CN105748518A - Anti-aging composition containing adipose-derived stem cell extract - Google Patents

Anti-aging composition containing adipose-derived stem cell extract Download PDF

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Publication number
CN105748518A
CN105748518A CN201610161141.0A CN201610161141A CN105748518A CN 105748518 A CN105748518 A CN 105748518A CN 201610161141 A CN201610161141 A CN 201610161141A CN 105748518 A CN105748518 A CN 105748518A
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stem cell
apolexis composition
cell extract
composition according
fat stem
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CN105748518B (en
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张静莹
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Beijing Jiamei Kanglian Medical Technology Co ltd
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Dalian University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Abstract

The invention belongs to the field of medicines and particularly relates to anti-aging composition containing an adipose-derived stem cell extract. Adipose-derived stem cells are extracted after subjected to ultraviolet radiation and Abeta slight damage.

Description

Comprise the anti-apolexis composition of fat stem cell extract
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of anti-apolexis composition comprising fat stem cell extract.
Background technology
Aging is the biological process of too many levels, is that body is in degeneration function reduction in period and disorderly general performance.Universality, Progressive symmetric erythrokeratodermia, degenerative and endogenous are the basic features of human senility.Just enter physiology, biochemistry the research of Aging mechanism to the forties in 20th century and form aspects systematic research.The basis of great many of experiments evidence proposes many new theories, such as free radical theory, genetic program theory, cross linkage theory, lipofuscin accumulation theory, hypoendocrinism theory etc., inquired into mechanism and the countermeasure of old and feeble generation respectively from different angles.
Antiaging agent be a class to improve life efficiency for final purpose, from multisystem, multi-level play it with the multistage and adjust function, delay senility or improve the medicine of quality of life in the life time that can define on hereditism.People have carried out a large amount of exploration, have accumulated some experiences and effective method, have also been developed some antiaging agents.Such as Folium Ginkgo extract, vitamin E and hormone etc., although having a lot of medicine to be all proved to be able to slow down aging at present, but antiaging agent can not be abused, the toxicity case that it causes day by day increases.
Fat stem cell (Adipose-derivedStemCells, ADSCs), is a kind of adult stem cell being now widely used for organizational project and regenerative medicine research field.Fat stem cell is the same with mesenchymal stem cells MSCs has multi-lineage potential, under given conditions can to multiple directions differentiation such as adipose cell, osteoblast, chondroblast, sarcoplast, one-tenth endotheliocyte and neuroblasts.In addition, fat stem cell also has the ability secreting various trophic factors, these factors include Brain Derived Neurotrophic Factor (BDNF), nerve growth factor (NGF), VEGF (VEGF), VEGF R2 (VEGFR2), basic fibroblast growth factor (bFGF) etc., and these factors can also participate in the injury repairing of histoorgan.
Summary of the invention
It is an object of the present invention to provide a kind of anti-apolexis composition comprising fat stem cell extract, the preparation method of described fat stem cell extract is as follows:
1) 0.1% gelatin paving T75 Tissue Culture Flask half an hour, blot and stand 5 minutes；
2) human adipose-derived stem cell serum-free medium is adjusted to 1 × 105The human adipose-derived stem cell suspension of individual/ml；Adding 15ml suspension in culture bottle to cultivate, after incubator is cultivated three days, serum-free medium changes the serum-free medium containing amyloid beta (A β), utilizes UV-crosslinked instrument that culture bottle is irradiated after changing liquid；
3) cultivate three days after irradiating；Sucking-off culture fluid, with PBS 2-3 time, then adds 2-3ml cell pyrolysis liquid dissolved cell, then collects in centrifuge tube, centrifuging and taking supernatant；
4) take supernatant and be placed in bag filter, dialyse 8 hours with 4 DEG C of PBS solution；By solution lyophilization in bag filter, it is saved in-80 DEG C of refrigerators.
In one embodiment of the invention, the concentration of described amyloid beta is 0.1 μ g/L.
In another embodiment of the invention, the actual conditions that culture bottle is irradiated by described UV-crosslinked instrument is for 1mJ/cm2Irradiate 10min.
In yet another embodiment of the present invention, centrifugal described in step 3 actual conditions is the centrifugal 10min of 12000rpm.
In yet another embodiment of the present invention, bag filter molecular cut off described in step 4 is 5000kD.
Further carrying out in scheme in the present invention, described anti-apolexis composition is made up of described fat stem cell extract and metformin, and the mass ratio of described fat stem cell extract and metformin is 10:0.3.
Described anti-apolexis composition can be formulated for oral administration, drug administration by injection, percutaneous dosing, implantation bank or rectally, or for being suitable to the form (by mouth or nose) by sucking or be blown into administration.
For oral administration, described anti-apolexis composition can take the form of such as tablet or capsule, described tablet or capsule are prepared with pharmaceutically acceptable excipient by conventional method, described pharmaceutically acceptable excipient is such as binding agent (such as, pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methyl cellulose)；Filler (such as, lactose, microcrystalline Cellulose or calcium hydrogen phosphate)；Lubricant (such as, magnesium stearate, Talcum or silicon dioxide)；Disintegrating agent (such as, potato starch or primojel)；Or wetting agent (such as, sodium lauryl sulfate).By means commonly known in the art described tablet can be carried out coating.The form of such as solution, syrup or suspending agent can be taked for the liquid preparation of oral administration, or they can provide for molten with water or other suitable vehicle weights before use by anhydrous Product Form.Such liquid preparation can be prepared with pharmaceutically acceptable additive by conventional method, and described pharmaceutically acceptable additive is such as suspending agent (such as, sorbitol syrups, cellulose derivative or hydrogenated edible lipid)；Emulsifying agent (such as, lecithin or arabic gum)；Non-aqueous vehicles (such as, almond oil, oily ester, ethanol or fractionated vegetable oil)；With preservative (such as, methyl parahydroxybenzoate or propyl p-hydroxybenzoate or sorbic acid).Described preparation contains buffer salt, correctives, coloring agent and sweeting agent also dependent on needs.Formulations for oral administration can through suitable preparation, to provide the control of reactive compound to discharge.
Described anti-apolexis composition can be formulated for percutaneous dosing.Percutaneous dosing can include topical application or transdermal administration.Transdermal application can be completed by suitable patch, Emulsion, ointment, solution, suspending agent, paste, foam, aerosol, lotion, ointment or gel, it is commonly known in the art, and be designed to transdermal delivery activating agent specially, and optional under the existence of concrete penetration enhancer.Topical composition can take one or more in these forms equally.One or more reactive compounds can combine existence with one or more pharmaceutically acceptable non-toxic excipients, and exist with other active ingredient combination as required, described pharmaceutically acceptable adjuvant, such as excipient, auxiliary agent (such as, buffer agent), carrier, inert solid diluent, suspending agent, preservative, filler, stabilizer, antioxidant, food additive, bioavailability reinforcing agent, coating substance, granulation agent and disintegrating agent, binding agent etc..
In the present invention, described human adipose-derived stem cell refers to people adipose stromal stem cell, it can be commercially available by commercial channel, prior art known method can also be passed through prepare voluntarily, such as, disclosed in patent CN2014103601433 preparation method is prepared (its description embodiment 1), as long as the fat stem cell obtained is through Detection of antigen, the fat stem cell with CD34, CD31 and CD45 accounts for total stem cell ratio lower than 1%；With CD29, CD73, CD90, CD105 and CD49d fat stem cell in the ratio of total stem cell higher than 95%.
Present invention discover that under the slight damage of ultra-vioket radiation and A β, it is possible to activate the defense mechanism within stem cell, so that it secretes useful somatomedin and related substances in a large number, so that the activity of fighting against senium of stem cell is greatly improved.It addition, in further research, present invention discover that described fat stem cell extract and metformin create obvious synergism；So that drug effect is greatly improved.
Detailed description of the invention
The detailed description present invention further below.It is pointed out that following description is only the illustration to claimed technical scheme, the not any restriction to these technical schemes.The content that protection scope of the present invention is recorded with appended claims is as the criterion.
The preparation of embodiment 1 fat stem cell extract
1) 0.1% gelatin paving T75 Tissue Culture Flask half an hour, blot and stand 5 minutes；
2) by human adipose-derived stem cell serum-free medium (modelMSCSFM, ThermoFisher company, the U.S., lower same) it is adjusted to 1 × 105The human adipose-derived stem cell suspension of individual/ml；Adding 15ml suspension in culture bottle to cultivate, after incubator is cultivated three days, serum-free medium changes the serum-free medium of the amyloid beta (A β) containing 0.1 μ g/L, utilizes UV-crosslinked instrument that culture bottle is irradiated after changing liquid；Condition is with 1mJ/cm2Irradiate 10min.
3) cultivate three days after irradiating；Sucking-off culture fluid, with PBS 2-3 time, then adds 2-3ml cell pyrolysis liquid dissolved cell, then collects in centrifuge tube, and 12000rpm is centrifuged 10min, takes supernatant；
4) take supernatant and be placed in bag filter (molecular cut off is 5000kD), dialyse 8 hours with 4 DEG C of PBS solution；By solution lyophilization in bag filter, it is saved in-80 DEG C of refrigerators.
Embodiment 2D-galactose causes senile cell scale-model investigation
Take H9C2 myocardial cell, PC12 neurocyte and human fibroblasts HDF respectively with 1 × 105Individual/ml is laid on 96 different orifice plates, and after incubator is cultivated 3 days, the culture medium adding fatty stem cell extract and D-galactose is hatched；The final concentration of 30mg/ml of every hole D-galactose；Matched group is without galactose and medicine, and model group adds galactose but without medicine, after cultivating 48h, adopts mtt assay to measure cytoactive, and adopts the content (the often multiple hole of group 5, parallel testing 3 times) of FDG method detection beta galactosidase.
Concrete outcome is as shown in the table:
OD590 Concentration H9C2 PC12 HDF
Matched group - 0.77±0.03 0.56±0.02 0.84±0.04
Model group - 0.52±0.02 0.31±0.01 0.61±0.03
Embodiment 1 10μg/L 0.69±0.03 0.48±0.02 0.76±0.03
30μg/L 0.78±0.03 0.54±0.03 0.83±0.04
Comparative example 1 10μg/L 0.58±0.02 0.37±0.01 0.68±0.02
30μg/L 0.62±0.03 0.45±0.02 0.74±0.03
Comparative example 2 10μg/L 0.60±0.03 0.41±0.03 0.71±0.03
30μg/L 0.67±0.03 0.47±0.03 0.78±0.02
Beta galactosidase Concentration H9C2 PC12 HDF
Matched group - 1764±87 892±42 1344±98
Model group - 7942±274 4672±148 6109±176
Embodiment 1 10μg/L 2542±119 1614±94 1952±83
30μg/L 1913±94 1035±75 1479±71
Comparative example 1 10μg/L 5274±189 3352±121 4743±194
30μg/L 3751±157 2514±108 2719±164
Comparative example 2 10μg/L 4359±172 2845±147 3259±176
30μg/L 3039±147 1861±113 2152±132
Comparative example 1 is fat stem cell extract, and preparation method, with embodiment 1, differs only in fat stem cell without ultra-vioket radiation and A β process；
Comparative example 2 is fat stem cell extract, and preparation method, with embodiment 1, differs only in fat stem cell without A β process.
Embodiment 3SAM aging mice scale-model investigation
Take Senescence-Accelerated Mouse SAM-P/8 (3 monthly age), male and female half and half, random packet, often group 15-20 is only, each administration group all carries out gastric infusion according to dosage 5mg/kg, and matched group gives normal saline, and every day, gavage was once until off-test, normal condition is raised, and adds up each group of mouse life and mortality rate and survival mice is carried out following test after raising 10 months:
1) water maze test: mice is put in the round pool containing circular platform, depth of water 30cm, if mice can not find platform more than 60s, is put into platform by experimenter by mice and is familiar with environment 10s, after training 3 days continuously, record every mice and find plateau time.
2) proliferation of bone marrow cells ability measures: aseptic take mice bilateral femur upper segment, after rinsing with Hanks liquid, cuts off two ends, and 1640 flush out medullary cell, centrifugal obtains cell, with 1640 cultivation keynote cell concentrations to 1 × 106Individual/ml, every hole 100 microlitre is seeded to 96 orifice plates, cultivates 3 days, adopts mtt assay to measure the OD value of 590nm.
3) index and spleen index and thymus index measure: take mouse spleen and thymus, weigh, and calculate the ratio of spleen and thymic weight and Mouse Weight.
Concrete outcome is as follows:
1, each group mice average life and mortality rate after raising 10 months
Mortality rate (%) Average life (my god)
Matched group 51.3 127.5
Embodiment 1 5.2 269.6
Comparative example 1 21.9 184.3
Comparative example 2 14.3 224.9
Test example 1 1.3 292.6
The comparative example 1 and 2 that comparative example 1 and 2 administration medicine is prepared referring to embodiment 3；
Test example 1 is fat stem cell extract and the compositions of metformin of embodiment 1 preparation, both is mixed, and the weight ratio of fat stem cell extract and metformin is 10:0.3.
2, water maze
Incubation period (s)
Matched group 61.9±6.7
Embodiment 1 30.9±4.8
Comparative example 1 49.5±4.2
Comparative example 2 40.3±3.9
Test example 1 25.2±4.4
3, proliferation of bone marrow cells ability
OD value
Matched group 0.24±0.05
Embodiment 1 0.41±0.04
Comparative example 1 0.29±0.05
Comparative example 2 0.32±0.04
Test example 1 0.54±0.06
4, index and spleen index and thymus index
Index and spleen index (mg/g) Thymus index (mg/g)
Matched group 3.9±0.7 1.7±0.3
Embodiment 1 5.7±1.1 2.8±0.5
Comparative example 1 4.4±0.6 1.9±0.4
Comparative example 2 4.8±0.5 2.2±0.4
Test example 1 6.1±1.2 3.2±0.6
Present invention merely illustrates some claimed specific embodiments; technical characteristic described in one of them or more technical scheme can be combined with arbitrary one or more technical schemes; the technical scheme that these are combined and obtain is also in the application protection domain, technical scheme that is combined just as these and that obtain specifically has been recorded in the disclosure of invention.

Claims (7)

1. comprising an anti-apolexis composition for fat stem cell extract, the preparation method of described fat stem cell extract is as follows:
1) 0.1% gelatin paving T75 Tissue Culture Flask half an hour, blot and stand 5 minutes；
2) human adipose-derived stem cell serum-free medium is adjusted to 1 × 105The human adipose-derived stem cell suspension of individual/ml；Adding 15ml suspension in culture bottle to cultivate, after incubator is cultivated three days, serum-free medium changes the serum-free medium containing amyloid beta (A β), utilizes UV-crosslinked instrument that culture bottle is irradiated after changing liquid；
3) cultivate three days after irradiating；Sucking-off culture fluid, with PBS 2-3 time, then adds 2-3ml cell pyrolysis liquid dissolved cell, then collects in centrifuge tube, centrifuging and taking supernatant；
4) take supernatant and be placed in bag filter, dialyse 8 hours with 4 DEG C of PBS solution；By solution lyophilization in bag filter, it is saved in-80 DEG C of refrigerators.
2. anti-apolexis composition according to claim 1, it is characterised in that the concentration of described amyloid beta is 0.1 μ g/L.
3. anti-apolexis composition according to claim 1, it is characterised in that the actual conditions that culture bottle is irradiated by described UV-crosslinked instrument is for 1mJ/cm2Irradiate 10min.
4. anti-apolexis composition according to claim 1, it is characterised in that actual conditions centrifugal described in step 3 is the centrifugal 10min of 12000rpm.
5. anti-apolexis composition according to claim 1, it is characterised in that bag filter molecular cut off described in step 4 is 5000kD.
6. anti-apolexis composition according to claim 1, it is characterised in that described anti-apolexis composition is made up of described fat stem cell extract and metformin.
7. anti-apolexis composition according to claim 6, it is characterised in that the mass ratio of described fat stem cell extract and metformin is 10:0.3.
CN201610161141.0A 2016-03-21 2016-03-21 Anti-apolexis composition comprising fat stem cell extract Active CN105748518B (en)

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CN114288202A (en) * 2021-12-06 2022-04-08 广州沛妍医疗科技有限公司 A sericin composition with antiaging effect, and its preparation method

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Publication number Priority date Publication date Assignee Title
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