CN108060119A - The method that micromolecular compound combines and prepares vascular smooth muscle cells using the cell that micromolecular compound combination induction is broken up - Google Patents

The method that micromolecular compound combines and prepares vascular smooth muscle cells using the cell that micromolecular compound combination induction is broken up Download PDF

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CN108060119A
CN108060119A CN201610975439.5A CN201610975439A CN108060119A CN 108060119 A CN108060119 A CN 108060119A CN 201610975439 A CN201610975439 A CN 201610975439A CN 108060119 A CN108060119 A CN 108060119A
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forskolin
vpa
chir99021
repsox
rolipram
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胡敏
李燕皎
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Yunnan Keats Institute Of Regenerative Medicine Co Ltd
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Abstract

The method for preparing vascular smooth muscle cells the invention discloses a kind of combination of micromolecular compound and using the cell that micromolecular compound combination induction is broken up.The small molecule combinatorial includes the first stage micromolecular compound being chronologically used separately and second stage micromolecular compound, and the first stage micromolecular compound includes at least one of DNMT inhibitor and lysine deacetylase inhibitors；The second stage micromolecular compound includes WNT/ β catenin agonists, cAMP agonists and TG F beta acceptor inhibitors.The present invention is induced stage by stage by micromolecular compound, can be vascular smooth muscle cells by the cells transdifferentiate of differentiation, each step can realize accurate Quality Control, convenient for normalizing operation and large-scale production.Method donor source provided by the invention is extensive, and sufferers themselves are preferable donor, can obtain the sufficient amount of vascular smooth muscle cells needed for basic research, clinical treatment or organizational project production within a short period of time.

Description

Micromolecular compound combines and utilizes the thin of micromolecular compound combination induction differentiation The method that born of the same parents prepare vascular smooth muscle cells
Technical field
The present invention relates to cell biology, organizational project and regenerative medicine field more particularly to a kind of micromolecular compound Combination and the method for preparing vascular smooth muscle cells using the cell that micromolecular compound combination induction is broken up.
Background technology
Since wound, angiocardiopathy, organ transplant, hemodialysis patients Population are huge, and autologous vein has Irreplaceability, allogeneic blood vessel has limited source, there are reasons such as transmission risk and immunological rejections, clinical It is huge to replacement vessels demand.Replacement vessels can be divided into high molecular polymer and 2 class of biomaterial by timbering material.Blood vessel Substitute need to have other biology of good intensity, toughness, compliance, not oozing of blood, histocompatbility and normal blood vessels Function.Acellular matrix artificial blood vessel's substitute has become current and mainstream development direction from now on.The artificial blood of acellular matrix Pipe substitute needs a large amount of vascular smooth muscle seed cells to provide extracellular matrix protein.The seed cell of tradition application is main From xenogenesis acquisition, allogeneic donations, adult stem cell directional induction, EC/IPS directional inductions, but there are limited source, Acquisition process is complicated, cell purity is low, has the problems such as immunogenicity or tumorigenesis risk, thus application is limited.
The cell of a variety of differentiation such as skin fibroblasts have abundance, are easily obtained and are easy to long-term big in vitro The advantages of measuring amplification cultivation.It can the cell such as skin fibroblasts of differentiation are straight currently with cells transdifferentiate technology Induction is connect as a variety of different types of functioning cells such as sarcoblast, neuron, liver cell, osteoblasts；Or first induction is more After energy stem cell, further directional induction is corresponding functioning cell.It is above-mentioned specific from certain using cells transdifferentiate technology The cell of differentiation such as skin fibroblasts directly or indirectly induce the functioning cell of acquisition no longer to keep point of source cell Subcharacter and function, but obtain the Typical molecular feature and cell function of respective objects cell.At present, above-mentioned induction sexual function Cell has gradually been applied to disease model research, clinical treatment research and Tissue Engineering Study.
Traditional cells transdifferentiate is needed to realize by importing specific allogenic gene, also needed to sometimes small point corresponding Sub- compound, cell factor or recombinant protein synergistic effect.Document has the specific allogenic gene of more use a certain differentiation Cell induction for another functional differentiation cell report.If document is existing using BMP-2, BMP-7, LMP3 are independent or assist Same-action makes skin fibroblasts transdifferentiation all to have the report of the osteoblast of bon e formation effect in vitro, in vivo.But Allogenic gene is imported there are tumorigenesis risk, and target cell may be made to generate immunogenicity, it is difficult to be promoted and applied.2013, Deng Hongkui reports can realize that the reprogramming of mouse skin fibroblasts is nerve cell only with micromolecular compound or its combination Transdifferentiation process, and confirm the cells transdifferentiate technology have Induction Process is short, inducible system is stable, is easy to Quality Control, cost It is low, be inserted into without allogenic gene caused by tumorigenesis risk, the target cell that obtains there is good security and stability and nothing Immunogenicity has potential clinical value and industrialization prospect.Hereafter, the China of Application No. 201410075246.5 Patent application provides a kind of the method and its application that the cell induction transdifferentiation of differentiation is neural stem cell.Specifically, should Application is related to is given birth to using histon deacetylase (HDAC) (HDACs) inhibitor, glycogen synthase kinase (GSK-3) inhibitor and conversion The combination of long factor-beta (TGF-β) signal pathway inhibitor, induced fibroblast, epithelium are thin under normal physiological low-oxygen environment The cells transdifferentiate of the differentiation such as born of the same parents is the neural stem cell with good versatility and mitotic stability.Application No. 201610213644.8 Chinese patent application provides the induction that a kind of induced fibroblast transdifferentiation is cardiac muscle cell and trains Base, method and its application are supported, the inducing culture includes basal medium and induction small molecule combinatorial, the induction small molecule Be combined as 6TCFOW or SCFOV, wherein 6 be E61541, T be tranylcypromine, C CHIR99021, F are forskolin, O is Dorsomorphin, W IWR-1, S SB431542, V are valproic acid.This application inducing culture can be by fibroblast Induction transdifferentiation is cardiac muscle cell.At present, by using simple micromolecular compound or the cell of its combination induction human differential As skin fibroblasts obtain schwann cell (THOMA EC, et al, 2014), nerve cell (HU W, et al, 2015), The achievement of islet cells (Sheng Ding, et al, 2015) is seen in report successively.
Since the mankind and mouse have about 25% gene difference, and succeed above-mentioned in mouse cell experiment The same cells transdifferentiation feasibility that patent application technical solution is applied to people is not high；On the other hand, due to inducing same cells Transdifferentiation obtains specific theoretical foundation and the technological means of different target cell and differs, applicant using above-mentioned report skill Art scheme repeats to test respectively, and the transdifferentiation technical solution applied to mouse cell had both been failed to be successfully applied to the similar thin of people Dysuria with lower abdominal colic is broken up, and it is vascular smooth muscle cells to also fail to the cell induction transdifferentiation of human differential.
The content of the invention：
Break up the present invention provides a kind of combination of micromolecular compound and using micromolecular compound combination induction thin The method that born of the same parents prepare vascular smooth muscle cells.The present invention combines chronologically treatment by stages by using micromolecular compound and breaks up Cell fast and stable sequencing obtain a large amount of vascular smooth muscle cells or its product.
First aspect present invention, provides a kind of micromolecular compound combination, and the small molecule combinatorial includes chronologically separating The first stage micromolecular compound and second stage micromolecular compound used, the first stage micromolecular compound include DNMT (dnmt rna) inhibitor and lysine deacetylase (Lysine deacetylases inhibitors, KDACIs) at least one of inhibitor；The second stage micromolecular compound includes WNT/ β-catenin agonists, CAMP agonists and TGF-beta acceptor inhibitors.
Further, the second stage micromolecular compound further includes lysine deacetylase inhibitors, RAR excitements Agent, DNMT inhibitor, HMT (histone methyltransferase) inhibitor, pkc inhibitor, ascorbate (ascorbic acid), JNK suppression Preparation, at least one of ROCK inhibitor and histone demethylase inhibitor.
Further, the lysine deacetylase inhibitors include sodium phenylbutyrate, Butyrate, sodium butyrate (NaB), MC1568, CI994 (Tacedinaline), chidamide, CAY10683 (SantacruzaMate A), CUDC-907, M344 (Histone Deacetylase Inhibitor III), LAQ824 (NVP-LAQ824, Dacinostat), Pracinostat (SB939), VPA, Scriptaid, Apicidin, LBH-589 (Panobinostat), MS-275, SAHA (Vorinostat), Trichostatin (TSA), Psammaplin A, PCI- 24781 (Abexinostat), Rocilinostat (ACY-1215), Mocetinostat (MGCD0103), 4- Phenylbutyrate (4PB), splitomicin, SRT1720, resveratrol, Sirtinol, APHA, CI-994, Depudecin, FK-228, HC-Toxin, ITF-2357 (Givinostat), Chidamide, RGFP 966, PHOB, BG45, Nexturastat A, TMP269, CAY10603, MGCD-0103, Niltubacin, PXD-101 (Belinostat), In Pyroxamide, Tubacin, EX-527, BATCP, Cambinol, MOCPAC, PTACH, MC1568, NCH51 and TC-H106 At least one；
The TGF-β acceptor inhibitor includes 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, At least one of SB525334, LY364947, ASP3029, D4476 and SB505124；
The pkc inhibitor includes Go6983, Ro31-8220Mesylate, Go6976 and Bisindolylmaleimide At least one of I (GF109203X)；
WNT/ β-catenin the agonists include MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763 and At least one of AR-A014418；
The cAMP agonists include EPAC/RAP1 agonists, 8-Bromo-cAMP, Dibutyryl-Camp and Sp-8- At least one of Br-cAMPs；
The EPAC/RAP1 agonists include Forskolin, IBMX, Prostaglandin E2 (PGE2), NKH477, 8-pCPT-2 '-O-Me-cAMP, GSK256066, Apremilast (CC-10004), Roflumilast, Cilomilast, At least one of Rolip ram and Milrinone；
The RAR agonists include TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, At least one of ATRA, 13-cis RA, Vitamin A and Vitamin A derivatives；
The ROCK inhibitor includes Y-27632, Y-27632 2HCl, Thiazovivin, Ripasudil (K-115), Fasu dil, Fasudil (HA-1077) HCl, GSK429286A, at least one of RKI-1447 and PKI-1313；
The jnk inhibitor includes SP600125, JNK Inhibitor IX, AS601245, AS602801 and JNK-IN- At least one of 8；
The DNMT inhibitor includes RG108, Thioguanine, 5-Aza-2'-deoxycytidine (Decitabine), at least one of SGI-1027, Zebularine and 5-Azacytidine (AZA)；
The HMT inhibitor includes EPZ004777, EPZ5676, GSK503, in BIX 01294 and SGC 0946 at least It is a kind of；
The histone demethylase inhibitor includes parnate (tranylcypromine), In Tranylcypromine (2-PCPA) HCl, SP2509,4SC-202, ORY-1001 (RG-6016), GSKJ1 and GSK-LSD1 At least one.
The micromolecular compound is combined as any one and second stage selected from following first stage micromolecular compounds The timing micromolecular compound combination of any one of micromolecular compound, the timing micromolecular compound combines affiliated Micromolecular compound must be contacted with corresponding cell or cellular products in sequence；
Preferably, the first stage micromolecular compound is using any one of following：
5-aza-2-deoxycytidine；
RG108；
TSA；
NaB；
VPA；
5-Aza-2'-deoxycytidine+NaB；
5-Aza-2'-deoxycytidine+VPA+NaB；
5-Aza-2'-deoxycytidine+TSA；
5-Aza-2'-deoxycytidine+TSA+VPA；
5-Aza-2'-deoxycytidine+VPA；
RG108+5-Aza-2'-deoxycytidine；
RG108+VPA；
RG108+NaB；
RG108+VPA+NaB；
RG108+TSA；
RG108+TSA+VPA；
RG108+5-Aza-2'-deoxycytidine+VPA；
RG108+5-Aza-2'-deoxycytidine+VPA+NaB；
The second stage micromolecular compound uses any one of following combination：
VPA+CHIR99021+Repsox+Forskolin；
VPA+CHIR99021+SB431542+Forskolin；
BIO+SB431542+Forskolin；
CHIR99021+Repsox+Forskolin+Rolipram；
CHIR99021+Repsox+Forskolin+Rolipram+SB431542；
CHIR99021+SB431542+Forskolin+Rolipram；
BIO+SB431542+Forskolin+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+NaB；
CHIR99021+Repsox+Forskolin+NaB；
VPA+BIO+SB431542+Rolipram；
BIO+SB431542+Rolipram+SP600125；
VPA+BIO+SB431542+Forskolin；
VPA+CHIR99021+SB431542+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+SP600125；
VPA+CHIR99021+Repsox+Forskolin+8-pCPT-2′-O-Me-cAMP；
VPA+CHIR99021+Repsox+Forskolin+SB431542；
VPA+CHIR99021+SB431542+Forskolin+Tranilast；
VPA+CHIR99021+Repsox+Forskolin+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Rolipram+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Forskolin+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Rolipram；
VPA+BIO+Repsox+Forskolin；
CHIR99021+Repsox+Forskolin；
CHIR99021+Repsox+Rolipram；
CHIR99021+SB431542+Forskolin；
CHIR99021+SB431542+Rolipram；
BIO+Repsox+Forskolin+Rolipram；
BIO+Repsox+Forskolin；
BIO+Repsox+Forskolin+SP600125；
BIO+SB431542+Rolipram；
BIO+SB431542+Rolipram+SP600125；
VPA+BIO+SB431542+Rolipram+SP600125；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+SP600125；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+SP600125；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+SP600125+Y- 27632；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+SP600125+Y-27632 +ascorbate；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+Y-27632+ ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983；
VPA+CHIR99021+SB431542+Forskolin+Go6983；
BIO+SB431542+Forskolin+Go6983；
CHIR99021+Repsox+Forskolin+Go6983+Rolipram；
CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542；
CHIR99021+SB431542+Forskolin+Go6983+Rolipram；
BIO+SB431542+Forskolin+Go6983+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+Go6983+NaB；
CHIR99021+Repsox+Forskolin+Go6983+NaB；
VPA+BIO+SB431542+Rolipram+Go6983；
BIO+SB431542+Rolipram+SP600125+Go6983；
VPA+BIO+SB431542+Forskolin+Go6983；
VPA+CHIR99021+SB431542+Rolipram+Go6983；
VPA+CHIR99021+Repsox+Forskolin+Go6983+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+8-pCPT-2′-O-Me-cAMP；
VPA+CHIR99021+Repsox+Forskolin+Go6983+SB431542；
VPA+CHIR99021+SB431542+Forskolin+Go6983+Tranilast；
VPA+CHIR99021+Repsox+Forskolin+Go6983+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Forskolin+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Rolipram+Go6983；
VPA+BIO+Repsox+Forskolin+Go6983；
CHIR99021+Repsox+Forskolin+Go6983；
CHIR99021+Repsox+Rolipram+Go6983；
CHIR99021+SB431542+Forskolin+Go6983；
CHIR99021+SB431542+Rolipram+Go6983；
CHIR99021+Repsox+Forskolin+SP600125+Go6983；
CHIR99021+Repsox+Forskolin+SP600125；
BIO+Repsox+Forskolin+Go6983+Rolipram；
BIO+Repsox+Forskolin+Go6983；
BIO+Repsox+Forskolin+Go6983+SP600125；
BIO+SB431542+Rolipram+Go6983；
VPA+SB431542+Rolipram+Go6983+Parnate；
BIO+SB431542+Rolipram+Go6983+SP600125；
VPA+BIO+SB431542+Rolipram+Go6983+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+ Y-27632；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+ Y-27632+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ ascorbate。
Second aspect of the present invention provides a kind of method for preparing vascular smooth muscle cells.It is a kind of to utilize the small molecule The method that the cell of compound combination induction differentiation prepares vascular smooth muscle cells, the method is by the cell of differentiation successively with the One stage micromolecular compound, the contact induction of second stage micromolecular compound, are prepared vascular smooth muscle cells.Described point For the cell derived of change in mammal such as people, the cell of the differentiation includes fibroblast, epithelial cell or adipocyte, excellent The cell broken up described in selection of land is fibroblast.
The time that the cell of the differentiation contacts induction with first stage micromolecular compound is 2~10 days, the first stage The time that cellular products after contact induction contact induction with second stage micromolecular compound is 5~20 days.
Some embodiments of the present invention, the minimum effective concentration of specific micromolecular compound is as follows, and given below is dense It spends scope simply to refer to, adaptation can be done on this basis, if other small molecules substitute following small molecule, concentration also may be used To do accommodation.
Forskolin concentration is 2 μM~20 μM；Repsox concentration is 2~15uM；CHIR99021 concentration is 1 μM~10 μ M；VPA concentration is 0.5mM~1.5mM；TTNPB concentration is 3 μM~8 μM；AM580 concentration is 0.03~0.08 μM；EPZ004777 Concentration is 3~8 μM；Go6983 concentration is 1~15 μM；Y-27632 concentration is 3~15 μM, L-Ascorbin acid 2- Phosphate concentration is 0.15~0.25mM；SP600125 concentration is 1~15 μM；5-Aza-2'-deoxycytidine concentration For 0.5~15 μM.
The method of the present invention can also be used or accommodation is done on the basis of the method for the present invention, induce the cell of differentiation Prepare other cell types such as vascular endothelial cell.If it is prepared using micromolecular compound provided by the invention combination except blood vessel Other cells outside smooth muscle cell, vascular endothelial cell can be adjusted corresponding small molecule chemical combination according to actual needs It the concentration of object and is adjusted in combination.
Third aspect present invention also provides a kind of kit or culture solution/base for including micromolecular compound combination.
Fourth aspect present invention also provides micromolecular compound combination, includes the reagent of micromolecular compound combination The application of box or culture solution/base is such as used to mobilize the vascular smooth muscle cells of induction or external evoked preparation in vivo or with it For regenerative medicine seed cell, tissue engineering seed cell and its product of source cell, available for basic research, clinical treatment And tissue engineering product research and development are with producing.
Fifth aspect present invention, also provides the vascular smooth muscle cells that the method is prepared, and the smooth muscle is thin Born of the same parents possess the molecular characterization of standard SMC, and the vascular smooth muscle cells can carry out trillion times amplification, and And purity is high, has good industrialization prospect.
Sixth aspect present invention also provides the application of the vascular smooth muscle cells and its product, is such as used to prepare tissue Engineered blood vessels or regenerative medicine seed cell can also prepare the micromolecular compound combination of vascular smooth muscle cells.
The method of the present invention carries out under conditions of generation inductivity vascular smooth muscle cells are suitble to, and the condition includes example Ingredient, concentration such as culture solution, cultivation temperature, incubation time and other conditions.Fully instructing and combining based on the prior art The exemplary illustration of the present invention, those skilled in the art are not required excessively experiment that can be readily determined above-mentioned condition of culture. Key is to select the required cell-signaling pathways inhibited or activate, and determines the order of function cells signal path.It is in addition, small The concentration and other conditions of molecular compound or its combination also can do accommodation on scope provided by the invention.
The mechanism that the present invention prepares vascular smooth muscle cells using the cell of micromolecular compound combination induction differentiation is as follows： The present invention is opened using lysine deacetylase inhibitors and DNMT inhibitor processing cell, the endogenous of activation into transdifferentiation The transcription of mover；Then the agonist of WNT/ β-catenin, the agonist of cAMP and TGF-beta inhibitor, lysine are utilized The inhibitor of deacetylase, RAR (Retinoic acid) agonist, dnmt rna (DNA Methyltransferas, DNMT) inhibitor, histone methyltransferase (Histone Methyltransferase, HMT inhibitor), the inhibitor of PKC, the inhibitor of ascorbate (ascorbic acid), JNK and ROCK (Rho-associated Protein kinase) inhibitor, promote the first stage obtained by differentiation cellular products continue transdifferentiation；Finally using conventional Vascular smooth muscle cells culture solution or it is commercially available commercialization vascular smooth muscle cells culture solution, the cell of induction is expanded It is cultivated with maintenance.By taking one of scheme as an example, the small molecule combinatorial and Forskolin of 5-AZA and TSA are utilized respectively, The small molecule combinatorial of CHIR99021, VPA, Repsox respectively act on 2~10 days and 5~20 days, fibroblast are oriented Induction obtains vascular smooth muscle cells.
The present invention has following technique effect：The present invention combines chronologically treatment by stages by using micromolecular compound A large amount of vascular smooth muscle cells or its product are obtained to the cell fast and stable sequencing of differentiation, required sample size is few, convenient for adopting Collection, donor source is extensive, is easy to normalizing operation and accurate Quality Control, can scale or personalized preparation vascular smooth muscle cells and The Related products such as engineering blood vessel.In addition, the research of the present invention or cardiovascular and cerebrovascular disease provides cell model or dry Pre- means are widely used in basic medical research, clinical research, clinical treatment or intravascular tissue engineering, have good application Prospect.
Description of the drawings
Fig. 1 is that human skin fibroblasts transdifferentiation is vascular smooth muscle cells method schematic diagram in embodiment 1；Wherein D0, D6, D9 and D23 represent culture 0 day, 6 days, 9 days and 23 days.
Fig. 2 to be induced by micromolecular compound, people source skin fibroblasts to vascular smooth muscle cells transdifferentiation figure, Wherein A figures are skin fibroblasts；B is treated first stage cellular products；C is the vascular smooth that obtains after transdifferentiation Myocyte's (embodiment 1), the cell are the 1st generation (P1) after transdifferentiation；D is the vascular smooth that obtains after 6 transdifferentiation of embodiment Myocyte, the cell are the 1st generation after transdifferentiation；E figures are the dyeing of the significant molecule Actin alpha of vascular smooth muscle cells Identification；
Fig. 3 is expression of the vascular smooth muscle cells molecular marker in the vascular smooth muscle cells of transdifferentiation, and A figures are blood The expression of the significant molecule Actin alpha (ACTA2) of pipe smooth muscle cell, wherein control are represented without small molecule The fibroblast of object combined treatment is closed, iVSMC represents the vascular smooth muscle cells of transdifferentiation acquisition, the two comparison, the gene It is substantially raised in the vascular smooth muscle cells obtained in transdifferentiation；B figures are the significant molecule myocardin of vascular smooth muscle cells Expression, wherein control represents the fibroblast without micromolecular compound combined treatment, and iVSMC represents transdifferentiation The vascular smooth muscle cells of acquisition, the two compare, and are substantially raised in the vascular smooth muscle cells which obtains in transdifferentiation；C Figure is the expression of the significant molecule smooth muscle myosin heavy chain (MHC-10) of vascular smooth muscle cells, Middle control represents the fibroblast without micromolecular compound combined treatment, and iVSMC represents the blood vessel of transdifferentiation acquisition Smooth muscle cell, the two compare, and are substantially raised in the vascular smooth muscle cells which obtains in transdifferentiation；
Fig. 4 is that the vascular smooth muscle cells iVSMC of transdifferentiation can be a large amount of to expand with continuous passage, so as to obtain a large amount of height Pure vascular smooth muscle cells.A figures are the significant molecule MHC-10 of vascular smooth muscle cells respectively in the vascular smooth of transdifferentiation The 1st generation of myocyte, the expression in the 5th generation and the 10th generation, using fibroblast (HuFib) as control；B figures are vascular smooth muscle Cell sign molecule ACTA2 respectively transdifferentiation vascular smooth muscle cells 1st generation, the expression in the 5th generation and the 10th generation, with Fibroblast (HuFib) is as control；C figures are the cellular morphology of the iVSMC in the 5th generation after passage expands, with P1 for phase Than cellular morphology has no significant change, and form is homogeneous；D figures are the cell shape of the iVSMC in the 10th generation after passage expands State, compared with P1 generations, cellular morphology has no significant change, and form is homogeneous；E figures are the growth curve of iVSMC in continuous 10 generation, IVSMC still can continuously rise in value, and be expanded more than upper trillion times；F figures are the separated skin fibroblasts out of adult human body (HuFib) under the sugared culture solution condition of culture of height containing 10% serum, the growth curve in continuous 10 generation.
Specific embodiment
Technical scheme is described in further details in the following with reference to the drawings and specific embodiments, but is not limited to In following experimental program.
Embodiment 1
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), it is first stage culture solution A to replace basic culture solution completely, cultivates cell 2 ~10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ Ml streptomysins (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine, the culture 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in system；100U/ Ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
After 2.2 by 2.1 step process cell 4~8 days, first stage culture solution B is replaced, cultivates cell 2~5 days, the One stage culture solution B refers to：+ 100 μ g/ml streptomysins of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma) (Sigma)+height sugar DMDM culture mediums (Gibco)+50nM~1 μM TSA, 10% hyclone can also be by the culture systems Serum replacement (invitrogen) is substituted with 10%~20% concentration；100U/ml penicillin (Sigma) and 100 μ g/ml chains Mycin (Sigma) can be without using.Cell is cultivated under 37 DEG C, 5%CO2 environment.Step 2.2 and step 2.1 can be closed And directly use the culture solution culture containing AZA and TSA.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (1 μM~30 μM)+Repsox (2~15 μM)+CHIR99021 (1 μM~15 μM)+VPA (0.5mM~1.5mM). 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in the culture systems； 100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2m ML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction, is shown in Fig. 1~Fig. 4,
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 2
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml The μ of streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine+50nM~1 M TSA, in the culture systems 10% hyclone can also by serum replacement (invitrogen) with 10%~20% it is dense Degree substitutes；100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Ring Cell is cultivated under border.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (1 μM~30 μM)+Repsox (2~15 μM)+CHIR99021 (1 μM~15 μM)+VPA (0.5mM~1.5mM). 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in the culture systems； 100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2mML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 3
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine+VPA (0.5mM~1.5mM), in the culture systems 10% hyclone can also by serum replacement (invitrogen) with 10%~ 20% concentration substitutes；100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (1 μM~30 μM)+Repsox (2~15 μM)+CHIR99021 (1 μM~15 μM)+VPA (0.5mM~1.5mM). 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in the culture systems； 100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2m ML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 3
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine+VPA (0.5mM~1.5mM), in the culture systems 10% hyclone can also by serum replacement (invitrogen) with 10%~ 20% concentration substitutes；100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (1 μM~30 μM)+Repsox (2~15 μM)+CHIR99021 (1 μM~15 μM)+VPA (0.5mM~1.5mM). 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in the culture systems； 100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2mML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 4
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine, the training system 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in system；100U/ml Penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (1 μM~30 μM)+Repsox (2~15 μM)+CHIR99021 (1 μM~15 μM)+VPA (0.5mM~1.5mM). 10% hyclone can also be put the concentration of % with 10%~20 by serum replacement (invitrogen) and replace in the culture systems Generation；100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2mML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 5
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine, the training system 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in system；100U/ml Penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (2 μM~20 μM)+Repsox (2~15uM)+CHIR99021 (1 μM~10 μM)+VPA (0.5mM~1.5mM)+ TTNPB (3 μM~8 μM)+AM580 (0.03~0.08 μM)+EPZ004777 (3~8 μM)+Go6983 (1~15 μM)+Y-27632 (3~15 μM)+L-Ascorbin acid 2-phosphate (0.15~0.25m)+SP600125 (8~12 μM).The training system 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in system；100U/ml Penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2mML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 6
1st, the separation of skin fibroblasts is enclosed)
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% C O2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：+ 100 μ g/m l of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma) Streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine, the training system 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in system；100U/ml Penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (2 μM~20 μM)+Repsox (2~15uM)+CHIR99021 (1 μM~10 μM)+VPA (0.5mM~1.5mM)+ TTNPB (3 μM~8 μM)+AM580 (0.03~0.08 μM)+EPZ004777 (3~8 μM).10% tire ox blood in the culture systems It can also be substituted by serum replacement (invitrogen) with 10%~20% concentration clearly；100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2mML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 7
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine, the training system 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in system；100U/ml Penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (2 μM~20 μM)+Repsox (2~15uM)+CHIR99021 (1 μM~10 μM)+VPA (0.5mM~1.5mM)+ TTNPB (3 μM~8 μM)+AM580 (0.03~0.08 μM)+EPZ004777 (3~8 μM)+Go6983 (1~15 μM)+Y-27632 (3~15 μM)+L-Ascorbin acid 2-phosphate (0.15~0.25m).10% hyclone in the culture systems It can be substituted by serum replacement (invitrogen) with 10%~20% concentration；100U/ml penicillin (Sigma) and 100 μ G/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2mML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
Embodiment 8
1st, the separation of skin fibroblasts
1.1 from donor any position obtain the skin histology block of diameter about 1cm, adherent method separates skin into fiber Cell, in basic culture solution, the basic culture solution refers to for separated cell culture：10% hyclone (Hyclone)+ + 100 μ g/ml streptomysins (Sigma) of 100U/ml penicillin (Sigma)+height sugar DMDM.
The a large amount of amplifications of 1.2 cells passage, cell algebraically are thin to vascular smooth muscle for carrying out between the 6th generation and the 12nd generation The transdifferentiation induction of born of the same parents.Start the previous day (Day-1) of activation, inoculating cell density 4~6 × 103/cm2It is incubated at 37 DEG C, 5% CO2Incubator in.
2nd, the activation of skin fibroblasts
During 2.1 startup Transactivation (Day0), basic culture solution is replaced completely as first stage culture solution, culture cell 2~ 10 days, the first stage, culture solution A referred to：10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+100 μ g/ml Streptomysin (Sigma)++ 1 μM~20 μM of height sugar DMDM culture mediums (Gibco) 5-Aza-2 '-deoxycytidine, the training system 10% hyclone can also be substituted by serum replacement (invitrogen) with 10%~20% concentration in system；100U/ml Penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.At 37 DEG C, 5%CO2Cell is cultivated under environment.
3rd, the directional induction (directional induction to vascular smooth muscle cells) of skin fibroblasts
After cell is by above-mentioned activation process, second stage culture solution is changed to completely and carries out cell culture, incubation time 10~15 days, at 37 DEG C, 5%CO2Cell is cultivated under environment.The second stage culture solution is：10% hyclone (Hyclone)+100 μ g/ml streptomysins (Sigma) of+100U/ml penicillin (Sigma)+height sugar DMDM culture mediums (Gibco)+ Forskolin (2 μM~20 μM)+Repsox (2~15uM)+CHIR99021 (1 μM~10 μM)+VPA (0.5mM~1.5mM)+ Y-27632 (3~15 μM).10% hyclone can also be by serum replacement (invitrogen) with 10% in the culture systems ~20% concentration substitutes；100U/ml penicillin (Sigma) and 100 μ g/ml streptomysins (Sigma) can be without using.
4th, the maintenance and amplification of the vascular smooth muscle cells of induction
Then it is changed to conventional vascular smooth muscle culture solution or commercially available commercialization vascular smooth muscle culture solution (Lonza), the cell of induction is carried out that culture and passage is maintained to expand.The conventional vascular smooth muscle culture solution refers to： + 100 μ g/ml streptomysins (Sigma) of 10% hyclone (Hyclone)+100U/ml penicillin (Sigma)+height sugar DMDM cultures Base (Gibco)+2mML-glutamine+40mM bicarbonate, wherein 100U/ml penicillin (Sigma) and 100 μ g/ml Streptomysin (Sigma) can be without using.
5th, the detection of the vascular smooth muscle cells of induction
By the method for immunohistochemical staining and RT-PCR, the molecular marker of vascular smooth muscle cells is detected, it is main Molecular marker is as follows：smooth muscle myosin heavy chain；actin alpha2；myocardin.
The vascular smooth muscle cells and detection that above example is prepared are shown in Fig. 1~Fig. 4, the skin in embodiment into The cell replacement of other differentiation, such as blood cell, adipocyte can be used in fibrocyte.
It is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention has in hereafter (such as embodiment) It can be combined with each other between the various technical characteristics of body description, so as to form new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Claims (10)

1. a kind of micromolecular compound combination, which is characterized in that the small molecule combinatorial includes chronologically be used separately first Stage micromolecular compound and second stage micromolecular compound, the first stage micromolecular compound include DNMT (DNA first Based transferase) inhibitor and lysine deacetylase (Lysine deacetylases inhibitors, KDACIs) inhibit At least one of agent；The second stage micromolecular compound include WNT/ β-catenin agonists, cAMP agonists and TGF-beta acceptor inhibitors.
2. micromolecular compound combination as described in claim 1, which is characterized in that the second stage micromolecular compound is also Including lysine deacetylase inhibitors, RAR agonists, DNMT inhibitor, HMT (histone methyltransferase) inhibitor, Pkc inhibitor, ascorbate (ascorbic acid), jnk inhibitor, in ROCK inhibitor and histone demethylase inhibitor At least one.
3. micromolecular compound combination as claimed in claim 2, which is characterized in that the lysine deacetylase inhibitors Including sodium phenylbutyrate, butyrate, sodium butyrate (NaB), MC1568, CI994 (Tacedinaline), chidamide, CAY10683 (SantacruzaMate A), CUDC-907, M344 (Histone Deacetylase Inhibitor III), LAQ824 (NVP-LAQ824, Dacinostat), Pracinostat (SB939), VPA, Scriptaid, Apicidin, LBH-589 (Panobinostat), MS-275, SAHA (Vorinostat), Trichostatin (TSA), Psammaplin A, PCI-24781 (Abexinostat), Rocilinostat (ACY-1215), Mocetinostat (MGCD0103), 4-Phenylbutyrate (4PB), splitomicin, SRT1720, resveratrol, Sirtinol, APHA, CI-994, Depudecin, FK-228, HC-Toxin, ITF-2357 (Givinostat), Chidamide, RGFP 966, PHOB, BG45, Nexturastat A, TMP269, CAY10603, MGCD-0103, Niltubacin, PXD-101 (Belinostat), Pyroxamide, Tubacin, EX-527, BATCP, Cambinol, At least one of MOCPAC, PTACH, MC1568, NCH51 and TC-H106；
The TGF-β acceptor inhibitor includes 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, At least one of SB525334, LY364947, ASP3029, D4476 and SB505124；
The pkc inhibitor includes Go6983, Ro31-8220Mesylate, Go6976 and Bisindolylmaleimide I At least one of (GF109203X)；
WNT/ β-catenin the agonists include MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, 216763 He of TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB At least one of AR-A014418；
The cAMP agonists include EPAC/RAP1 agonists, 8-Bromo-cAMP, Dibutyryl-Camp and Sp-8-Br- At least one of cAMPs；
The EPAC/RAP1 agonists include Forskolin, IBMX, Prostaglandin E2 (PGE2), NKH477,8- PCPT-2 '-O-Me-cAMP, GSK256066, Apremilast (CC-10004), Roflumilast, Cilomilast, At least one of Rolipram and Milrinone；
The RAR agonists include TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, AT RA, At least one of 13-cis RA, Vitamin A and Vitamin A derivatives；
The ROCK inhibitor includes Y-27632, Y-27632 2HCl, Thiazovivin, Ripasudil (K-115), Fasudil, Fasudil (HA-1077) HCl, GSK429286A, at least one of RKI-1447 and PKI-1313；
The jnk inhibitor includes SP600125, in JNK Inhibitor IX, AS601245, AS602801 and JNK-IN-8 At least one；
The DNMT inhibitor includes RG108, Thioguanine, 5-Aza-2'-deoxycytidine (Decitabine), At least one of SGI-1027, Zebularine and 5-Azacytidine (AZA)；
The HMT inhibitor includes EPZ004777, EPZ5676, GSK503, and at least one in BIX 01294 and SGC 0946 Kind；
The histone demethylase inhibitor includes parnate (tranylcypromine), Tranylcypromine (2- PCPA) HCl, SP2509,4SC-202, ORY-1001 (RG-6016), at least one of GSKJ1 and GSK-LSD1.
4. micromolecular compound combination as claimed in claim 2, which is characterized in that the micromolecular compound is combined as being selected from The timing small molecule of any one of following first stage micromolecular compounds and any one of second stage micromolecular compound Compound combination, micromolecular compound belonging to timing micromolecular compound combination must in sequence with accordingly Cell or cellular products contact；
The first stage micromolecular compound is using any one of following：
5-aza-2-deoxycytidine；
RG108；
TSA；
NaB；
VPA；
5-Aza-2'-deoxycytidine+NaB；
5-Aza-2'-deoxycytidine+VPA+NaB；
5-Aza-2'-deoxycytidine+TSA；
5-Aza-2'-deoxycytidine+TSA+VPA；
5-Aza-2'-deoxycytidine+VPA；
RG108+5-Aza-2'-deoxycytidine；
RG108+VPA；
RG108+NaB；
RG108+VPA+NaB；
RG108+TSA；
RG108+TSA+VPA；
RG108+5-Aza-2'-deoxycytidine+VPA；
RG108+5-Aza-2'-deoxycytidine+VPA+NaB；
The second stage micromolecular compound is using any one of following：
VPA+CHIR99021+Repsox+Forskolin；
VPA+CHIR99021+SB431542+Forskolin；
BIO+SB431542+Forskolin；
CHIR99021+Repsox+Forskolin+Rolipram；
CHIR99021+Repsox+Forskolin+Rolipram+SB431542；
CHIR99021+SB431542+Forskolin+Rolipram；
BIO+SB431542+Forskolin+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+NaB；
CHIR99021+Repsox+Forskolin+NaB；
VPA+BIO+SB431542+Rolipram；
BIO+SB431542+Rolipram+SP600125；
VPA+BIO+SB431542+Forskolin；
VPA+CHIR99021+SB431542+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+SP600125；
VPA+CHIR99021+Repsox+Forskolin+8-pCPT-2′-O-Me-cAMP；
VPA+CHIR99021+Repsox+Forskolin+SB431542；
VPA+CHIR99021+SB431542+Forskolin+Tranilast；
VPA+CHIR99021+Repsox+Forskolin+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Rolipram+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Forskolin+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Rolipram；
VPA+BIO+Repsox+Forskolin；
CHIR99021+Repsox+Forskolin；
CHIR99021+Repsox+Rolipram；
CHIR99021+SB431542+Forskolin；
CHIR99021+SB431542+Rolipram；
BIO+Repsox+Forskolin+Rolipram；
BIO+Repsox+Forskolin；
BIO+Repsox+Forskolin+SP600125；
BIO+SB431542+Rolipram；
BIO+SB431542+Rolipram+SP600125；
VPA+BIO+SB431542+Rolipram+SP600125；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+SP600125；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+SP600125；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+SP600125+Y-27632；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+SP600125+Y-27632+ ascorbate；
VPA+CHIR99021+Repsox+Forskolin+TTNPB+EPZ004777+AM580+Y-27632+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983；
VPA+CHIR99021+SB431542+Forskolin+Go6983；
BIO+SB431542+Forskolin+Go6983；
CHIR99021+Repsox+Forskolin+Go6983+Rolipram；
CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542；
CHIR99021+SB431542+Forskolin+Go6983+Rolipram；
BIO+SB431542+Forskolin+Go6983+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+Go6983+NaB；
CHIR99021+Repsox+Forskolin+Go6983+NaB；
VPA+BIO+SB431542+Rolipram+Go6983；
BIO+SB431542+Rolipram+SP600125+Go6983；
VPA+BIO+SB431542+Forskolin+Go6983；
VPA+CHIR99021+SB431542+Rolipram+Go6983；
VPA+CHIR99021+Repsox+Forskolin+Go6983+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+8-pCPT-2′-O-Me-cAMP；
VPA+CHIR99021+Repsox+Forskolin+Go6983+SB431542；
VPA+CHIR99021+SB431542+Forskolin+Go6983+Tranilast；
VPA+CHIR99021+Repsox+Forskolin+Go6983+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542+A8301；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Forskolin+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram；
VPA+CHIR99021+Repsox+Forskolin+Go6983+Rolipram+SB431542；
VPA+CHIR99021+Repsox+Rolipram+Go6983；
VPA+BIO+Repsox+Forskolin+Go6983；
CHIR99021+Repsox+Forskolin+Go6983；
CHIR99021+Repsox+Rolipram+Go6983；
CHIR99021+SB431542+Forskolin+Go6983；
CHIR99021+SB431542+Rolipram+Go6983；
CHIR99021+Repsox+Forskolin+SP600125+Go6983；
CHIR99021+Repsox+Forskolin+SP600125；
BIO+Repsox+Forskolin+Go6983+Rolipram；
BIO+Repsox+Forskolin+Go6983；
BIO+Repsox+Forskolin+Go6983+SP600125；
BIO+SB431542+Rolipram+Go6983；
VPA+SB431542+Rolipram+Go6983+Parnate；
BIO+SB431542+Rolipram+Go6983+SP600125；
VPA+BIO+SB431542+Rolipram+Go6983+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y- 27632；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y- 27632+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ ascorbate。
5. a kind of cell of micromolecular compound combination induction differentiation using as described in Claims 1 to 4 is any prepares blood vessel The method of smooth muscle cell, which is characterized in that the method by the cell of differentiation successively with first stage micromolecular compound, The contact induction of two-stage micromolecular compound, is finally prepared vascular smooth muscle cells.
6. vascular smooth muscle cells are prepared using the cell of micromolecular compound combination induction differentiation as claimed in claim 5 Method, which is characterized in that the cell of the differentiation contacted the time of induction with first stage micromolecular compound as 2~10 days, The time that cellular products after first stage contact induction contact induction with second stage micromolecular compound is 5~20 days.
7. a kind of kit or culture solution/base of the micromolecular compound combination comprising as described in Claims 1 to 4 is any.
8. a kind of micromolecular compound combination, the application of kit or culture solution/base comprising micromolecular compound combination.
9. a kind of vascular smooth muscle cells, which is characterized in that the vascular smooth muscle cells are any described by Claims 1 to 4 Micromolecular compound combination or prepared by any method of claim 5~6.
10. a kind of application of vascular smooth muscle cells as claimed in claim 9 and its product.
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