CN103784474A - Human fat mesenchymal stem cell extract and lyophilized powder and application thereof - Google Patents
Human fat mesenchymal stem cell extract and lyophilized powder and application thereof Download PDFInfo
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Abstract
The invention discloses a human fat mesenchymal stem cell extract and lyophilized powder and applications thereof. The human fat mesenchymal stem cell extract is prepared by the steps: collecting subcultured P3-P30 generations of human fat mesenchymal stem cells, culturing with cell growth liquid until 80% of cells are fused, removing culture liquid, washing the cells with a phosphate buffer solution for three times, adding into a serum-free culture solution, continuously culturing for 72 hours, collecting the serum-free culture solution, digesting uncollected cells with a digestion solution, crushing the cells with an ultrasonic crushing device, centrifuging, and then collecting supernatant; and mixing the collected serum-free culture solution and the collected supernatant, filtering, collecting filtrate, concentrating, and sterilizing. The human fat mesenchymal stem cell extract and the lyophilized powder thereof have multiple biological activities, can be used for preparing wound healing or cell repair medicaments and other biopharmaceutical products, and can also be used for preparing whitening products or skin anti-wrinkle products and other fine chemical products.
Description
Technical field
The present invention relates to the technology that stem cell is used in biological medicine or medical cosmetology, relate in particular to the use of human adipose mesenchymal stem cells in biological medicine or medical cosmetology.
Background technology
Adipose tissue-derived extensively, draw materials easily, cell separation efficiency wound high, that bring to patient is less and do not relate to ethical issues, therefore day by day in widespread attention.
Human adipose mesenchymal stem cells is the class multipotent stem cells in fatty tissue, it can secrete various bioactivators, comprise protein, polypeptide and cytokine etc., such as stem cell factor (SCGF), VEGF (VEGF), insulin like growth factor (IGF), hepatocyte growth factor (HGF) and transforming growth factor etc., wounded tissue cell can be controlled or maintain to these cell growth factor, guides and himself repair.In addition, in human adipose mesenchymal stem cells, also contain the Multiple components such as multiple film protein and bioactie agent, these compositions are coordinated mutually to supplement mutually, there is complicated physiological action, can exert an influence to many histoorgans, therefore there is the effects such as reasonable anti-light aging, antioxidation, crease-resistant, skin whitening, wound healing, cytothesis.
At present, although the existing external evoked stem cell secretion supernatant of the specific inductive condition of research and utilization is for aspects such as medical treatment, cosmetics, but this conditioned medium is the specific cytokine induction stem cell secretion of application, thereby some specific factor reaches curative effect, cytokine itself is expensive, and the application cost of stem cell is increased extremely.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide one to there is various biological activity, can be used for the human adipose mesenchymal stem cells extract of biological medicine, field of fine chemical.
Another object of the present invention is to provide above-mentioned human adipose mesenchymal stem cells extract and is preparing the biomedical product such as wound healing medicine or cytothesis medicine, or in the application of preparing in the fine chemical product such as whitening product or skin wrinkle resisting product.
Another object of the present invention is to provide the lyophilized powder of above-mentioned human adipose mesenchymal stem cells extract.
Another object of the present invention is to provide the preparation method of above-mentioned human adipose mesenchymal stem cells extract freeze-drying powder.
Another object of the present invention is to provide the lyophilized powder of above-mentioned human adipose mesenchymal stem cells extract and is preparing the biomedical product such as wound healing medicine or cytothesis medicine, or in the application of preparing in the fine chemical product such as whitening product or skin wrinkle resisting product.
Above-mentioned purpose of the present invention is achieved by following scheme:
Human adipose mesenchymal stem cells extract, this extract is prepare by the following method and obtain:
By human adipose mesenchymal stem cells after primary separation and Culture, the cultivation of going down to posterity, P3～P30 that getting goes down to posterity cultivates is for human adipose mesenchymal stem cells, while utilizing cell growth medium to be cultured to cell 80% to merge, discard culture fluid, first use phosphate buffer wash cell three times, then add serum-free medium to continue to cultivate after 72 hours, collect serum-free medium, and the cell not being collected carries out subsequent treatment;
The cell that step 1 is not collected utilizes after Digestive system digestion, Ultrasonic Cell Disruptor smudge cells, centrifugal rear collection supernatant;
After the supernatant that the serum-free medium that step 1 is collected and step 2 are collected mixes, filter, collect filtrate concentrated to it, gained concentrated solution carries out, after degerming processing, preparing required human adipose mesenchymal stem cells extract again.
In above-mentioned steps 1, described by human adipose mesenchymal stem cells in primary separation and Culture, primary separation method and cultural method adopt those skilled in the art's routine operation.
In above-mentioned steps 1, described cell growth medium refers to the DMEM/F12 culture fluid that contains 10% hyclone, and in this culture fluid, also contains antibacterium antibiotic; Described antibacterium antibiotic is two kinds of penicillin and streptomycins, and wherein the final concentration of penicillin in cell growth medium is 200 U/ml, and the final concentration of streptomycin in cell growth medium is 250 U/ml; The described DMEM/F12 culture fluid that contains 10% hyclone is that in commercially available basic DMEM/F12 culture fluid, to add final concentration be 10% hyclone, and to add final concentration be that the penicillin of 200 U/ml and final concentration are that the streptomycin of 250 U/ml is cell growth medium of the present invention.
In above-mentioned steps 1, described phosphate buffer is that pH value is 7.0～7.2 PBS solution.
In above-mentioned steps 1, described cell continues to cultivate 72 hours in serum-free medium, and the serum-free medium is here commercially available prod.
In above-mentioned steps 1, when concrete operations, by going down to posterity, the difference of cultivating is processed respectively for cell, as: when the human adipose mesenchymal stem cells in P3 generation is utilized cell growth medium to be cultured to cell 80% to merge, discard culture fluid, first use phosphate buffer wash cell three times, then cell is joined in serum-free medium and continue to cultivate after 72 hours, collect the serum-free medium that obtains P3; When the human adipose mesenchymal stem cells in P4 generation is utilized cell growth medium to be cultured to cell 80% to merge, discard culture fluid, first use phosphate buffer wash cell three times, then cell is joined in serum-free medium and continue to cultivate after 72 hours, collect and obtain the serum-free medium in P4 generation etc.; Then serum-free medium P3～P30 being obtained respectively for human adipose mesenchymal stem cells obtains the serum-free medium that embodiment 1 collects after collecting and merging; Meanwhile, the cell harvesting not being collected that P3～P30 is obtained respectively for human adipose mesenchymal stem cells carries out subsequent treatment after merging together.
In above-mentioned steps 2, described Digestive system is 0.25% trypsin-0.02%EDTA mixture slaking liquid, and this mixture slaking liquid first could be used for the present invention after the membrane filtration degerming of 0.22 μ m; Described 0.25% trypsin-0.02%EDTA mixture slaking liquid is those skilled in the art's conventional a kind of Digestive systems while carrying out cell experiment, and its compound method, according to the compound method providing on textbook, all can realize the present invention; As adopted preparation with the following method: the PBS that gets 0.25g trypsin powder, 20mgEDTA powder and 100ml 0.01mol/L, first dissolve trypsin powder with a small amount of PBS, then EDTA powder and remaining PBS are added after mixing, be placed in 37 ℃ of water-baths, treat thoroughly to dissolve liquid be transparent till, use G5 sucking filtration, subpackage is placed in 4 ℃ of refrigerators and preserves.
In above-mentioned steps 2, described Ultrasonic Cell Disruptor smudge cells, its broken condition is 4 ℃, 400W, ultrasonic 3s, interval 6s, 99 circulations.
In above-mentioned steps 2, described centrifugal be 17000g, 30min.
In above-mentioned steps 3, the supernatant that the described serum-free medium that step 1 is collected and step 2 are collected mixes, and the mixed proportion of the supernatant that the serum-free medium that step 1 is collected here and step 2 are collected is 1:1, volume ratio.
In above-mentioned steps 3, described filtration is that employing aperture is the membrane filtration of 0.45 μ m.
In above-mentioned steps 3, the described concentrated filter membrane that is employing molecular cut off is 3KD carries out concentrating and desalinating.
In above-mentioned steps 3, described concentrated referring to is concentrated into 1/10 of original volume, that is to say, after filtrate is concentrated, obtains concentrated solution, and the volume of described concentrated solution is 1/10 of filtrate.
In above-mentioned steps 3, described concentrated solution is carried out to degerming processing, the degerming processing is here that concentrated solution is adopted to 0.22 aseptic μ m membrane filtration degerming.
The human adipose mesenchymal stem cells extract of above-mentioned preparation confirms through experimentation, it has a variety of biologic activity, can be used for preparing the biomedical product such as wound healing medicine or cytothesis medicine, also can be used for preparing the fine chemical product such as whitening product or skin wrinkle resisting product.
Although human adipose mesenchymal stem cells extract of the present invention has various biological activity, application is extensive, but its biologic activity is limited by various external condition, save inconvenience, therefore the present invention also provides the lyophilized powder of human adipose mesenchymal stem cells extract, and this lyophilized powder is exactly by the human adipose mesenchymal stem cells extract lyophilized powder that through this area prepared by conventional method for preparing freeze-dried powder of the present invention; Lyophilized powder be under gnotobasis, medicinal liquid is frozen into solid-state, evacuation is by the dry water sublimed aseptic powder forming, the preparation method of lyophilized powder is the technology of this area comparative maturity, the present invention adopts the preparation method of any lyophilized powder, can obtain required human adipose mesenchymal stem cells extract freeze-drying powder.
In order to obtain better medicine and pharmacology effect, the present invention also, especially for the feature of above-mentioned human adipose mesenchymal stem cells extract, has researched and developed the preparation method of human adipose mesenchymal stem cells extract freeze-drying powder, and this preparation method comprises the steps:
In human adipose mesenchymal stem cells extract, add excipient and water, filtration sterilization after mixing, then carries out frozen dried, prepares required human adipose mesenchymal stem cells extract freeze-drying powder.
In the preparation method of above-mentioned lyophilized powder, described excipient adopts any one in mannitol, trehalose or sucrose.
In the preparation method of above-mentioned lyophilized powder, the consumption of described excipient: excipient accounts for 5～10% of human adipose mesenchymal stem cells extract gross weight.
In the preparation method of above-mentioned lyophilized powder, described hydromining ultra-pure water.
In the preparation method of above-mentioned lyophilized powder, adding ultra-pure water to final volume is 100ml.
In the preparation method of above-mentioned lyophilized powder, described frozen dried refers to that freeze temperature is that-20～-35 ℃, vacuum are 50～200Pa, lyophilizing 48 hours.
Human adipose mesenchymal stem cells extract freeze-drying powder of the present invention has various biological activity equally, can be used for preparing the biomedical product such as wound healing medicine or cytothesis medicine, also can be used for preparing the fine chemical product such as whitening product or skin wrinkle resisting product.
Human adipose mesenchymal stem cells extract freeze-drying powder preparation method of the present invention is simple, is easy to storage and transport.
Accompanying drawing explanation
Fig. 1 is the fluidic cell detection figure of human adipose mesenchymal stem cells;
What Fig. 2 was human adipose mesenchymal stem cells extract freeze-drying powder on HDF Growth of Cells affects result block diagram;
What Fig. 3 was human adipose mesenchymal stem cells extract freeze-drying powder on SOD enzymatic activity in cell affects result block diagram;
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml groups; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml groups; 3 is model group; 4 positive matched groups; 5 negative matched groups; Wherein: * * P≤0.01, * P≤0.05;
Fig. 4 is the testing result block diagram of SOD enzymatic activity in the mice plasma of 5 experimental grouies;
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml groups; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml groups; 3 is model group; 4 positive matched groups; 5 negative matched groups; Wherein: * * P≤0.01, * P≤0.05;
Fig. 5 is the testing result block diagram of GPx activity in the mice plasma of 5 experimental grouies;
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml groups; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml groups; 3 is model group; 4 positive matched groups; 5 negative matched groups; Wherein: * * P≤0.01, * P≤0.05;
Fig. 6 is the testing result block diagram of MDA content in the mice plasma of 5 experimental grouies.
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml groups; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml groups; 3 is model group; 4 positive matched groups; 5 negative matched groups; Wherein: * * P≤0.01, * P≤0.05.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is done further and described, but specific embodiment does not do any restriction to the present invention.
primary separation, cultivation and the evaluation of embodiment 1 human adipose mesenchymal stem cells
1, primary separation and the cultivation of human adipose mesenchymal stem cells
By centrifugal the human abdomen's subcutaneus adipose tissue extract obtaining by suction lipectomy (preoperative own endocrinopathy and the infectious disease got rid of) 800g, with the flushing of PBS buffer, soak, then 800g × 4min washs 3 times; Reject macroscopic blood vessel and knot hoof tissue, after with eye scissors, it fully being shredded, put into 50ml centrifuge tube, add 2 times of volume 0.2% collagenases, 1 times of volume 1%BSA and dual anti-, mix, sealing, puts into 37 ℃ of digestion 45min of shaking table, till pasty state;
Add isopyknic low sugar DMEM that is 10% hyclone containing volume fraction to stop digestion, the centrifugal 10min of 1800r/min, is divided into 3 layers after centrifugal, and upper strata is oils and fats and does not digest fatty tissue completely, middle level is supernatant, and lower floor is the precipitation of the cell mixings such as fat stem cell and erythrocyte;
Abandon upper strata and middle level, lower floor adds complete medium re-suspended cell precipitation, 70 μ m cell screen filtrations, and it is 5 × 10 that the filtrate after filtering is adjusted to cell concentration
5mononuclear cell/mL is inoculated in T-25cm
2in culture bottle, 37 ℃, 5%CO
2, saturated humidity cultivate, after 48h, change liquid, add fat stem cell complete medium continue cultivate.
2, the evaluation of human adipose mesenchymal stem cells
Adopt flow cytometer to carry out the evaluation of human adipose mesenchymal stem cells, result as shown in Figure 1, CD29, CD44, CD90 positive expression in cell, CD49d, CD71 are the weak positive, the expression and CD34, CD45 are negative, therefore can be judged to be human adipose mesenchymal stem cells.
The human adipose mesenchymal stem cells extract of the present embodiment, this extract is prepare by the following method and obtain:
P3～the P30 of the cultivation of going down to posterity that process is identified is for human adipose mesenchymal stem cells, while utilizing cell growth medium (described cell growth medium be add final concentration in commercially available basic DMEM/F12 culture fluid be that 10% hyclone and final concentration are that the penicillin of 200 U/ml and final concentration are that the streptomycin preparation of 250 U/ml obtains) to be cultured to cell 80% to merge, discard culture fluid, first use phosphate buffer (described phosphate buffer is that pH value is 7.0～7.2 PBS solution) washed cell three times, then add commercially available serum-free medium to continue to cultivate after 72 hours, collect serum-free medium merging that P3～P30 human adipose mesenchymal stem cells of each generation in generation obtains respectively, and the cell not being collected carries out subsequent treatment,
The cell that step 1 is not collected utilizes Digestive system, and (described Digestive system is 0.25% trypsin-0.02%EDTA mixture slaking liquid, and this mixture slaking liquid is through the membrane filtration degerming of 0.22 μ m) digestion after, add the cell growth medium of step 1 use to stop digestion, 4 ℃, 3000g, centrifugal 5min; PBS washed cell 3 times, and re-suspended cell, be put in and utilize Ultrasonic Cell Disruptor smudge cells on ice, and broken condition is 400W, ultrasonic 3s, interval 6s, 99 circulations; After liquid becomes clarification, 4 ℃, 17000g, centrifugal 30min are collected supernatant, by P3～P30 for each generation the supernatant that obtains respectively of human adipose mesenchymal stem cells collect and merge;
After the supernatant that the serum-free medium that step 1 is collected and step 2 are collected mixes with the volume ratio of 1:1, utilize 0.45 μ m filter filtering solution, collect filtrate and this filtrate is concentrated into 1/10th of original volume, 0.22 aseptic μ m membrane filtration degerming for gained concentrated solution, prepares the human adipose mesenchymal stem cells extract solution of the present embodiment.
The human adipose mesenchymal stem cells extract solution that adopts BCA method to prepare embodiment 2 carries out determination of protein concentration, microplate reader is measured the absorbance of A570, obtain the protein concentration of human adipose mesenchymal stem cells extract solution prepared by embodiment 2, then according to this protein concentration, add excipient mannitol (mannitol account for human adipose mesenchymal stem cells extract gross weight 5%) and the ultra-pure water of 50～60 ℃ to mix according to the final mass that needs Solutions in Freeze-drying, and to be adjusted to final concentration of protein be 50.0 ± 0.5 μ g/ml, filtration sterilization, then frozen dried, freeze temperature is-20～-35 ℃, vacuum is 50～200Pa, freeze-drying time 48 hours, make required human adipose mesenchymal stem cells extract freeze-drying powder, subpackage is measured/propped up to lyophilized powder according to 1ml.
The quality standard of human adipose mesenchymal stem cells extract freeze-drying powder is as follows:
(1) outward appearance
Observer's fat mesenchymal stem cell extract freeze-drying powder product is white, Powdered.After dissolving with 1ml ultra-pure water by labelled amount, be clarification, transparent pale red solution.
(2) mensuration of pH value
Adopt Accurate pH instrumentation to determine the pH value of human adipose mesenchymal stem cells extract freeze-drying powder.First utilize titer to proofread and correct pH meter according to operating instruction, then stem cell extract freeze-drying powder aqueous solution is measured.The pH value of human adipose mesenchymal stem cells extract freeze-drying powder aqueous solution is between 6.5～7.5.
(1) mtt assay is measured the impact of human adipose mesenchymal stem cells extract freeze-drying powder on fibroblasts of adult human dermis growth
Detect the human adipose mesenchymal stem cells extract freeze-drying powder prepared of sample: embodiment 3 (protein content be 50.0 ± 0.5 μ g /).
Biological subject body: HDF cell.
Concrete steps are as follows:
(1) utilize 10%(v/v) the DMEM/F12 culture medium (penicillin 200 U/ml, streptomycin 250 U/ml, pH7.2) of FBS cultivates HDF cell, in the time that cell density reaches 100% fusion, utilizes cell dissociation buffer peptic cell, adjusts cell concentration for being 1 × 10
5cell/ml, is inoculated in 96 orifice plates, and every hole 100 μ l set 4 multiple holes, in 37 ℃, the cell culture incubator of 5%CO2, saturated humidity, continues to cultivate;
(2) after 24h, discard culture medium, diluting stem cell extract to final concentration by the DMEM/F1 culture medium containing 1%FBS is 400 μ g/ml, 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, every hole adds 100 μ l, set 4 multiple holes, matched group adds the DMEM/F12 culture medium of 1%FBS.In 37 ℃, the cell culture incubator of 5%CO2, saturated humidity, continue to cultivate;
(3) after 48h, add the MTT solution in 20 μ l/ holes, continue to cultivate 4h, abandon supernatant, add DMSO100 μ l/ hole, lucifuge concussion 15min, utilize microplate reader at wavelength for 570nm, reference wavelength are 630nm place mensuration absorbance, and calculate cell survival rate.
Cell survival rate (%)=(experimental group mean light absorbency value/matched group mean light absorbency value) × 100%
Experimental result as shown in Figure 2, human adipose mesenchymal stem cells extract freeze-drying powder in the time of the μ g/ml of Nong Du≤400 to HDF cell without obvious inhibitory action, Microscopic observation cell is also without the obvious symptom such as dead.Experimental group is compared with matched group, and its cell survival rate does not have obvious difference, illustrates and does not occur significant cell death, and in the time of the μ g/ml of human adipose mesenchymal stem cells extract freeze-drying powder Nong Du≤100, can promote the growth of HDF cell.
(2) protective effect of human adipose mesenchymal stem cells extract freeze-drying powder to ultraviolet damage fibroblasts of adult human dermis
Detect the human adipose mesenchymal stem cells extract freeze-drying powder prepared of sample: embodiment 3 (protein content be 50.0 ± 0.5 μ g /).
Biological subject body: HDF cell.
Concrete steps are as follows:
(1) in the time that fibroblasts of adult human dermis grows to 80% fusion, with UVA ultraviolet radiation cell, irradiate every day 2 hours, concurrent irradiation 4 days, negative control group encases Tissue Culture Flask with tinfoil.Examine under a microscope the form of cell, find that cell starts to occur shrinkage, indivedual cells swim in culture medium; The 5th day, add human adipose mesenchymal stem cells extract freeze-drying powder, final concentration of protein is respectively 20 μ g/ml and 10 μ g/ml, and Catergen 0 μ g/ml, as positive control, continues to cultivate 48h, collecting cell;
(2) impact of human adipose mesenchymal stem cells extract freeze-drying powder on SOD enzymatic activity in cell: after trypsinization, cell is centrifugal 10min under 1500g, 4 ℃ of conditions.Cell precipitation is suspended in meets in cold buffer (Tris – HCl, 5 mM EDTA and the 1 mM DTT of 50mM) and carries out ultrasonicly, and lysate is centrifugal 10min under 10000g, 4 ℃ of conditions, and supernatant utilizes SOD detection kit to analyze according to its illustration method.
Result as shown in Figure 3; no matter human adipose mesenchymal stem cells extract freeze-drying powder protein concentration is 20 μ g/ml; or 10 μ g/ml have improved the activity of SOD enzyme in cell to some extent; thereby reduce attack and the infringement of the oxygen-derived free radicals producing because of ultraviolet radiation induction to cell; reduce the mortality rate of cell; thereby play the effect of Cell protection, reach effect of anti-light aging.
One, experimental program
1, detect the human adipose mesenchymal stem cells extract freeze-drying powder prepared of sample: embodiment 3 (protein content be 50.0 ± 0.5 μ g /).
2, test method:
2.1 animal feedings: the female kunming mice of SPF level in 4 week age (body weight 20 ± 2g), Guangdong Province's Medical experimental center provides, and adaptability was raised after one week, rejected mouse back hair, the about 2cm of baring skin cotton knot
2, test.Normal feedwater and feedstuff in process of the test.
2.2 experiment grouping and drug treating: mice is divided into five groups, 5 every group, proceeds as follows respectively:
The 1st group: with physiological saline solution human adipose mesenchymal stem cells extract freeze-drying powder, preparing human adipose mesenchymal stem cells extract freeze-drying powder concentration is the solution of 20 μ g/ml; It is upper that the solution that is 20 μ g/ml by aforementioned human adipose mesenchymal stem cells extract freeze-drying powder concentration is applied to mice, and applying amount is lml/, 1 times/day of (pre-irradiation), then ultra-vioket radiation;
The 2nd group: with physiological saline solution human adipose mesenchymal stem cells extract freeze-drying powder, preparing human adipose mesenchymal stem cells extract freeze-drying powder concentration is the solution of 10 μ g/ml; It is upper that the solution that is 10 μ g/ml by aforementioned human adipose mesenchymal stem cells extract freeze-drying powder concentration is applied to mice, and applying amount is lml/, 1 times/day of (pre-irradiation), then ultra-vioket radiation;
The 3rd group: model group, the mice of this group is not smeared human adipose mesenchymal stem cells extract freeze-drying powder, ultra-vioket radiation;
The 4th group: positive controls, prepare the solution that vitamine concentration is 20 μ g/ml with physiological saline solution vitamin C (VitC), this solution is applied in to mice upper, applying amount be lml/ only, 1 times/day of (pre-irradiation), then ultra-vioket radiation;
The 5th group: negative control group, the mice of this group is not smeared whatever, also ultra-vioket radiation not.
The concrete operations of ultraviolet radiation: mice administration 1h is carried out to ultra-vioket radiation after medicine is absorbed, irradiate 1h first week every day, second week irradiates 2h every day, irradiates 3h the 3rd week every day, irradiates 4h since the 4th week every day, total concurrent irradiation 42 days; Ultraviolet intensity is: UVA:0.25mJ/cm
2/ s, UVB:0.02mJ/cm
2/ s; After to be illuminated completing, mouse is put to death, carry out the detection of indices; Mouse is carried out to blood-letting, and serum is centrifugal 4min under 2500g, 4 ℃ of conditions, gets supernatant and carries out the detection of indices.
3, test item:
The mensuration of SOD enzyme, glutathion peroxidase (GPx) and malonaldehyde (MDA) content in blood plasma: mice is carried out to blood-letting, and centrifugal 4min under 2500g, 4 ℃ of conditions, collects supernatant;
The activity of SOD, the diagnostic kit that the activity of GPx and the content of MDA utilize Nanjing to build up Bioengineering Research Institute to be provided carries out determination and analysis according to supplier's description.
Skin slice HE dyeing, observes the variation of skin texture: get the skin of mouse back outshot wool, 10% formalin is fixed, routine paraffin wax section, the HE observation of dyeing.
4, experimental result
Fig. 4 is the testing result block diagram of SOD enzymatic activity in the mice plasma of 5 experimental grouies, as seen from Figure 4: the SOD enzymatic activity 7.26 ± 2.24U/ml in model group Mice Body, and SOD enzymatic activity is 53.3 ± 5.99U/ml in the 1st group of Mice Body, in the 2nd group of Mice Body, SOD enzymatic activity is 37.26 ± 2.24U/ml; Although SOD enzymatic activity does not return to Normal group level in the Mice Body of stem cell extract protection; but compare with model group and positive drug VitC group; all tool is greatly improved; illustrate that stem cell extract can more effectively remove or assist antioxidant system in animal body to remove the harmful substances such as oxygen-derived free radicals in body, thereby make antioxidation balance sysmte keep stable.
Fig. 5 is the testing result block diagram of GPx activity in the mice plasma of 5 experimental grouies, and as can be seen from Figure 5, the GPx activity of the 1st group of mice and the 2nd group of mice is all good than model group and positive drug VitC group, thereby has protected mouse skin; Wherein in the 1st group and the 2nd group of tested mice plasma, GPx enzymatic activity is respectively: 1125.33 ± 104.47U/ml, 811.27 ± 125.88U/ml; In model group mice plasma, GPx enzymatic activity is 591.55 ± 176.47U/ml.
Fig. 6 is the testing result block diagram of MDA content in the mice plasma of 5 experimental grouies, as can be seen from Figure 6, minimum 6.54 ± the 0.66nmol/ml of MDA content of negative control group, the highest 25.92 ± the 1.16nmol/ml of MDA content of model group, human adipose mesenchymal stem cells extract has reduced the content of MDA effectively, has kept antioxidation balance sysmte in body.
The testing result of the mouse skin tissue of 5 experimental grouies is as follows:
The 1st group: skin texture is complete and clear, and normal skin tissue do not have obvious difference, epidermis and skin corium are showed no obviously and thicken, and have no the phenomenons such as inflammatory cell infiltration;
The 2nd group: intradermal arrangement of collagen fibers is comparatively neat, the appendages of skin have the hypertrophy of a little;
The 3rd group (model group): mouse skin thickness obviously increases, and dermis thickness sharply reduces, collagen fiber supernormal growth and arrangement disorder, also there is obvious hypertrophy in the appendages of skin, presents aging feature of skin initial stage;
The 4th group (positive controls): epidermis has certain hypertrophy phenomenon, and deep skin arrangement of collagen fibers is comparatively loose;
The 5th group (negative control group): skin is normal, does not have obvious slight crack and hypertrophy phenomenon, there is no aging feature of initial stage.
Because vitamin C is the known medicine with good sun-proof result; therefore as the positive drug of this experiment; and final result shows that the various dose group of human adipose mesenchymal stem cells extract freeze-drying powder of the present invention has the protection or the repair that preferably ultraviolet are caused Mice Photoaging, effect is equal to or is greater than positive controls.
the toxicity assessment of embodiment 6 human adipose mesenchymal stem cells extract freeze-drying powders
The toxicity assessment of the human adipose mesenchymal stem cells extract freeze-drying powder of the present embodiment is divided into the test of chmice acute Oral toxicity (method of once limiting the quantity), the rat acute percutaneous toxicity test (method of once limiting the quantity) of human adipose mesenchymal stem cells extract freeze-drying powder and the rabbit skin Phototoxicity experiment of human adipose mesenchymal stem cells extract freeze-drying powder of human adipose mesenchymal stem cells extract freeze-drying powder.
One, the chmice acute Oral toxicity of human adipose mesenchymal stem cells extract freeze-drying powder test (method of once limiting the quantity)
Human adipose mesenchymal stem cells extract freeze-drying powder prepared by sample: embodiment 3.
Biological subject body: kunming mice.
Test method:
Early stage, the result of experiment in vitro showed, human adipose mesenchymal stem cells extract freeze-drying powder is substantially nontoxic, therefore adopt once limitation method to confirm its safety to organism, once maximum gavage dosage adopts the tested material of its 10 times of actual effects sign doses (50.0 ± 0.5 μ g/prop up × 10) to 10 kunming mice (male and female half and half, body weight is 20.0g ± 0.2g) carry out gavage, observe its toxic reaction, when not causing animal dead, no longer carry out the acute oral toxicity test of multiple dosage.
Observe the time limit: 14 days.
Experimental result is observed: not observing experimental animal has any untoward reaction, also there is no animal dead.
Two, the rat acute percutaneous toxicity test of human adipose mesenchymal stem cells extract freeze-drying powder (method of once limiting the quantity)
Human adipose mesenchymal stem cells extract freeze-drying powder prepared by sample: embodiment 3.
Biological subject body: SD rat (zoopery center, Guangdong Province).
Test method:
Early stage, the result of experiment in vitro showed, human adipose mesenchymal stem cells extract freeze-drying powder is substantially nontoxic, therefore adopt the method for once limiting the quantity further to confirm its safety to organism, use 10 animal (male and female half and half, body weight is 149.1 ± 12.4g), skin is smeared the stem cell solution of extract of 2000mg/kg body weight dosage, when not causing animal dead, no longer carries out the acute dermal toxicity test of multiple dosage.
Observe the time limit: 14 days.
Observed result: not observing experimental animal has any untoward reaction, also there is no animal dead.
Three, the rabbit skin Phototoxicity experiment of human adipose mesenchymal stem cells extract freeze-drying powder
Human adipose mesenchymal stem cells extract freeze-drying powder prepared by sample: embodiment 3.
Biological subject body: 50 of the white rabbit (zoopery center, Guangdong Province) of growing up, female half and half.
UV light source: the UVA that wavelength is 320nm～400nm.
Irradiation time: 1.5h.
Test procedure:
Carry out the front 18～24h of the malicious test of formal light, by rabbit spinal column both sides skin unhairing, test position skin needs intact, harmless injuring extremely; Standby 4 unhairing districts, every unhairing area is about 2cm × 2cm, and the test arrangement of unhaired hide is as shown in table 1; Animal is fixed, according to following table, apply respectively 1ml in animal unhairing district 1 and 2 containing 10 μ g/ml, 20 μ g/ml tested material aqueous solutions; After 30min, left side (unhairing district 1 and 3) is topped with aluminium foil, and adhesive tape is fixed, right side is irradiated with UVA, after end respectively at 1,24,48 and 72h observe dermoreaction, judge whether that according to skin irritation reaction grade form skin is had to irritant reaction, result is as shown in table 2.
The test arrangement in table 1 animal unhairing district
| Unhairing district numbering | Test is processed |
| 1 | Be coated with tested material, do not irradiate |
| 2 | Be coated with tested material, irradiate |
| 3 | Be not coated with tested material, do not irradiate |
| 4 | Be not coated with tested material, irradiate |
Table 2 skin irritation is commented reaction grade form
Result of the test shows: 60 tested rabbit 1h, 24h, 48h and 72h after processing all do not observe skin irritation reaction, show that human adipose mesenchymal stem cells extract freeze-drying powder of the present invention is without phototoxic action.
embodiment 7 human adipose mesenchymal stem cells extract freeze-drying powder hand cream
The present embodiment divides into groups hand cream composition, as shown in table 3, prepares the hand cream that contains human adipose mesenchymal stem cells extract freeze-drying powder prepared by embodiment 3 of 3 kinds of formulas.
Table 3 hand cream formula table
The preparation technology of the present embodiment hand cream comprises the steps:
A component, after 70～80 ℃ of dissolvings, is added to B component homogenizing (negative pressure, homogenizing 1～2 time, each 15～-30 minutes) emulsifying, after cooling to 37～50 ℃, add C and D component, stir, vacuum outgas, obtains the human adipose mesenchymal stem cells extract freeze-drying powder hand cream of the present embodiment.
The present embodiment divides into groups emollient cream composition, as shown in table 4, prepares emollient cream that contains human adipose mesenchymal stem cells extract freeze-drying powder prepared by embodiment 3 of 3 kinds of formulas:
Table 4 emollient cream formula table
The preparation technology of the present embodiment emollient cream comprises the steps:
By A component after 70～80 ℃ of water-baths are dissolved, after B component is uniformly dispersed HANSHENGJIAO and disodiumedetate with glycerol, water-bath is dissolved, A is poured in B, homogenizing (negative pressure, homogenizing 1～2 time, each 15～-30 minutes) emulsifying, after cooling to 37～50 ℃, add C component and D component, homogenizing is even, prepares human adipose mesenchymal stem cells extract freeze-drying powder emollient cream of the present embodiment.
embodiment 9 human adipose mesenchymal stem cells extract freeze-drying powder eye creams
The present embodiment divides into groups eye cream composition, as shown in table 5, prepares the eye cream that contains human adipose mesenchymal stem cells extract freeze-drying powder prepared by embodiment 3 of 3 kinds of formulas.
Table 5 eye cream formula table
The preparation technology of the present embodiment eye cream comprises the steps:
By part deionized water heating for dissolving for B component, stir, A component is in 70～80 ℃ of dissolvings, A component is poured in B component, stir, after being cooled to 37～50 ℃, add C component and D component, stir, prepare the human adipose mesenchymal stem cells extract freeze-drying powder eye cream of the present embodiment.
Claims (10)
1. human adipose mesenchymal stem cells extract, is characterized in that this human adipose mesenchymal stem cells extract is prepare by the following method and obtain:
Step 1.
By human adipose mesenchymal stem cells after primary separation and Culture, the cultivation of going down to posterity, P3～P30 that getting goes down to posterity cultivates is for human adipose mesenchymal stem cells, while utilizing cell growth medium to be cultured to cell 80% to merge, discard culture fluid, first use phosphate buffer wash cell three times, then add serum-free medium to continue to cultivate after 72 hours, collect serum-free medium, and the cell not being collected carries out subsequent treatment;
Step 2.
The cell that step 1 is not collected utilizes after Digestive system digestion, Ultrasonic Cell Disruptor smudge cells, centrifugal rear collection supernatant;
Step 3,
After the supernatant that the serum-free medium that step 1 is collected and step 2 are collected mixes, filter, collect filtrate concentrated to it, gained concentrated solution carries out, after degerming processing, preparing required human adipose mesenchymal stem cells extract again.
2. human adipose mesenchymal stem cells extract according to claim 1, it is characterized in that in described step 1, cell growth medium refers to the DMEM/F12 culture fluid that contains 10% hyclone, and in this culture fluid, also containing penicillin and the final concentration that final concentration is 200 U/ml is the streptomycin of 250 U/ml.
3. human adipose mesenchymal stem cells extract according to claim 1, is characterized in that in described step 2, Digestive system is through the 0.25% trypsin-0.02%EDTA of the membrane filtration degerming of 0.22 μ m mixture slaking liquid.
4. human adipose mesenchymal stem cells extract according to claim 1, is characterized in that in described step 2, and the condition of Ultrasonic Cell Disruptor smudge cells is 4 ℃, 400W, ultrasonic 3s, interval 6s, 99 circulations.
5. human adipose mesenchymal stem cells extract according to claim 1, is characterized in that in described step 3, the mixed proportion of the supernatant that the serum-free medium that step 1 is collected and step 2 are collected is 1:1, volume ratio.
6. human adipose mesenchymal stem cells extract according to claim 1, it is characterized in that in described step 3, filtration is that employing aperture is the membrane filtration of 0.45 μ m, concentrated be adopt filter membrane that molecular cut off is 3KD be concentrated into original volume 1/10, degerming processing is to adopt 0.22 aseptic μ m membrane filtration degerming.
Described in claim 1-6 the human adipose mesenchymal stem cells extract of any one in the application of preparing in wound healing medicine, cytothesis medicine, whitening product or skin wrinkle resisting product.
8. human adipose mesenchymal stem cells extract freeze-drying powder, is characterized in that this lyophilized powder is to be obtained through frozen dried preparation by the human adipose mesenchymal stem cells extract of any one described in claim 1-6.
9. the preparation method of human adipose mesenchymal stem cells extract freeze-drying powder described in claim 8, is characterized in that this preparation method comprises the steps:
In human adipose mesenchymal stem cells extract, add excipient and water, filtration sterilization after mixing, then carries out frozen dried, prepares human adipose mesenchymal stem cells extract freeze-drying powder; Described excipient adopts any one in mannitol, trehalose or sucrose, and excipient accounts for 5～10% of human adipose mesenchymal stem cells extract gross weight; The freeze temperature of described frozen dried is that-20～-35 ℃, vacuum are 50～200Pa, lyophilizing 48 hours.
Described in claim 8 human adipose mesenchymal stem cells extract freeze-drying powder in the application of preparing in wound healing medicine, cytothesis medicine, whitening product or skin wrinkle resisting product.
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| Publication number | Priority date | Publication date | Assignee | Title |
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