CN104450608A

CN104450608A - Human mesenchymal stem cell proliferation promotion adjuvant, human mesenchymal stem cell amplification, pharmaceutical composition, growth factor and use

Info

Publication number: CN104450608A
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: 2015-03-25
Anticipated expiration: 2033-09-12
Also published as: CN104450608B

Abstract

The invention relates to a human mesenchymal stem cell in-vitro rapid proliferation adjuvant used to solve the problem of poor cell amplification efficiency of human mesenchymal stem cell culture. The human mesenchymal stem cell culture adjuvant comprises at least an antioxidant and a second type fibroblast growth factor (FGF-2). Thereby, after the adjuvant is added into a medium containing human mesenchymal stem cells for culture in normoxic environment (with the oxygen partial pressure being about 21%), phenomena such as rapid cell division, increase of cell cycle period synthesis phase proportion, reduction of aging, improvement of the differentiation potential, and the like, the human mesenchymal stem cell in-vitro rapid proliferation adjuvant can not only rapidly expand the human mesenchymal stem cells and obtain the growth factor, can still keep the more functional characteristic of the stem cells, and can be used for the purposes of human mesenchymal stem cell culture and amplification.

Description

The adjuvant, amplifying human interstital stem cell method, medical composition, somatomedin and uses thereof of short Human Meta's stem cell proliferation

Technical field

The present invention has about a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant, espespecially a kind of adjuvant comprising antioxidant and somatomedin to make an addition in the substratum of Human Meta stem cell, in order to make Human Meta stem cell can rapid multiplication obtain somatomedin in first generation or succeeding transfer culture.

Background technology

Press, current scientific circles have descended some basic definitions for stem cell and have had characteristic: refer to that a group has cell self-renewal (Self-renewal), hyperplasia (Proliferation) ability, energy long term maintenance is at the cell not breaking up (Undifferentiation) state simultaneously, and can be divided into multi-differentiation (multi-differentation) characteristic of the cell mass of different pedigree and the tissue of specific function after being subject to suitably induction stimulation.

The difference of foundation source of human stem cell and differentiation potential thereof, can be categorized as:

1, totipotency stem cell (Totipotent stem cell): it has the ability that can develop into complete independent biochemical body, if zygote (Zygote) or fetal development are to the cell mass of about eight cell stage.

2, multi-functional stem cell (Pluripotent stem cell): zygote (Zygote) fertilization is after about 4 days, fully functioning stem cell starts differentiation and enters the blastaea body phase, blastaea body can be divided into two portions: outer trophocyte (outer layer of cells) and inner cell mass (inner cell mass), in embryo development procedure, outer trophocyte can form placenta and fetus is attached to sustentacular tissue required in uterus, inner cell mass then can form ectoderm, mesoderm and entoderm, each germinal layer is down divided into various different system and organ again.Although the ability of each part of the tangible adult body of inner cell mass, if but separately inner cell mass is put into the intrauterine of human mature's women's health, in default of outer trophocyte, the external supporting system needed for placenta and fetal growth and environment cannot be formed, so cannot develop into a complete individuals, therefore its multifunctionality still has part restriction.

3, multipotency stem cell (Multipotent stem cell): the stem cell being current most study, down break up again from above-mentioned multi-functional stem cell, specialization can become the stem cell of particular organization, as hemopoietic stem cell (hematopoietic stem cell), mesenchymal stem cell (mesenchymal stem cells) etc., wherein, hemopoietic stem cell comes from peripheral blood, Cord blood, marrow etc., various blood cell and lymph can be divided into, and mesenchymal stem cell can come from fat, periosteum (periosteum), membrana articulata (synovial membrane), the stroma (mesenchymal tissue) of marrow and some internal organs, as placenta etc.For hemopoietic stem cell, hemopoietic stem cell can down be divided into lymphoid stem cell and bone marrow stem cell again, and wherein lymphoid stem cell can be divided into lymph corpuscle, killer cell line etc., and bone marrow stem cell can be divided into red blood corpuscle, white cell and thrombocyte etc.; All can find multipotency stem cell in adult and child's body, by the ability that stem cell regenerates voluntarily, multipotency stem cell mainly plays to be supplemented and upgrades normal used up cell in our body.The multipotency stem cell be separated at present comprises brain, retina, marrow, liver, skeletal muscle, skin, umbilical cord, Cord blood and fatty tissue etc.

4, single-minded/single type stem cell (Unipotent stem cell): general reference is had the ability to be divided into the stem cell of specific a certain tissue or is referred to as precursor cell (progenitor cell), this kind of cell is prevalent in the tissue of each several part, the stem cell be the most easily found, as liver precursor cell, neural precursor cell etc.

Mesenchymal stem cell is academicly being defined as the micro-parent cell of fibre (colony-forming unit of fibroblast-CFU-Fs) that can form settlement unit for the first time.In the process of cultivating, plastic culture dish surface can be attached to by individual layer, the micro-parent cell of the similar fibre of form, present fusiform, settlement can be formed by rapid multiplication in vitro, and possess the potential being divided into osteoblast (osteoblast), adipocyte (adipocyte), chondrocyte (chondrocyte).Also research is had to point out can be divided into (Minguell et al., 2001) such as liver cell, myocardial cell, neurocyte, islet cellss in recent years.

The source of mesenchymal stem cell can be separated from the many different tissues of human body, such as directly excised by operation or be the abundant source of a stem cell via the fatty tissue that liposuction obtains, separable go out fat stem cell (Adipose tissue-derived stem cells, ADSCs), its advantage had has: acquisition mode invasive is low, little to human injury and the amount once obtained is more, can in external hyperplasia cultivation etc.; Add that fat stem cell also has the potentiality (Zuk et al., 2002) that can be divided into os osseum, cartilage, muscle and adipocyte, therefore, fat stem cell is considered to one of stem cell of most researchdevelopment potentiality.

The research and apply of stem cell, no matter on basic medical research or on clinical treatment, all need enough cell count, and must cultivate under a suitable environment, comprise the stimulations such as suitable microenvironment, somatomedin, to avoid stem cell at cultivation, premature aging in hyperplastic process, lose activity or be divided into other cells.But, the difference due to cell separation technology, growth medium and culture condition cause stem cell in hyperplasia from differentiation capability has significantly different (Pittenger et al., 2008).In addition, many reported in literature all unanimously notice that interstital stem cell is cultivating aging Problem of trend (Bonab et al., 2006 in hyperplastic process; Shibata et al., 2007; Wagner et al., 2008).Therefore, in stem cell proliferation process run into cell performance and aging on difference, urgently may hinder the clinical application of interstital stem cell.Therefore, how can effectively and fast the stem cell obtained not easily cultivated and amplify number, can keep again undifferentiated state simultaneously and reduce aging phenomenon, and still having multi-functional characteristic, be that scientists does one's utmost pursuit always.

And in recent years, research report is also pointed out, cultivated in the conditioned medium of bone marrow interstital stem cell (BMMSC), be found to have and the paracrine factor of wound healing can be impelled in a large number to exist, such as: vascular endothelial growth factor (VEGF), insulin-like growth factor 1(IGF-I), Urogastron (EGF), keratinocyte growth factor (KGF), angiogenin (angiopoietin-1), stromal-derived factor (stromal-derived factor-1), scavenger cell inflammatory albumen (macrophage inflammatory protein-1 α, macrophage inflammatory protein-1 β), with (Martin et al. such as erythropoietin (erythropoietin), 1997).And adipose stromal stem cell (ADSC) has been proved with bone marrow interstital stem cell, Cord blood interstital stem cell, Periosteal Mesenchymal Stem Cells, synovial membrane interstital stem cell and muscle interstital stem cell on gene expression and phenotype, there is no significant difference (Sheehy et al., 2012; Hung et al., 2012; Hung et al., 2007).In view of adipose stromal stem cell has better separation and external hyperplasia condition compared to bone marrow interstital stem cell, be very hopefully at present applied to wound repair and regeneration.Known at present, in conditioned medium, the following somatomedin of adipose stromal stem cell secretion, as: Second-Type fibroblast growth factor (bFGF), keratinocyte (KGF), somatomedin transition (TGF-β), pHGF (HGF), vascular endothelial growth factor (VEGF) etc., and these somatomedins all may be relevant with wound healing.Therefore how to make interstital stem cell rapid multiplication and produce the raised growth factor, in fact for one is worth the problem of making great efforts research.

Just like TaiWan, China patent No. 201331366 one " external serum-free adult stem cell amplification culture technology ", a kind of method of external serum-free adult stem cell amplification culture is provided, its method utilizes adds first culture and the succeeding transfer culture that autologous growth factor (PRGF) carries out human adult stem cell in serum-free stem cell medium, human adult stem cell cultivated in this approach is after succeeding transfer culture repeatedly, and human adult stem cell still remains on the undifferentiated state of essence.Only, the stem cell population obtained through its cultural method just reaches about 55 at succeeding transfer culture to the third generation (P3) is front, 000 cell count/every square centimeter, to obtain more stem cell population, the algebraically that may improve cultivated days and succeeding transfer culture satisfies the demands, relative, also must bear repeatedly the risk that succeeding transfer culture may be polluted.

Again just like TaiWan, China patent No. 201118172 one " low density merges the method that hypoxemia cultivates amplification interstital stem cell ", the lower fast and efficiently amplifying human interstital stem cell that the method can not affect cell proliferation and stem cell properties, being included in external or body increases propagation, reduces aging and increase differentiation potential.Only mostly be because most of research unit is used for the incubator of culturing cell partial pressure of carbon dioxide and humidity adjustment function are only provided, if need cultivate under low-oxygen environment, then need zero type to purchase the incubator of tool oxygen partial pressure adjustment function; Be one to bear greatly in fact for the research unit that reasearch funds are very insufficient.

For another No. 201231087, TaiWan, China patent " a kind of skin care products processing procedure containing multiple somatomedin ", first take out healthy fat; Again the solvent of a predetermined volumes is mixed into fatty tissue, to clean fatty tissue; Add the fatty tissue of predetermined volumes by the reagent of predetermined dose, and carry out centrifugation; The fatty tissue produced after being separated then is mixed into the ferment of predetermined dose in a centrifuge tube, and shakes this centrifuge tube; The throw out of generation is carried out cell cultures to produce somatocyte; And after obtaining this somatocyte, be rich in the somatomedin (as: VEGF, HGF, b-FGF, TGF, IGF) of emiocytosis through the cell culture fluid cultivated acquired by aforesaid way, to complete efficient skin care products raw material.Precisely because the cultural method disclosed somatocyte is inserted one to comprise in the basic medium DMEM (Dulbecco`s Modified Eagle Medium) of percent 10 foetal calf serums (10%FBS) and cultivate, this somatocyte proliferation rate is slow, somatomedin secreted by it is relatively less, to obtain a large amount of somatomedins, the algebraically that just must improve cultivated days and succeeding transfer culture is reached, therefore, the risk also had because repeatedly succeeding transfer culture may be polluted exists.

Comprehensively above-mentioned, in order to make external mankind's interstital stem cell can rapid multiplication, and still can possess the feature of stem cell multifunctional, and the somatomedin of Human Meta's stem cell secretion can be obtained in a large number simultaneously, we need badly and want a Human Meta stem cell to cultivate adjuvant and cultural method, with amplifying cells number obtain the raised growth factor efficiently again fast.

Summary of the invention

The present inventor is because existing cultivation for Human Meta stem cell is studied with the disappearance of amplifying cells number and the somatomedin that obtains Human Meta stem cell secretion again, there is provided a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant and cultural method and a kind of external rapid amplifying Human Meta stem cell thereof to obtain method of somatomedin and uses thereof, to reaching more fast and amplifying human interstital stem cell obtain the object of somatomedin efficiently.

For reaching above-mentioned purpose, solution of the present invention is:

The invention provides a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant, it comprises at least one antioxidant 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: adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord mesenchymal stem cells form one of group.

Further, aforementioned antioxidant comprises the combination of a long-acting type ascorbic acid derivates (Long-acting ascorbic acid derivative) and an ACETYLCYSTEINE (N-acetyl-L-cysteine, NAC).

Further, aforementioned long-acting type ascorbic acid derivates is an ascoltin-2-phosphoric acid salt (L-ascorbic acid-2-phosphate, AsA2P).

Further, aforementioned Second-Type fiber mother cell growth factor working concentration is that 1 ng/ml (ng/mL) is to 20 ngs/ml (ng/mL).

Further, it is by the cell within a cell cyclin-dependent kinase inhibition suppressing aforementioned human interstital stem cell: the performance of p21 and p27 albumen is to improve cell cycle protein dependent kinase-2(CDK-2), cell cycle protein dependent kinase-4(CDK-4) and the performance of cell division cycle protein (CDC2).

The invention provides a kind of method of external rapid amplifying Human Meta stem cell, comprise and aforesaid adjuvant is added one comprise in the substratum of Human Meta stem cell, with amplifying human interstital stem cell, and obtain the undifferentiated Human Meta stem cell of essence.

The present invention reoffers a kind of method of external rapid amplifying Human Meta stem cell, comprise in the substratum aforesaid adjuvant being added and comprises a serum additive and a Human Meta stem cell, with the aforementioned human's interstital stem cell that increases, and obtain the undifferentiated Human Meta stem cell of essence.

Further, aforementioned serum additive is serum human or foetal calf serum, and the concentration expressed in percentage by volume used is 2 to percent 10 percent.

Further, the method for foregoing in vitro rapid amplifying Human Meta stem cell more comprises the Human Meta stem cell step of the aforementioned amplification of a freezen protective, for further using.

Further, utilize 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 comprises execution one extraction step to obtain the cell extraction thing of a Human Meta stem cell.

Further, the method for foregoing in vitro rapid amplifying Human Meta stem cell more comprises the differentiation-inducing step of execution one, to obtain a cell broken up from aforementioned human's interstital stem cell.

Further, the aforementioned cell broken up from Human Meta stem cell, comprises osteocyte, one of adipocyte or chondrocyte.

The present invention separately provides a kind of medical composition, its to comprise by the method for foregoing in vitro rapid amplifying Human Meta stem cell obtain the undifferentiated Human Meta stem cell of essence or a cell broken up from aforementioned human's interstital stem cell.

Further, aforementioned medical composition in conjunction with a biocompatible material to be applied to regenerative medicine or organizational project.

The present invention reoffers a kind of external rapid amplifying Human Meta stem cell to obtain the method for somatomedin, after the method for foregoing in vitro rapid amplifying Human Meta stem cell is cultivated, to obtain a somatomedin in prior culture media.

Further, aforementioned growth factors comprises: 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-R β, PDAF-AA, PDGF-AB, PDGF-BB, PIGF, SCF, TGF-β 3, VEGF, VEGF R2.

The invention provides a kind of somatomedin, obtained with the method obtaining somatomedin according to foregoing in vitro rapid amplifying Human Meta stem cell.

The invention provides a kind of medical composition, include the somatomedin that foregoing in vitro rapid amplifying Human Meta stem cell obtains with the method obtaining somatomedin.

The invention provides a kind of including if aforementioned growth factors is in the purposes of the conglutinant medicine of preparation.

The invention provides a kind of including if aforementioned growth factors is as the purposes of skin care product.

Effect of the present invention is:

One, cell rapid multiplication: by interpolation Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention in substratum, Human Meta's stem cell body is made to occur that synthesis phase cell cycle (S phase) ratio improves when first generation or succeeding transfer culture, and impel the phenomenon of the quick division and proliferation of cell, and still can possess the potential of stem cell multifunctional differentiation.

Two, cell senescence is reduced: by interpolation Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention in substratum, aforementioned human's interstital stem cell body telomerase reverse transcription ferment (Telomerase Reverse Transcriptase can be made, TERT) improve, the time that cell telomere (Telomere) shortens can be extended, to slow down cell senescence and to extend life-span of cell.

Three, the quick obtaining raised growth factor: by interpolation Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention in substratum, more knownly can being incubated at one, to comprise concentration expressed in percentage by volume be in the substratum of percent 10% foetal calf serums, obtains somatomedin [as IGF-1 (IGF-1), pHGF (HGF) and Urogastron (the EGF)] content of more than at least 2 to 3 times.

Four, reduce pathogeny to pollute: Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention, when serum human of arranging in pairs or groups is cultivated, can be avoided having the risk of allosome or xenogeneic pathogeny cross staining because using the serum of allosome or xenogenesis to carry out cultivating.

Accompanying drawing explanation

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

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

Fig. 2 B illustrates that in the present invention, fat stem cell is after different condition is cultivated, and the performance for the albumen of responsible cell cycle regulation affects;

Fig. 3 be in the present invention fat stem cell through different condition cultivate after, for the impact of cell relative telomere length;

Fig. 4 A be in the present invention fat stem cell through different condition cultivate after, the graph of a relation of cultivated days and total cellular score;

Fig. 4 B is that in the present invention, fat stem cell is after different condition is cultivated, and the performance for the albumen of responsible cell cycle regulation affects;

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

Fig. 5 B-Fig. 5 D be in the present invention fat stem cell through different condition cultivate after, cell cultures number of days and cell quantity graph of a relation;

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

Fig. 6 B be in the present invention fat stem cell through different condition cultivate after, its stem cell gene relative performance analysis chart;

Fig. 7 A-Fig. 7 B be in the present invention fat stem cell after different condition is cultivated and is induced to differentiate into os osseum cell, through chemical staining result figure;

Fig. 7 C be in the present invention fat stem cell after different condition is cultivated and is induced to differentiate into os osseum cell, its os osseum cellular elements mark relative performance analysis chart;

Fig. 8 A be in the present invention fat stem cell after different condition is cultivated and is induced to differentiate into chondrocyte, through chemical staining result figure;

Fig. 8 B be in the present invention fat stem cell after different condition is cultivated and is induced to differentiate into chondrocyte, its chondrocyte's molecular marker relative performance analysis chart;

Fig. 8 C be in the present invention fat stem cell after different condition is cultivated and is induced to differentiate into adipocyte, through chemical staining result figure;

Fig. 8 D be in the present invention fat stem cell after different condition is cultivated and is induced to differentiate into adipocyte, its adipocyte molecular marker relative performance analysis chart;

Fig. 9 A be in the present invention fat stem cell through different condition cultivate after, cytokine array analysis chart;

Fig. 9 B is the cytokine array analytical results comparison diagram of Fig. 9 A in the present invention;

Fig. 9 C-Fig. 9 D is relative performance's quantitative analysis figure of cytokine in Fig. 9 B;

Fig. 9 E be in the present invention fat stem cell through different condition cultivate after, relative performance's analysis chart of cytokine in its cell;

Fig. 9 F be in the present invention fat stem cell through different condition cultivate after, cytokine array analysis chart;

Fig. 9 G is the cytokine array analytical results comparison diagram of Fig. 9 F in the present invention;

Figure 10 A is the opticmicroscope striograph that in the present invention, adipose stromal stem cell is incubated at a microcarrier;

Figure 10 B is the fluoroscopic image figure of Figure 10 A in the present invention.

Embodiment

In order to above and other object of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and coordinate appended diagram, be described in detail below:

The invention provides a kind of Human Meta's stem cell in vitro rapid multiplication adjuvant, it comprises at least one antioxidant and a Second-Type fiber mother cell growth factor (FGF-2); Wherein aforementioned antioxidant comprises the combination of a long-acting type ascorbic acid derivates and an ACETYLCYSTEINE (NAC).It is preferred that this long-acting type ascorbic acid derivates is a L-AA-2-phosphoric acid salt (Asc 2-P), and Second-Type fiber mother cell growth factor working concentration is 1 to 20 ng/ml.

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 mesenchymal stem cells form one of group.

Preferably, Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention is by the cell within a cell cyclin-dependent kinase inhibition suppressing aforementioned human interstital stem cell: the performance of p21 and p27 albumen is to improve cell cycle protein dependent kinase-2(CDK-2), cell cycle protein dependent kinase-4(CDK-4) and the performance of cell division cycle protein (CDC2), and then the ratio promoting the cell cycle to enter synthesis phase (S phase) improves, thus make the quick division and proliferation of cell.

The present invention provides a kind of method of external rapid amplifying Human Meta stem cell simultaneously, comprise and aforesaid adjuvant added one and comprise in the substratum of Human Meta stem cell and cultivate, with amplifying human interstital stem cell, and obtain the undifferentiated Human Meta stem cell of essence.

It is preferred that aforementioned serum additive is serum human or foetal calf serum, the concentration expressed in percentage by volume used is 2 to percent 10 percent.

It is preferred that the method for foregoing in vitro rapid amplifying Human Meta stem cell comprises execution one extraction step further 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 more comprises the Human Meta stem cell step of the aforementioned amplification of a freezen protective, for further using.

Preferably; " freezen protective " as used herein one word typically refer to method cell being added cryoprotectant and preserve as being cooled to subzero temperature after methyl-sulphoxide (DMSO) or glycerine, be such as negative 80 DEG C or negative 196 DEG C (boiling point of liquid nitrogen).Freezen protective can as know the personage of this skill the method for carrying out and flow process, only therefore it will not go into details (consults HummaPress company in 1997 and publish the 2nd edition Pollard for non-invention demand emphasis, J.W. flow process is cultivated with basal cell that Walker, J.M.. show; Within 2000, Wiley-Liss company publishes the 4th edition Freshney, R.I., show the cultivation of zooblast).

The invention provides a kind of cell bank, it comprises the Human Meta stem cell by the amplification of the method institute freezen protective of foregoing in vitro rapid amplifying Human Meta stem cell.

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

It is preferred that the aforementioned cell broken up from Human Meta stem cell, comprise osteocyte, one of adipocyte or chondrocyte.

The present invention reoffers a kind of medical composition, its to comprise by the method for foregoing in vitro rapid amplifying Human Meta stem cell obtain the undifferentiated Human Meta stem cell of essence or a cell broken up from aforementioned human's interstital stem cell.

It is preferred that this medical composition comprises aforementioned human's interstital stem cell, from its cell broken up, cell secreta or cell extraction thing one or a combination set of and an acceptable carrier/excipient in suitable treatment.Wherein, cell secreta in certain embodiments can in cell culture medium purifying and concentrated after obtain.

The present invention reoffers a kind of external rapid amplifying Human Meta stem cell to obtain the method for somatomedin, after the method cultivation of foregoing in vitro rapid amplifying Human Meta stem cell, cell is while rapid multiplication, also secrete various kinds of cell somatomedin in substratum, aforementioned growth factors can be obtained in prior culture media whereby.Wherein, aforementioned growth factors comprises: 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-R β, PDAF-AA, PDGF-AB, PDGF-BB, PIGF, SCF, TGF-β 3, VEGF, VEGF R2.

The invention provides a kind of medical composition, include foregoing in vitro rapid amplifying Human Meta stem cell to obtain the somatomedin that method obtains of somatomedin, can be used for but not limit treatment skin wound.

The invention provides a kind of skin care product including aforementioned growth factors, in order to repair skin sunburn or delaying skin cell senescence.

Alleged " Human Meta stem cell " word of the present invention refers to the cell that any Human Meta of being obtained from organize and has the unlimited ability that upgrades voluntarily and can be divided into various kinds of cell or organizational form, forms one of group such as but not limited to adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord mesenchymal stem cells, Cord blood interstital stem cell, Periosteal Mesenchymal Stem Cells, synovial membrane interstital stem cell and muscle interstital stem cell.Embodiments of the invention give demonstration with the embodiment of human adipose's interstital stem cell and illustrate, but the present invention not limited by following embodiment.

Experiment one: adjuvant of the present invention cultivates the impact of cell growth for different mankind's interstital stem cell under normal oxygen and low-oxygen environment.

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

This experiment is ratified to carry out by the inside examination board (IRB100-102) of Buddhism Tzu Chi general hospital polyclinic, respectively with human adipose tissue, umbilical cord and myeloid tissue for stem cell obtains source, only herein the separation method of Human Meta stem cell be known techniques and non-this case demand emphasis therefore it will not go into details, main purpose all acquisition Human Meta stem cell to carry out first culture; The cell culture condition of this experiment is divided into three groups: basic medium adds in normal oxygen environment cultivation group, basic medium, FGF-2 adds in low-oxygen environment cultivation group and basic medium, FGF-2 adds that adjuvant of the present invention is in normal oxygen environment cultivation group; Under wherein alleged normal oxygen environment refers to about percent 21 oxygen partial pressure, and under low-oxygen environment refers to 5 oxygen partial pressure of about percentage; This basic medium is IMDM(Iscove`s modified Dulbecco`s medium, GIBCO-Invitrogen) 10% foetal calf serum (FBS is added again, MSC-Qualified, GIBCO-Invitrogen) and the left-handed bran amido acid (L-glutamine, GIBCO-Invitrogen) of 2 millimolar concentrations (mM) formed; And the concentration that aforementioned growth factors FGF-2 uses is 10 ng/mL(R & D Systems); and aforementioned adjuvant of the present invention comprises 2 millimolar concentrations (mM) ACETYLCYSTEINE (NAC; and 0.2 millimolar concentration (mM) ascoltin-2-phosphoric acid salt (AsA2P, Sigma) Sigma).Often organize stem cell with 3000 every square centimeter cell (3000 cells/cm ²) cell density be incubated at (6-well plates in 6 porocyte culture plates, Becton Dickinson), and all cells is all incubated at 37 degree Celsius, incubator (Forma Series II Model 3110 under percent 5 partials pressure of carbon dioxide and percent 95 humidity environments, Thermo), and every 3 days change a subculture; The culture experiment of another hypoxemia culture environment is then carry out in another incubator (MCO-18M, Sanyo).To observe in 7 days different mankind's interstital stem cell whereby under different culture condition, on the impact of hyperplasia.

1.1 cell density analyses

By each group of stem cell with phosphate buffer soln (PBS) cleaned once after, carefully remove with cell spatula again after acting on 5 minutes with trypsin-EDTA (Trypsin-EDTA) solution in 37 degree Celsius and be not applied cell completely, add equal proportion and contain in the substratum of foetal calf serum and tryptic ferment action activity.All cells number all calculates with cell counter (Vi-CELL AS, Beckman Coulter).The cell of survival is with 0.4% trypan blue (Trypan-blue, GIBCO-Invitrogen) come to distinguish with dead cell, during calculating, judge that the parameter setting of the interstital stem cell of work is as 100 images, Size 10 – 30 microns, 75% on-the-spot brightness, 5% field regions.The presenting of result is often organized experimental data and measures three repetitions, represents it with mean value ± standard deviation.

1.2 experimental result

Above experimental data tests (t-test) statistical study through Microsoft Excel Software t, with p < 0.05 for conspicuous level, and result quantities is turned to statistical graph.First, see Figure 1A, for the growth situation of fat stem cell under above-mentioned 3 kinds of different culture condition, wherein basic medium adds with basic medium, FGF-2 adds that FGF-2 compares in low-oxygen environment cultivation group two groups in normal oxygen environment cultivation group, find that stem cell hyperplasia under low-oxygen environment is comparatively quick, especially the 3rd day time, there were significant differences, and within the 4th day, start obvious especially, so proliferation rate more normal oxygen environment under low-oxygen environment of display adipose stromal stem cell gets off good; Another basic medium adds in low-oxygen environment cultivation group and basic medium, FGF-2 adds that adjuvant of the present invention compares in normal oxygen environment cultivation group two groups, add adjuvant of the present invention under finding normal oxygen environment to cultivate, can reach with low-oxygen environment under cultivate close proliferation rate, even within the 5th day, rise, the proliferation rate cultivated under surmounting low-oxygen environment, it can thus be appreciated that, the interpolation of adjuvant of the present invention, can make adipose stromal stem cell under normal oxygen environment is cultivated, reach the effect of rapid multiplication.Join Figure 1B and Fig. 1 C again, identical result is also found in the culture experiment of bone marrow interstital stem cell or umbilical cord mesenchymal stem cells; Known by this experiment, the interpolation of adjuvant of the present invention under normal oxygen environment is cultivated, all can reach the effect of rapid multiplication for adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord mesenchymal stem cells.

2. cell cycle analysis

By each experimental group be incubated at by above-mentioned adipose stromal stem cell under different culture condition, add one and only comprise the basic medium of 10% foetal calf serum as a control group, the change situation of its cell cycle is analyzed with flow cytometer and software (Phoenix Flow Systems) detecting thereof, each group of cultivation sampled after three days, often organize experiment three to repeat, after alcohol fixation, the DNA of propidium iodide (propidium iodide, Sigma) stain transfect cell is used to carry out the change in analysis of cells cycle.

2.1 experimental result

Ginseng Fig. 2 A, for control group is under above-mentioned three groups of different culture condition, to the Cell cycle influences of adipose stromal stem cell, wherein * P < 0.05, * * P < 0.01, * * * P < 0.005.Found by experimental result, basic medium adds in low-oxygen environment cultivation group and basic medium, FGF-2 adds that adjuvant of the present invention is in normal oxygen environment cultivation group etc. two groups, the per-cent of the synthesis phase (S phase) that its adipose stromal stem cell was in the cell cycle compares this control group and this basic medium adds that FGF-2 is in normal oxygen environment cultivation group, significantly improve many, and basic medium adds in low-oxygen environment cultivation group and basic medium, FGF-2 adds that comparatively this control group and this basic medium add that FGF-2 comes much lower in normal oxygen environment cultivation group to the per-cent of adjuvant of the present invention in the G0/G1 phase (stop division stage/vegetative period) of normal oxygen environment cultivation group etc. two groups, it can thus be appreciated that, under low-oxygen environment or add adjuvant of the present invention and cultivate under normal oxygen environment, all the cell cycle can be made to be in synthesis phase, promote that cell DNA constantly synthesizes, and then make the quick division and proliferation of cell.Wherein, add the adipose stromal stem cell that adjuvant of the present invention is cultivated under normal oxygen environment, the per-cent that its cell cycle is in synthesis phase exceeds cultivation group under low-oxygen environment especially, has significant difference; Therefore, add adjuvant of the present invention under normal oxygen environment is cultivated, rapid multiplication effect can not only be reached, and even can surmount the effect of cultivating under low-oxygen environment and can improve hyperplasia speed.

3. the performance situation of the associated protein of cell cycle regulation is analyzed with the west point method of the use of ink and water

The known west point method of the use of ink and water is the characteristic utilizing antibody, antigenic specificity to combine, and coordinate SDS-PAGE colloid electrophoresis to detect the quantification and qualification of specific protein performance, only the method is the technology widely known, its details just repeats no more at this.This experiment for by reported in literature for the relevant albumen of cell cycle regulation, such as: the albumen such as Cyclin A2, Cyclin D1, Cyclin D3, CDK2, CDK4, CDK6, CDC2, p21 and p27, the specificity of above-mentioned each albumen with between its antibody is utilized to be combined, come in the adipose stromal stem cell that analysis and observation cultivates via different culture condition through the above-mentioned west point method of the use of ink and water, it is for the impact of the relevant protein expression of cell cycle regulation; Wherein with a β-actin protein expression amount for contrast, be quantified as the standardized benchmark foundation of data as above-mentioned each protein expression; Because β-actin albumen be a kind of house-keeping gene (Housekeeping gene) the protein of transcribing, translating, by cell maintenance normal physiological phenomena is necessary, and do not have too large change because of various experiment condition, therefore be suitable as the standardized benchmark foundation of quantized data.

3.1 experimental result

Continuous ginseng Fig. 2 B, for above-mentioned Fig. 2 A tetra-groups of experimental group, the associated protein performance of its cell cycle regulation is analyzed, by the performance situation of protein each in figure in different experiments group, find to be thought by reported in literature the Cyclin dependent kinase inhibitor that the T suppression cell cycle carries out: the performance amount of p21 and p27 albumen, add that FGF-2 is in normal oxygen environment cultivation group compared to above-mentioned control group and basic medium, this basic medium adds in low-oxygen environment cultivation group and this basic medium, FGF-2 adds that adjuvant of the present invention is in normal oxygen environment cultivation group, obviously in down state, even this basic medium adds that adjuvant of the present invention is in normal oxygen environment cultivation group, the performance amount of itself p21 and p27 albumen presents close or adds that FGF-2 is in the situation of low-oxygen environment cultivation group lower than this basic medium, another cell cycle protein dependent kinase-2(CDK-2), cell cycle protein dependent kinase-4(CDK-4) and the performance amount of cell division cycle protein (CDC2), add that FGF-2 is in normal oxygen environment cultivation group compared to above-mentioned control group and basic medium, this basic medium adds in low-oxygen environment cultivation group and this basic medium, FGF-2 adds that adjuvant of the present invention is in normal oxygen environment cultivation group, obviously in raising state, even this basic medium adds that adjuvant of the present invention is in normal oxygen environment cultivation group, its CDK-2, the performance amount of CDK-4 and CDC2 albumen presents close or adds that FGF-2 is in the situation of low-oxygen environment cultivation group higher than this basic medium.It can thus be appreciated that, add adjuvant of the present invention and under normal oxygen environment, cultivate adipose stromal stem cell to reach the effect of rapid multiplication, by T suppression cell cyclin-dependent kinase inhibition: the performance of p21 and p27 albumen, to improve the performance amount of CDK-2, CDK-4 and CDC2 albumen, and then promote cell quick copy, division, to reach the effect of rapid multiplication.

4. the relative telomere of cell (Telomere) length analysis

In cell, chromosome structure is organized into by DNA, and telomere is the repetitive dna sequence of end of chromosome, and effect is the chromosomal integrity of protection.Karyomit(e) can first copy before fissional, and DNA copies at every turn, and telomere will shorten a bit, when telomere shortens to a certain degree, just cannot remain chromosomal stable, cell finally can deathward, so can predict the age of cell according to the length of telomere.After the four groups of adipose stromal stem cells comprising aforementioned three groups of different culture condition and above-mentioned control group (comprising the basic medium of 10% foetal calf serum) are cultivated 14 days, utilize the technological method (Cawthon that be can be used to measure cell relative telomere length (T/S ratio) by reported in literature, 2002), under observing different culture condition, for the impact of the telomere of adipose stromal stem cell.

4.1 experimental result

Shown in Figure 3, via the method for above-mentioned known measurement cell relative telomere length (T/S ratio), under being attained at aforementioned four groups of experimental group culture condition, the T/S ratio of its cell telomere, wherein * P < 0.05.By found that, during with this control group for benchmark, this basic medium adds in low-oxygen environment cultivation group and this basic medium, FGF-2 adds compared to this basic medium, adjuvant of the present invention adds that FGF-2 comparatively improves in normal oxygen environment cultivation group in normal oxygen environment cultivation group, and tool significant difference; It can thus be appreciated that, cultivate or add adjuvant of the present invention under low-oxygen environment and cultivate under normal oxygen environment, all can improve the ratio of relative telomere length, and then reach the aging effect delaying cell.

5. the adipose stromal stem cell impact of cultivating under normal oxygen or oxygen environment

Culture condition is divided into four groups by this experiment, it is respectively: basic medium adds in normal oxygen environment cultivation group, basic medium, FGF-2 adds in oxygen environment cultivation group, basic medium, FGF-2 adds in normal oxygen environment cultivation group and basic medium, adjuvant of the present invention adds that adjuvant of the present invention is in oxygen environment cultivation group etc., under wherein oxygen environment refers to 37.5 about percent oxygen partial pressure, and other culture condition parameter (as basic medium, incubator and cell culture density) is all identical with 1. contents of previous experiments one.

The cell density analysis of 5.1 adipose stromal stem cells

By cell with phosphate buffer soln (PBS) cleaned once after, carefully remove with cell spatula again after acting on 5 minutes with trypsin-EDTA (Trypsin-EDTA) solution in 37 degree Celsius and be not applied cell completely, add equal proportion and contain in the substratum of foetal calf serum and tryptic ferment action activity.All cells number all calculates with cell counter (Vi-CELL AS, Beckman Coulter).The cell of survival is with 0.4% trypan blue (Trypan-blue, GIBCO-Invitrogen) come to distinguish with dead cell, during calculating, judge that the parameter setting of the interstital stem cell of work is as 100 images, Size 10 – 30 microns, 75% on-the-spot brightness, 5% field regions.The presenting of result is often organized experimental data and measures three repetitions, represents it with mean value ± standard deviation.

5.1.1 experimental result

Shown in ginseng Fig. 4 A, for adipose stromal stem cell is under different culture condition, the relation of cultivated days and total cell count, by found that, along with cultivated days increases, this basic medium adds that the cell number of adjuvant of the present invention in normal oxygen environment cultivation group just rose afterwards rapidly from second day, reaches 1.5 × 10 in the 5th day ⁶above quantity, compares this basic medium and adds that FGF-2 to significantly improve and fast in normal oxygen environment cultivation group; This basic medium adds that FGF-2 then presents in oxygen environment cultured tissue cell number slowly to be increased, and even the total cellular score of the 7th day reaches 2.5 × 10 not yet ⁵quantity, known oxygen environment has adverse influence for the hyperplasia of cell; And this basic medium adds that adjuvant of the present invention was compared this basic medium in oxygen environment cultured tissue total cellular score and added that FGF-2 presents the trend improved fast in oxygen environment cultivation group after second day, during to the 6th day, its total cellular score reaches 1.0 × 10 ⁶above quantity, compares this basic medium and adds that FGF-2 to significantly improve and fast in oxygen environment cultured tissue total cellular score.It can thus be appreciated that, add adjuvant of the present invention to cultivate under normal oxygen environment and can not add adjuvant person of the present invention, its hyperplasia speed is comparatively quick and number is many, and this basic medium adds that adjuvant of the present invention is in oxygen environment cultivation group, though cultivate under being in oxygen environment, have adverse influence for hyperplasia, but adjuvant of the present invention can slow down the disadvantageous effect because oxygen environment causes hyperplasia really, thus make part cell can continue to maintain the phenomenon of hyperplasia.

5.2 adipose stromal stem cells under different culture condition on the impact of p21 albumen and CDK2 protein expression

The culture condition of adipose stromal stem cell is divided into four groups with described in aforementioned 5., and in utilize the aforementioned west point method of the use of ink and water adipose stromal stem cell that analysis and observation cultivates via different culture condition, its impact that albumen (as p21, CDK2) that cell cycle regulation is relevant is showed, equally with β-actin protein expression amount for contrast, be quantified as the standardized benchmark foundation of data as above-mentioned each protein expression.

5.2.1 experimental result

Ginseng Fig. 4 B, result can find, this basic medium adds in normal oxygen environment cultivation group and this basic medium, FGF-2 adds that adjuvant of the present invention has the phenomenon of attenuating in the p21 protein expression amount of normal oxygen environment cultivation group, and the performance amount of this CDK2 albumen has the phenomenon of increase; This basic medium adds FGF-2 in oxygen environment cultivation group then because oxygen environment makes its p21 protein expression amount significantly improve many, thus curbs the performance of its CDK2 albumen, has a negative impact to hyperplasia; It should be noted that, this basic medium adds that adjuvant of the present invention is in oxygen environment cultivation group, though cultivate under being in oxygen environment, but owing to the addition of adjuvant of the present invention, so add that FGF-2 has the effect obviously reduced in oxygen environment cultivation group to p21 protein expression amount compared to this basic medium, thus the performance of its CDK2 albumen is also significantly improved.Therefore, adding adjuvant of the present invention under normal oxygen environment or under oxygen environment no matter reconfirm, all comparatively without adding adjuvant person of the present invention, the proliferation rate of interstital stem cell be significantly improved.

Experiment two: the impact of adipose stromal stem cell on hyperplasia under different serum additive is cultivated

The cell culture condition of this experiment is divided into four groups, and it is respectively: basic medium adds 10% foetal calf serum group, basic medium adds 10% serum human group, basic medium adds that 10% serum human adds that adjuvant group of the present invention and basic medium add that 2% serum human adds adjuvant group of the present invention etc. four groups; This basic medium is IMDM(Iscove`s modified Dulbecco`s medium; GIBCO-Invitrogen) substratum adds 2 millimolar concentrations (mM) left-handed bran amido acid (L-glutamine again; GIBCO-Invitrogen) formed; and aforementioned adjuvant of the present invention comprises 2 millimolar concentrations (mM) ACETYLCYSTEINE (NAC; and 0.2 millimolar concentration (mM) ascoltin-2-phosphoric acid salt (AsA2P, Sigma) Sigma).Often organize stem cell with 3000 every square centimeter cell (3000 cells/cm ²) cell density be incubated at (6-well plates in 6 porocyte culture plates, Becton Dickinson), and all cells is all incubated at 37 degree Celsius, incubator (Forma Series II Model 3110 under percent 5 partials pressure of carbon dioxide and percent 95 humidity environments, Thermo), in, cultivate one week and change a subculture in every 3 days; To observe in 7 days different adipose stromal stem cell whereby under different culture condition, on the impact of hyperplasia.

1. cell density analysis

By each group of stem cell with phosphate buffer soln (PBS) cleaned once after, carefully remove with cell spatula again after acting on 5 minutes with trypsin-EDTA (Trypsin-EDTA) solution in 37 degree Celsius and be not applied cell completely, add equal proportion and contain in the substratum of foetal calf serum and tryptic ferment action activity.All cells number all calculates with cell counter (Vi-CELL AS, Beckman Coulter).The cell of survival is with 0.4% trypan blue (Trypan-blue, GIBCO-Invitrogen) come to distinguish with dead cell, during calculating, judge that the parameter setting of the interstital stem cell of work is as 100 images, Size 10 – 30 microns, percent 75 on-the-spot brightness, percent 5 field regions.The presenting of result is often organized experimental data and measures three repetitions, represents it with mean value ± standard deviation.

1.1 experimental result

Above experimental data tests (t-test) statistical study through Microsoft Excel Software t, with p < 0.05 for conspicuous level, and result quantities is turned to statistical graph, wherein * P < 0.05, * P < 0.01, * * * P < 0.005.First, ginseng Fig. 5 A, be adipose stromal stem cell under different culture condition, under microscope, knit cell kenel, scale is 500 microns (μm).Find in observation, each histocyte kenel is rather similar, all has the cell kenel of shuttle shape.See Fig. 5 B, for this basic medium adds that 10% foetal calf serum group and this basic medium add cultivated days and the cell density graph of a relation of 10% serum human group, from latter four days of cultivation this basic medium add 10% serum human histocyte density comparatively this basic medium add that 10% foetal calf serum group significantly improves the cell number of 28 to percent 74 about percent, and there is significant difference; Learn thus, with serum human as serum additive during cell cultures, comparatively can add foetal calf serum and more can make adipose stromal stem cell rapid multiplication.See Fig. 5 C, for this basic medium adds that 10% serum human group and this basic medium add that 10% serum human adds the cell density comparison diagram of adjuvant group of the present invention, by found that, this basic medium add 10% serum human add adjuvant group of the present invention in cultivate cell density one day after comparatively this basic medium add that 10% serum human group significantly improves the cell number of 155 to percent 324 about percent, and there is significant difference; Learn thus, under interpolation adjuvant of the present invention is cultivated, more can improve the speed of adipose stromal stem cell proliferation, with under identical cultivated days, obtain more a large amount of cell numbers.Join Fig. 5 D again, for this basic medium adds that 10% serum human group and this basic medium add that 2% serum human adds comparing of adjuvant group of the present invention, by found that, though this basic medium adds that 2% serum human adds that adjuvant group of the present invention only has 2% serum human, but after cultivating by interpolation adjuvant of the present invention, still can make adipose stromal stem cell under the cultivation of the first five day, the speed of hyperplasia still adds with this basic medium that 10% serum human group is close; Learn that the interpolation of adjuvant of the present invention not only can reduce the usage quantity of serum human thus, also can possess the effect promoting adipose stromal stem cell rapid multiplication simultaneously.

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

This experiment carries out the mensuration of cell-surface antigens with flow cytometer (FACSCalibur, Becton Dickinson).By the adipose stromal stem cell attachment removal that is incubated at respectively under aforementioned four groups of different culture condition and with after phosphoric acid buffer cleaning, back dissolving is in appropriate phosphoric acid buffer, respectively different antigen is dyeed with corresponding immunofluorescence Primary antibodies, comprise the antibody (Becton Dickinson) such as CD13, CD34, CD44, CD73, CD90, CD105, β2-microglobulin (B2M) and HLA-DR.After lucifuge dyes 15 minutes under room temperature, after adding appropriate phosphoric acid buffer, upper machine analysis, collects after data through flow cytometer, analyzes with flow cytometry analysis software (FACSCalibur, Becton Dickinson).Wherein negative control group is omit the staining procedure of above-mentioned Primary antibodies.

2.1 experimental result

See Fig. 6 A, the adipose stromal stem cell under aforementioned four groups of different culture condition can be found by result, its cell populations be CD13+, CD34-, CD44+, CD73+, CD90+, CD105+, for the cell populations of similar interstital stem cell, that is expression is said, the adipose stromal stem cell under aforementioned four groups of different culture condition still possesses the surface antigen feature of similar interstital stem cell.Wherein, there are the three groups of experiments adding serum human, the performance amount of itself CD44 and CD73 adds 10% foetal calf serum group higher than this basic medium, this result also illustrates the three groups of experiments added serum human and cultivate, and the effect that its effect of possessing the surface antigen feature of similar interstital stem cell comparatively adds foetal calf serum cultivation is come better.

3. stem cell gene performance is analyzed

This experiment, by being incubated at the adipose stromal stem cell of gained under aforementioned four groups of different culture condition respectively, analyzes the performance of the genes involved of undifferentiated stem cell with real time aggregation enzyme chain reaction instrument (Real-Time PCR System).After the cell of turning out is cleaned with phosphoric acid buffer, be collected in 1.5 ml eppendorf, add 1 ml TriZol(10296-010, Invitrogen) reagent, place five minutes in room temperature, add 100 μ l BCP(BP. 151, MRC) solution, after Vortex mixing to one-tenth pink solution, room temperature places 15 minutes, again with 15, centrifugal 15 minutes of 000 g is at 4 DEG C.Centrifugal complete after, can divide three layers in eppendorf, lower floor is red color layer, the white layer of middle thin layer, and upper strata is transparent layer, and upper strata sucking-off is put into 1.5 milliliters of new centrifuge tubes (eppendorf), being careful in suction process, it is two-layer in addition not to be drawn onto.In new centrifuge tube, add 0.5 milliliter of Virahol (isopropanol), after shaking up, room temperature places 30 minutes, afterwards again with 15, supernatant liquor is extracted out at 4 DEG C, is not drawn onto agglomerate (pellet) by 000 g for centrifugal 10 minutes, add 1 milliliter of 75% alcohol (ethanol) cleaning, again with 15, centrifugal 10 minutes of 000 g at 4 DEG C, after taking out alcohol, dry air 10 minutes, with containing suppress RNase(DEPC) water back dissolving after namely complete RNA extraction.Draw about 10 micrograms (μ g) RNA, add Reverse Transcription group (RT-for-PCR kit, Clontech), polysaccharase (GoTaq Green Master Mix is added after completing reverse transcription with polymerase chain reaction device (PCR machine), M7122, Promega) carry out Polymerase Chain Reaction, imposing a condition of its reaction of P slightly adjusts because of the different Tm value of different introduction (Primer).Analyzing and to be correlated with undifferentiated genes involved with stem cell, similarly is Nanog, SOX2, CXCR4, TERT etc., this experimental analysis Nanog, SOX2, CXCR4, TERT and control group β-actin gene.Analyze introduction that each gene uses as shown in the following chart:

3.1 experimental result

See Fig. 6 B, via result, we find, there are three groups of experiment (10% serum humans adding serum human, 10% serum human+adjuvant, 2% serum human+adjuvant), its stem cell gene (Nanog, SOX2, CXCR4, TERT) performance all adds 10% foetal calf serum group apparently higher than this basic medium, and can observe equally, though this basic medium adds that 2% serum human adds that adjuvant group of the present invention only has 2% serum human, but after cultivating by interpolation adjuvant of the present invention, it still can as added as 10% serum human cultivation, keep the performance of above-mentioned stem cell gene, therefore known, add adjuvant of the present invention and carry out cell cultures, really the usage quantity of serum human can be reduced.

4. the differentiation of adipose stromal stem cell

Reported in literature is pointed out, adipose stromal stem cell has and is divided into mesoblastemic ability, similarly is fat and bone cell.This experiment with as experiment two by adipose stromal stem cell after cultivating six days under four groups of different culture condition, by its differentiation-inducing one-tenth os osseum cell, chondrocyte and adipocyte, to confirm, after cultivating six days under above-mentioned four groups of different culture condition, whether still there is stem cell multifunctional differentiation capability.The differentiation-inducing system of stem cell (Kanda et al., 2011 that have been commonly used in the known document of differentiation-inducing experimental basis in the present invention; Song et al., 2010), because of non-this case demand emphasis, therefore it will not go into details, and its main purpose to be to confirm in the present invention, after cultivating under above-mentioned four groups of different culture condition, whether still having stem cell multifunctional differentiation capability.

The chemical staining of 4.1 cells and molecular marker analysis

The os osseum cell of differentiation is with alkaline phosphatase (Alkaline phosphatase, ALP) dye, this alkaline phosphatase is the important indicator of ripe osteoblast differentiation, and its dyeing process carries out (Yoshimura et al. according to known staining technique, 2011), do not repeat them here; Separately carry out a known Von-kossa dyeing again, to confirm the existence having calcium phosphate.The chondrocyte of differentiation confirms the existence (Song et al., 2010) of the proteoglycan (Proteoglycan) had in cartilaginous tissue with the blue staining (Alcian blue) of A Erxin.Differentiation adipocyte with oil red O stain (Oil red O staining), to be confirmed whether the existence (Kanda et al., 2011) of lipid cavity (Lipid vacuoles).Molecule marker [the core binding factor (cbfa1) that this experiment another has for os osseum cell, osteocalcin (Osteocalcin, OC) and the gene such as the first collagen type (COL IA1)], [cartilage gathers glucoprotein (ACAN) to the molecule marker that chondrocyte has, the genes such as Second-Type collagen protein (COL IIA1)] and the fat synthesis related gene [peroxisome proliferators activated receptor γ (PPAR γ) of adipocyte, fat associated proteins (aP2)] performance, and with β-actin gene for control group, carry out real time aggregation enzyme chain reaction (Real-Time PCR) and analyze its relative performance amount.Analyze introduction that each gene uses as shown in the following chart:

4.2 experimental result

See Fig. 7 A, for alkaline phosphatase staining result, scale is 500 microns (μm), adipose stromal stem cell under aforementioned four groups of different culture condition, through being induced to differentiate into os osseum cell, after alkaline phosphatase staining, all there is the part presenting black crystalline, that is represent the existence of alkaline phosphatase, further illustrate these four groups, adipose stromal stem cell all can be made to possess the ability of differentiation of stem cells, continuous see Fig. 7 B, for Feng Kusa (Von-kossa) coloration result, scale is 500 microns (μm), can find that aforementioned four groups all have black or umbrinaceous calcium phosphate crystal, again illustrate these four groups, adipose stromal stem cell all can be made to possess the ability of differentiation of stem cells, Fig. 7 C again, for the molecular marker performance analytical results of os osseum cell, can find in osteocalcin (OC) relative performance amount, this basic medium adds that 10% serum human adds that adjuvant group of the present invention and this basic medium add that 2% serum human adds adjuvant group of the present invention etc. two groups, than this basic medium, the molecular marker performance of its os osseum cell all adds that 10% foetal calf serum group and this basic medium add that 10% serum human group significantly improves, wherein * P < 0.05, * P < 0.01, * * P < 0.005, especially this basic medium adds that 2% serum human adds adjuvant group of the present invention, and its cbfa1, OC and COL IA1 three relative performance amount adds 10% foetal calf serum group apparently higher than this basic medium especially.It can thus be appreciated that, add adjuvant of the present invention and add that serum human carries out the cultivation of adipose stromal stem cell, the differentiation capability that stem cell is original can be possessed, and effect through being induced to differentiate into os osseum cell is better.

Refer to Fig. 8 A, for the blue coloration result of A Erxin, scale is 500 microns (μm), can find that aforementioned four groups all present blue proteoglycan coloration result, especially this basic medium adds that 2% serum human adds that the blue protein polysaccharide pigmented section of adjuvant group of the present invention far exceedes other three groups.Continuous ginseng Fig. 8 B, for the molecule marker performance of chondrocyte is analyzed, can find that this basic medium adds that 10% serum human adds that adjuvant group of the present invention and this basic medium add that 2% serum human adds that adjuvant group of the present invention is in the isogenic relative performance's amount of ACAN and COL IIA1, all add that 10% foetal calf serum group and this basic medium add 10% serum human group apparently higher than this basic medium, wherein * P < 0.05, * P < 0.01, * * * P < 0.005; Especially this basic medium adds that 2% serum human adds adjuvant group of the present invention, and the molecular marker performance of its chondrocyte significantly improves many especially.It can thus be appreciated that, add adjuvant of the present invention and add that serum human carries out the cultivation of adipose stromal stem cell, the differentiation capability that stem cell is original can be possessed, and effect through being induced to differentiate into chondrocyte is better.Join Fig. 8 C again, be oil red O stain result, scale is 500 microns (μm), can find that aforementioned four groups all present red lipid cavity coloration result.Continuous see Fig. 8 D, for the relative performance of fat synthesis related gene PPAR γ and aP2 measures, wherein * P < 0.05, * * P < 0.01.Can find that this basic medium adds that 10% serum human adds adjuvant group of the present invention, the relative performance of its PPAR γ and aP2 measures comparatively this basic medium and adds that 10% foetal calf serum group and this basic medium add that 10% serum human group significantly improves.It can thus be appreciated that, add adjuvant of the present invention and add that serum human carries out the cultivation of adipose stromal stem cell, the differentiation capability that stem cell is original can be possessed, and effect through being induced to differentiate into adipocyte is better.

5. the analysis of cytokine array shows with Relative gene and analyzes

This analysis is tested under three kinds of states (before 10% serum human cultivation, 10% serum human cultivates and 10% serum human adds that adjuvant is cultivated) of adipose stromal stem cell, get its medium supernatant respectively to utilize and commercially availablely comprise 41 kinds of Human cytokine's array analysis cover group (Cat #AAH-GF-1, RayBiotech, Norcross, GA) analyze, to observe adipose stromal stem cell under aforementioned three kinds of states, on the impact of its secrete cytokines and somatomedin; Therefore it will not go into details for analytical procedure non-this case demand emphasis of aforementioned array analysis cover group, its detailed content can join RayBiotech company the specification sheets of Cat #AAH-GF-1 is provided.This experiment another also by above-mentioned adipose stromal stem cell after cultivation under four groups of different culture condition with such as test two 3. analytical procedure for the particular growth factor as genes such as insulin-like growth factor (IGF-1), pHGF (HGF) and Urogastrons (EGF), with β-actin gene for control group, carry out real time aggregation enzyme chain reaction (Real-Time PCR) and analyze its relative performance amount.Analyze introduction that each gene uses as shown in the following chart:

In addition, this experiment also for adipose stromal stem cell following three kinds of culture condition as: under (low-oxygen environment of 5% oxygen partial pressure) group that basic medium adds 10% foetal calf serum group, basic medium adds 10% foetal calf serum and basic medium add that 10% foetal calf serum adds that adjuvant group of the present invention etc. three groups is cultivated, get its medium supernatant respectively and carry out Human cytokine's array analysis (Cat #AAH-GF-1, RayBiotech, Norcross, GA), to observe the impact on adipose stromal stem cell secretion cytokine and somatomedin.

5.1 experimental result

Join Fig. 9 A figure and Fig. 9 B in the lump, for cytokine array analytical results compares schematic diagram with cytokine array analytical results, wherein POS represents positive regulation group, NEG represents negative control group, * P < 0.05, * P < 0.01, * * * P < 0.005.Can be found by results contrast, adipose stromal stem cell is after interpolation adjuvant of the present invention is cultivated, and in conditioned medium, secretion has following cytokine and somatomedin: 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-R β, PDAF-AA, PDGF-AB, PDGF-BB, PIGF, SCF, TGF-β 3, VEGF, VEGF R2.Be that the block of oblique line is to left down as Second-Type fibroblast growth factor (bFGF), Urogastron (EGF) in the comparison diagram that wherein 10% serum human shown in Fig. 9 B is cultivated with 10% serum human before cultivating, Thr6 PDGF BB (PDGF-AA, AB, BB) and vascular endothelial growth factor (VEGF R3) etc. present the situation utilized by Cell uptake; And in descending the block of oblique line to the right as pHGF (HGF), binding protein of insulin-like growth factor (IGFBP-1, IGFBP-4, IGFBP-6), insulin-like growth factor 1(IGF-1 in figure), vascular endothelial growth factor (VEGF) etc. present by emiocytosis to the situation in substratum.Join Fig. 9 C and Fig. 9 D again, for aforementioned 10% serum human cultivation and 10% serum human add the quantification schematic diagram that adjuvant cultivation results compares, can find that Thr6 PDGF BB (PDGF-AA, AB, BB) continues to present the situation utilized by Cell uptake, pHGF (HGF), binding protein of insulin-like growth factor (IGFBP-1) and vascular endothelial growth factor (VEGF) are then not only and are continued to present by emiocytosis to the situation in substratum, and add that the result that adjuvant is cultivated significantly improves through 10% serum human.It can thus be appreciated that, add that serum human carries out the cultivation of adipose stromal stem cell by interpolation adjuvant of the present invention, there is the secretion of some somatomedin of raising as HGF, IGFBP-1 and VEGF and tell on.

Continuous ginseng Fig. 9 E, for adipose stromal stem cell is after cultivating under four groups of different culture condition, the relative performance of its growth factors I GF-1, HGF and EGF measures result, wherein * P < 0.05, * P < 0.01, * * * P < 0.005.Learnt by result, there is the relative performance adding three groups of (10% serum human, 10% serum human+adjuvant, 2% serum human+adjuvant) its IGF-1, HGF and EGF that serum human is cultivated to measure all comparatively this basic medium and add that 10% foetal calf serum group significantly improves, especially add that 2% serum human adds that relative performance's amount of adjuvant group of the present invention is the highest with this basic medium.It can thus be appreciated that, add after adjuvant of the present invention adds that serum human carries out the cultivation of adipose stromal stem cell, somatomedin such as the relative performance of IGF-1, HGF and EGF can be improved and measure.

See also Fig. 9 F and Fig. 9 G again, for cytokine array analytical results figure and above-mentioned cytokine array analytical results comparison diagram, wherein in Fig. 9 G, if show central clear pie chart sample person in each cytokine block, represent this basic medium and add that the adipose stromal stem cell of 10% foetal calf serum group has this cytokine of secretion; In like manner, if show complete black pie chart sample person, representing the adipose stromal stem cell that this basic medium adds that 10% foetal calf serum (low-oxygen environment of 5% oxygen partial pressure) is organized has this cytokine of secretion; And show oblique line pie chart sample person, represent this basic medium and add that 10% foetal calf serum adds that the adipose stromal stem cell of adjuvant group of the present invention has this cytokine of secretion or somatomedin.Can be found by Fig. 9 G, under adipose stromal stem cell is incubated at condition, namely can secrete cytokine or the somatomedins such as bFGF, EGF, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-6, IGF-II, M-CSF, M-CSF R, NT-4, PDGF R β, PIGF, TGF-β 3, VEGF.It should be noted that, though this basic medium adds that 10% foetal calf serum (low-oxygen environment of 5% oxygen partial pressure) group and basic medium add that 10% foetal calf serum adds that adjuvant group of the present invention can increase the cytohormone kind of adipose stromal stem cell secretion more, but this basic medium adds that 10% foetal calf serum adds the kind that adjuvant group of the present invention increases and measures 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, PDGF-BB, SCF, VEGF R2, VEGF R3, the cytokines such as VEGF-D or somatomedin.It can thus be appreciated that, add adjuvant of the present invention carry out the cultivation of adipose stromal stem cell can comparatively under low-oxygen environment be cultivated, more increase kind and the amount of cytokine secreted by adipose stromal stem cell or somatomedin.

No matter it is be incubated at interpolation adjuvant of the present invention add the basic medium of serum human or only add adjuvant of the present invention under the basic medium containing 10% foetal calf serum that comprehensive the above results describes adipose stromal stem cell, not only all can reach the effect of rapid multiplication, simultaneously also can obtain the somatomedin of secreting in a large number between adipose stromal stem cell incubation period, wherein add under the basic medium condition of serum human as optimal culture condition to be incubated at interpolation adjuvant of the present invention again; Whereby using as follow-up for the preparation of wound healing promoting medicine and the purposes for the preparation of wound healing promoting medicine, or as preparation skin care product and the purposes for the preparation of skin care product.

Experiment three: adipose stromal stem cell microcarrier is cultivated

The Human Meta stem cell cultivated in vitro carries out mainly with the training method of sticking type (Anchorage-dependent cells), so use a kind of culture systems meeting sticking type to produce a large amount of and cell of uniform quality for the application of regenerative medicine and organizational project is needs.This experiment utilizes stirring-type microcarrier cultivation reactor (Spinner Flasks, Bellco Glass, Inc., Vineland, NJ, USA) to carry out the cultivation of adipose stromal stem cell.Before inoculating cell enters reactor, first aforementioned cultivation inside reactor surface was processed with silica gel (Sigmacote, Sigma, St. Louis, MO, USA); And the microcarrier used (CultiSpher-G; HyClone, Logan, UT, USA) then according to the step on operational manual sequentially weighing, add hydration it, and use Sterilization Kettle 121 DEG C of process in 15 minutes; Before being mixed into cell, first removing unnecessary phosphate buffer soln, then adding the balance about 24 hours that the nutrient solution for culturing cell cell makes.Adipose stromal stem cell is added containing pre-balance nutrient solution and microcarrier altogether the stirring-type of about 50 milliliters cultivate in reactor, open external-added electromagnetic stirring system in step mode at first, rotate 2 hours with the frequency that each 25 r.p.m had a rest 10 to 20 minutes after 30 minutes; After aforementioned 2 hours with the rotating speed of 25 r.p.m start cultivate, and every 3 days change once accompany nutrient solution, more exchange treaties at every turn 50 to 70 percent nutrient solution, and in replacing before first stop stir about 5 minutes, make cell and microcarrier drop down onto reactor bottom.Wherein microcarrier culturing process 37 DEG C, cultivate 7 days under the environment of humidity 95% and 5% partial pressure of carbon dioxide, and prior culture media comprises IMDM and adds 10% serum human, 2 millimolar concentrations (mM) left-handed bran amido acid and adjuvant of the present invention again and formed; And aforementioned adjuvant comprises 10 ng/mL FGF-2,2 millimolar concentration ACETYLCYSTEINEs and 0.2 millimolar concentration ascoltin-2-phosphoric acid salt.

For observation of cell is in grown on microcarriers and distribution scenario, take out 1 milliliter of enchylema containing microcarrier every day, and centrifugally remove substratum and with phosphate buffer solution cleaning once; Continue with 10 formalin stationary liquids after room temperature fixes 10 minutes, remove stationary liquid and clean with phosphate buffer solution; Viable cell and dead cell is contaminated respectively again with 5 mg/ml green fluorescence sodium Diacetates (FDA) and 2 mg/ml propidium iodides (PI), in room temperature lucifuge after 5 minutes, stain is removed and cleans three times with phosphate buffer solution, again cell and microcarrier are evenly distributed on slide, use fluorescence microscope.

Experimental result

Refer to Figure 10 A and Figure 10 B, for adipose stromal stem cell is incubated at microcarrier 7 days, the image under opticmicroscope under image and fluorescent microscope, wherein green fluorescence represents viable cell, and red fluorescence represents dead cell.Learnt by the Imaging Study of Figure 10 B, adipose stromal stem cell is more easily attached at microcarrier, and forms micro-assembly robot state.Known whereby, adipose stromal stem cell is incubated at a biocompatible material, as microcarrier, micro-assembly robot state can be formed, using the purposes as subsequent regeneration medical science or organizational project.

Comprehensive above-mentioned experimental result is known, Human Meta's stem cell in vitro rapid multiplication adjuvant of the present invention is added in the substratum comprising Human Meta stem cell, after normal oxygen environment (about 21% oxygen partial pressure) is cultivated, occur that cell divides fast as can cultivating under low-oxygen environment (about 5% oxygen partial pressure) as being same as, synthesis phase cell cycle (S phase) ratio improves, reduce aging and improve the phenomenons such as differentiation potential, and collocation uses serum human to replace foetal calf serum when cultivating, its hyperplasia speed is better, and the somatomedin amount of emiocytosis improves, kind increases, make the present invention not only can fast and efficiently amplifying human interstital stem cell, and still can possess the feature of stem cell multifunctional, in order to reach the rapid amplifying of Human Meta stem cell and to obtain effect of somatomedin.

Above-described embodiment and graphic and non-limiting product form of the present invention and style, any person of an ordinary skill in the technical field, to its suitable change done or modification, all should be considered as not departing from patent category of the present invention.

Claims

1. Human Meta's stem cell in vitro rapid multiplication adjuvant, is characterized in that: comprise at least one antioxidant and a Second-Type fiber mother cell growth factor.

2. Human Meta's stem cell in vitro rapid multiplication adjuvant as claimed in claim 1, is characterized in that: Human Meta's stem cell body is selected from: adipose stromal stem cell, bone marrow interstital stem cell or umbilical cord mesenchymal stem cells form one of group.

3. Human Meta's stem cell in vitro rapid multiplication adjuvant as claimed in claim 1, is characterized in that: antioxidant comprises the combination of a long-acting type ascorbic acid derivates and an ACETYLCYSTEINE.

4. Human Meta's stem cell in vitro rapid multiplication adjuvant as claimed in claim 3, is characterized in that: long-acting type ascorbic acid derivates is a L-AA-2-phosphoric acid salt.

5. Human Meta's stem cell in vitro rapid multiplication adjuvant as claimed in claim 1, is characterized in that: Second-Type fiber mother cell growth factor working concentration is 1 ng/ml to 20 ngs/ml.

6. Human Meta's stem cell in vitro rapid multiplication adjuvant as claimed in claim 1, it is characterized in that: the cell within a cell cyclin-dependent kinase inhibition by suppressing aforementioned human's interstital stem cell: the performance of p21 and p27 albumen, in order to improve the performance of cell cycle protein dependent kinase-2, cell cycle protein dependent kinase-4 and cell division cycle protein.

7. the method for an external rapid amplifying Human Meta stem cell, it is characterized in that: comprise and the adjuvant described in claim 1 to 6 is added one comprise in the substratum of Human Meta stem cell, with the aforementioned human's interstital stem cell that increases, and obtain the undifferentiated Human Meta stem cell of essence.

8. the method for an external rapid amplifying Human Meta stem cell, it is characterized in that: comprise in the substratum adjuvant described in claim 1 to 6 being added and comprises a serum additive and a Human Meta stem cell, with the aforementioned human's interstital stem cell that increases, and obtain the undifferentiated Human Meta stem cell of essence.

9. the method for external rapid amplifying Human Meta stem cell as claimed in claim 8, it is characterized in that: serum additive is serum human or foetal calf serum, the concentration expressed in percentage by volume used is 2 to percent 10 percent.

10. the method for the external rapid amplifying Human Meta stem cell as described in claim 7 to 9, is characterized in that: the Human Meta stem cell step more comprising the aforementioned amplification of a freezen protective, for further using.

11. 1 kinds of cell banks, is characterized in that: comprise by the Human Meta stem cell of the amplification of the method institute freezen protective of external rapid amplifying Human Meta stem cell as claimed in claim 10.

The method of 12. external rapid amplifying Human Meta stem cells as described in claim 7 to 9, is characterized in that: perform an extraction step further to obtain the cell extraction thing of a Human Meta stem cell.

The method of 13. external rapid amplifying Human Meta stem cells as described in claim 7 to 9, is characterized in that: perform a differentiation-inducing step further, to obtain a cell broken up from aforementioned human's interstital stem cell.

14. 1 kinds of medical compositions, is characterized in that: to comprise by the method for external rapid amplifying Human Meta stem cell as claimed in claim 13 obtain the undifferentiated Human Meta stem cell of essence or a cell broken up from aforementioned human's interstital stem cell.

The method of 15. external rapid amplifying Human Meta stem cells as claimed in claim 13, is characterized in that: the cell that Human Meta stem cell breaks up, comprises osteocyte, one of adipocyte or chondrocyte.

16. 1 kinds of external rapid amplifying Human Meta stem cells, to obtain the method for somatomedin, is characterized in that: after the method for claim 7 to 9 is cultivated, to obtain a somatomedin in prior culture media.

17. external rapid amplifying Human Meta stem cells as claimed in claim 16, to obtain the method for somatomedin, is characterized in that: somatomedin comprises: 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-R β, PDAF-AA, PDGF-AB, PDGF-BB, PIGF, SCF, TGF-β 3, VEGF, VEGF R2.

18. 1 kinds of somatomedins, is characterized in that: be that external rapid amplifying Human Meta stem cell according to claim 16 obtained with the method obtaining somatomedin.

19. 1 kinds of medical compositions, is characterized in that: include somatomedin as claimed in claim 18.

20. 1 kinds include the purposes at the conglutinant medicine of preparation of somatomedin as claimed in claim 18.

21. 1 kinds include as claimed in claim 18 somatomedin as the purposes of skin care product.

22. medical compositions as claimed in claim 14, is characterized in that: further combined with a biocompatible material to be applied to regenerative medicine or organizational project.