CN105769869B - Application of the Oltipraz in human child's early ageing disease senescent phenotypes are reversed - Google Patents
Application of the Oltipraz in human child's early ageing disease senescent phenotypes are reversed Download PDFInfo
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- CN105769869B CN105769869B CN201610265387.2A CN201610265387A CN105769869B CN 105769869 B CN105769869 B CN 105769869B CN 201610265387 A CN201610265387 A CN 201610265387A CN 105769869 B CN105769869 B CN 105769869B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/4965—Non-condensed pyrazines
- A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
Abstract
The invention discloses application of the Oltipraz in human child's early ageing disease senescent phenotypes are reversed.The new application of Oltipraz provided by the present invention is specially following (I) or (II)：(I) application in the product for delaying or reversing human child's early ageing disease senescent phenotypes is prepared；(II) application in delaying or reversing human child's early ageing disease senescent phenotypes.Experiment proves, Oltipraz can reduce the expression quantity of the cell proportion of beta galactosidase stained positive in HGPS MSCs, up-regulation HGPS MSCs center embrane-associated proteins LAP2 and/or LaminB1, and improve the survival abilities of HGPS MSCs in vivo, i.e. Oltipraz can improve the senescent phenotypes of HGPS MSCs, to delaying or reversing human child's early ageing disease senescent phenotypes to play an important roll.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of new application of Oltipraz, and specially Oltipraz is reversing
Application in human child's early ageing disease senescent phenotypes.
Background technology
Aging is that human body gradually moves towards disorderly process, it is considered to be to a variety of sickness influences such as tumour, angiocarpy maximum
Pathogenic factor.Human body has certain self-renewal capacity, while this self-renewal capacity is also strictly regulated and controled.With
The aging of body, stem cell self-control balance are destroyed, and tissue and organ regeneration ability declines, so as to cause aging correlation disease
Disease.Therefore, stem cell aging is considered as one of major incentive of body aging at present, and how to be delayed or reversing stem cell declines
Also become the research hotspot in global aging basis and Study on Transformation field always.Children's early ageing disease (Hutchinson-Gilford
Progeria syndrome, HGPS) be a kind of extremely one among a thousand's class early ageing disease, patient's average life span it is only 13 years old.In recent years
Come, scientific research finds that HGPS pathogenesis, should for the lamin Lamin A producers mutation in human pluripotent stem cells
Mutation has activated abnormal RNA montages, so as to cause the expression of abnormal Lamin A misfolded proteins Progerin, due to
Progerin has lacked degradable signal, therefore leads to its abnormal accumulation in cell, and then causes a series of cell and lack
Fall into, including nuclear membrane exception, the loss of heterochromatin, DNA damage repair ability be damaged with cellular anti-oxidant stress access change
Deng, eventually lead to stem cell cross presenility and exhaust (Kudlow, B.A., Kennedy, B.K., and Monnat, R.J., Jr.
(2007).Werner and Hutchinson-Gilford progeria syndromes:mechanistic basis of
human progeroid diseases.Nat Rev Mol Cell Biol 8,394-404.).Internal multipotential stem cell carries
Preceding aging and exhaustion cause HGPS patient to show the clinical phenotypes for accelerating aging, and it is relevant dynamic that most of patients ultimately succumbs to aging
The angiocardiopathy that pulse atherosclerosis causes, it can be seen that, HGPS is the day for studying mankind's naturally-aged mechanism and Intervention Strategy
Right model and important breakthrough mouth.
Have a small amount of fibroblasts model for utilizing mouse model or obtaining from HGPS patient's separation at present to decline to HGPS
Old process carries out the research report of micromolecular compound intervention, and the micromolecular compound used includes rapamycin
(Rapamycin), sulforaphen (Sulforaphane) and farnesyl transferase inhibitor (Farnesyltransferase
Inhibitors) etc..However have no that the adult stem cell that HGPS patient source is effectively improved using micromolecular compound is declined so far
The in vitro and in vivo research of male cousin's type.
Oltipraz (Oltipraz), trade name Oltipraz come out the seventies in France, are inhaled for treating Egyptian blood
Worm and Manson's schistosomiasis.It there is no any report that Oltipraz is used for HGPS correlative studys at present.
Invention content
The object of the present invention is to provide a kind of new applications of Oltipraz.
The new application of Oltipraz provided by the present invention, the specifically application in following (I) or (II)：
(I) product for delaying or reversing human child's early ageing disease senescent phenotypes is prepared；
(II) delay or reverse human child's early ageing disease senescent phenotypes.
Further, it is the application in following (A) or (B)：
(A) prepare for improve human child early ageing disease patient source adult stem cell senescent phenotypes product；
(B) improve the senescent phenotypes of the adult stem cell in human child early ageing disease patient source.
More specifically, it is the application in following (a) or (b) or (c) or (d)：
(a) it prepares for reducing beta galactosidase dyeing sun in the adult stem cell in human child early ageing disease patient source
The product of the cell proportion of property；Or it reduces beta galactosidase in the adult stem cell in human child early ageing disease patient source and dyes
Positive cell proportion；
(b) prepare for maintain human child early ageing disease patient source adult stem cell nuclear membrane integrality product；
Or maintain the nuclear membrane integrality of the adult stem cell in human child early ageing disease patient source；
(c) the adult stem cell center embrane-associated protein LAP2 for raising human child early ageing disease patient source is prepared
And/or the product of the expression quantity of LaminB1；Or nuclear membrane is related in the adult stem cell in up-regulation human child early ageing disease patient source
The expression quantity of albumen LAP2 and/or LaminB1；
(d) production for improving the survival ability of the adult stem cell in human child early ageing disease patient source in vivo is prepared
Product；Or improve the survival ability of the adult stem cell in human child early ageing disease patient source in vivo.
Certainly, the new application is alternatively the application in following (1) or (2)：
(1) product for inhibiting adult stem cell aging is prepared：
(2) inhibit adult stem cell aging.
Correspondingly, a kind of product is also claimed in the present invention, active constituent is Oltipraz, and the product has as follows
At least one of function：
(a1) delay or reverse human child's early ageing disease senescent phenotypes；
(a2) improve the senescent phenotypes of the adult stem cell in human child early ageing disease patient source；
(a3) the thin of beta galactosidase stained positive in the adult stem cell in human child early ageing disease patient source is reduced
Born of the same parents' ratio；
(a4) the nuclear membrane integrality of the adult stem cell in human child early ageing disease patient source is maintained；
(a5) the adult stem cell center embrane-associated protein LAP2 in up-regulation human child early ageing disease patient source and/or
The expression quantity of LaminB1；
(a6) survival ability of the adult stem cell in human child early ageing disease patient source in vivo is improved；
(a7) inhibit the aging of adult stem cell.
In the present invention, the adult stem cell in human child's early ageing disease patient source is specially human child's early ageing disease
The mescenchymal stem cell in patient source.The adult stem cell is specially mescenchymal stem cell.
More specifically, the mescenchymal stem cell in human child's early ageing disease patient source is according to including the following steps
Method prepare：
(1) multipotential stem cell in human child early ageing disease patient source is subjected to embryoid body differentiation (such as differentiation 14 days), obtained
Obtain embryoid body；
(2) embryoid body is inoculated in matrigel (matrigel) coated culture plate and cultivated, cultivated to fibre
It ties up shape cell and (incubation time such as 2 weeks) occurs；After primary passage, sorting wherein CD73, CD90, CD105 is the positive
Cell population, the mescenchymal stem cell in as described human child's early ageing disease patient source.
In one embodiment of the invention, the multipotential stem cell in human child's early ageing disease patient source is the mankind
The induced multi-potent stem cell in virgin early ageing disease patient source；Specially " Liu, G.H., Barkho, B.Z., Ruiz, S., Diep, D.,
Qu,J.,Yang,S.L.,Panopoulos,A.D.,Suzuki,K.,Kurian,L.,Walsh,C.,et al.(2011a)
.Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford
" HGPS-iPSCs " disclosed herein progeria syndrome.Nature 472,221-225. " one.
Wherein, the product can be drug.
In an embodiment of the present invention, a concentration of 25 μM of the use of the Oltipraz.
The present invention generates corresponding MSCs using acquired HGPS-iPSC and cHGPS-iPSCs differentiation, and using this two
MSCs of the kind with identical genetic background has by the screening of in vitro and in vivo research method and delays or reverse HGPS senescent phenotypes
Micromolecular compound Oltipraz.It is demonstrated experimentally that Oltipraz can reduce beta galactosidase dyeing sun in HGPS-MSCs
Property cell proportion, up-regulation HGPS-MSCs center embrane-associated proteins LAP2 and/or LaminB1 expression quantity, and improve HGPS-
The survival abilities of MSCs in vivo, i.e. Oltipraz can improve the senescent phenotypes of HGPS-MSCs, to delaying or reversing the mankind
Virgin early ageing disease senescent phenotypes play an important roll.
Description of the drawings
Fig. 1 is that present invention produces the MSC that the induced multi-potent stem cell differentiation for carrying LMNA (C1824T) mutation obtains.Its
In, A is the data of flow cytometer showed MSC surface moleculars label expression；B determines that carrying LMNA (C1824T) is mutated for genotype identification
(GC-MSC represents cHGPS-MSC)；C is identification (the GC expressions cHGPS-MSC that Progerin expression rises；HGPS represents HGPS-
MSC).In figure, * * * represent p<0.001.
Fig. 2 is the phenotype that MSC derived from HGPS-iPSC of the present invention has aging.Wherein, A for aging correlation β-
Galactosidase coloration result；B is the qualification result (cylindricality of aging correlation nuclear membrane marker LAP2 and Lamin B1 expressions
In figure, expression quantity is relatively low for HGPS-MSC；Expression quantity is relatively high for cHGPS-MSC)；C is MSC after transplanting in vivo
The result of cell viability measurement detection.In figure, * represents p<0.05；* represents p<0.01；* * represent p<0.001.GC is represented
cHGPS-MSC；HGPS represents HGPS-MSC.
Fig. 3 is the senescent phenotypes that Oltipraz (Oltipraz) can reverse HGPS-MSC.Wherein, A for aging correlation β-
Galactoside enzyme dyeing coloration result；B is the identification of aging correlation nuclear membrane molecular marked compound LAP2 and Lamin B1 expressions
As a result；C is the data of survival ability enhancing in the HGPS-MSC bodies of Oltipraz processing.In figure, " control (i.e. Vehicle) " table
Show and be not added with Oltipraz.In figure, * * represent p<0.01；* * represent p<0.001.GC-MSC represents cHGPS-MSC.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Cell culture condition in following embodiments is 37 DEG C unless otherwise specified, 5%CO2。
Quantitative experiment in following embodiments is at least set to be repeated three times, as a result takes mean value.
Oltipraz (Oltipraz)：Sigma Products, catalog number O9389.>=98% (HPLC), not
Name：4-Methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione；BRN 0978110；CCRIS 4048；NSC
347901；RP 35972；Empirical formula (Xi Er representations) C8H6N2S3；Molecular weight 226.34；EINECS numbers 264-
736-6。
The iPSC (abbreviation HGPS-iPSCs) in patient HGPS source：Be recorded in " Liu, G.H., Barkho, B.Z., Ruiz,
S.,Diep,D.,Qu,J.,Yang,S.L.,Panopoulos,A.D.,Suzuki,K.,Kurian,L.,Walsh,C.,et
al.(2011a).Recapitulation of premature ageing with iPSCs from Hutchinson-
Gilford progeria syndrome.Nature 472,221-225. " text, the public can obtain at applicant, only use
It is used in repeating present invention experiment.
Be recorded in the control cell lines (abbreviation cHGPS-iPSCs) that HGPS-iPSC has identical genetic background " Liu,
G.H.,Suzuki,K.,Qu,J.,Sancho-Martinez,I.,Yi,F.,Li,M.,Kumar,S.,Nivet,E.,Kim,J.,
Soligalla,R.D.,et al.(2011b).Targeted gene correction of laminopathy-
associated LMNA mutations in patient-specific iPSCs.Cell Stem Cell 8,688-
694. " one texts, the public can obtain at applicant, be only used for repeating present invention experiment use.
1st, the fluorescent labeled antibody for flow cytometry sorting MSC is as follows：
The anti-human cell surface identification molecule CD90 antibody of fluorescein FITC labels, BD Biosciences, article No.：
555595。
The anti-human cell surface identification molecule CD73 antibody of fluorescein PE labels, BD Biosciences, article No.：
550257。
The anti-human cell surface identification molecule CD105 antibody of fluorescein APC labels, BD Biosciences, article No.：17-
1057-42。
Fluorescein APC marks isotype control Ab, BD Biosciences, article No.：555751.
Fluorescein PE marks isotype control Ab, BD Biosciences, article No.：555749.
Fluorescein FITC marks isotype control Ab, BD Biosciences, article No.：555742.
2nd, it is as follows for the antibody of immunofluorescence：
Anti-human LAP2 antibody, BD Biosciences, article No.：611000.
Anti-human Lamin B1 antibody, Santa Cruz Biotechnology, article No.：sc-6216.
3rd, the culture medium prescription in following embodiments is as follows：
1) induced multi-potent stem cell (iPSC) culture medium prescription：80% (volumn concentration) DMEM/F12 culture mediums
(Invitrogen), 20% (volumn concentration) Knockout serum substitutes (Invitrogen), 0.1mM non-essential aminos
Sour (Invitrogen), 1mM GlutaMAX (Invitrogen), 1% (1g/100ml) penicillin/streptomycin
(Invitrogen), 55 μM of beta -mercaptoethanols (Invitrogen), 10ng/ml people recombinate FGF2 (Joint Protein
Central)。
2) mescenchymal stem cell (MSC) culture medium prescription：MEM culture mediums (Invitrogen, 12571071)；10% (body
Product percentage composition) fetal calf serum (Invitrogen, 10091148)；1% (1g/100ml) penicillin/streptomycin
(Invitrogen, 15070-063)；10ng/ml recombinant human fibroblast growth factors (JPC, bFGF)；5ng/ml TGFβ
(Humanzyme, HZ1131).
Embodiment 1, the identification for obtaining HGPS-MSC and its senescent phenotypes
1st, the culture of the induced multi-potent stem cell in patient HGPS source and the differentiation of MSC
Mutation (LMNA genome sequences have occurred in the iPSC (HGPS-iPSCs) in patient HGPS source, wherein LMNA genes
For GenBank：NG_008692.2；CDNA sequence is GenBank：NM_170707.3), for C1824T, (GGCGG's mutation type dashes forward
Become GGTGG), which is LMNA cDNA sequences GenBank：CDS regions in NM_170707.3.This implementation
The further vitro directed differentiations of HGPS-iPSCs are mescenchymal stem cell (HGPS-MSC) by example.Specific method is as follows：
HGPS-iPSCs is carried out embryoid body (EB) to break up, is broken up 14 days, EB is inoculated in matrigel (matrigel) packet
It is cultivated in 6 orifice plates of quilt, continues to cultivate 2 weeks to fibrous cell's appearance.After primary passage, fluidic cell is utilized
Art sorting wherein CD73, CD90 and CD105 are the mescenchymal stem cell of positive cell population, as patient HGPS source
(being denoted as HGPS-MSC) (see A in Fig. 1).Experiment sets the MSC conducts that the cHGPS-iPSCs differentiation after gene correction obtains simultaneously
Control, gained mescenchymal stem cell are denoted as cHGPS-MSC.
2nd, the senescent phenotypes identification of HGPS-MSC
(1) genotype and the identification of Progerin protein accumulations
The genome of two kinds of MSC cells (HGPS-MSC and cHGPS-MSC) obtained using primer (P1+P2) to step 1
PCR is carried out respectively, and the PCR product for obtaining purifying is sequenced.The results show that HGPS-MSC carries the special mutation of HGPS
(C1824T), and the mutation has been corrected (B in see Fig. 1) in cHGPS-MSC.
P1：5’-TACAAGCTTGCTCCCGTTCTCTCTTCTTTTCCTCTTAAGCT-3’；
P2：5’-GAAGGGAGGTAGCATCTCCCCCATCCCTCACACTC-3’.
Further, the two kinds of MSC cells obtained by real-time fluorescence quantitative RT-PCR (primer P3+P4) detecting step 1
The expression of Progerin genes in (HGPS-MSC and cHGPS-MSC), internal reference are 18S rRNA, primer sequence 18S-F
And 18S-R.The results show that HGPS-MSC is compared to the big panel height expression Progerin of cHGPS-MSC (see C in Fig. 1).Patient HGPS
Meet the feature of patient's HGPS cell model in special mescenchymal stem cell containing excessive Progerin toxalbumin.
P3：5’-GCGTCAGGAGCCCTGAGC-3’；
P4：5’-GACGCAGGAAGCCTCCAC-3’.
18S-F：5’-GTAACCCGTTGAACCCCATT-3’；
18S-R：5’-CCATCCAATCGGTAGTAGCG-3’.
Wherein, primer sequence P3 and P4 has been recognized from having delivered document, source for " Liu, G.H.,
Barkho,B.Z.,Ruiz,S.,Diep,D.,Qu,J.,Yang,S.L.,Panopoulos,A.D.,Suzuki,K.,Kurian,
L.,Walsh,C.,et al.(2011a).Recapitulation of premature ageing with iPSCs from
A Hutchinson-Gilford progeria syndrome.Nature 472,221-225. " texts.
(2) senescence associated-β-galactosidase dyeing identification
The dyeing of cell ageing beta galactosidase is that one kind is based on senescence associated-β-galactosidase dyeing during aging
(senescence-associated beta-galactosidase) activity level raises and senile cell or tissue is carried out
The method for dyeing detection.
The HGPS-MSC cells and cHGPS-MSC cells obtained respectively using step 1 sucks 6 holes as examination cell (the 5th generation)
The cell culture fluid for examination cell cultivated in plate, is washed 1 time with PBS, adds dyeing fixer (4% paraformaldehyde), room
Temperature fixes 15 minutes.Fixer is discarded, is washed 1 time with PBS, 1ml dyeing working solutions are added in per hole.Using X-Gal as substrate, declining
Navy blue product can be generated under the beta galactosidase catalysis of old specificity.It can be observed under common light microscope
The aging situation of cell or tissue, and it is further thin to the senescence associated-β-galactosidase dyeing stained positive in two groups of cells
Born of the same parents' ratio carries out quantitative statistical analysis.
As a result as shown in A in Fig. 2, the senescence associated-β-galactosidase dyeing staining positive cells ratio of HGPS-MSC groups
(51.6 ± 6.1%) are significantly higher than the senescence associated-β-galactosidase dyeing staining positive cells ratio of cHGPS-MSC groups
(36.0 ± 6.2%) (P<0.05).As it can be seen that HGPS-MSC cells shows go out the symptom of serious aging.
(3) nuclear structures damage situations are identified in the special mescenchymal stem cell of patient HGPS
Nuclear membrane differential protein in mescenchymal stem cell is marked using immunofluorescence technique, to evaluate the complete of nuclear membrane
Property.It is as follows：The poly first for examination cell (HGPS-MSC and cHGPS-MSC) 4% that will be incubated on coverslip
Aldehyde room temperature fixes 30 minutes, after PBS rinsings (3 times, 5 minutes/time), using containing 0.4% (volumn concentration) Triton X-
100 PBS is incubated at room temperature 30 minutes, then uses 10% (volumn concentration) donkey serum (Jackson instead
ImmunoResearch Laboratories, Inc. article No.s：017-000-121) room temperature is closed 1 hour.It uses instead and adds respectively later
Add primary antibody (anti-LaminB1, rabbit source, abcam companies, ab16048)；Anti-LAP2 alpha, rabbit source, abcam companies,
Ab5162 confining liquid) is in 4 DEG C of overnight incubations.After PBS rinsings (3 times, 5 minutes/time), corresponding secondary antibody (anti-is then added in
LaminB1：The donkey anti-rabbit antibody of Alexa555 labels, Invitrogen companies, article No. A-31572；anti-LAP2
alpha：The donkey anti-rabbit antibody of Alexa488 labels, Invitrogen companies, article No. A-21206), it is incubated at room temperature 1 hour.
After PBS rinsings (3 times, 5 minutes/time), with Hoechst 33258 (Invitrogen, the article No. of a concentration of 2g/ml of work：
H3569 it) is incubated at room temperature 15 minutes, last mounting and observation.
As a result it shows：In the special mescenchymal stem cell of patient HGPS (HGPS-MSC) nuclear membrane differential protein LaminB1 and
There is exception in the nuclear membrane of LAP2 labels, shows as fluorescence intensity reduction and defect (as shown in B in Fig. 2) occurs in nuclear membrane,
It is found by statistics, the nuclear membrane indicated in HGPS-MSC by nuclear membrane GAP-associated protein GAP LAP2 and LaminB1 immunofluorescence dyeing is different
Normal nucleus amount is respectively cHGPS-MSC original 39.0 ± 2.4% and 63.8 ± 1.6%, shows that patient HGPS is special
Nuclear structures are damaged in mescenchymal stem cell (HGPS-MSC).
(4) transfer ability detection in vivo
It is obtained using slow virus carrier virus (brightness fine horse biology, article No. FT124) infection step 1 of expressing luciferase
HGPS-MSC cells and cHGPS-MSC, infection titer 109Slow virus carrier infection 5 × 105Cell, the specific steps are：First
By 5 × 105Cell inoculation in six orifice plates, is changed liquid culture by/hole after 8-12 hours, change liquid after 1 hour by 109Slow virus carrier
It adds in culture medium, weak vibrations mixing, liquid is changed after continuing thereafter with culture 12 hours, after changing liquid 36 hours, i.e. virus infection 48
Cell is collected after hour and is resuspended in the PBS containing 5% glucose, cell density 106/ 50 μ l then infect virus
CHGPS-MSC pallium cell injections enter NOD/SCID immunodeficient mouses (Beijing tie up the limited public affairs of tonneau China's experimental animal technology
Department) in left leg tibialis anterior, the HGPS-MSC pallium cell injections of virus infection are entered into NOD/SCID immunodeficient mouses (Beijing
Tie up experimental animal Technology Co., Ltd. of tonneau China) in right leg tibialis anterior, per injection volume is 50 μ l.After cell transplantation 5 days
It is detected with small animal imaging system (IVIS Spectrum Pre-clinical In Vivo Imaging System).
Specific practice is as follows：Then the 200 μ L/ mouse of fluorescein substrate of 300 μ g/ml are injected intraperitoneally in anesthetized animal first.15 minutes
Afterwards, mouse is put into instrument, and the automatic fluorescence signal for collecting luciferase is evaluated by the signal value of the left and right leg of same animal
Cell viability measurement.Cell survival is more, then fluorescence signal is stronger, since two groups of MSC are transplanted to mouse by this experiment respectively
Left and right leg, therefore by comparing the fluorescence signal value of left and right, it is possible to judge which group MSC has stronger internal survival energy
Power, with the increase of size of animal, it is possible to complete numerical statistic, judge that two groups of MSC whether there is significant difference.
As a result it shows：The fluorescent value that survivaling cell generates after transplanting in HGPS-MSC bodies be cHGPS-MSC 29.3 ±
15.4%.C is fluorescence signal direct result figure in Fig. 2；The left hand view of column diagram is the fluorescence signal to C in Fig. 2 in C in Fig. 3
The statistical result of value.
Result above fully shows that the HGPS-MSC cells that step 1 is built can show typical children's early ageing disease
Symptom.
Embodiment 2, the micromolecular compound using HGPS-MSC screening anti-aging phenotypes
The present inventor is established when cultivating HGPS-MSC prepared by embodiment 1 for the small of anti-aging phenotype
The screening technique of molecular compound finds that Oltipraz (Oltipraz) can reverse the senescent phenotypes of HGPS-MSC.Specifically such as
Under：
By HGPS-MSC cells with 5 × 104It is laid in six orifice plates per hole, cell is adherent to be started for second day, in experimental group
It adds in and continues culture (while the HGPS- that setting is handled without Oltipraz containing 25 μM Oltipraz (Oltipraz) culture medium
MSC cells are as control), 21 are continuously cultivated, during which cell passes on experience three times, then to through the Oltipraz
(Oltipraz) HGPS-MSC of processing and control carry out analysis below：
(1) senescence associated-β-galactosidase dyes
Specific method is carried out with reference to 1 step 2 of embodiment (2).
As a result as shown in A in Fig. 3, compared to control, Oltipraz is added in HGPS-MSC cell culture systems can be with
The cell proportion of beta galactosidase stained positive is made to be reduced to 44.1 ± 15.1%.
(2) Immunofluorescence test nuclear membrane molecular marker
Specific method is carried out with reference to 1 step 2 of embodiment (3).LAP2 and Lamin B1 immunofluorescence dyeing signals are stronger, together
When the nuclear membrane that indicates it is more complete, then illustrate nuclear membrane integrality higher, so if the LAP2 and Lamin of two groups of cells
There were significant differences for the nuclear membrane integrality of B1 dyeing marks, it is possible to judge that drug-treated can reverse the nuclear membrane of HGPS-MSC
Abnormal senescent phenotypes.
The results show that compared to HGPS-MSC and not adding Oltipraz control group, it is added to the experimental group of Oltipraz
In increase by 3.3 ± 0.2 and 1.3 respectively by the nucleus amount with complete nuclear membrane of LAP2 and Lamin B1 dyeing marks ±
0.1 times (B in Fig. 3).
(3) transplanting detection in vivo
Specific method is carried out with reference to 1 step 2 of embodiment (4), and the difference lies in will infect the slow of expressing luciferase
The HGPS-MSC cell infusions handled by Oltipraz of virus vectors virus enter the tibialis anterior of the right leg of immunodeficient mouse
In, by the HGPS-MSC cell infusions handled without Oltipraz of the slow virus carrier for having infected expressing luciferase virus
In the tibialis anterior for entering the left leg of immunodeficient mouse.
The results show that for HGPS-MSC and not adding Oltipraz control group, it is added to the experimental group body of Oltipraz
The survival ability enhancing 2.8 ± 1.1 times (C in Fig. 3) of cell after interior transplanting.
The result explanation of the present embodiment, Oltipraz can reduce beta galactosidase stained positive in HGPS-MSCs
The expression quantity of cell proportion, up-regulation HGPS-MSCs center embrane-associated proteins LAP2 and/or LaminB1, and improve HGPS-MSCs
Survival ability in vivo, i.e. Oltipraz can improve the senescent phenotypes of HGPS-MSCs, early to delaying or reversing human child
The disease senescent phenotypes that decline play an important roll.
Claims (9)
1. application of the Oltipraz in the product for delaying or reversing human child's early ageing disease senescent phenotypes is prepared.
2. Oltipraz prepare for improve human child early ageing disease patient source adult stem cell senescent phenotypes production
Application in product.
3. application of the Oltipraz in following (a) or (b) or (c) or (d)：
(a) it prepares for reducing beta galactosidase stained positive in the adult stem cell in human child early ageing disease patient source
The product of cell proportion；
(b) prepare for maintain human child early ageing disease patient source adult stem cell nuclear membrane integrality product；
(c) prepare for raise human child early ageing disease patient source adult stem cell center embrane-associated protein LAP2 and/or
The product of the expression quantity of LaminB1；
(d) product for improving the survival ability of the adult stem cell in human child early ageing disease patient source in vivo is prepared.
4. application of the Oltipraz in the product for inhibiting adult stem cell aging is prepared.
5. the application according to Claims 2 or 3, it is characterised in that：The adult in human child's early ageing disease patient source
Stem cell is the mescenchymal stem cell in human child early ageing disease patient source.
6. application according to claim 4, it is characterised in that：The adult stem cell is mescenchymal stem cell.
7. application according to claim 5：The mescenchymal stem cell in human child's early ageing disease patient source be according to
What the method included the following steps prepared：
(1) multipotential stem cell in human child early ageing disease patient source is subjected to embryoid body differentiation, obtains embryoid body；
(2) embryoid body is inoculated in the coated culture plate of matrigel and cultivated, cultivated to fibrous cell and occur；Again
After once passing on, the cell population that wherein CD73, CD90, CD105 are the positive, as described human child's early ageing are sorted
The mescenchymal stem cell in disease patient source.
8. application according to claim 7, it is characterised in that：The multipotency in human child's early ageing disease patient source is done carefully
Born of the same parents are the induced multi-potent stem cell in human child early ageing disease patient source.
9. according to the application any in claim 1-4, it is characterised in that：The product is drug.
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| CN114432302A (en) * | 2020-11-05 | 2022-05-06 | 中国科学院上海营养与健康研究所 | Application of small molecule SR9009 in resisting aging and relieving chronic inflammation caused by aging |
| CN112546216B (en) * | 2020-11-20 | 2022-10-28 | 西湖大学 | Application of small molecular compound oltipraz in preparation of medicine for enhancing humoral immune response |
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| Adult stem cell and mesenchymal progenitor theories of aging;So-ichiro Fukada 等;《Frontiers in Cell and Developmental Biology》;20140328;第2卷;第1-9页 * |
| Nrf2对肿瘤的双重作用;潘灏 等;《医学研究生学报》;20120531;第25卷(第5期);第549-551页 * |
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