CN106318905A - Target for treating ischemic disease by thromboxane A2 receptor serving as adipose-derived stem cells - Google Patents
Target for treating ischemic disease by thromboxane A2 receptor serving as adipose-derived stem cells Download PDFInfo
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Abstract
The invention relates to a Target for treating the ischemic disease by thromboxane A2 receptor (TP) serving as adipose-derived stem cells. Particularly, the TP of the adipose-derived stem cells is inactivated or inhibited, and/or calpain is inactivated or the activity of the calpain is reduced, and/or the activity of beta-chain protein is improved. The invention further provides a method for improving the capacity of the adipose-derived stem cells for promoting angiogenesis or prompting differentiation of the adipose-derived stem cells to vascular endothelial cells. The method comprises improving the activity of the beta-chain protein of the stem cells. The invention further relates to application of a TP antagonist and/or a calpain inhibitor in preparation of a medicine for promoting the differentiation capacity of the adipose-derived stem cells to the vascular endothelial cells and improving the angiogenesis capacity, application of the adipose-derived stem cells in preparation of a medicine for preparing the ischemic disease, and a method for preparing the vascular endothelial cells or a blood vessel from the adipose-derived stem cells.
Description
Technical field
The present invention relates to the thromboxane element A2 receptor target as fat stem cell treatment ischemic diseases.
Background technology
Adipose-derived stem cell (adipose-derived stem cell, ADSC) is in recent years from fat group
Knit a kind of stem cell with multi-lineage potential of middle isolated.Separate the fatty tissue obtaining ADSC
The fatty tissue discarded after being mainly derived from liposuction the earliest and cut in anaplasty.At present it has proven convenient that permissible
ADSC is obtained from the fatty tissue of the different plant species such as people, Mus, rabbit, pig, sheep and Canis familiaris L.；Derive from not
Population doubling time with individual ADSC is basically identical, the most about 60h；Research shows, ADSC has
There are stronger self-renewal capacity and low Aging feature, are preferable source of human stem cell (land in organizational project
Big, " progress of adipose tissue-derived stem cell ", " China's clinicist's magazine " (electronic edition),
In October, 2013, volume 7, the 20th phase).
(Lu F, Mizuno H, Uysal AC etc., the Improved viability of random pattern such as Lu
Skin flaps through the use of adipose-derived stem cells, Plast Reconstr Surg, 2008,
121:50-58) research discovery, ADSC possesses the function promoting angiogenesis.
Nakagami etc. (Hironori Nakagami, Ryuichi Morishita, Kazuhisa Maeda etc.,
Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy,
Ather Thromb, 2006, l3 (2): 77-81) think, ADSC angiogenesis promoting ability and BMSC do not have
There is difference.ADSC can promote angiogenesis not only due to it has the potential being divided into vascular endothelial cell
And and then participate in blood vessel composition, it addition, its secretory action plays an important role the most wherein.Rehman
Showing Deng the experiment of [33], the secretory volume of the ADSC in low-oxygen environment, its VEGF is about normoxia
In environment 5 times；ADSC supernatant in low-oxygen environment can be obviously promoted the growth of vascular endothelial cell and subtract
Few endothelial cell apoptosis.
(Rehman J, Traktuev D, Li J etc., the Secretion of angiogenic and such as Kim
Antiapoptotic factors by human adipose stromal cells, Circulation, 2004,
109:1292-1298) Induction experiments that breaks up to blood vessel endothelium of the stem cell that carries out finds, ADSC compares BMSC
More complete tubular structure can be formed.
Thromboxane element A2 receptor is again TP, belongs to G-protein coupling receptor, has seven cross-film features.People
The TP of class has two kinds of hypotypes: TP α and TP β, is made up of 343 and 407 aminoacid respectively, although
TP α and TP β is the most only that the existence of C end afterbody is different, but has some researchs to show, both are in merit
There is also some on can different, being TP β as it turned out now, rather than TP α is raw at suppression blood vessel endothelium
Migration that the long factor (VEGF) is induced and angiogenesis are worked (Yang Guibao etc., thromboxane A2 receptor with
The relation of cardiovascular disease, " Chinese Medicine ", volume 2011,6, the 5th phase).
Summary of the invention
The present invention uses construction of recombinant plasmid technology, laser scanning confocal microscopy, siRNA knockdown
Technology, Real-time round pcr, flow cytometry, immunoblot assay and laser-Doppler imaging system
The biology techniques means that system etc. are advanced, with wild type and thromboxane element A2 receptor (TP) knock out little
Mus adipose-derived stem cells (ADSC) is object, and research TP signal path is to ADSC angiogenesis promoting
Impact and molecular mechanism thereof.Result of study shows: after the ADSC induction differentiation that TP knocks out or is suppressed
The ADSC that endothelial cell marker thing CD31 positive rate is originated apparently higher than wild type, and at body and in vitro base
One-tenth pipe ability on matter glue is remarkably reinforced, and endothelial cell marker thing CD31 expresses and dramatically increases.Equally, exist
In lower limb ischemia model, TP knocks out the therapeutic effect that can significantly improve ADSC, and new vessels substantially increases
Many.In vitro culture finds, TP knocks out and causes ADSC intracellular β-catenin (beta chain albumen) level to raise,
Strike low β-catenin to reduce differentiation efficiency and become pipe ability with ADSC, reduce ADSC to lower limb ischemia
Therapeutic effect.Finding, TP knocks out the work of rear ADSC intracellular calcium dependent proteolytic enzyme calpain simultaneously
Property be remarkably decreased, process LAN TP, calpain activity rising, β-catenin level decline, endothelial differentiation
Efficiency reduces.Further, TP knocks out and causes intracellular free calcium level to decline, the stream respond decline of calcium wink,
Calcium ion level after suppression TP process LAN can recover activity and the ADSC differentiation capability of calpain.Cause
This, our result indicate that suppression TP is expected to become and improve with ADSC for material treatment ischemic diseases
New Policy.
Therefore, first aspect present invention provides a kind of adipose-derived stem cell or the combination containing this stem cell
Thing, described stem cell:
(1) TP inactivates or is suppressed；And/or
(2) calpain activity inactivation or reduction；And/or
(3) beta chain protein active improves.
In a specific embodiment, the gene expressing TP in described stem cell is knocked or causes TP
The sudden change that activity reduces or loses.
In a specific embodiment, the gene expressing calpain in described stem cell is knocked or leads
Cause calpain activity to reduce or loss mutation.
In a specific embodiment, described stem cell contains the expression vector expressing beta chain albumen, thus β
-catenin process LAN.
In a specific embodiment, described stem cell from mammal such as people, Mus, rabbit, pig, sheep and
The fatty tissue of Canis familiaris L. etc..
The present invention also provides for ability or the promotion of the adipose-derived stem cell angiogenesis promoting of a kind of external raising
The method that adipose-derived stem cell breaks up to vascular endothelial cell, described method includes improving described stem cell
The activity of beta chain albumen.
In a specific embodiment, the activity of the beta chain albumen of the described stem cell of described raising includes:
(1) reduce, suppress or eliminate the TP activity of described stem cell；And/or
(2) reduce, suppress or eliminate the activity of the calpain of described stem cell；And/or
(3) the beta chain albumen in stem cell described in process LAN.
In a specific embodiment, the TP activity reducing, suppress or eliminating described stem cell includes: strike
Except described stem cell is expressed the gene of TP, or the gene expressing TP in described stem cell is made to undergo mutation,
Or make described stem cell contact with TP antagonist.
In a specific embodiment, the calpain activity reducing, suppress or eliminating described stem cell includes:
Knock out the gene expressing calpain in described stem cell, or make described stem cell is expressed the base of calpain
Because undergoing mutation, or described stem cell is made to contact with calpain inhibitor.
In a specific embodiment, TP antagonist selected from AH 29848, SQ-29548, AH-23848,
GR-32191、ONO-11120、BM-13177、BM-13505、SKF-88046、L-655240、S-145、
TMQ, R-68070, CV-4151 and Bay-K 8644.
In a specific embodiment, calpain inhibitor is selected from: the N-acetyl group-L-bright ammonia of leucyl-L-
The brightest ammonium aldehyde of acyl-L-(ALLN), methyl fluoride ketone (Z-LLY-FMK), calpastatin (Calpastatin),
α-one base amide-type inhibitor and Calpeptin (CPT).
Present invention additionally comprises TP antagonist and/or calpain inhibitor and promote adipose-derived doing carefully in preparation
Born of the same parents are to the differentiation capability of vascular endothelial cell and/or the purposes in improving the medicine of its angiogenesis ability.
Present invention additionally comprises the adipose-derived stem cell of the present invention or the compositions containing this stem cell is controlled in preparation
Treat the purposes in the medicine of ischemic diseases.
Present invention additionally comprises a kind of external prepare by adipose-derived stem cell or containing the compositions of this stem cell
Vascular endothelial cell or the method for blood vessel, the stem cell that described method includes making the present invention adipose-derived with
VEGF contacts.
Accompanying drawing explanation
Fig. 1: process ADSC with VEGF and can activate TXA2/TP signal path.A, flow cytometry
Analyze the surface marker of ADSC；The expression of prostaglandin receptor in b, ADSC；c、VEGF
After process, immune-blotting method COX-1 and COX-2 expression in ADSC；D-f, VEGF process
After, the mRNA relative expression change of Real-time round pcr detection TxB2, TxAS and TP.
Fig. 2: TP knock out can improve VEGF induction ADSC to the differentiation of endotheliocyte.A, at VEGF
In process group and matched group, CD34 (green, the first row), CD31 (red, the second row), nucleus (DAPI,
Indigo plant, the third line) immunofluorescence photograph；B, to a scheme CD34+/CD31+The quantitative statistics of cell；c、
In VEGF process group and matched group, the relative mRNA level in-site of the endothelial marker of ADSC；D, at VEGF
CD31 in process group and matched group, in Flow cytometry ADSC+Cell；E, to d scheme CD31+
Cell quantification statistics；F, in VEGF process group and matched group, the ADSC of WT and TP KO
One-tenth pipe experiment；G, to tubular structure quantitative in f figure；H, in VEGF process group and matched group,
The relative mRNA level in-site of the angiogenesis promoting factor in the ADSC of WT and TP KO；I, WT with
And the generation blood vessel experiment in the ADSC of TP KO matrigel in vivo；J, the glue in i figure is cut
The representative graph of sheet soldier's immunofluorescence dyeing；K, to EGFP in j figure+/CD31+Blood vessel quantitative.
Fig. 3: TP knocks out the therapeutic efficiency that can improve ADSC in mice lower limb ischemia model.a、ADSC
The laser-Doppler photo (arrow, ischemic limb) for the treatment of mice lower limb ischemia；B, to a figure lower extremity blood flow
Statistics, by calculating the blood flow ratio calculating of ischemic limb and normal lower limb；C, to ADSC treat mice
The immunostaining of the section of the ischemic limb of lower limb ischemia；D-g, to CD31 in c figure+, PCNA+,
RFP+/CD31+/PCNA+And RFP-/CD31+/PCNA+The quantitative statistics of cell.
Fig. 4: TP knock out make β-catenin albumen VEGF process after substantially raise.A, at VEGF
In reason group and matched group, the β-catenin in the ADSC of WT and TP KO, c-myc, CD31 exempt from
Epidemic disease trace detects；B-d, to a scheme β-catenin, the protein level quantitative statistics of c-myc, CD31；e、
In VEGF process group and matched group, the relative mRNA level in-site of β-catenin genes of interest in ADSC；
F, the mrna expression change of TP after process LAN TP adenovirus and empty carrier in the ADSC of TP KO；
G, β-catenin after process LAN TP adenovirus, c-myc, CD31 immunity in the ADSC of TP KO
Trace detects；H-j, to g scheme β-catenin, the protein level quantitative statistics of c-myc, CD31；K,
In VEGF process group and matched group, reticent β-catenin (Si-β-cat) β-catenin, c-myc afterwards,
The immune-blotting method of CD31 and VE-cadherin (VE-cad)；L-m, reticent β-catenin (Si-
β-cat) after, the relative level detection to the mRNA of VEGF-A and bFGF.
Fig. 5: strike low β-catenin and can eliminate the stronger angiogenesis energy of the ADSC after TP knocks out
Power.A, the shadow becoming pipe ability of ADSC of reticent β-catenin (Si-β-cat) WT and TP KO
Ring；B, quantitative statistics to a figure tubular structure；C, reticent β-catenin (Si-β-cat) WT and
The impact forming vessel patency in matrigel of the ADSC of TP KO；D, the glue in c figure is cut
Sheet the representative graph of immunofluorescence dyeing, EGFP+Cell derived in the transgenic mice of EGFP；E, right
D schemes EGFP+/CD31+Cell quantification statistics.
Fig. 6: strike low β-catenin and can reduce after TP knocks out the therapeutic effect to mice lower limb ischemia.a、
To in ADSC silence β-catenin (Si-β-cat) or matched group (Scram), treat mice lower limb
The laser-Doppler photo (arrow, ischemic limb) of ischemia；B, to a figure lower extremity blood flow add up, by meter
Calculate the blood flow ratio calculating of ischemic limb and normal lower limb；C, to ADSC treatment mice lower limb ischemia lack
The immunostaining of the section of blood lower limb, white arrow represents RFP+/CD31+/PCNA+Cell；D-g, right
CD31 in c figure+, PCNA+, RFP+/CD31+/PCNA+And RFP-/CD31+/PCNA+Determining of cell
Amount statistics.
Fig. 7: suppression Calpain activity can strengthen the angiogenesis ability of the ADSC of β-catenin mediation.
A, carry out immunoblotting assay β-catenin WT and TP KO's with β-catenin C end antibody
Change in ADSC；B, to transfection TP adenovirus (Adeno-TP) and comparison adenovirus (Vector)
TP KO ADSC in β-catenin change-detection；C, whether there iing U46619 (TP specificity
Agonist) in the presence of, in the ADSC of WT and TP KO Calpain activity detection；
D, in the ADSC of TP KO, to infecting TP adenovirus or comparison disease in the case of whether having U46619
After poison, the detection of Calpain activity；E, in the ADSC of TP KO, with the Calpain of various dose
After inhibitor (CPT) processes, the albumen change of β-catenin, cyclinD1 and CD31；F, excessively table
After reaching TP, the CPT impact on ADSC angiogenesis ability in matrigel；G, to the glue in f figure
Carry out the representative graph of section immunofluorescence dyeing, GFP+Cell derived in the transgenic mice of GFP；h、
D is schemed GFP+/CD31+Cell quantification statistics；After i, j, process LAN TP, ADSC is expressed by CPT
The impact of the mRNA level in-site of VEGF-A and bFGF；K, in VEGF process group and matched group, WT
And the protein level change of u-calpain and m-calpain in the ADSC of TP KO；L, different agent
U46619 is on the impact of the protein level of u-calpain and m-calpain in ADSC for amount.
Fig. 8: TP affects its differentiation by coupling Gq in ADSC.A, WT and TP KO's
The immunofluorescence representative graph of the Fluo-3 (green) of ADSC；B, the fluorescence intensity in a figure is quantitatively united
Meter；C, U46619 process under, to the change of calcium current in the ADSC of WT and TP KO；D,
In the ADSC of TP KO, after infecting TP adenovirus or comparison virus in the case of whether having U46619,
The immunofluorescence photograph of Rhod (red, middle column)；E, the fluorescence intensity in d figure is carried out quantitative statistics；
F, in the ADSC of TP KO, to infecting TP adenovirus or comparison disease in the case of whether having U46619
The change of calcium current after poison；G, co-immunoprecipitation detection TP and Gq/11Interact；H, to TP KO's
After ADSC infects TP adenovirus, U-73122 is to m-calpain, β-catenin, cyclinD1 and CD31
The impact of albumen；The one-tenth pipe ability of the ADSC of i, U73122 TP KO to having transfected TP adenovirus
Impact；J, to the quantitative statistics of tubular structure in i figure；K, TP pass through calcium dependent calpain/ β-catenin
The path regulation ADSC ideograph to endothelial cell differentiation.
Fig. 9: suppression TP can strengthen the ADSC of people to endothelial cell differentiation.A, SQ29548 be (TP's
Inhibitor) people ADSC after, flow cytometer detection CD31+The representative graph of cell；B, to the CD31 in a figure+
Cell is added up；C, SQ29548 become the impact of pipe ability to the ADSC of people；D, to tubulose in c figure
The quantitative statistics of structure；E, SQ29548 on the ADSC of people in the impact of matrigel medium vessels Forming ability；
F, the glue in e figure is carried out section the representative graph of immunofluorescence dyeing；G, to (h) of people in f figure with
And (m) CD31 of mice+The statistics of cell；H, i, SQ29548 are to Angiogensis in the ADSC of people
The impact of the mRNA level in-site of factor Ⅴ EGF-A and bFGF.
Detailed description of the invention
The present invention relates to using TP or beta chain albumen as the target of fat stem cell treatment ischemic diseases.Logical
Cross and reduce, suppress or eliminate TP activity, or by improving beta chain protein active, it is possible to provide adipose-derived
The ability of stem cell angiogenesis promoting, or promote that adipose-derived stem cell breaks up to vascular endothelial cell, from
And improve the curative effect of adipose-derived stem-cell therapy ischemic diseases.
Technology well known in the art can be used to reduce, suppress or eliminate TP activity, come including knocking out fat
The stem cell in source is expressed TP gene, make shown gene cause TP activity reduce or lose sudden change,
Or make adipose-derived stem cell contact with the antagonist of TP.
Such as, can be by technology such as CRISPR-Cas9 or TALEN, to external adipose-derived doing carefully
Born of the same parents carry out TP fixed point and knock out.Also TP implementation section can be suddenlyd change, so that its activity reduces or loses.
Or, TP antagonist known in the art can be used to reduce, suppress or eliminate TP activity.These
TP antagonist include but not limited to AH 29848, SQ-29548, AH-23848, GR-32191,
ONO-11120、BM-13177、BM-13505、SKF-88046、L-655240、S-145、TMQ、
R-68070, CV-4151 and Bay-K 8644 etc., the structure of part of compounds is as follows:
Can be found in, such as Chen is newborn, Wan Weiqin, " thromboxane A2 and receptor antagonist thereof ", " China
Medical industry magazine ", the 2nd phase of volume 21 nineteen ninety, the 88-93 page；Yin Kaisheng, Zhao Lin, " blood
The progress of bolt element A2 receptor antagonist seratrodast ", " world's clinical medicine ", 2005 01
Phase.Herein entire content of these documents is included in by reference herein.
Improve beta chain protein active may be accomplished by:
(1) reduce, suppress or eliminate the TP activity of described stem cell；
(2) reduce, suppress or eliminate the activity of the calpain of described stem cell；And/or
(3) process LAN beta chain albumen in described stem cell.
As it was previously stated, can by knock out adipose-derived stem cell is expressed TP gene, make shown gene
Cause the reduction of TP activity or the sudden change lost or make adipose-derived stem cell connect with the antagonist of TP
Touch and realize the reduction of TP activity, suppress or eliminate.
For the activity of the calpain of stem cell, it can be made to contact with the inhibitor of calpain, thus real
Now this calpain reduction, suppress or eliminate.
The inhibitor of calpain known in the art can be used, include but not limited to the N-bright ammonia of acetyl group-L-
The acyl the brightest ammonium aldehyde of-L-leucyl-L-(ALLN), methyl fluoride ketone (Z-LLY-FMK), calpain press down
Element (Calpastatin), α-one base amide-type inhibitor and calpeptin (CPT).Can be found in, such as Zhang Yong,
" design, synthesis and the activity research of α-one base amide-type Calpain inhibitor ", thesis for the doctorate, 2008
Year, herein entire contents is included in by reference herein.
As an example, the structural formula of CPT is as follows:
Or, can by knocking out or the gene of mutation expression calpain (Calpain), thus realize described
The reduction of the calpain of stem cell, suppress or eliminate.Can use and strike described in technology realization well known in the art
Remove or sudden change.Sudden change is typically to make the active structure domain of calpain to morph, so that it is as calcium egg
The activity of white enzyme reduces or loses；Or make its expressing gene undergo mutation so that its gene is not expressed or table
The amount of reaching reduces.
Or, such as siRNA technology can be used to reduce the expression of the gene expressing calpain or TP.
Can transfect adipose-derived in vitro by building the plasmid containing beta chain albumen or adenovirus vector
Stem cell, and then make beta chain protein overexpression.Can be found in Sawant DA, Tharakan B, Hunter FA,
Smythe WR, Childs EW, " Role of β-catenin in regulating microvascular
Endothelial cell hyperpermeability ", J Trauma, in February, 2011,70 (2): 481-7.
Should be understood that when adipose-derived stem cell is contacted with various antagonisies or inhibitor, antagonist or
The amount of inhibitor can be according to selected antagonist or inhibitor, the amount of adipose-derived stem cell, suppression
Effect etc., and combine prior art and determined.
By above-mentioned means, the ability that can prepare angiogenesis promoting improves and/or divides to vascular endothelial cell
Change the adipose-derived stem cell improved.Preferably, described adipose-derived stem cell from mammal,
The especially fatty tissue of people, Mus, rabbit, pig, sheep and Canis familiaris L. etc., particularly preferably from the fatty tissue of people.
Described adipose-derived stem cell preferably has following one or more feature:
(1) gene expressing TP is knocked or undergos mutation, and causes TP activity to reduce or loses；
(2) express calpain gene be knocked or undergo mutation, cause calpain activity reduce or
Lose；With
(3) expression vector containing expression beta chain albumen, so that beta chain protein overexpression.
Due to its have raising angiogenesis promoting ability and/or to vascular endothelial cell differentiation ability, this
The adipose-derived stem cell of invention can be used to treat to be needed angiogenesis and/or needs the disease of blood vessel endothelium,
Include but not limited to various diseases and diabetes that ischemia causes.Under this kind of disease or disease include but not limited to
Limb ischemic diseases, cerebral thrombosis, myocardial infarction, diabetes, neurodegenerative diseases etc..
Therefore, the present invention includes that TP antagonist and/or calpain inhibitor promote adipose-derived in preparation
Stem cell is to the differentiation capability of vascular endothelial cell and/or the purposes in improving the medicine of its angiogenesis ability.
Present invention additionally comprises a kind for the treatment of or the method for prevention ischemic diseases, described method includes the present invention
Adipose-derived stem cells give need object.In a specific embodiment, described method includes this
The adipose-derived stem cells of invention is cultivated with matrigel, altogether then by containing adipose-derived stem cells of the present invention
Matrigel gives (such as, being injected at sufferer) object.During injection, VEGF can be given simultaneously.Or,
Directly the adipose-derived stem cells of the present invention can be injected at the sufferer of object.Object can be that suckling is moved
Thing, such as people, Mus, rabbit, pig, sheep and Canis familiaris L. etc..
Present invention additionally comprises the ability of the adipose-derived stem cell angiogenesis promoting of external raising or promote fat
The method that the stem cell in source breaks up to vascular endothelial cell, described method includes the β improving described stem cell
The activity of-catenin.
Present invention additionally comprises the present invention adipose-derived stem cell medicine in preparation treatment ischemic diseases
In purposes.
Present invention additionally comprises the external method being prepared vascular endothelial cell or blood vessel by adipose-derived stem cell,
Described method includes that the stem cell making the present invention adipose-derived contacts with VEGF.The concentration of VEGF during contact
Can be determined by practical situation, this is within the scope of those skilled in the art grasp.
Present invention additionally comprises a kind of compositions, said composition contains the adipose-derived stem cell of the present invention.Should
Compositions can be a kind of pharmaceutical composition, also can be containing the most pharmaceutically acceptable in addition to described stem cell
Carrier.Or, said composition can be the culture of adipose-derived stem cells of the present invention, in this culture
In addition to described stem cell, also can be containing being suitable for the described stem cell culture medium with undifferentiated state propagation.This
A little culture medium can be the various trainings for maintaining the undifferentiated breeding of adipose-derived stem cells of this area suppression
Support base.
The present invention will be described in the way of specific embodiment below.Should be understood that these embodiments are only to illustrate
Property, not limit the scope of the invention.In embodiment, unless otherwise stated, use ability
Territory routine techniques means implement various methods, step and the various reagent of use.
Experiment material and method
Animal
All of mice is all C57BL/6 genetic background.Redness or the transgenic mice of green fluorescent protein
The mice that or green fluorescence/TP red with TP knock-out mice copulation generation knocks out.All of mice use with
And in raising, all have followed the article of Chinese Academy of Sciences nutrition science institute management of laboratory animal committee.
Reagent
Calpeptin, U46619, arachidonic acid is bought in Cayman chemical reagents corporation (Cayman
Chemical, Ann Arbor, MI, USA).The VEGF (VEGF) of mice and people
Buy in Peprotech (Peprotech Inc., Rocky with bFGF (bFGF)
Hill, NJ, USA).Matrigel is bought in BD biotech firm (San Jose, CA, USA).
The separation of adipose-derived stem cells (ADSCs) and cultivation
The ADSCs of people buys from Cyagen biotech firm (article No.: HUXMD-01001), uses OriCell
The ADSC growth medium (article No.: HUXMD-90011) of people is cultivated.The ADSCs of mice is isolatable from
The epididymal adipose tissues pad fat of mice.It is summarized as follows: fatty tissue is cut into small pieces, with the collagenase of 0.2%
(Sigma-Aldrich, St.Louis, MO, USA) digests 1 hour in 37 DEG C of water-baths；Then will disappear
Change liquid 100um sieve (BD Biosciences) to filter to remove fragment of tissue；Then by cell suspension
Dilute with PBS and be centrifuged to remove collagenase.Cell 160mM NH under being centrifuged4Cl is resuspended, room
Temperature places 10min to remove erythrocyte.Finally by gained cell Dulbecco ' s modified Eagle ' s
medium/Ham’s F12(DMEM/F12；Invitrogen, Carlsbad, CA, USA) culture medium adds 10%
Hyclone adds 1% penicillin/streptomycin, at 37 DEG C of 5%CO2Under the conditions of cultivate.Change liquid every other day.All little
The ADSCs of Mus is used for testing in the second filial generation.Endothelial differentiation is tested, 200ul matrigel is laid on
In one hole of 12 orifice plates, hatch 1 hour for 37 DEG C；Then by 1 × 105Individual ADSCs kind is in culture medium.
Flow cytometry
The ADSCs of mice BD flow cytometer (FACScan, BD Biosciences) carries out streaming
Analyze.In short, hatch 30 minutes in the PBS containing 1% calf serum anti-containing after receipts cell.
The CD29, CD90, Sca-1, CD34 and the CD31 that use in experiment buy in eBioscience
(eBioscience, San Diego, CA, USA).Stream data FlowJo 8.3.3 software is carried out point
Analysis.
Immunofluorescence dyeing
Before dyeing, section or cell climbing sheet being fixed with cold third, PBS washes away acetone, then with containing 0.25%
The PBS permeable membrane of Triton X-100 10 minutes, then closes 30 minutes with 3%BSA/PBS, and afterwards one
Anti-4 DEG C of overnight incubation.Anti-CD34 (the extension rate 1:50 including mice of used in experiment；
EBioscience), CD31 (extension rate 1:50；EBioscience), CD31 (extension rate 1:500；BD
Biosciences), GFP (extension rate 1:1000；Abcam, Cambridge, MA, USA), RFP is (dilute
Release multiple 1:1000；Abcam), and PCNA (extension rate 1:1000；Cell Signaling
Technology, Danvers, MA, USA) and (the extension rate 1:200 of people；R&D Systems,
Minneapolis, MN, USA).After overnight, PBS washes away Excess antibody, two anti-incubated at room 1 hour.
The fluorescence of the two anti-couplings that this experiment is used includes Alexa Fluor 488, Alexa Fluor 594, or Alexa
Fluor 633(Invitrogen).With the mountant mounting containing DAPI.Finally use laser confocal microscope
(Carl Zeiss, Oberkochen, Germany) takes pictures, statistics.
The extraction of prostaglandin and analysis
The ADSCs cultivated is stimulated with the arachidonic acid of 30 μm ol/L, receipts supernatant after 30 minutes, 12000g,
4 DEG C, centrifugal 15 minutes, extract prostaglandin and analyze its content with Liquid Chromatography-Tandem Mass Spectrometry.
Become pipe experiment
Enter 200 μ L matrigels at 12 orifice plate each hole middle berths, hatch 1 hour for 37 DEG C.The most each hole
Plant 2 × 105ADSCs.Cell culture incubator is cultivated 12 hours, inverted microscope (IX51；Olympus,
Center Valley, PA, USA) under observe and take pictures.
Matrigel angiogenesis experiment in vivo
With comprising ADSCs (0.5 × 106) 100 μ L matrigels be subcutaneously injected into the mice of C57BL/6
Lateral side regions.The ADSCs (1 × 10 of people6) with 500ng/mL VEGF (Peprotech) and 2 μMs
Add in 100 μ L matrigels after SQ29548 (Cayman Chemical) mixing, be then subcutaneously injected into naked
Mus lateral side regions.After two weeks, take out, take pictures, and carry out morphological analysis.
Lower limb ischemia model
Proximal femoral and far-end to the left lower extremity of eight weeks big male mices ligature respectively, centre cut off with
Cause lower limb ischemia.After one day, in ischemic limb sura intramuscular injection 106Individual ADSCs.7 days and 14 days
Time with laser Doppler flowmetry (LDI；Moor Instruments Ltd., UK) detection restoration of blood flow situation.
Adenovirus
Adenovirus is to utilize pAd-track-CMV GFP carrier to contain encoding murine total length TPcDNA to produce
It is raw, produce in 293A Viral packaging cell system and expand.Through the amplification of several recombinant adenoviruss taken turns,
Caesium chloride density gradient centrifugation purification.Its efficiency of infection is estimated by measuring the fluorescence of green fluorescent protein.
The extraction of RNA and realtime quantitative inspection
Trizol reagent (Invitrogen) is used to provide method to extract cell total rna according to manufacturer.Will
The RNA extracted is cDNA with Reverse Transcription box (Takara company, Dalian, China) reverse transcription.Real
Time quantitative PCR SYBR Green (Takara) mixed liquor.With the expression of actin to target base
The expression of cause carries out specification.PCR program is as follows: 95 DEG C, within 5 minutes, is a cycle, 40 subsequently
The individual cycle 95 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, last extend 72 DEG C 5 minutes.
The period of each PCR primer is obtained by solubility curve.
RNA interference experiment
Use the method that RNAiFect transfection reagent (Qiagen, Crawley, UK) provides according to manufacturer
ADSCs is transfected, including specific sequence β-catenin (100nM) or compare unordered siRNA
(Genepharma, Shanghai, China).The siRNA transfection reagent of one microgram and RNAiFect mixing chamber
Temperature is hatched 10 to 15 minutes, is added dropwise in the cell cultivated, is divided by immunoblotting after 48 hours
Analysis transfection efficiency.
The calcium imaging of laser co-focusing
With laser scanning co-focusing microscope (Carl Zeiss, Inc, Germany), ADSCs calcium transient is carried out
Record.Briefly, after cell is loaded 6 μ g/ml Fluo-3 or Rhod 4, place 30 minutes at 37 DEG C.
After HBSS washs, Fluo-3 excites at 488nm, and Rhod 4 excites at 594nm.Use linear scanning
(X-T) image is scanned by the speed of 512 every row pixels.Quick with multichannel by 1 μM of U46619
Application system evaluates the Ca in endoplasmic reticulum2+Load.
Co-immunoprecipitation
With carrying empty carrier or TP cDNA adenovirus infection ADSCs.With containing 5 μ L HA tag antibodies
Or normal IgG (Cell Signaling Technology) co-immunoprecipitation buffer and 1 milligram of full cell split
The mixing of the thing system of solution, hatches 3 hours at 4 DEG C, subsequently with protein A/G agarose (Invitrogen) 4
DEG C hatch and be gently mixed overnight.Fully after washing, immune complex is carried out denatured by boiling, immunoblotting
Analyze the antibody of HA labelling or anti-Gq/11 protein antibodies (Santa Cruz Biotechnology, Santa Cruz,
CA, USA).
Immunoblot experiment
With the lysate containing protease inhibitor, ADSCs is extracted albumen.By using Piece BCA
The BCA method of determination of protein concentration test kit measures total protein of cell (Pierce, Rockford, IL, USA).
By the protein denaturation of equivalent, by 10% concentration by SDS-polyacrylamide acrylamide gel
(SDS-PAGE) electrophoresis, transfers on nitrocellulose filter afterwards, and 5% skim milk is closed 1 hour,
Then resist with one 4 DEG C of overnight incubation.What this experiment was used one anti-includes: anti-mouse actin (1:2000；
Sigma-Aldrich), anti-c-myc (1:1000；GeneTex), anti-cyclin D1 (1:1000；Abcam),
Anti-CD31 (1:500；Abgent), anti-HA-tag, anti-m-calpain, anti-μ-calpain, anti-
-VE-cadherin, anti-phospho-GSK3 β S9, anti-total-GSK3 β, anti-phospho-β
-cateninS33(1:1000；Cell Signaling Technology), anti-C-terminal β-catenin
(1:1000；BD Biosciences), anti-N-terminal β-catenin (1:1000；Abcam), and resists
-β-catenin(1:1000；Millipore).The two anti-room temperatures to film horseradish peroxidase-labeled afterwards
Hatch 2 hours.Then with enhancement mode luminescence reagent (Piece), film is developed the color.
Data analysis
Result mean+/-standard error (SEM) represents.Use 16.0 version SPSS software (SPSS
Inc., Chicago, IL, USA) data are carried out statistical analysis, with t inspection or variance analysis.Analyze
In less than 0.05 P value think statistically significant.
Result
1, TXA2 and TP signal is activated during endothelial differentiation at ADSC
Fig. 1 shows, ADSC has dryness labelling, and relatively high expressed prostaglandin receptor TP.At VEGF
Induction Process in, the upstream enzyme system COX1 of prostaglandin, 2 and TXAS all raise, the TXB2 of secretion
Increase, and prostaglandin synthetase TxAS and receptor TP up-regulated, illustrate that TXA2/TP axle exists
ADSC is activation in endothelium atomization.
2, knock out TP and accelerate the ADSC differentiation to endotheliocyte
As seen from Figure 2, compared with the ADSC in WT mice source, TP knocks out and makes ADSC lure
Endothelial cell marker CD31 and CD34 during leading expresses rising；The labelling Tie2 that endothelium is relevant,
CD31 etc. are the most significantly raised in mRNA level in-site；Flow cytometry shows that mature endothelial cell ratio shows
Write and increase；Pipe ability is become to strengthen；Promote that the mRNA level in-site of angiogenesis factor VEGF-A and bFGF is bright
Aobvious rising；Three-dimensional one-tenth pipe experiment in internal matrigel also indicates that TP has higher angiogenesis after knocking out
Ability.
3, TP knocks out and makes the efficiency of ADSC treatment lower limb ischemia improve
Fig. 3 shows, compared with the ADSC in WT mice source, the ADSC of TP knock-out mice is in treatment
During mice lower limb ischemia 7 days and 14 days, blood perfusion degree substantially increases.Immunofluorescence shows, TP knocks out
After ADSC treatment, ischemic muscle endotheliocyte substantially increases；The ADSC using RFP source follows the trail of display,
Derive from the CD31 of external source ADSC+Endotheliocyte (the PCNA of endotheliocyte and propagation+, CD31+)
Substantially increase, illustrate that TP promotes ADSC angiogenesis after knocking out, mainly broken up by induction ADSC
Realize for endotheliocyte.
4, β-catenin raises is that TP knocks out the key factor promoting that ADSC breaks up to blood vessel endothelium
Fig. 4 result shows, compared with WT, TP knocks out so that ADSC intracellular β-catenin level increases,
Target molecule c-myc level increases downstream, and CD31 level increases；TP knocks out the purpose of rear β-catenin
Gene substantially increases in mRNA level in-site；By Adenovirus Transfection, process LAN TP makes above-mentioned phenotype lose；
By siRNA strike low β-catenin then can stop ADSC to the expression of endothelial cell differentiation correlation molecule,
The mRNA level in-site promoting angiogenesis factor VEGF-A and bFGF declines.
5, strike low β-catenin and can reduce ADSC angiogenesis ability
Fig. 5 shows, strikes low β-catenin by siRNA and can reduce the one-tenth pipe ability of ADSC；Reduce ADSC
Three-dimensional one-tenth pipe ability in matrigel；Immunofluorescence shows, strikes low β-catenin and makes to come from external source
ADSC effect during blood vessel is constituted substantially weakens, and endothelial differentiation ability declines.
6, strike low β-catenin and can reduce the effect of ADSC treatment lower limb ischemia
Fig. 6 shows, after striking low β-catenin, the therapeutic effect of the ADSC that TP knocks out is remarkably decreased, 14
It time blood perfusion no longer recover；Immunofluorescence results shows, ischemic region endotheliocyte significantly reduces, its
In be divided into endotheliocyte (RFP from the cell of external source ADSC+, CD31+) and have multiplication capacity
Endotheliocyte (PCNA+, CD31+) ratio be decreased obviously.
7, calpain activity decrease makes ADSC angiogenesis ability strengthen
Fig. 7 shows, by using the antibody of C end, the different bands of β-catenin, process LAN detected
TP makes degraded increase；Finding, TP relies on the proteolytic enzyme calpain (calcium of calcium ion after knocking out simultaneously
Protease) activity be decreased obviously, covering TP after, calpain activity rise；Use calpain inhibitor
After CPT, TP covering can reduce the degraded of β-catenin and promote endothelium correlative protein expression；Simultaneously
The ADSC three-dimensional one-tenth pipe ability in matrigel strengthens, and endothelial differentiation ability strengthens, angiogenic growth factor
The mRNA level in-site of VEGF-A and bFGF raises.
8, TP knocks out and causes intracellular calcium concentration to decline
Fig. 8 shows, TP knocks out rear intracellular free calcium level and declines, and cell calcium wink, stream was in the stimulation of TP agonist
Lower forfeiture；Process LAN TP calcium wink flows recovery；Co-immunoprecipitation result display TP really can and Gq/11Phase
Interaction, can significantly affect the albumen table of the relevant molecule of TP-mediation with the inhibitor U-73122 of PLC
Reach change, improve the one-tenth pipe ability caused due to TP process LAN and decline；Therefore, TP is logical in ADSC
Cross Gq and cause Ca2+ influx, after activation calpain, the mediation regulation and control to β-catenin, and then affect ADSC
To the differentiation of endotheliocyte.
9, suppression TP can improve the angiogenesis ability of ADSC of people
Fig. 9 shows, processes the ADSC of people with the inhibitor SQ2958 of TP, and it is to endothelial cell differentiation
Ability strengthens；Pipe ability is become to strengthen；The three-dimensional experiment of internal matrigel shows, TP inhibitor processes descendant's
The angiogenesis ability of ADSC strengthens；The ability of endothelial differentiation strengthens；Angiogenic growth factor VEGF-A
Raise with the mRNA level in-site of bFGF.
Claims (10)
1. an adipose-derived stem cell or the compositions containing this stem cell, it is characterised in that described dry
Cell:
(1) TP inactivates or is suppressed；And/or
(2) calpain activity inactivation or reduction；And/or
(3) beta chain protein active improves.
The most adipose-derived stem cell or the compositions containing this stem cell, it is special
Levy and be, described stem cell:
(1) gene expressing TP is knocked or causes the sudden change that TP activity reduces or loses；With/
Or
(2) gene expressing calpain is knocked or causes calpain activity to reduce or lose prominent
Become；And/or
(3) expression vector containing expression beta chain albumen, thus beta chain protein overexpression.
The most adipose-derived stem cell or the compositions containing this stem cell,
It is characterized in that, described stem cell is from fatty group of mammal such as people, Mus, rabbit, pig, sheep and Canis familiaris L. etc.
Knit.
4. the ability of the stem cell angiogenesis promoting that an external raising is adipose-derived or promote adipose-derived
The method that stem cell breaks up to vascular endothelial cell, described method includes the beta chain egg improving described stem cell
White activity.
5. method as claimed in claim 4, it is characterised in that the β of the described stem cell of described raising-
The activity of catenin includes:
(1) reduce, suppress or eliminate the TP activity of described stem cell；And/or
(2) reduce, suppress or eliminate the activity of the calpain of described stem cell；And/or
(3) the beta chain albumen in stem cell described in process LAN.
6. method as claimed in claim 5, it is characterised in that
(1) the TP activity reducing, suppress or eliminating described stem cell includes: knock out in described stem cell
Express the gene of TP, or make the gene expressing TP in described stem cell undergo mutation, or make described dry thin
Born of the same parents contact with TP antagonist；With
(2) the calpain activity reducing, suppress or eliminating described stem cell includes: knock out described dry thin
Born of the same parents express the gene of calpain, or make the gene expressing calpain in described stem cell undergo mutation,
Or make described stem cell contact with calpain inhibitor.
7. method as claimed in claim 6, it is characterised in that
(1) described TP antagonist selected from AH 29848, SQ-29548, AH-23848, GR-32191,
ONO-11120、BM-13177、BM-13505、SKF-88046、L-655240、S-145、TMQ、
R-68070, CV-4151 and Bay-K 8644；With
(2) described calpain inhibitor is selected from: the N-acetyl group-L-leucyl-L-the brightest ammonia of leucyl-L-
Aldehyde (ALLN), methyl fluoride ketone (Z-LLY-FMK), calpastatin (Calpastatin), α-
Keto amide class inhibitor and Calpeptin (CPT).
In preparation, 8.TP antagonist and/or calpain inhibitor promote that adipose-derived stem cell is to blood vessel
The differentiation capability of endotheliocyte and/or the purposes in improving the medicine of its angiogenesis ability.
9. stem cell adipose-derived described in claim 1-3 or containing the compositions of this stem cell or employing
The adipose-derived stem cell that method according to any one of claim 4-7 obtains is at preparation treatment glycosuria
Sick and ischemic diseases such as ischemic disease of lower extremity, cerebral thrombosis, myocardial infarction and neurodegenerative diseases are used
Medicine in purposes.
10. the external method being prepared vascular endothelial cell or blood vessel by adipose-derived stem cell, institute
The method of stating include making stem cell adipose-derived described in claim 1-3 or the compositions containing this stem cell or
The adipose-derived stem cell using the method according to any one of claim 4-7 to obtain connects with VEGF
Touch.
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| PCT/CN2016/089295 WO2017008686A1 (en) | 2015-07-10 | 2016-07-08 | Thromboxane a2 receptor as target of adipose-derived stem cell for treating ischemic diseases |
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Non-Patent Citations (6)
| Title |
|---|
| AMY L. STRONG ET AL.: "Obesity-Associated Dysregulation of Calpastatin and MMP-15 in Adipose-Derived Stromal Cells Results in Their Enhanced Invasion", 《STEM CELLS》 * |
| GUANGNAN LI ET AL.: "Calpain as an effector of the Gq signaling pathway for inhibition of Wnt/β-catenin-regulated cell proliferation", 《PNAS》 * |
| HUI-XIALI ET AL.: "Roles of Wnt/β-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells", 《MOLECULAR AND CELLULAR ENDOCRINOLOGY》 * |
| VIJAY EASWARAN ET AL.: "β-Catenin Regulates Vascular Endothelial Growth Factor Expression in Colon Cancer", 《CANCER RESEARCH》 * |
| 林明辉 等: "前列腺素代谢与心血管疾病", 《生命科学》 * |
| 申毓军 等: "血栓素受体敲除对脂肪干细胞向内皮细胞定向分化的影响", 《中国病理生理杂志》 * |
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