CN101473030A - Cosmetic or plastic composition comprising multipotent stem cells derived from human adipose tissue, fibroblast, and adipose or adiopocyte - Google Patents
Cosmetic or plastic composition comprising multipotent stem cells derived from human adipose tissue, fibroblast, and adipose or adiopocyte Download PDFInfo
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- CN101473030A CN101473030A CNA2007800223436A CN200780022343A CN101473030A CN 101473030 A CN101473030 A CN 101473030A CN A2007800223436 A CNA2007800223436 A CN A2007800223436A CN 200780022343 A CN200780022343 A CN 200780022343A CN 101473030 A CN101473030 A CN 101473030A
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Abstract
The present invention relates to a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes, and a method for preparing the same. The composition for cosmetic or plastic surgery according to the present invention can be prepared by culturing an adipose-containing suspension obtained by liposuction and then collecting cell layers containing both adipose-derived adult stem cells and fibroblasts which are attached to the surface of the culture flask, followed by adding adipocytes to the collected cell layers. The composition according to the present invention is useful for cosmetic purposes such as skin tightness improvement, wrinkle and flabby skin improvement, and depressed area restoration through plastic surgery as well as useful for the formation of new breast tissue.
Description
Technical field
The present invention relates to a kind of composition and method of making the same that is used for lift face or plastic surgery operations, described composition contains adult stem, inoblast and fat or the adipocyte (Adiopocyte) from human adipose tissue.
Background technology
Stem cell refers to not only has the of self-replication capacity but also have the cell that is divided at least two kinds of cell abilities, and it can be divided into myeloid-lymphoid stem cell (totipotent stem cell), multipotential stem cell (pluripotent stem cells) and many plasticity stem cell (multipotent stem cells).
Recently, find that fatty tissue is new many plasticity stem cell source (people such as Cousin, BBRC., 301:1016,2003; People such as Miranville, Circulation, 110:349,2004; People such as Gronthos, J.Cell Physiol., 189:54,2001; People such as Seo, BBRC., 328:258,2005).That is to say, reported that the human adipose tissue that obtains by liposuction contains one group of undifferentiated cell, they have ability (people such as Zuk, the Tissue Eng. that is divided into elaioplast, scleroblast, flesh source cell and chondroblast, 7:211,2001; People such as Rodriguez, BBRC., 315:255,2004).And know to have regeneration muscle and stimulation is divided into neurovascular ability by animal model experiment from the cell of fatty tissue.Because the advantage that this fatty tissue has is that it can be extracted on a large scale, it has caused as the new stem cell source that overcomes existing shortcoming attractes attention.
Up to the present, known stem cell from fatty tissue comprises can be divided into the adipose-derived adult stem of the epithelial mankind (people such as Brzoska, BBRC, 330:142,2005), can be divided into the human adipose-derived adult stem (people such as Cao of scleroblast and elaioplast, BBRC, 332:370,2005), can be divided into human adipose-derived adult stem (people such as Safford, BBRC, the 294:371 of neurocyte, 2002), can be divided into adipose-derived stem cell (people such as Ogawa, BBRC, the 319:511 of mouse of elaioplast, 2004), can be divided into adipose-derived stem cell (people such as Ogawa, BBRC, the 313:871 of mouse of scleroblast and chondroblast, 2004), can be divided into human adipose-derived stem cell (people such as Awad, Biomaterials, the 25:3211 of chondroblast, 2004), can be divided into adipose-derived stem cell (people such as Fujimura, BBRC, the 333:116 of mouse of neurocyte, 2005), with can be divided into scleroblast, the adipose-derived stem cell of chondroblast and neurocyte or muscle cell (United States Patent (USP) 6,777,231).
Though up to the present paid many effort adipose-derived stem cell is used for the disease that medical field comprises that treatment can not healing property, less effort goes to attempt using stem cell to carry out lift face and plastic surgery operations.
Therefore; the present inventor has paid great effort and used stem cell in lift face and art of orthopedic surgery (protection and the similar problem that comprise wrinkle improvement, lax skin); by cultivating the suspension that contains adipocyte that obtains by liposuction; collection contains adult stem and fibroblastic cellular layer; and they are mixed with adipocyte experimentize; and find that the composition that obtains is useful for cosmetic treatment and plastic surgery operations, thereby finished the present invention.
Summary of the invention
The object of the present invention is to provide a kind of composition and method of making the same that is used for lift face or plastic surgery operations, described composition contains adult stem, inoblast and fat or the adipocyte from human adipose tissue.
To achieve these goals, the invention provides the method for compositions that a kind of preparation is used for lift face or plastic surgery operations, described composition contains (i) adult stem from human adipose tissue, (ii) inoblast and (iii) fat or adipocyte, described method comprise the following steps: (a) in culturing bottle, cultivate by liposuction from human adipose tissue obtain contain fat suspension with collection contain from fat attached to the adult stem on the culturing bottle and fibroblastic cellular layer; And adult stem and fibroblastic collecting cell layer that (b) will contain from fat mix with fat or adipocyte.
The present invention also provides a kind of preparation to be used for the method for compositions of lift face or plastic surgery operations, described composition contains (i) adult stem from human adipose tissue, (ii) inoblast and (iii) fat or adipocyte, described method comprises the following steps: that (a) is by the adult stem of the tissue-derived particulate preparation of cultivator class in the substratum that contains N-acetyl-L-cysteine (NAC) fat from fat, to collect from the stem cell and the inoblast of fat; And (b) selected adult stem and inoblast from fat mixed with fat or adipocyte.
In addition, the invention provides a kind of composition that is used for lift face or plastic surgery operations, described composition contains (i) adult stem from human adipose tissue, (ii) inoblast and (iii) fat or adipocyte.
By following detailed description and appending claims, other features of the present invention and embodiment will be clearer.
Description of drawings
Fig. 1 has shown the picture attached to the cell on the culturing bottle, wherein contains with good grounds many plasticity stem cell, inoblast and analogue from human adipose tissue of the present invention (original magnification 100 *).
Fig. 2 shows and uses the expression of results (original magnification 100 *) of immunostaining sphere is cultivated in containing the MEBM substratum of CORM-2 (sphere-cultured) from Nestin, Oct4, SH2, SH3/4 in many plasticity stem cell of human adipose tissue.
Fig. 3 has shown the picture (A: the differing of differentiation state of the adipocyte that is become by the many plasticity differentiation of stem cells from human adipose tissue according to the present invention; And B: use the painted adipocyte of oil red O stain method, original magnification 200 *).
Embodiment
In one aspect, the present invention relates to a kind of composition that is used for lift face or plastic surgery operations, it contains stem cell, inoblast and fat or adipocyte from human adipose tissue.
Preferably can be used to increase or the wrinkle removal according to composition of the present invention, but be not limited to this purpose by the breast that forms new breast tissue.For example, its frown line and nasolabial groove that can be used to keep compacting of skin of face and remove glabella by injection of micro-fat and autologous fat transplantation.The adult stem from human adipose tissue that it is desirable to contain in composition according to the present invention is from the body derived cell.
Can be by cultivating the suspension that contains fat that obtains by liposuction according to the composition that is used for lift face or plastic surgery operations of the present invention from human adipose tissue, and mixing with other cell or tissues from the adult stem of fatty tissue and inoblast of will obtaining prepares.
In one embodiment, the composition that is used for lift face or plastic surgery operations according to the present invention can prepare through the following steps: (a) culturing bottle cultivate the suspension that contains fat that obtains from human adipose tissue by liposuction with collection contain from fat attached to the adult stem on the culturing bottle and fibroblastic cellular layer; And adult stem and fibroblastic collecting cell layer that (b) will contain from fat mix with fat or adipocyte.
Particularly, described composition prepares by following manner: cultivate by what liposuction obtained and be suspended in the suspension that contains fat in the physiological saline, and after washing is attached to culture vessel such as stem cell in the culturing bottle and inoblast, collect, before collecting them, use physiological saline their to be washed 1 time so that remove fat and hemocyte remaining in the supernatant liquor, and trypsinized comprises stem cell and fibroblastic cellular layer attached to the bottom, directly collect the cell that is suspended in a small amount of physiological saline by scraping cellular layer, perhaps, then the cell of collecting is mixed with fat by centrifugal collection centrifugal cell.
In the present invention, adult stem is preferably from mesoblastic cell, is preferably elaioplast from mesoblastic cell.
In the present invention, adult stem is preferably cultivated to be divided into elaioplast in containing the a-MEM substratum of dexamethasone, INDOMETHACIN, Regular Insulin and IBMX.
In another embodiment, the composition that is used for lift face or plastic surgery operations according to the present invention can prepare through the following steps: (a) by prepare the adult stem from fat at the tissue-derived particulate of substratum cultivator class fat that contains N-acetyl-L-cysteine (NAC), to collect from the stem cell and the inoblast of fat; And (b) selected adult stem and inoblast from fat mixed with fat or adipocyte.
Liposuction refers to by vacuum pump or vacuum syringe and extracts fat via the otch in the donor areas as the lift face that is used for the fat removal or the technology of plastic surgery operations.
In the present invention, for fat or adipocyte, can use by subsidiary fat or the adipocyte that from human adipose tissue, obtains of liposuction, but be not limited to this.In the present invention, fat comprises fatty tissue.
When fat was used as the composition that is used for according to lift face of the present invention or plastic surgery operations, preferred every 1ml fatty tissue contained 1 * 10
4～1 * 10
7Individual stem cell and the inoblast that comes from fat, but when the use adipocyte, the preferred use recently from the stem cell of fat and the many 1-50 of fibroblastic total cell number times adipocyte.
In addition, inoblast is the cell that constitutes the important component of fiber conjunctive tissue, is also referred to as fibrocyte.Fibroblastic tissue slice is flat strip, has irregular expansion usually.Inoblast has the tenuigenin that contains plastosome, Golgi complex, centrosome, little matrix etc., but does not show other specificitys differentiation except that them.Nucleus is also phosphorous for the weak ellipse of dyeing.They are named as inoblast is because they accumulate in around the collegen filament and are considered to relevant with the formation of collegen filament.In the present invention, inoblast can be by separating stem cell separation steps from fatty tissue.
Simultaneously, the many plasticity stem cell that obtains expression target cell surface antigen from the stem cell broth from human adipose tissue that obtains comprises the method (Int.Immunol. that uses the flow cytometry with sorting function, 10 (3): 275,1998), use the method for magnetic bead and use antibody specificity to discern elutriator (panning the method) (J.Immunol. of many plasticity stem cell, 141 (8): 2797,1998).And the method that obtains many plasticity stem cell from a large amount of culture broth comprises wherein discerns the method that molecule (being called as " surface antigen " here) is used alone or is used in combination with post at the antibody specificity of cell surface expression.
The flow cytometry separating method comprises water droplet charge method and cell capture method and similar approach, the lip-deep antigenic antibody of specificity recognizing cells is converted into electrical signal by fluorescent mark and from the fluorescence intensity that is labeled antigen-antibody complex, thereby the antigen amount of expressing is carried out quantitatively.Can also make up by the fluorescence types that will use to separate and express the antigenic cell of kinds of surface.The fluorescent substance that can use in this case comprises FITC (lsothiocyanates), PE (phycoerythrin), APC (different phycocyanin), TR (Texas Red), Cy3, CyChrome, and Red 613, Red670, TRI-Color, Quantum Red or the like.
Use the FACS method of flow cytometry to comprise: the method that wherein above-mentioned stem cell broth is collected, by this method by the isolated cell that dyes such as centrifugal and facedown body; And wherein cell is cultivated in suitable medium and is bred then that antagonist carries out painted method.The dyeing of cell was undertaken by 1 hour by cultivating on ice with the antigenic primary antibody of identified surface and target cell sample mix and with mixture in 30 minutes.After elementary antibody was by fluorescent mark, cell separated with flow cytometry after washing.When elementary antibody during not by fluorescent mark, cell and primary antibody reaction and will be fluorescently-labeled primary antibody be had and combine active second antibody and after washing, mix, and cultivation 30 minutes to 1 hour on frozen water.After the washing, the cell with painted primary antibody and second antibody separates with flow cytometry.
Owing to have the skin-tightening of raising degree, improve the frown function of line and lax skin of wrinkle such as nasolabial groove and glabella, and by plastic surgery operations recovery sunk area, the composition of the present invention that contains adult stem, inoblast and fat from human adipose tissue is useful for the lift face purpose.In addition, owing to can be divided into adipocyte from the adult stem of human adipose tissue, composition according to the present invention is useful for new breast tissue forms.
Embodiment
After this, will describe the present invention by embodiment.But it will be apparent to those skilled in the art that these embodiment only are used for illustrative purposes, and do not constitute the restriction of scope of the present invention.
Embodiment 1: from the separation of many plasticity adult stem of fatty tissue be used for the preparation of compositions of plastic surgery operations
Preparation embodiment 1
Pass through physiological saline that the subsidiary suspension that contains adipocyte that obtains of liposuction uses appropriate amount resuspending equably with being suspended in the physiological saline, and the suspension of the resuspending of q.s placed Tissue Culture Flask or shake that bottle leaves standstill or wave and culture.For leaving standstill cultivation, need 6-12 hour at least.Then, attached to the lip-deep cellular layer of culturing bottle (from MSC, the inoblast of fat) by collect (Fig. 1) with trypsin treatment.At this moment, the cell that is suspended in a small amount of physiological saline directly is collected so that use, perhaps the cellular layer of collecting from physiological saline under the 1000rpm centrifugal 10 minutes so that only use precipitation bead under the situation that the volume of cellular layer wherein should be reduced.The isolated cells layer contains adult stem and inoblast.The composition that is used for lift face or plastic surgery operations that contains adult stem, inoblast, fat and adipocyte according to the present invention prepares by the isolated cells layer is mixed with fat.
Preparation embodiment 2
Many plasticity stem cell separates and purifying from human adipose tissue.That is to say, isolating human adipose tissue is washed fine cut then 3 times with PBS.The tissue of cutting is with the ratio (fatty tissue: DMEM) digested 1 hour down at 37 ℃ in DMEM-LG (low sugar) substratum that has added 1 Collagen Type VI enzyme (1mg/ml) of 1:3.With PBS washing by the digestion tissue after, under 1000rpm centrifugal 5 minutes.Supernatant liquor is removed in suction, and goes up remaining bead with 10% FBS and DMEM-LG (low sugar) with the mixed solution washing bottom of the mixed of 1:1, then with 1000rpm centrifugal again 5 minutes.The bead that obtains by 100 μ m net filtrations to remove residue and to wash with PBS.The cell that obtains is cultivated in DMEM substratum (10% FBS, 2mM NAC, 0.2mM xitix).After one night, wash the cell that does not adhere to PBS, and the cell that cultivation is adhered in K-NAC substratum (keratinocyte-SFM substratum+2mM NAC+0.2mM xitix+0.09mM calcium+5ng/mlrEGF+50 μ g/ml BPE+5 μ g/ml Regular Insulin+74ng/ml hydrocortisone), in culturing process, changed a subculture in per two days, thereby obtain from many plasticity of human adipose tissue stem cell and inoblast.Mix with fat by the cell that will obtain, prepared and contained adult stem, inoblast according to the present invention and make or the composition that is used for lift face or plastic surgery operations of adipocyte.
Embodiment 2: from the immunological characteristic of many plasticity stem cell of fat
The adult stem from fatty tissue that obtains in embodiment 1 washs and uses trypsin treatment with PBS.The cell of handling is collected and under 4 ℃ with 1000rpm centrifugal 5 minutes.Abandoning supernatant and cell with 2% be dissolved in FBS washing among the PBS after, under 4 ℃ centrifugal 5 minutes with 1000rpm.Supernatant discarded, and particulate suspends to make in each sample with PBS and contains 1 * 10
5Individual cell, under 4 ℃ centrifugal 5 minutes afterwards with 1000rpm.Supernatant discarded, and cell and 150 μ l confining liquids (5% be dissolved in FBS among the PBS) are mixed, and under 4 ℃ with centrifugal 5 minutes of 1000rpm supernatant discarded and cell and 100 μ l confining liquids (5% be dissolved in FBS among the PBS) are mixed so that they were cultivated 30 minutes down at 4 ℃ once more.Afterwards, solution centrifugal 5 minutes of 4 ℃ of following 1000rpm and remove supernatant liquor, is then joined 150 μ l confining liquids (5% be dissolved in FBS among the PBS) in the mix aperture.Antibody (R-phycoerythrin bonded mouse-anti human monoclonal antibodies) is inserted in each hole and at 4 ℃ to descend to cultivate 30 minutes.After the cultivation, with they under 4 ℃ centrifugal 5 minutes with 1000rpm.Remove supernatant liquor, and with cell with PBS washing and under 4 ℃ centrifugal 5 minutes with 1000rpm.Remove supernatant liquor once more, with cell with PBS washing and under 4 ℃ centrifugal 5 minutes with 1000rpm.After abandoning supernatant, cell is mixed with 200 μ lPBS and fix with 1% Paraformaldehyde 96, use flow cytometry analysis at last.
Table 1: from the facs analysis of the surface antigen of the stem cell of fat
| Antigen | AD-MSCs |
| CD73 | + |
| CD90 | + |
| CD29 | + |
| CD44 | + |
| CD105 | + |
| CD33 | - |
| CD34 | - |
| CD45 | - |
| CD4 | - |
| CD31 | - |
| CD62p | - |
| CD14 | - |
| HLA-DR | - |
As a result, as shown in table 1, the adult stem from fat according to the present invention show to CD73 91%, to 97% of CD90, to 96% of CD29, to 83% of CD44, and to 80% positive response of CD105.And, the result of other antigenic facs analysis is shown stem cell of the present invention shows all CD33, CD34, CD45, CD4, CD31, CD62p, the negative immunne response of CD14 and HLA-DR.
Embodiment 3: from the immunostaining analysis of the stem cell of fatty tissue
The stem cell from fatty tissue that will obtain in embodiment 1 is fixed 30 minutes with PBS washing 3 times and with the PBS that contains 4% Paraformaldehyde 96.Cell is with PBS washing 3 times and with containing 0.1%Triton-X100 infiltration 10 minutes.After using PBS to wash 3 times, cell was cultivated 1 hour with 10%NGS, then with the PBS overnight incubation that contains primary antibody.Cell is washed 3 times with PBS again, and allow it in the darkroom, to react 1 hour with second antibody.After with PBS washing 3 times, with cell fixation (mounted).
As a result, as shown in Figure 2, many plasticity stem cell spheroid according to the present invention shown to all can be considered to undifferentiated cell marker Oct4 and as the SH2 (CD105) of the marker of mesenchymal cell stem cell and the positive response of SH3/4 (CD73).
Embodiment 4: from many plasticity differentiation of stem cells lipoblast of fat
Many plasticity adult stem from fatty tissue that will obtain in embodiment 1 is containing 5% FBS, 1 μ M dexamethasone, 200 μ M INDOMETHACIN, cultivated for two weeks in the α of 10 μ g/ml Regular Insulin and 0.5mM IBMX (3-isobutyl-1-methylxanthine)-MEM substratum, induced lipolysis forms differentiation, uses the oil red O stain method to analyze then.As a result, as shown in Figure 3, find to become elaioplast from many plasticity differentiation of stem cells of human adipose tissue.
Embodiment 5: use the animal analysis from many plasticity stem cell of fat
Use mouse to detect by the ability that keeps adipose tissue mass from the stem cell of fat.Use 12 mouse to organize in contrast, 12 mouse are as experimental group.In experimental group with fat (1ml)+from the many plasticity stem cell and the inoblast (5 * 10 of fatty tissue
5/ 200 μ l salt) be expelled in the head corium, do not have subcutaneus adipose tissue in the mouse, and fat (1ml)+vehicle (200 μ l salt) is expelled in the control group.
Inject after 15 days, by the head otch isolated adipose tissue of mouse, and the amount of mensuration fatty tissue.As a result, the fat quantity of injecting in control group has reduced 31%, is maintained and injected 45% of the fat quantity injected in the experimental group of the composition by embodiment 1 preparation therein.Found that by this stem cell and inoblast from fatty tissue according to the present invention has the effect of minimizing by the fat quantity reduction of liposuction generation.
Industrial applicibility
That as above describes in detail is such, and the present invention has provides a kind of effect that is used for the composition of lift face or plastic surgery operations, and described composition contains adult stem, inoblast and fat or the adipocyte from human adipose tissue.The composition that is used for lift face or plastic surgery operations according to the present invention is useful for the lift face purpose not only, such as the degree of compacting that improves skin, improve wrinkle and lax skin, and recovery is by the zone of plastic surgery operations reduction, and also be useful for the formation of new breast tissue, because can be divided into adipocyte from the adult stem of human adipose tissue.
Though the present invention is described in detail, it will be apparent to those skilled in the art that this description only is used for preferred implementation, rather than limit the scope of the invention with reference to special characteristic.Therefore, essential scope of the present invention will be limited by appending claims and equivalent thereof.
Claims (6)
1, a kind of composition that is used for lift face or plastic surgery operations, it contains (i) adult stem from human adipose tissue, (ii) inoblast and (iii) fat or adipocyte.
2, composition according to claim 1 is characterized in that, composition is used for the formation or the wrinkle of new breast tissue and removes.
3, a kind of preparation is used for the method for compositions of lift face or plastic surgery operations, and described composition contains (i) adult stem from human adipose tissue, (ii) inoblast and (iii) fat or adipocyte, and described method comprises the following steps:
(a) in culturing bottle, cultivate the suspension that contains fat that obtains from human adipose tissue by liposuction with collection contain from fat attached to the adult stem on the culturing bottle and fibroblastic cellular layer; And
(b) adult stem and fibroblastic collecting cell layer that will contain from fat mixes with fat or adipocyte.
4, method according to claim 3 is characterized in that, adult stem is from mesoblastic cell.
5, method according to claim 3 is characterized in that, cultivates in containing the α of dexamethasone, INDOMETHACIN, Regular Insulin and IBMX-MEM substratum from the adult stem of fat.
6, a kind of preparation is used for the method for compositions of lift face or plastic surgery operations, and described composition contains (i) adult stem from human adipose tissue, (ii) inoblast and (iii) fat or adipocyte, and described method comprises the following steps:
(a) by the adult stem of the tissue-derived particulate preparation of cultivator class in containing the substratum of N-acetyl-L-cysteine fat, collect then from the stem cell and the inoblast of fat from fat; And
(b) adult stem and the inoblast from fat that will collect mix with fat or adipocyte.
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| CN101785853A (en) * | 2010-03-24 | 2010-07-28 | 晏泽 | Mesenchymal stem cell biological winkle removing agent and preparation method thereof |
| CN104114691A (en) * | 2011-12-01 | 2014-10-22 | K-干细胞有限公司 | Culture-medium composition for rejuvenating stem cells |
| CN104814788A (en) * | 2015-03-21 | 2015-08-05 | 李玉欣 | Plastic-surgery treatment device |
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| EP2039348A1 (en) * | 2007-09-21 | 2009-03-25 | Jürgen Schliefelbein | Cosmetic preparation and method to obtain a somatic stem cell preparation |
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| KR101237430B1 (en) * | 2008-05-07 | 2013-02-26 | 남명진 | A cosmetic composition comprising stem cell culture medium and a process for producing the same |
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| JP2010178830A (en) * | 2009-02-04 | 2010-08-19 | Olympus Corp | Live organ transplant material and method for manufacturing the same |
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| KR101523040B1 (en) * | 2012-06-26 | 2015-05-26 | 라정찬 | Method for Preparing High Concentration of Stem Cells |
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| CN101785853B (en) * | 2010-03-24 | 2013-02-27 | 晏泽 | Mesenchymal stem cell biological winkle removing agent and preparation method thereof |
| CN104114691A (en) * | 2011-12-01 | 2014-10-22 | K-干细胞有限公司 | Culture-medium composition for rejuvenating stem cells |
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| WO2007145438A1 (en) | 2007-12-21 |
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