiently expanded Human Meta Stem cell, and can still possess the feature of stem cell multifunctional, rapid amplifying and acquisition to reach Human Meta stem cell Effect of growth factor.

Above-described embodiment and schema and non-limiting product form of the invention and style, any art it is common Appropriate change or modification that technical staff is done to it, all should be regarded as not departing from the patent category of the present invention.

Claims

1. a kind of Human Meta's stem cell in vitro rapid multiplication culture medium, it is characterised in that：Including Human Meta's stem cell in vitro Rapid multiplication adjuvant and human serum, the external rapid multiplication adjuvant of aforementioned human's interstital stem cell include a long-acting type ascorbic acid The combination of derivative and an ACETYLCYSTEINE and a Second-Type fiber mother cell growth factor.

2. Human Meta's stem cell in vitro rapid multiplication culture medium as claimed in claim 1, it is characterised in that：Human Meta does Cell is selected from：Adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord mesenchymal stem cells constitute one of group.

3. Human Meta's stem cell in vitro rapid multiplication culture medium as claimed in claim 1, it is characterised in that：Long-acting type is anti-bad Hematic acid derivative is a L-AA -2- phosphate.

4. Human Meta's stem cell in vitro rapid multiplication culture medium as claimed in claim 1, it is characterised in that：Second fiber type Mother cell growth factor concentration is 1 ng/ml to 20 ngs/ml.

5. Human Meta's stem cell in vitro rapid multiplication culture medium as claimed in claim 1, it is characterised in that：Between aforementioned human Matter stem cell in vitro rapid multiplication adjuvant is by the cell within a cell cyclin dependent kinase for suppressing aforementioned human's interstital stem cell Kinase inhibitor：The performance of p21 and p27 albumen, to improve cell cycle protein dependent kinase -2, cyclin according to Rely the performance of property kinases -4 and cell division cycle protein.

6. a kind of method of external rapid amplifying Human Meta stem cell, it is characterised in that：Including claim 1 to 5 is any Described adjuvant is added in the culture medium comprising human serum and a Human Meta stem cell, dry thin to expand aforementioned human's interstitial Born of the same parents, and obtain the undifferentiated Human Meta stem cell of essence.

7. the method for external rapid amplifying Human Meta stem cell as claimed in claim 6, it is characterised in that：Aforementioned human's blood Clear concentration expressed in percentage by volume is percent 2 to percent 10.

8. the method for external rapid amplifying Human Meta stem cell as claimed in claims 6 or 7, it is characterised in that：Further include Human Meta's stem cell step of the foregoing amplification of one freezen protective, for further using.

9. a kind of cell bank, it is characterised in that：Comprising by external rapid amplifying Human Meta stem cell as claimed in claim 8 Method institute freezen protective amplification Human Meta stem cell.

10. the method for external rapid amplifying Human Meta stem cell as claimed in claims 6 or 7, it is characterised in that：Further An extraction step is performed to obtain the cell extraction thing of a Human Meta stem cell.

11. the method for external rapid amplifying Human Meta stem cell as claimed in claims 6 or 7, it is characterised in that：Further Perform one and induce differentiation step, to obtain a cell broken up from aforementioned human's interstital stem cell.

12. a kind of medical composition, it is characterised in that：Comprising by external rapid amplifying Human Meta as claimed in claim 11 The undifferentiated Human Meta stem cell of essence acquired in the method for stem cell one is broken up from aforementioned human's interstital stem cell Cell.

13. the method for external rapid amplifying Human Meta stem cell as claimed in claim 11, it is characterised in that：Human Meta One of the cell that stem cell is broken up, including osteocyte, fat cell or cartilage cell.

14. a kind of method of external rapid amplifying Human Meta stem cell to obtain growth factor, it is characterised in that：Through right It is required that after 6 or 7 method culture, to obtain a growth factor in prior culture media.

15. external method of the rapid amplifying Human Meta stem cell to obtain growth factor as claimed in claim 14, it is special Levy and be：Growth factor includes：FGF-2、EGF、FGF-4、FGF-6、FGF-7、HB-EGF、HGF、IGFBP-1、IGFBP-2、 IGFBP-3、IGFPB-4、IGFBP-6、IGF-I、IGF-I SR、IGF-II、M-CSF、M-CSF R、PDGF Rα、PDGF-Rβ、 PDAF-AA、PDGF-AB、PDGF-BB、PIGF、SCF、TGF-β3、VEGF、VEGF R2。

16. medical composition as claimed in claim 12, it is characterised in that：Further combined with a biocompatible material with should For regenerative medicine or organizational project.