CN1969039A - Tissue material and muscle derived matrix - Google Patents

Abstract

The present invention relates generally to a tissue preparation including tissue cells and extracts thereof useful for promoting or facilitating the growth, development and differentiation of cells and tissues. More particularly, the present invention provides muscle-derived material comprising intact or extracted extracellular matrix and/or cells as well as cytokines, growth factors and other components. The muscle preparations of the present invention resemble basement membrane and are derived from cellular-based material. The muscle preparation may be used in vitro or in vivo as inter alia, a cellular scaffold in various tissue engineering applications and in other cell culture systems for nurturing and enriching a range of cell types including, but not limited to, precursor and stem cells such as pre-adipogenic cells. The muscle preparation is also useful as a base for creams, such as in the cosmetic and topical therapeutic industries and as a matrix or additive in the food industry.

Description

Organization material and muscle derived matrix

Background of invention

Invention field

Generally speaking, the present invention relates to promote or be beneficial to growth, growth and the differentiation of cell and tissue useful organize preparation, comprise histocyte and extract thereof.More specifically, the invention provides and comprise complete or the extracellular matrix that extracts and/or the muscle derived material of cell and cytokine, somatomedin and other components.The similar basilar membrane of muscle preparation of the present invention and derive from material based on cell.This muscle preparation can use in external or body, especially in using, various organizational projects are used for nutrition and a series of cell types of enrichment as cytoskeleton and in other cell culture systems, include but not limited to for example preceding elaioplast (pre-adipogenic ceu) of precursor and stem cell.This muscle preparation also is used as the basis of emulsifiable paste and is used as matrix or additive in foodstuffs industry in for example makeup and local therapeutical agent industry.

Description of the Prior Art

The catalogue details of the reference in this specification sheets also is listed in ending place of this specification sheets.

Not should not be regarded as admitting or the described prior art of any type of hint constitutes the part of the common practise of any country to the reference of any prior art in this specification sheets yet.

Basilar membrane is the successive thin slice that epithelial cell and matrix and nerve, myofiber, smooth muscle cell and adipocyte are on every side separated.Electron microscopic analysis shows that the component of basilar membrane is cancellated filament, and described filament interacts and forms film.This reticulated structure is caused by the existence of collagen iv molecule that partly described collagen iv molecule is by intermolecular disulfide linkage interconnection (people such as Inoue, J Cell Biol, 97:1524-1539,1983).

The various components of known basilar membrane are interact with each other.For example, one of component of basilar membrane, ln combines with collagen protein IV and heparan sulfate proteoglycan.

The basilar membrane preparation can provide the relevant environment of the physiology with cell growth, growth and differentiating characteristic.These preparations are heterogeneous on composition and activity usually.For example, some preparation be solubility and lack as cytostromatic suitability people such as (, Cell22:719-726,1980) Terranova.

The preparation of a kind of Engelbreth of deriving from Holm-Swarm (EHS) rat meat knurl is with trade(brand)name " Matrigel " [trade mark, BD Biosciences] matrix that is rich in basilar membrane of selling and by people such as Kleinman, Biochem 21:8188-6193,1982 describe.Matrigel has become and has promoted cell growth, the useful products of growing and breaking up.Yet in some cases, may there be species specificity difference in the Matrigel in some cell and muroid source on synergistic level, and this makes this product not be suitable for for example people's cell of non-murine cell.It can also be in non-murine host wrongful initiation immunne response.

According to the present invention, be provided at a series of cells and comprise that having useful especially growth, morphology and differentiation in people's cell promotes the active novel preparation of organizing that is rich in basilar membrane.

Summary of the invention

This specification sheets from start to finish, unless context is requirement otherwise, word " comprises " or " comprising " will be understood that to hint and comprise the element of being stated or a whole or set of pieces or whole but do not get rid of other any elements or whole or a set of pieces or integral body.

The invention provides cell and complete and the cell epimatrix material that extracts, it as support with growth, growth and differentiation and the support of sustenticular cell or realize the morphological change of cell.Organization material preferably derives from muscle tissue and comprises the preparation that comprises basement membrane composition.These components comprise one or more following ingredients, described composition such as but not limited to, ln, collagen protein I, collagen protein IV, sense of touch albumen/nidogen, heparan sulfate proteoglycan and one or more cytokine or somatomedin, described cytokine or somatomedin are such as but not limited to, Urogastron (EGF), Prostatropin (bFGF), nerve growth factor (NGF), the somatomedin (PDGF) in thrombocyte source, insulin-like growth factor-i (IGF-1), transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-α).Usually, organization material comprises preparation or the extracellular matrix complete or that extract based on cell.For example urea or SDS extract or freezing/melt general using or lyophilize prepares cell-free preparation complete or that extract with the method for after scouring.General using is for example shredded, glutaraldehyde is fixed and/or in dimethyl sulfoxide (DMSO) (DMSO) or other freezing preservativess the refrigerated technology prepare preparation based on cell.Lyophilize can not preserve complete cell but critical point drying is passable.

For convenience's sake, organization material is also referred to as muscle matrix, muscle basement membrane matrix, myomatrix, myotrix, muscle support, myogel and cell culture compositions in this article.Term " muscle matrix " is for convenience succinct and use, and should understand it and contain preparation and acellular preparation based on cell.Cell-free preparation comprises complete and the extracellular matrix that extracts.

Muscle matrix of the present invention has various uses, for example promotes a large amount of tissues to produce in organizational project and is used for tissue repair, increase and/or alternative medicine.Also as the support of engineering tissue, described tissue is such as but not limited to muscle and fat for this muscle matrix.It also is used as enrichment and the nutrition means from the suitably preceding elaioplast at suitable stem cell position.As research tool, muscle matrix of the present invention is used for growth, growth and the differentiation research of cell, and described cell is endotheliocyte, epithelial cell, neuroglia, neurone, myocyte and preceding adipocyte for example.In makeup and foodstuffs industry, this muscle preparation as emulsifiable paste the basis and as foodstuff additive and the treatment on as the cytothesis composition.

Muscle matrix of the present invention is better than other basilar membrane preparations especially, because it is induced or promotes that otherwise the cytoactive of broad range also can the conservative mode of kind be used more.

The employed abbreviation of this paper is defined in the table 1.

Table 1

Abbreviation

Abbreviation	Describe
		bFGF	Prostatropin
DMEM	Da Erbaishi (Dulbecco) improves basic medium
		EGF	Urogastron
FCS	Foetal calf serum
		HUVEC	Human umbilical vein endothelial cells
IBMX	Isobutyl methylxanthine
		IGF-1	Type-1 insulin like growth factor
NaCl	Sodium-chlor
		NGF	Nerve growth factor
PDGF	Thr6 PDGF BB
		SDS-PAGE	Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
TGF-β	Transforming growth factor-beta
		TIPS	Thermally induced phase separation
TNF-α	Tumor necrosis factor-alpha
		VEGF	Vascular endothelial growth factor

The accompanying drawing summary

The photo of accompanying drawing 1 has shown that the SDS PAGE of Matrigel and various other periplasts compares.(from left to right) swimming lane 1 and the prestained molecular weight standard of 2 representatives; The Metrigel (BD Biosciences) that swimming lane 3 and 4 representatives are purchased; Swimming lane 5 is represented rat muscle matrix; Swimming lane 6 is represented pig muscle matrix; Swimming lane 7 representative's muscle matrix.All samples is all with sample on about 10 μ g total protein concentrations.

The photo of accompanying drawing 2 has shown (a) muscle matrix formulations from people's skeletal muscle.Figure (b) and (c) for example understand a kind of other form of muscle matrix, promptly muscle matrix is handled the sponge of formation through TIPS.Figure (c) is the scanning electron photomicrograph of sponge in the accompanying drawing 2 (b).

The photo of accompanying drawing 3 is the thick muscle matrix in (a) pig muscle source and (b) Matrigel.

The Photomicrograph of accompanying drawing 4 has shown the successful generation of tissue (comprising fatty tissue) in rat.Accompanying drawing 4 (a) and (b) be that described model covers with MyoGel before implantation from the representative slice of rat tissue's engineering chamber model.

The photo of accompanying drawing 5A has shown adipocyte before the contrast on tissue culturing plastic's goods, shows differentiation.5B is presented at the preceding adipocyte on the Myogel, shows that lipid gathers and to the differentiation of mature fat cell.The figure that inserts shows that individual cells gathers the high magnification map picture of lipid.5C shows the low power Photomicrograph of mouse chamber, and described chamber has been full of the muscle extract that induced lipolysis forms.

The photo of accompanying drawing 6 is representative example of the western blotting of various ECM components in the different MyoGel kinds.All arrows and digitized representation molecular weight (MW) level.6A relates to the immunoblotting of collagen protein I in the rat muscle extract, 3 chains of mark (100,200,300kD).Swimming lane 1-10 is ten kinds of different rat muscle extract formulations.6B shows ln α 4 (180kD) in the rat muscle extract and α 2 (80kD fragment and in a little disperse at 300kD place).Swimming lane 1 is for Matrigel contrast swimming lane 2-11 is identical rat muscle extract formulation, shows particularly the strong band about α 4 chains.6C is presented at the heparan sulfate proteoglycan combination in rat MyoGel heparan sulfate proteoglycan (HSPG) gel, swimming lane 1 is a molecular weight standard, swimming lane 2 is Matrigel, perlecan dyeing is arranged on the 200kD band and swimming lane 3-11 is the rat muscle extract.6D is presented at bonded CH-296 in the rat muscle extract sample, and swimming lane 1 is identical rat muscle extract for Matrigel contrast swimming lane 2-11.

Detailed description of the preferred embodiments

The invention provides especially lipogenesis is used and other application relevant with organizational project, increase, reparation and research are useful organizes preparation.Said preparation also has application in topical therapeutic agent, makeup and foodstuffs industry.A kind of preferred form of material comprises the basilar membrane material that derives from muscle of dissolved, extraction.Another form comprises the complete matrix that contains dissolved cell and basilar membrane material.A preferred form comprises complete tissue and cell again.Therefore, organization material is called as muscle extract material, muscle matrix, myomatrix, myotrix, muscle basement membrane matrix, muscle support, myogel and cell composition or preparation in a different manner.These terms exchange use from start to finish at this specification sheets but are contained by term " muscle matrix ".

Therefore, the muscle matrix formulations can be based on extracellular matrix cell or complete or that extract.General using for example urea or SDS extracting method prepares cell-free extract.Generally by freezing/melt with after scouring and prepare complete cell extract material, but can comprise the residue after the extraction.General using is for example shredded, glutaraldehyde is fixed and/or Freeze Drying Technique prepares preparation based on cell.Also can adopt other similar approach and all these class methods to contain by the present invention.These methods comprise critical point drying, utilize the protein cross and the fresh muscle of Mechanical Crushing of the fixing agent except that glutaraldehyde.In addition, have the preceding back technology of extracting of various extractions, described technology can be used for further strengthening product or can use separately.These technology comprise by various physical chemistry programs (for example grind, pulverizing, TIPS etc.) handle material, after freezing and rinsing and the freezing after scouring fixing organization that changes.Even can change initial organizing and for example use unstriated muscle or cardiac muscle.All change all within the scope of the present invention.

Preferred organization material of the present invention generally comprises one or more following components, described component such as but not limited to, ln, collagen protein I, collagen protein IV, sense of touch albumen/nidogen, heparan sulfate proteoglycan and other components, for example comprise cytokine and somatomedin, but be not limited to one or more EGF, bFGF, NGF, PDGF, IGF-1, TGF-β, VEGF and TNF-α.Organization material is rich in the muscle basement membrane composition.Organization material of the present invention has a series of effectiveness, comprises particularly fabric study and organizational project, and described tissue includes but not limited to fat, muscle, liver and pancreas.It also provides the foundation for the lipogenesis potentiality of vitro bioassay source material, and described material is promptly from the fat and the fatty precursor cell of different sites.Said preparation further has application in topical therapeutic agent, makeup and foodstuffs industry.

Correspondingly, the invention provides the cell growth that composition of matter is used for promoting cell or tissue, comprise differentiation, propagation, division and/or morphological change, described composition comprises based on muscle tissue preparation cell or cell-free extract, this preparation provides the source of following component, described component such as but not limited to, ln, collagen protein I, collagen protein IV, sense of touch albumen/nidogen, heparan sulfate proteoglycan and other components, for example comprise cytokine and somatomedin, but be not limited to one or more EGF, bFGF, NGF, PDGF, IGF-1, TGF-β, VEGF and TNF-α or its homologue.

In a preferred embodiment, organization material comprises ln, sense of touch albumen/nidogen, heparan sulfate proteoglycan, collagen protein IV, bFGF, PDGF, TGF-β, VEGF and TNF-α.

For convenience's sake, term " cytological effect " will be used to contain growth and division, propagation and the morphological change of cytodifferentiation.

The source of organization material can be from any animal and preferred mammal, for example, but be not limited to people, non-human primates (for example gorilla (gorilla), marmoset monkey (marmoset) or orangutan (orangoutang)), livestock animal (for example milk cow, sheep, pig, horse, donkey, goat, camel), laboratory test animal (for example mouse, rat, rabbit, cavy, hamster) or companion animals (for example dog, cat).The present invention also extends to birds sources, for example chicken, duck, goose, turkey and other poultry or hunt fowl, Reptilia for example originate snake and lizard and Amphibians originate for example frog and toad.

In an especially preferred embodiment, use is from pig, mouse, rat or people's muscle tissue.

Correspondingly, another aspect of the present invention provides the composition of matter that comprises from mammiferous muscle preparation, and described composition comprises:

(i) based on cell or acellular material;

(ii) be selected from the component of tabulation, described tabulation comprises one or more, but is not limited to, ln, collagen protein IV, sense of touch albumen/nidogen, heparan sulfate proteoglycan;

(iii) be selected from the cytokine or the somatomedin of tabulation, described tabulation comprises one or more, but is not limited to, EGF, bFGF, NGF, PDGF, IGF-1, TGF-β, VEGF and TNF-α or its homologue; And

The total protein content of the about 100mg/ml of (iv) about 1 μ g/ml-.

For example ln, collagen protein IV, sense of touch albumen/nidogen, heparan sulfate proteoglycan or EGF, bFGF, NGF, PDGF, IGF-1, IGF-2, TGF-β, VEGF and TNF-α should be considered to those components to mention said components, but be not necessarily limited to those components, in other words, said preparation can comprise other unlisted components.

The component of organization material can derive from muscle tissue fully or can add extraneous factor in preparation process, such as but not limited to, extra jelling agent (for example salt solute and/or sugar), cytokine, microbiotic, growth intensifying factor, gene expression enhancer, antiblastic and/or differentiation of stem cells promote the factor.

In one embodiment, organization material can be considered to be in and promote cell cultures, cytodifferentiation, the composition that dedifferentes or grow in external or the body.

In an especially preferred embodiment, organization material promotes cell growth and differentiation, and described cell is selected from particularly stem cell, epithelial cell, skin cells, organ cell and endotheliocyte.

" acellular material " comprises independent extraction matrix or cell is dissolved and the preparation of most of removal.

The present invention further provides cell culture compositions is used to promote cell growth and differentiation or realizes the cell or tissue morphological change, wherein said cell culture compositions comprises one or more following components, described component such as but not limited to, ln, collagen protein I, collagen protein IV, sense of touch albumen/nidogen, heparan sulfate proteoglycan and other components, comprise cytokine and somatomedin, such as but not limited to, one or more EGF, bFGF, NGF, PDGF, IGF-1, IGF-2, TGF-β, VEGF and TNF-α or its homologue, and cell culture compositions aggregates into gel.

" cytokine " comprises the single or multiple cytokines that the tabulation that provides is provided.Certainly, also can there be or comprises other cytokine.Similarly, can exist one of ln, collagen protein IV, sense of touch albumen/nidogen and/or heparan sulfate proteoglycan maybe can have two or more of these components.Also can there be or adds other cell epimatrix material.

Polymerization generally occurs in about 15 ℃-Yue 50 ℃ temperature, for example 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50 ℃.Can also adopt fluctuating temperature.

Term " gel " is with its implication use the most widely and comprise semi-fluid, semihard formula material, deformable material, thick liquid, emulsifiable paste, solid support or its combination, comprises the material that is suitable as foodstuff additive.

In a preferred embodiment, the amount of the cytokine of existence is as follows:

BFGF: the about 4ng/ml of about 0.3ng/ml-for example 0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0ng/ml.

PDGF: the about 3000pg/ml of about 1pg/ml-for example 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1010,1020,1030,1040,1050,1060,1070,1080,1090,1100,1110,1120,1130,1140,1150,1160,1170,1180,1190,1200,1210,1220,1230,1240,1250,1260,1270,1280,1290,1300,1310,1320,1330,1340,1350,1360,1370,1380,1390,1400,1410,1420,1430,1440,1450,1460,1470,1480,1490,1500,1510,1520,1530,1540,1550,1560,1570,1580,1590,1600,1610,1620,1630,1640,1650,1660,1670,1680,1690,1700,1710,1720,1730,1740,1750,1760,1770,1780,1790,1800,1810,1820,1830,1840,1850,1860,1870,1880,1890,1900,1910,1920,1930,1940,1950,1960,1970,1980,1990,2000,2010,2020,2030,2040,2050,2060,2070,2080,2090,2100,2110,2120,2130,2140,2150,2160,2170,2180,2190,2200,2210,2220,2230,2240,2250,2260,2270,2280,2290,2300,2310,2320,2330,2340,2350,2360,2370,2380,2390,2400,2410,2420,2430,2440,2450,2460,2470,2480,2490,2500,2510,2520,2530,2540,2550,2560,2570,2580,2590,2600,2610,2620,2630,2640,2650,2660,2670,2680,2690,2700,2710,2720,2730,2740,2750,2760,2770,2780,2790,2800,2810,2820,2830,2840,2850,2860,2870,2880,2890,2900,2910,2920,2930,2940,2950,2960,2970,2980,2990,3000pg/ml.

TGF-β: the about 2000pg/ml of about 1pg/ml-for example 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1010,1020,1030,1040,1050,1060,1070,1080,1090,1100,1110,1120,1130,1140,1150,1160,1170,1180,1190,1200,1210,1220,1230,1240,1250,1260,1270,1280,1290,1300,1310,1320,1330,1340,1350,1360,1370,1380,1390,1400,1410,1420,1430,1440,1450,1460,1470,1480,1490,1500,1510,1520,1530,1540,1550,1560,1570,1580,1590,1600,1610,1620,1630,1640,1650,1660,1670,1680,1690,1700,1710,1720,1730,1740,1750,1760,1770,1780,1790,1800,1810,1820,1830,1840,1850,1860,1870,1880,1890,1900,1910,1920,1930,1940,1950,1960,1970,1980,1990,2000pg/ml.

EGF: the about 100ng/ml of about 0ng/ml-for example 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100ng/ml.

VEGF: the about 3000pg/ml of about 1pg/ml-for example 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1010,1020,1030,1040,1050,1060,1070,1080,1090,1100,1110,1120,1130,1140,1150,1160,1170,1180,1190,1200,1210,1220,1230,1240,1250,1260,1270,1280,1290,1300,1310,1320,1330,1340,1350,1360,1370,1380,1390,1400,1410,1420,1430,1440,1450,1460,1470,1480,1490,1500,1510,1520,1530,1540,1550,1560,1570,1580,1590,1600,1610,1620,1630,1640,1650,1660,1670,1680,1690,1700,1710,1720,1730,1740,1750,1760,1770,1780,1790,1800,1810,1820,1830,1840,1850,1860,1870,1880,1890,1900,1910,1920,1930,1940,1950,1960,1970,1980,1990,2000,2010,2020,2030,2040,2050,2060,2070,2080,2090,2100,2110,2120,2130,2140,2150,2160,2170,2180,2190,2200,2210,2220,2230,2240,2250,2260,2270,2280,2290,2300,2310,2320,2330,2340,2350,2360,2370,2380,2390,2400,2410,2420,2430,2440,2450,2460,2470,2480,2490,2500,2510,2520,2530,2540,2550,2560,2570,2580,2590,2600,2610,2620,2630,2640,2650,2660,2670,2680,2690,2700,2710,2720,2730,2740,2750,2760,2770,2780,2790,2800,2810,2820,2830,2840,2850,2860,2870,2880,2890,2900,2910,2920,2930,2940,2950,2960,2970,2980,2990,3000pg/ml.

TNF-α: the about 1000pg/ml of about 0pg/ml-for example 0,1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000pg/ml.

In one embodiment, lack IGF-1.

Therefore the invention provides tissue-derived material based on cell or cell-free preparation form, be used for the cell g and D and be used to realize the cell or tissue morphological change, described material source is in the people, non-human primates, livestock animals, companion animals, birds, the muscle of Reptilia or Amphibians also comprises cytokine and/or the somatomedin that one or more are selected from tabulation, described tabulation comprises, but be not limited to, the about 4ng/ml bFGF of about 1.0ng/ml-, the about 3000pg/ml PDGF of about 1pg/ml-, the about 2000pg/ml TGF-of about 1pg/ml-β, the about 3000pg/ml VEGF of about 1pg/ml-, the about 100ng/ml EGF of about 0pg/ml-about 1000pg/ml TNF-α and about 0ng/ml-, described material further comprises one or more components, described component is selected from, but be not limited to ln, collagen protein IV, sense of touch albumen/nidogen and/or heparan sulfate proteoglycan.

Organization material is preferably the gel form of the laminate structure with similar basilar membrane and/or its component.Organization material can be that gel form or it can be that polymerizable is that " precursor " form of gel form or it can be based on cell.Expediently, when organization material was precursor forms, it can be redeveloped into gel or matrix form.Even more expediently, the matrix form of rebuilding precursor is called as " muscle matrix " in this article.The muscle matrix of the present invention of gel form or precursor forms (comprising based on cell preparation) also can be made into or be admixed in pearl, sponge, the emulsifiable paste etc.Although gel form is a kind of preferred form of preparation, the present invention has also expected to have and the preparation based on cell of gel-like like " pectisation " feature.

Correspondingly, muscle matrix of the present invention is used to promote the cell growth and the differentiation of various cells and realize the cell or tissue morphological change.Epithelial cell, endotheliocyte, neurocyte and stem cell respond muscle matrix especially and grow and break up.It also helps cell adhesion and helps the cell growth, grows, breaks up and/or breed, and described cell is selected from, but is not limited to, neurone, liver cell, sustenticular cell, hair follicle, thyroid cell etc.As mentioned above, " muscle matrix " should be understood that to contain preparation and the acellular preparation based on cell.

Cell can be cultivated and get back to subsequently on external muscle matrix in the animal of animal that they were derived from or immunosuppression or tissue compatible.In this context, " animal " comprises people, non-human mammal, livestock animals, companion animals or birds, reptiles or batrachians.Similarly, muscle matrix can be used for promoting in the body cell growth or tissue growth at specific site or in chamber or in the support of other implantation healths separately.

Muscle matrix of the present invention is preparation at low temperatures generally, for example about 1 ℃-Yue 10 ℃ of for example 1,2,3,4,5,6,7,8,9,10 ℃ or the non-integer temperature in this scope.4 ℃ temperature is particularly useful.Collect fresh muscle.The about 100g of about 10-is convenient to handle.Cut off visible fat and allow from muscle and be organized in (for example NaCl damping fluid) contactin enzyme inhibitors the damping fluid.In order to prepare cell-free extract, resulting tissue is carried out homogenized and carries out centrifugal to remove supernatant liquor subsequently.To organize pellet resuspended in damping fluid (for example NaCl), also to carry out homogenized again.Repeat this two washing steps.After the washing, resuspension precipitates in urea buffer solution the last time.The tissue homogenate stirring is spent the night and is finished a series of washings, collects supernatant liquor at every turn.Use the filtered through gauze supernatant liquor to remove the floating fat of free subsequently.Subsequently to solvent (for example chloroform) dialysed overnight with sterile material.Change solvent into damping fluid and continue dialysis to remove solvent.After the last dialysis, the final damping fluid of use is DMEM or its equivalent.Substrate material before resulting is distributed and be kept at-20 ℃.

When needing, the sample of substrate material and hatch for about 50 ℃ for example about 37-42 ℃ before fetching at about 20-, matrix aggregates into the gel form under described temperature.Can also add the additive that helps pectisation, include but not limited to blood plasma.

Utilize SDS also can prepare extract.Alternately, can utilize lyophilize/melt to prepare preparation with after scouring.This is called as complete matrix.Utilize the fixing preparation (being complete tissue) that can prepare easily of chopping, lyophilize and/or glutaraldehyde based on cell.

The form of matrix or matrix was sold before tissue extract material of the present invention or muscle matrix can be packaged as, and can be with supplying about the specification sheets how to use.Annexing ingredient can and be comprised before use in packing or test kit or be mixed in use.

As mentioned above, muscle matrix is particularly suitable for cultivating various cells, and described cell includes but not limited to adipocyte, 3T3-L1 cell, HUVEC, MCF-7 and MDA-MB-231 breast cancer cell, PC-12 cell and NG-108 neurocyte and a series of by the isolating preceding adipocyte of standard program.

Muscle matrix also is used for the lipogenesis potentiality of vitro bioassay source material.Said preparation also has for example ability of cancer and diabetes of the differentiation of other basilar membranes of vitro bioassay response cell types and many morbidity states, and described cell is epithelial cell, neurone, endotheliocyte for example.

According to this aspect, muscle matrix is implemented external test to measure the moiety that induced lipolysis forms, comprise cell and/or molecular components.Mensuration comprise with one or more layers potential lipogenesis component to be measured for example the extract of muscle matrix cover vessel surface, the inoculation potentialization is with the cell of experience lipogenesis differentiation and check lipogenesis subsequently.Alternately, muscle matrix is covered from the teeth outwards and add other compounds and check the lipogenesis that strengthens or reduce in this system subsequently.In another embodiment, the cell that potentialization is experienced lipogenesis differentiation maintains in the suspension culture and replenish potential lipogenesis the component to be measured for example extract or the fraction of muscle matrix in substratum.The advantage of cell suspension is to allow whether the sharp separation cell has experienced differentiation to measure them from substratum.Alternately, mensuration comprises that generation comprises the three-dimensional rack of potential lipogenesis component or extract, and the cell of inoculation potentialization experience lipogenesis differentiation is also checked lipogenesis subsequently.Therefore, mensuration of the present invention system also can have attendant advantages, and described advantage is for providing or select or develop adipocyte colony before the optimization that is used for organizational project.Mensuration can be carried out in external proper container easily.In this case, can cover the surface of matrix with the cell material preparation.Container also can be with the packaged sale of working instructions.

Correspondingly, the present invention expects that external test regulates adipogenic component, extract or cell system, described mensuration comprises checks that the muscle preparation has the cell mass of adipocyte differentiation tendency with discriminating, produce or obtain the adipogenic component of potential adjusting or extract or cell system, described cell mass with adipocyte differentiation tendency is seeded on described component or the extract, described cell hatched the chien shih lipogenesis produces when enough, and in described cell, check the differentiation of adipocyte subsequently." cell system " in this context have with based on the identical implication of the preparation of cell.

In certain embodiments, adipogenic component of potential adjusting or extract or cell system promote lipogenesis.In other embodiments, regulate adipogenic component or extract or cell system and suppress lipogenesis.

" adjusting " is meant increases or reduces the lipogenesis level directly or indirectly.

Can obtain or produce adipogenic component of potential adjusting or extract or cell system layer.Alternately, can obtain or produce the three-dimensional supported matrix that comprises the adipogenic component of potential adjusting or extract or cell system.In certain embodiments, the cell that potentialization can be experienced adipocyte differentiation maintains in the suspension culture and replenish adipogenic component of potential adjusting or extract in substratum.Term " layer " comprises two-layer or multilayer.Present method extends to and add potential lipogenesis promotor in muscle matrix.

Of the present invention provide on the one hand again to produce be fit to be transplanted to the method that is subjected to intravital donor blood vessel tissue, described method comprise make comprise the vessel pedicle of the function recycle system and in a organized way or tissue extract or its component inject, adhere to or otherwise related with described vessel pedicle; Vessel pedicle is combined in supported matrix inside and/or top; The separated cell or tissue fragment of inoculation in supported matrix, described cell or tissue utilize external test to differentiate to promoting lipogenesis; Or some other useful end points, the supported matrix that will comprise vessel pedicle is implanted to certain position of acceptor, the function recycle system and partial artery or venous anastomosis on described position; And supported matrix stayed implant site enough periods to allow the vascularization growth of new tissue, wherein select injecting material or inoculation material according to certain criteria, for example, it promotes lipogenesis when measuring by assay method, described assay method comprises checks that tissue or tissue extract have the cell mass of lipogenesis differentiation tendency with discriminating, produce or obtain adipogenic component of potential promotion or extract, the cell mass that will have adipocyte differentiation tendency is seeded on described component or the extract, and described cell is hatched the differentiation that adipocyte takes place and checks subsequently for chien shih lipogenesis when enough in described cell.

In a preferred embodiment, vessel pedicle comprises the tissue that adheres to fat or other fatty tissues or comprise following cell, and described cell has sarcoplast, inoblast, preceding adipocyte and adipocyte, the myocardial cell, keratinocyte, endotheliocyte, smooth muscle cell, the chondrocyte, pericyte, the matrix precursor cell of derived from bone marrow, embryonic stem cell, mescenchymal stem cell or hemopoietic stem cell, other cells of Xu Wang (Schwann) cell and periphery and central nervous system, olfactory cell, liver cell and other liver cells, glomerular mesangium and other nephrocytes, beta Cell of islet and vessel cell, thyroid cell, other endocrine organs' the cell and the spherule of above-mentioned cell.Before selecting, all these cells are in its potentiality of growing on matrix/surviving of vitro test or the ability that is divided into other useful tissues lipogenesis potentiality for example.The existence of adhering tissue further promotes new fats to be organized in the inner or growth on every side of supported matrix on the vessel pedicle.In an alternate embodiment, the reorganization of tissue extract or tissue, synthetic or purified components and vessel pedicle link.For example, in a preferred embodiment, these components and extract derive from substrate material and in vitro examination lipogenesis potentiality.

Matrix is allowed to set and will have the cell of adipocyte differentiation capability to cover on the individual layer matrix in the presence of perfect medium (DMEM that for example contains FCS) or division culture medium (being supplemented with the perfect medium of 1 μ M dexamethasone, Regular Insulin, indomethacin and IBMX).Time period through 14 days is observed lipogenesis.The example of suitable adipocyte comprises the 3T3-L1 cell or passes through the isolating preceding adipocyte of standard program.

Therefore, the present invention expects that external test promotes adipogenic component or extract, described mensuration is included in that vessel surface produces or obtains potential lipogenesis extract layer from muscle matrix, inoculation has the cell mass of adipocyte differentiation tendency on described layer, and described cell is hatched the differentiation that adipocyte takes place and checks subsequently for chien shih lipogenesis when enough in described cell.

In another embodiment, be determined in the three-dimensional supported matrix and carry out, described three-dimensional supported matrix can be made of or comprise the support that is covered by muscle matrix basically muscle matrix, carry out three-dimensional cell culture technique well known by persons skilled in the art about this point, for example rolling bottle technology (Mueller-Klieser J Cancer Res Clin Oncol 13:101-122,1986), (Yuhas waits the people to the fluid coating technology, Cancer Res 37:3639-3643,1977).Another example of three-dimensional cell culture technique is the rotating and culturing bottle of transforming through special, by around the culturing bottle of horizontal rotational shaft fluid filled so that minimum hydrodynamic shear suspension cell and cell mass are used in the gravitational vector randomization simultaneously.These devices are in U.S. Patent number 5,153,131; 5,153,132; 5,153,133; Obtain in 5,153,034 and 5,155,035 describing.

It is longer than single-layer system that one of advantage of three dimensional matrix is that it keeps period of the active propagation of culturing cell.This may be partly because the area increase of three dimensional matrix causes cytoactive to prolong proliferating cycle.Matrix provides keeps required upholder, somatomedin and the regulatory factor of culturing cell long period of activity propagation.By adding protein, glycoprotein, glycosaminoglycan, cell matrix and other materials to upholder itself or growing by the cell that can further strengthen in the presence of this upholder with these materials covering upholders.The three-dimensional of matrix allows to be formed with the microenvironment that helps cell maturation and migration.In the time of in being grown in this three dimension system, proliferative cell is similar to intravital mature tissue component ripe also suitably the separation with formation.

In order to make three-dimensional structure can keep the activity of viable cell, three dimensional matrix should be showed suitable space and component characteristic.This type of matrix comprise hydrogel or porous matrix for example based on fiber or sponge sample matrix.Common used material is natural polymer or " bio-matrix ", synthetic polymer and inorganic synthetics in the three dimensional matrix.In the present embodiment, when this method expection three dimensional matrix was used for external test, the biocompatibility of matrix was not a particularly important.

The preferred embodiment of bio-matrix be extract from muscle matrix those or with like the muscle matrix phase those or have the cell system that comprises muscle matrix.

The present invention has further contemplated that the purposes of muscle matrix in the composition product that promotes the cell growth.Muscle preparation of the present invention also helps based on selected the special growth of muscle preparation and the morphological feature concrete cell type of cell mixture selectivity purifying (adipocyte for example) to complexity.

Article " a " and " an " are used to refer to the article that surpass (being an at least one) grammer target in this article.For example, " component " refers to one and the component above.

Further describe the present invention by following non-limiting examples.

Embodiment 1

Matrix is extracted I

Collect from the animal (rat and pig) slaughtered recently or from the patient's of experience plastic surgery operations muscle samples.All samples all under the approval of suitable Ethics Committee and fully informed consent collect.All programsteps are all carried out on ice or at 4 ℃.

Muscle is collected, weighs and cut off fat before extracting matrix.Sample is washed and homogenate in the ice-cold 3.4M NaCl damping fluid that adds proteinase inhibitor (0.5mM PMSF, 2mM EDTA, 0.1M EACA, 2mM NEM) subsequently.Tissue homogenate is subsequently at 4 ℃ 10, centrifugal 15 minutes of 000rpm, subsequently abandoning supernatant and in 3.4M NaCl damping fluid the resuspension precipitation.This step repeats 2-3 time.

Precipitation subsequently with the 2M urea buffer solution of original structure volume equal volume in resuspension, homogenate is also spent the night 4 ℃ of stirrings.After this, extract is at 4 ℃ 14, and centrifugal 30 minutes of 000RPM also preserves supernatant liquor.Be deposited in half the 2M urea buffer solution of initial volume the repeated centrifugation step subsequently of homogenate again.Subsequently supernatant liquor and the supernatant liquor of preserving are in the past merged and filter.

Extract subsequently in 0.05M Tris/0.15M NaCl damping fluid (TBS) 4 ℃ to 0.5% v/v chloroform dialysed overnight as sterilization steps.Extract is only dialysed to TBS subsequently several times, dialyses for DMEM subsequently.The extract of aliquot is stored in-20 ℃.

Embodiment 2

Matrix is extracted II

Institute is in steps all at 4 ℃ or carry out on ice.Collect fresh as far as possible muscle tissue (preferred minimum about 20-30g) and weigh.In the steel Dissecting tray, cut off all as seen fat from muscle as quickly as possible.Add in the beaker on ice with 2: 1 volume ratios (for example the 100ml damping fluid is to 50g muscle) and to contain the cold 3.4M NaCl damping fluid of proteinase inhibitor and to add muscle.Sample is thoroughly homogenate subsequently.Muscle tissue homogenate is at 4 ℃ 10, centrifugal 15 minutes of 000RPM.Subsequently abandoning supernatant and in the 3.4M of equivalent NaCl damping fluid with pellet resuspended and homogenate again.This step repeats 2 times, makes and washs 3-4 time altogether in NaCl.The 3rd washing postprecipitation contains resuspension and homogenate (for example 50ml is to 50mg muscle) among the 50mMTris-HCl (pH7.4) of 0.5M NaCl, proteinase inhibitor at 1: 1 volume.Utilize magnetic stirring apparatus that the sample rotation is spent the night at 4 ℃.Sample is subsequently at 4 ℃ 14, centrifugal 30 minutes of 000RPM, and abandoning supernatant is also preserved sample.Precipitation is containing the 2.0M Guanidinium hydrochloride subsequently), carry out homogenate among the 50mM Tris-HCl (pH7.4) of 0.2mM dithiothreitol (DTT).Tissue homogenate stirred spend the night and at 4 ℃ 14, centrifugal 30 minutes of 000rpm also removes supernatant liquor and preserves.

All following steps are all carried out in two kinds of supernatant liquors, and described two kinds of supernatant liquors keep separating in these steps from start to finish.

By the filtered through gauze supernatant liquor to remove the floating fat of free etc.Extract is 0.2% v/v chloroform dialysed overnight in 4 ℃ of TBS damping fluids (5mls/ liter) that 1-2 is risen on magnetic stirring apparatus.This is a sterilization steps.Changing damping fluid into clean TBS also dialysed 8 hours at least.This step repeats 3 times.When changing dialysis buffer liquid, will manage terminal rotation for several times to guarantee mixing.After the last TBS dialysis, change damping fluid into DMEM and dialysed overnight.Thicker solution for example pig muscle matrix needs the longer time.Subsequently these two kinds of extract mixtures are lumped together the matrix that produces different structure so that improve pectisation.Subsequently with sample aliquot to sterile test tube.In the circulation cupboard, carry out this operation on ice.Sample is stored in-20 ℃ subsequently.

Embodiment 3

SDS-PAGE

Measure protein concn by bicinchoninic acid (BCA protein determination test kit).The matrix sample was prepared into 0.5-1.0mg/mL and boiled 5 minutes in Laemmli solution (Laemmli, Nature227:680-685,1970) before separating on the SDS-PAGE gel.On the polyacrylamide gel (Invitrogen) of 4-12%w/v gradient, will separate in the sample adding swimming lane of 15 μ L volumes and by the SDS-polyacrylamide gel electrophoresis.Gel electrophoresis 50 minutes under the constant voltage of 200V takes off gel and is used for immunoblotting assay with Xylene Brilliant Cyanine G (BrilliantBlue) dyeing or transfer after this.

Embodiment 4

Immunoblotting assay

As described in the embodiment 3 on the SDS-PAGE gel isolated protein and the gel of no dyeing is transferred to the nitrocellulose diaphragm.By applying the 100V constant voltage 1 hour, initial current is 220mA, shifts according to the wet method branching program.At first the Tris buffer saline (TBS) that contains 5%w/v non-fat dried milk, 0.1%v/v Tween 20 (TTBS) in the trace overnight incubation to reduce nonspecific reaction.First antibody was at room temperature hatched 1 hour, with TTBS rinsing 3 times and use second antibody (1: 5000-1: 10000) hatch 1 hour in conjunction with peroxidase subsequently.Before immunoreactive protein manifests by enhanced chemiluminescence type immunoblotting detection system (Amersham Pharmacia), trace rinsing 3 times in TTBS once more.Used six kinds of first antibody: HSPG 1: 5000 (Seikagaku Corporation), ln 1: 5000 (Dako), nidogen 1: 10000 (Chemicon) and collagen protein IV 1: 10000 (Dako), fibronectin 1: 10000 and SPARC 1: 10000.

Embodiment 5

Muscle matrix is analyzed

To muscle matrix with carry out the component comparative analysis from the preparation (Matrigel) of BD Biosciences and the results are shown in the table 2,3 and 4.

The evaluation of somatomedin:

Utilize QuantikineELISA test kit (R﹠amp; D System) and according to the test kit specification sheets measure somatomedin/cytokine levels.Somatomedin to be measured comprises vascular endothelial growth factor (VEGF), Thr6 PDGF BB (PDGF), transforming growth factor-beta (TGF-β), Prostatropin (bFGF), tumor necrosis factor alpha (TNF-α), Urogastron (EGF), type-1 insulin like growth factor (IGF-1), leukaemia inhibitory factor (LIF) and nerve growth factor (Chemicon test kit) (NGF).In brief, extract is diluted (1: 5-1: 10) and with standard and contrast in the antibody coating plate, hatched 2-3 hour.Plate is washed and adds polyclone second antibody in conjunction with horseradish peroxidase (HRP).After hatching, remove unnecessary binding substances and plate is carried out the 3rd time and hatch with the color substrate.After adding stop bath, (Axion, Mutliskan USA) read plate, and the absorbancy of described microplate reader is set in λ 450nm place, the correction reading is set in λ 550nm place to utilize the microplate reader.Utilize all measurements and calculations of Genesis 2.0 plate ocr software (detailed) execution, and numerical value is changed into pg/mg matrix.Table 2 comprises the growth factor levels among the Matrigel (registered trademark) that reports as manufacturer and measure according to ELSA.

Table 2

	BD Full (sample strip)	BD GRF (sample strip)	BD Full (ELISA)	BD GFR (ELISA)	KK Full (ELISA)	KK GFR (ELISA)
							bFGF	0.1pg/ml	0.1pg/ml	48pg/ml	-	304pg/ml	0pg/ml
PDGF	12pg/ml	＜5pg/ml	0pg/ml	0pg/ml	12pg/ml	0pg/ml
							TGFβ	2.3ng/ml	1.7ng/ml	2.3ng/ml	1.8ng/ml	4.5ng/ml	1.6ng/ml
EGF	0.5-1.3 ng/ml	＜0.5-1.3 ug/ml	0ng/ml	0ng/ml	0ng/ml	0ng/ml
							VEGF	NR	NR	8.2ng/ml	2.9ng/ml	8.3ng/ml	0.8ng/ml
TNFα	NR	NR	0pg/ml	0pg/ml	0pg/ml	0pg/ml

BD Full=is purchased Matrigel (registered trademark)

The Matrigel (registered trademark) that BD GFR=somatomedin reduces

Our Matrigel of KK Full=(registered trademark) preparation

The preparation that our somatomedin of KK GFR=reduces

NR=is record not

Table 3 and table 4 comprise the growth factor levels in the muscle matrix of measuring according to ELISA of being made by rat, pig and people.Table 3 is represented numerical range.The mean value of table 4 representative data and average standard error (SEM).

Table 3

Somatomedin	The rat extract	The pig extract	People's extract	The mouse extract	Matrigel
						(pg/mg)	(n＝10)	(n＝10)	(n＝6)	(n＝3)##	(n＝3-7)
FGF2	27-297	14-1281g#	41-250		0-18
						VEGF	7-64	NM	NM	34-45	801-1703
PDGF	2-45	1-8	5-12	NM	0-2.7
						NGF	2-100	13-22	18-71	NM
TGF-β	0-9	NB	0-45	NM	32-88
						TNFα	3-21	3-12	2-11	NM	2-6
EGF	NB	NB	2-18	NM
						LIF	NB	NB	NB	3-9

Table 4

The matrix source	The people	Pig	Rat	Mouse	Matrigel
							(n＝6)	(n＝10)	(n＝10)	(n＝3)	***
bFGF	131.16±36	217.9±121	113.5±32.6		6.17±2.3
						VEGF	1.05±0.4	0.58±0.25	23.15±5.8	41.3±	1145±165
TGF-β	24.75±8	NB	1.7±94		68.5±17.8
						TNF-α	7.38±1.3	7.15±1.14	7.1±1.7		2.8±1.7
PDGF	7.90±0.8	3.50±0.6	10.56±4.1		0.9±0.9
						NGF	33.9±7.9	18.2±1.9	28.5±9.1		15.03
EGF	9.3±2.4	NB	NB		NB
						LIF	NB	NB	NB		56.7

Very high reading of #=-other all readings all are lower than 339pg/mg.

Whether ## tests to these samples and watches and stride kind of combination and exist

NB: use these ELISA in these samples, not observe combination

NM: do not measure

The measurement of total protein concentration

Utilize bicinchoninic acid to measure that (Amersham Biosciences Corporation NewJersey USA) measures the total protein level of matrix.Sample is measured and is contrasted a series of BSA standards according to the test kit specification sheets with dilution in 1: 10 and measures.Utilize spectrophotometer t to analyze sample at 562nm wavelength place after hatching.Rat and pig muscle matrix are analyzed 10 samples for every kind, and people's muscle matrix is then analyzed 6.Table 5 shows according to the BCA total protein measures the muscle matrix of measurement and the total protein level among the Matrigel (registered trademark)

Table 5

The matrix source	Pig muscle	Rat muscle	People's muscle	Matrigel
					Total protein	4-15mg/ml	6-12mg/ml	4-10mg/ml	8-10mg/ml

Muscle matrix has also been described in the accompanying drawing 1 to 4.Accompanying drawing 1 provides the SDS-PAGE of Matrigel and other periplasts to compare.Accompanying drawing 2 is the photos from the muscle matrix of skeletal muscle.Accompanying drawing 3 is photos of the muscle matrix in pig muscle source.The Photomicrograph of accompanying drawing 4 has shown the successful generation of organizing in the rat.Accompanying drawing 5.

The measurement of ECM component

1,9-dimethylated methylene base indigo plant, Direct Red 80 (Sirius Red), chondroitin sulfate A (CSA), papoid, dithiothreitol (DTT) and collagen protein I are all available from Sigma-Aldrich.Sulfated glycosaminoglycans (GAGs) by with dyestuff 1, the precipitating action of 9-dimethylated methylene base indigo plant (DMMB) in the MyoGel sample by quantitatively.Digest interference albumen by the 40mM sodium phosphate buffer (pH6.8) that adds the isopyknic 0.6mg/ml of comprising papoid, 2mM EDTA and 4mM DTT, hatched 60 minutes at 60 ℃ subsequently.(people Biochimica ET Biophysica Acta 882:173-177 such as Farndale, 1986).Every kind of sample of aliquot (100uL) is hatched 30 minutes with the DMMB solution (the 16mg/L DMMB in 0.2M salt GuHCl, 1g/L sodium formiate and 1ml/L formic acid) of 1mL subsequently, and mixes continuously on swiveling wheel.GAG-DMMB mixture post precipitation separates insoluble material and supernatant liquor and removes supernatant liquor by centrifugal (10,000 * g 10 minutes).From precipitation, discharge dyestuff people Glycobiology 13:647-653 such as (, 2003) Barbosa by the decomplexing damping fluid (50mM sodium acetate buffer, pH6.8 contain 10% n-propyl alcohol and 4M GuHCl) that adds 1mL.Subsequently at microplate reader (Multiskan RC; Labsystems) measure the absorbancy (λ 650nm) of damping fluid in.Adopt chondroitin sulfate A (CSA) as standard.

With ln from 1mL heparin affinity chromatography post (Amersham Biosciences; Uppsala assesses people EMBO J18:863-870 such as (, 1999) Talts people J Biol Chem 275:35192-35199 such as (, 2000) Talts to it behind the wash-out in Sweden).In brief, dissolved the MyoGel sample in 24 hours by hatching at 4 ℃ with the 50mMTris-HCl (pH7.4) of the isopyknic 4M of containing GuHCl+2mM DTT.After 50mMTris-HCl (pH7.4)+0.15M NaCl dialysed overnight, the aliquot of 1mL is applied to affinity column.50mM Tris-HCl (pH7.4)+0.15M NaCl with 5mL flushes out non-ln from post, and goes out ln with the 50mMTris-HCl that contains 0.5M NaCl (pH7.4) wash-out of 5mL.Utilize aforesaid microplate micrometering to decide program with the Bio-Rad protein determination subsequently and come the evaluation layer Fibronectin

Precipitating action by itself and polyazo dye Sirius Red is measured collagen protein (Marotta and Martino Analytical Biochemistry 150:86-90,1985).The MyoGel of aliquot (100 μ L) was at room temperature hatched 30 minutes with the 0.5M acetate that contains 50 μ M Sirius Red of 1mL.After centrifugal (10,000 * g 10 minutes), in the microplate reader, measure the absorbancy (λ 550nm) of supernatant liquor.Use from the collagen protein I of rat tails as standard.

(Echelon, UT USA) measure the level of hyaluronan according to the test kit specification sheets by ELISA.

Table 6 and 7 shows the extracellular matrix components of the muscle matrix of being made by rat, pig and people.Table 6 is represented numerical range, and the mean value of table 7 representative data and average standard error (SEM).

Table 6

Matrix	The rat extract	The pig extract	People's extract
				GAG	0-5μg/mg	0.5-22μg/mg	0.6-1.8μg/mg
Ln	223-700μg/mg	62-1000μg/mg	375-1000μg/mg
				Collagen protein	49-532μg/mg	45-1000μg/mg	54-1249μg/mg
Hyaluronan	5-10ng/mg	0-5ng/mg	0-5ng/mg

Table 7

μ g/mg matrix	People's extract	The pig extract	The rat extract	Per-cent
					GAG	0.86±0.2	6±2.8	2.45±0.6	0.4％
Ln	865.57±150	442±120	422.8±67	45％
					Collagen protein	437.61±175	486.75±133	254.28±46.7	27％
Ng/mg matrix
					Hyaluronan	5-l0	＜5	＜5	NS

The SDS-PAGE/ immunoblotting assay

The matrix sample was prepared into 0.5-1.0mg/mL and boiled 5 minutes in Laemmli solution (Laemmli, 1970) before separating on the SDS-PAGE gel.The polyacrylamide gel of 3-8% or 4-12% gradient (Invitrogen, Carlsbad, CA, USA) on, sample that will 10 μ L volumes adds in the swimming lane and by the SDS-polyacrylamide gel electrophoresis and separates.Gel electrophoresis 45 minutes under the constant voltage of 200V takes off gel and is used for immunoblotting assay with coomassie brilliant blue staining or transfer after this.

For immunoblotting, isolated protein and the gel of no dyeing is transferred to the nitrocellulose diaphragm on the SDS-PAGE gel as mentioned above.Apply the 30V constant voltage by the wet method branching program shifted in 1 hour.After the transfer, contain 5% non-fat dried milk (Homebrand, SafewaysAUS], the phosphate buffered saline (PBS) (PBS) of 0.1% Tween 20 (TPBS) in the trace overnight incubation to reduce non-specific binding.Trace was at room temperature hatched in first antibody 1 hour, with TPBS rinsing 3 times and second antibody (Molecular Probes, UT, the USA or the Rocklands of suitable infrared markers with being suitable for used one anti-use subsequently, CA, USA) hatched 1 hour (1: 10000).Immunoreactive protein by the Odyssey infrared detection system (Licor Biosciences, USA) manifest before, trace rinsing 3 times in TPBS once more.The first antibody that uses comprises anti-HSPG 1: 5000 (Seikagaku Corporation, Japan), ln α 4 and α 21: 1000 (Dr Lydia Soroken friendship is gifted), 1: 3000 (Chemicon of nidogen, USA), fibronectin 1: 5000, collagen protein I 1: 10000, collagen protein IV 1: 10000 and SPARC 1: 10000 (Dr HKleinman friendship provides, NIH USA).

Accompanying drawing 6 is representative example of the western blotting of various ECM components in the different MyoGel kinds.All arrows and digitized representation molecular weight level.6A relates to the immunoblotting of collagen protein I in the rat muscle extract, 3 chains of mark (100,200,300kD).Swimming lane 1-10 is ten kinds of different rat muscle extract formulations.6B shows ln α 4 (180kD) in the rat muscle extract and α 2 (80kD fragment and in a little disperse at 300kD place).Swimming lane 1 is for Matrigel contrast swimming lane 2-11 is identical rat muscle extract formulation, shows particularly the strong band about α 4 chains.6C is presented at bonded heparan sulfate proteoglycan in the rat MyoGel HSPG gel, and swimming lane 1 is a molecular weight standard, and swimming lane 2 is Matrigel, perlecan dyeing is arranged on the 200kD band and swimming lane 3-11 is the rat muscle extract.6D shows bonded CH-296 in the rat muscle extract sample, and swimming lane 1 is identical rat muscle extract for Matrigel contrast swimming lane 2-11.

The cell in vitro differentiation assays

Utilize the preceding adipocyte of rat epididymis to carry out mensuration.In 24 orifice plates, carry out the mensuration that is used for morphological analysis.In 24 orifice plates, add 300 μ l extracellular matrix (ECM)/holes.Matrix is in 37 ℃ of 20-30.Subsequently, in each hole, add cell (is 0.3 * 106 cells/well for 24 well culture plates).37 ℃/5%CO ₂Under spend the night and allow cell attachment on matrix.Observe differentiation through 14 days time periods (following photograph in every 4-5 days).Use the cell be seeded in separately on tissue culturing plastic's goods in contrast.

Accompanying drawing 5 has shown adipocyte before the contrast on tissue culturing plastic's goods, and showing does not have differentiation.5B is presented at the preceding adipocyte on the MyoGel, shows that lipid gathers and to the differentiation of mature fat cell.The figure that inserts shows that individual cells gathers the high magnification map picture of lipid.5C shows the low power Photomicrograph of mouse chamber, and described chamber has been full of the muscle extract that induced lipolysis forms.

It will be appreciated by those skilled in the art that the present invention as herein described allows except that specifically described change and modification those.It should also be understood that and the present invention includes all this type of change and modifications.The present invention also comprise the institute mentioning individually or jointly in this specification sheets or hint in steps, feature, composition and compound, and any and all any two or more described step or combination of features.

BIBLIOGRAPHY

Barbosa et al.Glycobiology 13：647-653，2003

Farndale et al.Biochimica ET Biophysica Acta 882：173-177，1986

Inoue et al.，J Cell Biol，97：1524-1539，1983

Kleinman et al.，Biochem 21：6188-6193，1982

Laemmli，Nature 227：680-685，1970

Marotta and Martino Analytical Biochemistry 150：86-90，1985

Mueller-Klieser，J Cancer Res Clin Oncol 13：101-122，1986

Talts et al.EMBO J 18：863-870，1999

Talts et al.J Biol Chem 275：35192-35199，2000

Terranova et al.，Cell 22：719-726，1980

Yuhas，et al.，Cancer Res 37：3639-3643 1977

Claims (18)

1. composition of matter that is used to promote cytological effect, described composition comprises based on muscle tissue preparation extract cell or acellular, described preparation provides the source of following component, described component such as but not limited to, ln, collagen protein I, collagen protein IV, sense of touch albumen/nidogen, heparan sulfate proteoglycan and other components, comprise cytokine and somatomedin, such as but not limited to, one or more in EGF, bFGF, NGF, PDGF, IGF-1, TGF-β, VEGF and TNF-α or its homologue.

2. the composition of claim 1, wherein said preparation is the source of ln, sense of touch albumen/nidogen, heparan sulfate proteoglycan, collagen protein IV, bFGF, PDGF, TGF-β, VEGF and TNF-α.

3. the composition of claim 1, wherein said cell is from muscle tissue.

4. the composition of claim 3, wherein said muscle tissue is from people, non-human primates, livestock animals, laboratory test animal, companion animals, birds, Reptilia or Amphibians.

5. the composition of claim 3, wherein said muscle tissue is from people, pig or rat.

6. the composition of claim 1, wherein said preparation is based on the preparation of cell.

7. the composition of claim 1, wherein said preparation comprises complete extracellular matrix.

8. the composition of claim 1, wherein said preparation comprises the extracellular matrix of extraction.

9. the composition of claim 1, wherein said preparation comprises the total protein content of the about 100mg/ml of about 1 μ g/ml-.

10. the composition of any one among the claim 1-9, wherein said preparation aggregates into gel.

11. the composition of claim 1, it comprises that further one or more exogenous cytokines, microbiotic, growth intensifying factor, gene expression enhancer, antiblastic and/or differentiation of stem cells promote the factor.

12. the composition of claim 1, wherein said composition promotes cytological effect, and described cell is selected from stem cell, epithelial cell, skin cells, organ cell and endotheliocyte.

13. the composition of claim 1, wherein the bFGF of Cun Zaiing is the about 4ng/ml of about 0.3ng/ml-; PDGF is the about 3000pg/ml of about 1pg/ml-; TFG-β is the about 2000pg/ml of about 1pg/ml-; EGF is the about 100ng/ml of about 0ng/ml-; VEGF is the about 3000pg/ml of about 1pg/ml-; And TNF-α is the about 1000pg/ml of about 0pg/ml-.

14. the composition of any one is in the purposes of the lipogenesis potentiality of some material being carried out in the biological assay among the claim 1-13.

15. the purposes of claim 14, be used for external test and regulate adipogenic component, extract or cell system, described mensuration comprises that examination muscle preparation has the cell mass of adipocyte differentiation tendency with discriminating, produce or obtain the adipogenic component of potential adjusting or extract or cell system, described cell mass with adipocyte differentiation tendency is seeded on described component or the extract, described cell is hatched the differentiation that adipocyte takes place and checks subsequently for chien shih lipogenesis when enough in described cell.

16. the purposes of claim 15, be used to produce and be fit to be transplanted to the method that is subjected to intravital donor blood vessel tissue, described method comprises makes the vessel pedicle comprise the function recycle system, described vessel pedicle in a organized way or tissue extract or its component inject, adhere to or otherwise link with described vessel pedicle; With described vessel pedicle be combined in supported matrix inner and/or above; The separated cell or tissue fragment of inoculation in described supported matrix, described cell or tissue fragment utilize external test to differentiate to promoting lipogenesis; Or some other useful end points, the described supported matrix that will comprise described vessel pedicle is implanted to certain position of acceptor, the described function recycle system and partial artery or venous anastomosis at described position; And described supported matrix stayed described implant site enough periods to allow the vascularization growth of new tissue, wherein select described injecting material or inoculation material according to certain criteria, for example, it promotes lipogenesis when measuring by assay method, described assay method comprises checks that tissue or tissue extract have the cell mass of adipocyte differentiation tendency with discriminating, produce or obtain adipogenic component of potential promotion or extract, the cell mass that will have adipocyte differentiation tendency is seeded on described component or the extract, and described cell is hatched the differentiation that adipocyte takes place and checks subsequently for chien shih lipogenesis when enough in described cell.

17. the method for claim 16, wherein said vessel pedicle comprises fat or other fatty tissues that adhere to or the tissue that comprises following cell, and described cell is a sarcoplast, inoblast, preceding adipocyte and adipocyte, the myocardial cell, keratinocyte, endotheliocyte, smooth muscle cell, the chondrocyte, pericyte, the matrix precursor cell of derived from bone marrow, embryonic stem cell, mescenchymal stem cell or hemopoietic stem cell, other cells of schwann cell and periphery and central nervous system, olfactory cell, liver cell and other liver cells, glomerular mesangium and other nephrocytes, beta Cell of islet and vessel cell, thyroid cell, other endocrine organs' the cell and the spherule of above-mentioned cell.

18. the method for claim 17, wherein said vessel pedicle constitutes for the external test that promotes adipogenic component or extract, described mensuration is included in that vessel surface produces or obtains potential lipogenesis extract layer from muscle matrix, inoculation has the cell mass of adipocyte differentiation tendency on described layer, and described cell is hatched the differentiation that adipocyte takes place and checks subsequently for chien shih lipogenesis when enough in described cell.