CN1560233A - Growing promoter of eukaryocyte - Google Patents
Growing promoter of eukaryocyte Download PDFInfo
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- CN1560233A CN1560233A CNA2004100061637A CN200410006163A CN1560233A CN 1560233 A CN1560233 A CN 1560233A CN A2004100061637 A CNA2004100061637 A CN A2004100061637A CN 200410006163 A CN200410006163 A CN 200410006163A CN 1560233 A CN1560233 A CN 1560233A
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Abstract
The invention discloses a eukaryotic cell growth accelerant, including active components oligomeric 3-hydroxy fatty acid and/or its monomer 3-hydroxy fatty acid, which both have an alkyl group with a straight chain or side chain composed of 1-10 carbons. The preferred alkyl group is methyl, ethyl, propyl, butyl, amyl or heptyl. The accelerant can be widely applied to cell engineering and tissue engineering as well as the culture in vitro of eukaryotic cells and tissues and organs.
Description
Technical field
The present invention relates to a kind of growth promoter in cell engineering and the field of tissue engineering technology, particularly a kind of eukaryotic cell growth stimulant.
Background technology
In recent years along with the development of related disciplines such as cytobiology, molecular biology, biomaterial and transplantation immunology, clinical medicine, achievement and technology that various fields are obtained are constantly intersected, are merged and interpenetrate, formed a new subject gradually---organizational engineering (Fishman J.How to build a body part.Time, 1999,153 (8): 54-55; Cowley G, Underwood A.Replacement parts.Newsweek, 1997,129 (4): 66-69; Stix G.Growing a new field.Scientific American, 1997,277 (4): 15-16).
Organizational project (Tissue Engineering) speech is the earliest by the formal proposition of the American National science fund council and definite.Its ultimate principle and method are that the normal tissue cell with cultured and amplified in vitro is adsorbed in and a kind ofly has good cell compatibility and can be formed mixture above the biomaterial of body degraded and absorbed, then with cell, biomaterial composites implant into body tissue, the disease of organ is decreased the position, as the biomaterial of cell growth support gradually by the body degraded and absorbed in, cell is constantly bred, differentiation, form new and its form, function aspects and respective organization, the tissue of organ unanimity, thereby reach purpose (the Langer R that repairs wound and rebuild function, Vacanti J P.Artificial organs.Scientific American, 1995,273 (3): 130-133; Keith T, Cima L G, Yaremchuk, et al.Injectable cartilage.Plast ReconstrSurg, 1995,96 (6): 1390-1398; Schultz O, Keuszer G, Zacher J, et al.Arithritis Rheum, 1997; 40 (8): 1420-1433.).
Polyhydroxyalkanoate (PHA) is a lot of bacteriums a kind of bacterium born of the same parents of synthetic inner macromolecule polyester (Chen GQ, Wu Q, Zhao K, Yu HP ﹠amp under given conditions; Chan A.Chinese J of Polymer Science18 (2000) 389-396), it is one of bio-energy source of newfound bacterium, has very good character, characteristics such as its biodegradability, bio-compatible have caused academia, the great interest on biomedical boundary (Deng Y, Zhao K, Zhang XF particularly, Hu P, Chen GQ.Biomaterials 23 (20) (2002) 4049-4056; Zheng Z, Deng Y, Lin XS, Zhang LX, Chen GQ.J Biomater Sci.PolymerEdn 14 (2003) 615-624).It launches just in the world in the application and development aspect the high added values such as medicament slow release material, human implantable biomaterial, at artificial organs, plastic surgery, wound. the application prospect in the healing especially wide (Deng Y, Lin XS, Zheng Z, Deng JG, Chen JC, Ma H, Chen GQ.Biomaterials 24 (2003) 4273-4281; Wang YW, Chen GQ, Wu Q.Biomaterials 24 (2003) 4621-4629).
PHA discharges oligomerization PHA (OHA) and 3-hydroxy fatty acid (HA) monomer in its biodegradation process, as, 3-hydroxybutyric acid (HB) and oligomer (OHB) thereof, 3-hydroxypentanoic acid (HV) and oligomer (OHV) thereof, 3-hydroxycaproic acid (HHx) and oligomer (OHHx) thereof, 3-hydroxydecanoic acid (HD) and oligomer (OHD) thereof.Studies show that organoid of higher animal and endochylema the inside also exist OHA (oligomerization PHA), relevant with calcium channel and DNA transhipment etc.Experimental result shows that OHB forms non-selective ionic channel in manual simulation's planar lipid bilayer, can transport the duplicature of calcium ion by vesica.In preliminary study, the mixture of PHA, OHA and albumen etc. is considered to and human two kinds of principal disease arteriosclerosis relevant (Reusch RN, Sadoff HL.Proc NatlAcad Sci USA 1988 with diabetes; 85 (12): 4176-80).
The innovation and creation content
The purpose of this invention is to provide a kind of eukaryotic cell growth stimulant.
Eukaryotic cell growth stimulant provided by the present invention, its activeconstituents are oligomerization 3-hydroxy fatty acid and/or monomer whose 3-hydroxy fatty acid, and described oligomerization 3-hydroxy fatty acid and monomeric chemical structural formula thereof are as follows:
Wherein, R is that n is the natural number of 1-1000 by the alkyl of one to ten straight chain that carbon is formed or side chain.In actual applications, R is preferably methyl, ethyl, propyl group, butyl, amyl group, hexyl or heptyl; N is preferably the natural number of 1-200.
The activeconstituents of above-mentioned eukaryotic cell growth stimulant also can be the salt of oligomerization 3-hydroxy fatty acid and/or monomer whose 3-hydroxy fatty acid.
When the R=methyl, oligomerization 3-hydroxy aliphatic acid mono is 3-hydroxybutyric acid (3-hydroxybutyrate or HB); During the R=ethyl, oligomerization 3-hydroxy aliphatic acid mono is 3-hydroxypentanoic acid (3-hydroxyvalerate or HV); During the R=propyl group, oligomerization 3-hydroxy aliphatic acid mono is 3-hydroxycaproic acid (3-hydroxyhexanoate or HHx); During the R=butyl, oligomerization 3-hydroxy aliphatic acid mono is 3-hydroxyl enanthic acid (3-hydroxyheptanoate or HHp); During the R=amyl group, oligomerization 3-hydroxy aliphatic acid mono is 3-Hydroxyoctanoic acid (3-hydroxyoctanoate or HO); During the R=hexyl, oligomerization 3-hydroxy aliphatic acid mono is 3-hydroxyl n-nonanoic acid (3-hydroxynonanoate or HN); During the R=heptyl, oligomerization 3-hydroxy aliphatic acid mono is 3-hydroxydecanoic acid (3-hydroxydecanoate or HD).
The monomer of described oligomerization 3-hydroxy fatty acid and 3-hydroxy fatty acid can be the compounds of single three-dimensional arrangement, as the R type, or the S type, even can be the mixture of R and S type, also can be the compound of racemize structure.Oligomerization 3-hydroxy fatty acid comprises 3-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxycaproic acid, 3-hydroxyl enanthic acid, 3-Hydroxyoctanoic acid, the 3-hydroxyl n-nonanoic acid of oligomerization, the simple oligomer or the copolymerization oligomer of 3-hydroxydecanoic acid, also comprises the mixture of these oligomers.Described 3-hydroxy fatty acid can be 3-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxycaproic acid, 3-hydroxyl enanthic acid, 3-Hydroxyoctanoic acid, 3-hydroxyl n-nonanoic acid, 3-hydroxydecanoic acid, also can be these sour mixtures.Described eukaryotic cell is inoblast, scleroblast, chondrocyte, neurocyte, skin cells, blood vessel epithelial cell, hemopoietic stem cell, liver cell, lymphocyte, corneal epithelial cell, nephrocyte etc.
Above-mentioned eukaryotic cell growth stimulant can promote the histoorgan of vitro culture, as becoming the growth of fibrous tissue, body of gland, lung, ovary, nerve, osseous tissue, cerebral tissue, blood vessel, liver, kidney.
Above-mentioned oligomerization 3-hydroxy fatty acid and monomer whose 3-hydroxy fatty acid can obtain by hydrolysis or enzymolysis and genetically engineered microorganism fermentation process (Xi Jianzhong. microbial process synthesis of chiral hydroxy fatty acid. Tsing-Hua University's Master's thesis (2000); Gao HJ, Wu Q, Chen GQ.FEMS Microbiol Lett 213 (2002) 59-65; Zhao K, Tian G, Zheng Z, Chen JC, Chen GQ.FEMS Microbiol Lett 218 (2003) 59-64; Zheng Z, Zhang MJ, Zhang G and Chen GQ.Antonie van Leeuwenhoek (2004) in press).Can be by the purity of liquid chromatographic detection oligomerization 3-hydroxy fatty acid.
The working concentration of eukaryotic cell growth stimulant of the present invention is the 0.001-0.1g/L substratum.Experimental results show that, eukaryotic cell growth stimulant of the present invention is within the specific limits along with the rising of concentration, the increment of cell is also improving, but too high concentration can not make the increment of cell continue to increase, and pair cell system and primary cell have shown same growth promoting function effect, and have the low advantage of cost, can be widely used in the vitro culture of cell engineering and organizational project and eukaryotic cell and histoorgan.
Description of drawings
Fig. 1 a is the influence histogram of different concns 3-hydroxybutyric acid eukaryotic cell growth stimulant to inoblast L929 growth.
Fig. 1 b is that different concns R type 3-(R)-hydroxybutyric acid is as the influence histogram of eukaryotic cell growth stimulant to inoblast L929 growth.
Fig. 2 a is the influence histogram of different concns 3-hydroxybutyric acid eukaryotic cell growth stimulant to newborn rabbit knee cartilage cell growth.
Fig. 2 b is that different concns 3-(R)-hydroxybutyric acid is as the influence histogram of eukaryotic cell growth stimulant to newborn rabbit knee cartilage cell growth.
Fig. 3 a is the influence histogram of different concns 3-hydroxybutyric acid eukaryotic cell growth stimulant to the human vascular endothelial growth.
Fig. 3 b is the influence histogram of different concns 3-(R)-hydroxybutyric acid eukaryotic cell growth stimulant to the human vascular endothelial growth.
Embodiment
R among the following embodiment represents opticity, is not substituting group.
(3-(R)-HB) eukaryotic cell growth stimulant is to the influence of inoblast L929 propagation for embodiment 1, racemize 3-hydroxybutyric acid (HB) eukaryotic cell growth stimulant and photoactive 3-(R)-hydroxybutyric acid
Clone: inoblast L929 (purchasing) in Inst. of Viruses, China Preventive Medicine Science Academy.
Substratum: DMEM (Dulbecco ' s Modified Eagle Medium) (GIBCO), 216mg/LL-glutamine, the acid of 36mg/L altheine, 4.766g/L Hepes (dihydroxy ethyl croak piperazine ethane sulfonic acid), 2g/L NaHCO
3, 100mg/L penicillin, 100mg/L Streptomycin sulphate, 10% foetal calf serum (three profits).
Serum free medium: DMEM (GIBCO), 216mg/L L-glutaminate, the acid of 36mg/L altheine, 4.766g/L Hepes, 2g/L NaHCO
3, 100mg/L penicillin, 100mg/L Streptomycin sulphate.
Digestive system: DMEM (GIBCO), 216mg/L L-glutaminate, the acid of 36mg/L altheine, 4.766g/LHepes, 2g/L NaHCO
3, 100mg/L penicillin, 100mg/L Streptomycin sulphate, 0.2% trypsinase.
Washing fluid: PBS damping fluid (8.0g/L sodium-chlor, 0.20g/L Repone K, 1.44g/L Sodium phosphate dibasic, 0.24g/L potassium primary phosphate), 100mg/L penicillin, 100mg/L Streptomycin sulphate.
MTT (Thiazolyl blue) dye liquor: 5mg/ml (5mg MTT is dissolved in 1ml PBS).
Lysate: DMSO (dimethyl sulfoxide (DMSO))
Racemize 3-hydroxybutyric acid HB eukaryotic cell growth stimulant: form by racemize 3-Sodium (available from Sigma company) single component.
3-(R)-hydroxybutyric acid ((R) HB) eukaryotic cell growth stimulant: form by 3-(R)-HB or 3-(R)-Sodium (available from Sigma company) single component.
The interpolation concentration of above-mentioned two kinds of eukaryotic cell growth stimulants is respectively: 0.005g/L, 0.01g/L, 0.02g/L, 0.05g/L, 0.1g/L.
Culture condition: 37 ℃, 5%CO
2Constant incubator, inoculum size are 10
4Individual/hole (24 orifice plate)
Measure the increment of cell with mtt assay:
1) dyeing: cell is cultivated certain hour in 24 orifice plates after, take out sample, inhale and remove nutrient solution, the aseptic PBS (PH=7.4) of every hole adding 0.5ml swings and washes 3 times.Add 0.1ml MTT dye liquor and 1ml again and do not contain the substratum of serum, place incubator to preserve 4 hours.
2) supernatant is removed in suction, adds 1ml DMSO, places 30 minutes under the room temperature, and colored particle is fully dissolved.
3) get 0.2ml liquid to be measured and join in the 96 hole enzyme plates, microplate reader 550nm measures absorbance down.
The result is shown in Fig. 1 a and Fig. 1 b, show in the effect group of having added bromoacetoxylphenylhexan-hydroxybutyric acid (HB) 3-hydroxybutyric acid eukaryotic cell growth stimulant or 3-(R)-hydroxybutyric acid eukaryotic cell growth stimulant, all produce certain cell growth-promoting effect, the best of the two is added concentration and is respectively 0.05g/L, 0.01g/L the growth increasing amount of pair cell has reached 98.5 ± 1.5% and 50.3 ± 2.6% respectively.
(3-(R)-HB) eukaryotic cell growth stimulant is to the influence of newborn rabbit knee cartilage cell proliferation for embodiment 2,3-hydroxybutyric acid (HB) eukaryotic cell growth stimulant and 3-(R)-hydroxybutyric acid
Passage cell: newborn rabbit knee cartilage cell
Substratum: DMEM (GIBCO), 216mg/L L-glutaminate, the acid of 36mg/L L-N, 4.766g/L Hepes, 2g/L NaHCO
3, 100mg/L penicillin, 100mg/L Streptomycin sulphate, 10% foetal calf serum (three profits)
Serum free medium: DMEM (GIBCO), 216mg/L L-glutaminate, the acid of 36mg/L L-a N, 4.766g/L Hepes, 2g/L NaHCO
3, 100mg/L penicillin, 100mg/L Streptomycin sulphate
Digestive system: DMEM (GIBCO), 0.125% trypsinase, 0.02%EDTA, 100mg/L penicillin, 100mg/L Streptomycin sulphate
Washing fluid: PBS, 100mg/L penicillin, 100mg/L Streptomycin sulphate
MTT dye liquor: 5mg/ml (5mg MTT is dissolved in 1ml PBS)
Lysate: DMSO
3-hydroxybutyric acid (HB) eukaryotic cell growth stimulant: form by 3-Sodium (available from Sigma company) single component.
3-(R)-hydroxybutyric acid eukaryotic cell growth stimulant: form by 3-(R)-HB or 3-(R)-Sodium (available from Sigma company) single component.
The interpolation concentration of above-mentioned two kinds of eukaryotic cell growth stimulants is respectively: 0.005g/L, 0.01g/L, 0.02g/L, 0.05g/L, 0.1g/L.
Culture condition and mtt assay are with embodiment 1.The result is shown in Fig. 2 a and Fig. 2 b, show in the effect group of having added 3-hydroxybutyric acid eukaryotic cell growth stimulant or 3-(R)-hydroxybutyric acid eukaryotic cell growth stimulant, all produce certain cell growth-promoting effect, the best of the two is added concentration and is respectively 0.05g/L, 0.005g/L the growth increasing amount of pair cell has reached 66.5 ± 1.4% and 28.4 ± 1.6% respectively.
(3-(R)-HB) eukaryotic cell growth stimulant is to the influence of human umbilical vein endothelial cell propagation for embodiment 3,3-hydroxybutyric acid (HB) eukaryotic cell growth stimulant and 3-(R)-hydroxybutyric acid
Passage cell: human umbilical vein endothelial cell
Former generation substratum: M199 (GIBCO), 2.3g/L NaHCO
3, 15mmol/L HEPES, 2mg/L ECGF, 5mg/L heparin, 2mmol/L L-Gln, 1mmol/L Sodium.alpha.-ketopropionate, 100mg/L penicillin, 100mg/L Streptomycin sulphate, 20% foetal calf serum (three profits).
Subculture medium: M199 (GIBCO), 2.3g/L NaHCO
3, 15mmol/L HEPES, 2mg/L ECGF, 5mg/L heparin, 2mmol/L L-Gln, 1mmol/L Sodium.alpha.-ketopropionate, 100mg/L penicillin, 100mg/L Streptomycin sulphate, 15% foetal calf serum (three profits).
Serum free medium: M199 (GIBCO), 2.3g/L NaHCO
3, 15mmol/L HEPES, 2mg/L ECGF, 5mg/L heparin, 2mmol/L L-Gln, 1mmol/L Sodium.alpha.-ketopropionate, 100mg/L penicillin, 100mg/L Streptomycin sulphate.
Washing fluid: PBS, 100mg/L penicillin, 100mg/L Streptomycin sulphate
Digestive system: PBS, 0.125% trypsinase, 0.02%EDTA, 100mg/L penicillin, 100mg/L Streptomycin sulphate
Stop buffer: PBS, 5% foetal calf serum (three profits)
MTT dye liquor: 5mg/ml (5mg MTT is dissolved in lml PBS)
Lysate: DMSO
3-hydroxybutyric acid (HB) eukaryotic cell growth stimulant: form by 3-Sodium (available from Sigma company) single component.
3-(R)-hydroxybutyric acid ((R) HB) eukaryotic cell growth stimulant: form by 3-(R)-HB or 3-(R)-Sodium (available from Sigma company) single component.
The interpolation concentration of above-mentioned two kinds of eukaryotic cell growth stimulants is respectively: 0.005g/L, 0.01g/L, 0.02g/L, 0.05g/L, 0.1g/L.Culture condition and mtt assay are with embodiment 1.The result is shown in Fig. 3 a and Fig. 3 b, show in the effect group of having added 3-hydroxybutyric acid eukaryotic cell growth stimulant or 3-(R)-hydroxybutyric acid eukaryotic cell growth stimulant, all produce certain cell growth-promoting effect, the best of the two is added concentration and is respectively 0.05g/L, 0.01g/L the growth increasing amount of pair cell has reached 57 ± 1.6% and 31.6 ± 1.8% respectively.
Embodiment 4,3-hydroxypentanoic acid (HV) eukaryotic cell growth stimulant, 3-(R)-hydroxypentanoic acid ((R) HV) eukaryotic cell growth stimulant and oligomer OHV eukaryotic cell growth stimulant are to the influence of human umbilical vein endothelial cell propagation
Passage cell: human umbilical vein endothelial cell
3-hydroxypentanoic acid (HV) eukaryotic cell growth stimulant: form by 3-hydroxypentanoic acid (HV) single component.
3-(R)-hydroxypentanoic acid ((R) HV) eukaryotic cell growth stimulant: form by 3-(R)-hydroxypentanoic acid ((R) HV) single component.
Oligomer OHV eukaryotic cell growth stimulant: form by oligomer OHV single component.
Wherein, 3-hydroxypentanoic acid (HV) or 3-(R)-hydroxypentanoic acid ((R) HV) or its oligomer OHV obtain in acidity or alkaline hydrolysis PHV by having a liking for water pseudomonas Aeromonas hydrophila synthetic poly-3-hydroxypentanoic acid PHV in undeeanoic acid.Concrete grammar reference literature: D Seebach, M G Fritz.InternationalJournal of Biological Macromolecules 25 (1999) 217-236.Wherein, have a liking for water pseudomonas Aeromonas hydrophila available from Chinese Academy of Sciences microbial strains preservation center.
The interpolation concentration of above-mentioned three kinds of eukaryotic cell growth stimulants is respectively: 0.005g/L, 0.01g/L, 0.02g/L, 0.05g/L, 0.1g/L.
Cultural method, condition and mtt assay are with embodiment 1.Experimental result shows in the effect group of having added 3-hydroxypentanoic acid (HV) eukaryotic cell growth stimulant, 3-(R)-hydroxypentanoic acid (R) HV eukaryotic cell growth stimulant, oligomer OHV eukaryotic cell growth stimulant, all produce certain cell growth-promoting effect, the best of three is added concentration and is respectively 0.05g/L, 0.01g/L, 0.04g/L the growth increasing amount of pair cell has reached 48 ± 1.5%, 24 ± 2.5%, 45 ± 2.1% respectively.
Embodiment 5,3-hydroxycaproic acid (HHx) eukaryotic cell growth stimulant, 3-(R)-hydroxycaproic acid ((R) HHx) eukaryotic cell growth stimulant and oligomer (OHHx) eukaryotic cell growth stimulant are to the influence of inoblast L929 propagation
Clone: inoblast L929 (purchasing) in Inst. of Viruses, China Preventive Medicine Science Academy
Cultural method is with embodiment 1.
3-hydroxycaproic acid (HHx) eukaryotic cell growth stimulant: form by 3-hydroxycaproic acid (HHx) (available from Sigma company) single component.
3-(R)-hydroxycaproic acid ((R) HHx) eukaryotic cell growth stimulant: form by 3-(R)-hydroxycaproic acid ((R) HHx) (available from Sigma company) single component.
Oligomerization 3-(R)-hydroxycaproic acid (OHHx) eukaryotic cell growth stimulant: form by oligomerization 3-(R)-hydroxycaproic acid (OHHx) single component.
Wherein, oligomerization 3-(R)-hydroxycaproic acid (OHHx) is by have a liking for water pseudomonas Aeromonas hydrophila synthetic 3-(R)-hydroxybutyric acid HB and 3-(the R)-hydroxycaproic acid copolymer p HBHHx that grow in lauric acid, obtain in acidity or alkaline hydrolysis PHBHHx, concrete grammar reference literature: D Seebach, M G Fritz.InternationalJournal of Biological Macromolecules 25 (1999) 217-236.Wherein, have a liking for water pseudomonas Aeromonas hydrophila available from Chinese Academy of Sciences microbial strains preservation center.
The interpolation concentration of above-mentioned three kinds of eukaryotic cell growth stimulants is respectively: 0.005g/L, 0.01g/L, 0.02g/L, 0.05g/L, 0.1g/L.
Culture condition and mtt assay are with embodiment 1.Experimental result shows in the effect group of having added 3-hydroxycaproic acid (HHx) eukaryotic cell growth stimulant, 3-(R)-hydroxycaproic acid ((R) HHx) eukaryotic cell growth stimulant and oligomer (OHHx) eukaryotic cell growth stimulant, all produce certain cell growth-promoting effect, the best concentration of adding is respectively 0.05g/L, 0.01g/L, 0.03g/L the growth increasing amount of pair cell has reached 32 ± 2.4%, 29 ± 2.6%, 68 ± 1.9% respectively.
Embodiment 6,3-hydroxydecanoic acid (HD) eukaryotic cell growth stimulant or 3-(R)-hydroxydecanoic acid ((R) HD) eukaryotic cell growth stimulant and oligomer OHD eukaryotic cell growth stimulant are to the influence of newborn rabbit knee cartilage cell proliferation
Passage cell: newborn rabbit knee cartilage cell
3-hydroxydecanoic acid (HD) eukaryotic cell growth stimulant: form by 3-hydroxydecanoic acid (HD) (available from Sigma company) single component.
3-(R)-hydroxydecanoic acid ((R) HD) eukaryotic cell growth stimulant: form by 3-(R)-hydroxydecanoic acid ((R) HD) (available from Sigma company) single component.
Oligomer OHD eukaryotic cell growth stimulant: form by oligomer OHD single component.
Wherein, oligomer OHD is grown in glucose by pseudomonas Pseudomonas stutzeri 1317 and synthesizes 3-(R)-Hydroxyoctanoic acid HO and 3-(R)-hydroxydecanoic acid copolymer p HOD, obtains in acidity or alkaline hydrolysis PHOD.Concrete grammar reference literature: D Seebach, M G Fritz.International Journal ofBiological Macromolecules 25 (1999) 217-236.Wherein, pseudomonas Pseudomonasstutzeri 1317 is available from Chinese Academy of Sciences microbial strains preservation center.
The interpolation concentration of above-mentioned three kinds of eukaryotic cell growth stimulants is respectively: 0.005g/L, 0.01g/L, 0.02g/L, 0.05g/L, 0.1g/L.
Cultural method is with embodiment 2, and culture condition and mtt assay are with embodiment 1.Experimental result shows in the effect group of having added 3-hydroxydecanoic acid (HD) eukaryotic cell growth stimulant or 3-(R)-hydroxydecanoic acid ((R) HD) eukaryotic cell growth stimulant and oligomer OHD eukaryotic cell growth stimulant, all produce certain cell growth-promoting effect, the best concentration of adding is respectively 0.02g/L, 0.005g/L, 0.02g/L, the growth increasing amount of pair cell has reached 58 ± 1.3%, 26 ± 1.7%, 57 ± 2.2% respectively.
Embodiment 7,3-(R)-hydroxybutyric acid ((R) HB) and 3-(R)-hydroxycaproic acid ((R) HHx) mixture eukaryotic cell growth stimulant and copolymerization oligomer OHBHHx eukaryotic cell growth stimulant are to the influence of inoblast L929 propagation
Clone: inoblast L929 (purchasing) in Inst. of Viruses, China Preventive Medicine Science Academy
Cultural method is with embodiment 1.
3-(R)-hydroxybutyric acid ((R) HB) and 3-(R)-hydroxycaproic acid ((R) HHx) mixture eukaryotic cell growth stimulant: mix (the blended mass ratio is 50: 50) by 3-(R)-hydroxybutyric acid ((R) HB) and 3-(R)-hydroxycaproic acid ((R) HHx) (all available from Sigma company).
3-(R)-hydroxybutyric acid ((R) HB) and 3-(R)-hydroxycaproic acid ((R) HHx) copolymerization oligomer OHBHHx eukaryotic cell growth stimulant: form by 3-(R)-hydroxybutyric acid (R) HB and 3-(R)-hydroxycaproic acid (R) HHx copolymerization oligomer OHBHHx single component.
Wherein, 3-(R)-hydroxybutyric acid ((R) HB) and 3-(R)-hydroxycaproic acid ((R) HHx) copolymerization oligomer OHBHHx obtain in acidity or alkaline hydrolysis PHBHHx by have a liking for water pseudomonas Aeromonas hydrophila synthetic 3-(R)-hydroxybutyric acid HB and 3-(the R)-hydroxycaproic acid copolymer p HBHHx that grow in lauric acid.Concrete grammar reference literature: D Seebach, M G Fritz.International Journal of BiologicalMacromolecules 25 (1999) 217-236.Wherein, have a liking for water pseudomonas Aeromonas hydrophila available from Chinese Academy of Sciences microbial strains preservation center.
The interpolation concentration of above-mentioned two kinds of eukaryotic cell growth stimulants is respectively: 0.005g/L, 0.01g/L, 0.02g/L, 0.05g/L, 0.1g/L.
Culture condition and mtt assay are with embodiment 1.Experimental result shows in the effect group of having added 3-(R)-hydroxybutyric acid ((R) HB) and 3-(R)-hydroxycaproic acid ((R) HHx) mixture eukaryotic cell growth stimulant and oligomer OHBHHx eukaryotic cell growth stimulant, all produce certain cell growth-promoting effect, the best concentration of adding is respectively 0.001g/L, 0.01g/L the growth increasing amount of pair cell has reached 30 ± 2.2%, 22 ± 2.0% respectively.
Claims (8)
1, a kind of eukaryotic cell growth stimulant, its activeconstituents are oligomerization 3-hydroxy fatty acid and/or monomer whose 3-hydroxy fatty acid, and described oligomerization 3-hydroxy fatty acid and monomeric chemical structural formula thereof are as follows:
Wherein, R is that n is the natural number of 1-1000 by the alkyl of one to ten straight chain that carbon is formed or side chain.
2, eukaryotic cell growth stimulant according to claim 1 is characterized in that: described R is a methyl, ethyl, propyl group, butyl, amyl group, hexyl or heptyl; N is the natural number of 1-200.
3, eukaryotic cell growth stimulant according to claim 2 is characterized in that: the monomer of described oligomerization 3-hydroxy fatty acid and 3-hydroxy fatty acid are the mixture of compound, R and S type of single three-dimensional arrangement or the compound of racemize structure.
4, eukaryotic cell growth stimulant according to claim 3 is characterized in that: the monomer of the oligomerization 3-hydroxy fatty acid of described composition eukaryotic cell growth stimulant and 3-hydroxy fatty acid are selected from one or more in 3-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxycaproic acid and the 3-hydroxydecanoic acid.
5, method according to claim 4 is characterized in that: the mixture of simple oligomer that described oligomerization 3-hydroxy fatty acid is 3-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxycaproic acid, 3-hydroxydecanoic acid or copolymerization oligomer or described simple oligomer and copolymerization oligomer.
6, method according to claim 5 is characterized in that: described eukaryotic cell is inoblast, scleroblast, chondrocyte, neurocyte, skin cells, blood vessel epithelial cell, hemopoietic stem cell, liver cell, lymphocyte, corneal epithelial cell or nephrocyte.
7, the application of the described eukaryotic cell growth stimulant of each claim of claim 1-6 in the vitro culture histoorgan.
8, method according to claim 7 is characterized in that: described histoorgan comprises into fibrous tissue, body of gland, lung, ovary, nerve, osseous tissue, cerebral tissue, blood vessel, liver, kidney.
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| CN101293831B (en) * | 2007-04-27 | 2013-09-18 | 汕头大学 | Uses of 3-hydroxy fatty acid and its derivative in preparing calcium ion duct modifying agent |
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| CN101293831B (en) * | 2007-04-27 | 2013-09-18 | 汕头大学 | Uses of 3-hydroxy fatty acid and its derivative in preparing calcium ion duct modifying agent |
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