CN103784474B - 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 uses in biological medicine or medical cosmetology, particularly relate to the use of human adipose mesenchymal stem cells in biological medicine or medical cosmetology.
Background technology
Adipose tissue-derived extensively, draw materials easily, cellular segregation efficiency is high, the wound brought to patient is less and do not relate to ethics problem, 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), vascular endothelial growth factor (VEGF), rhIGF-1 (IGF), pHGF (HGF) and transforming growth factor etc., these cell growth factors can control or maintain wounded tissue cell, guide and himself repair.In addition, also containing the Multiple components such as multiple film protein and biologically active factors in human adipose mesenchymal stem cells, these compositions are coordinated mutually to supplement mutually, there is complicated physiological action, can have an impact to many histoorgans, therefore there is the effects such as reasonable anti-light aging, anti-oxidant, 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 used for the aspects such as medical treatment, makeup, but this conditioned medium is application some specific factor of specific cytokine induction stem cell secretion thus 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 have 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 at biomedical product such as preparation wound healing medicine or cytothesis medicine etc., or is preparing the application 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 at biomedical product such as preparation wound healing medicine or cytothesis medicine etc., or is preparing the application 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 prepares by the following method and obtains:
Step 1.
By human adipose mesenchymal stem cells after primary separation and Culture, carry out Secondary Culture, get the P3 ~ P30 of Secondary Culture for human adipose mesenchymal stem cells, cell growth medium is utilized to be cultured to cell 80% when merging, discard nutrient solution, first use phosphate buffer wash cell three times, then add serum-free medium and continue cultivation after 72 hours, collect serum-free medium, and the cell be not collected carries out subsequent disposal;
Step 2.
After cell step 1 be not collected utilizes Digestive system to digest, Ultrasonic Cell Disruptor smudge cells, collected after centrifugation supernatant;
Step 3,
After the supernatant mixing that serum-free medium step 1 collected and step 2 are collected, filter, collect filtered liquid and it is concentrated, after gained concentrated solution carries out bacteria removing again, then prepare required human adipose mesenchymal stem cells extract.
In above-mentioned steps 1, described by human adipose mesenchymal stem cells in primary separation and Culture, primary separation method and cultural method adopt the routine operation of those skilled in the art.
In above-mentioned steps 1, described cell growth medium refers to the DMEM/F12 nutrient solution containing 10% foetal calf serum, and also containing antibacterium microbiotic in this nutrient solution; Described antibacterium microbiotic is penicillin and Streptomycin sulphate two kinds, and wherein the final concentration of penicillin in cell growth medium is 200 U/ml, and the final concentration of Streptomycin sulphate in cell growth medium is 250 U/ml; The described DMEM/F12 nutrient solution containing 10% foetal calf serum is add the foetal calf serum that final concentration is 10% in commercially available basic DMEM/F12 nutrient solution, and to add penicillin that final concentration is 200 U/ml and final concentration be that the Streptomycin sulphate of 250 U/ml is cell growth medium of the present invention.
In above-mentioned steps 1, described phosphate buffered saline buffer to be pH value be 7.0 ~ 7.2 PBS solution.
In above-mentioned steps 1, described cell continues cultivation 72 hours in serum-free medium, and serum-free medium is here commercially available prod.
In above-mentioned steps 1, during concrete operations, the difference of Secondary Culture is processed respectively for cell, as: the human adipose mesenchymal stem cells in P3 generation is utilized cell growth medium be cultured to cell 80% merge time, discard nutrient solution, first use phosphate buffer wash cell three times, then cell is joined in serum-free medium and continue cultivation after 72 hours, collect the serum-free medium obtaining P3; Cell growth medium is utilized by the human adipose mesenchymal stem cells in P4 generation to be cultured to cell 80% when merging, discard nutrient solution, first use phosphate buffer wash cell three times, then cell is joined in serum-free medium and continue cultivation after 72 hours, collect and obtain the serum-free medium in P4 generation etc.; Then after P3 ~ P30 being collected merging for the serum-free medium that human adipose mesenchymal stem cells obtains respectively, then the serum-free medium that embodiment 1 is collected is obtained; Meanwhile, after being merged for the cell harvesting be not collected that human adipose mesenchymal stem cells obtains respectively by P3 ~ P30, subsequent disposal is carried out together.
In above-mentioned steps 2, described Digestive system is 0.25% trypsinase-0.02%EDTA mixture slaking liquid, and this mixture slaking liquid first could be used for the present invention after the membrane filtration of 0.22 μm is degerming; Described 0.25% trypsinase-0.02%EDTA mixture slaking liquid is a kind of Digestive systems conventional when carrying out cell experiment of those skilled in the art, and its compound method, according to the compound method that textbook provides, all can realize the present invention; Prepare with the following method as adopted: the PBS getting 0.25g trypsinase powder, 20mgEDTA powder and 100ml 0.01mol/L, first dissolve trypsinase powder with a small amount of PBS, then after EDTA powder and remaining PBS being added mixing, be placed in 37 DEG C of water-baths, treat thoroughly to dissolve liquid transparent till, use G5 suction filtration, packing is placed in 4 DEG C of refrigerators and preserves.
In above-mentioned steps 2, described Ultrasonic Cell Disruptor smudge cells, its broken condition is 4 DEG C, 400W, ultrasonic 3s, interval 6s, 99 circulations.
In above-mentioned steps 2, described centrifugal be 17000g, 30min.
In above-mentioned steps 3, the supernatant mixing that the described serum-free medium step 1 collected and step 2 are collected, the blending ratio of supernatant that the serum-free medium collected of step 1 and step 2 are collected here is 1:1, volume ratio.
In above-mentioned steps 3, described filtration adopts aperture to be the membrane filtration of 0.45 μm.
In above-mentioned steps 3, described concentrated be adopt molecular weight cut-off to be that the filtering membrane of 3KD carries out concentrating and desalinating.
In above-mentioned steps 3, described concentrated referring to is concentrated into 1/10 of original volume, and that is, obtain concentrated solution after filtered liquid is concentrated, the volume of described concentrated solution is 1/10 of filtered liquid.
In above-mentioned steps 3, described concentrated solution is carried out bacteria removing, bacteria removing is here adopted by concentrated solution 0.22 μm of aseptic membrane filtration degerming.
The human adipose mesenchymal stem cells extract of above-mentioned preparation confirms through experimental study, it has a variety of biologic activity, can be used for biomedical product such as preparation wound healing medicine or cytothesis medicine etc., 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 Areas is extensive, but its biologic activity limits by various ambient conditions, save inconvenience, therefore the present invention also provides the lyophilized powder of human adipose mesenchymal stem cells extract, and this lyophilized powder is exactly the lyophilized powder prepared by the method for preparing freeze-dried powder that human adipose mesenchymal stem cells extract of the present invention is commonly used through this area; Lyophilized powder be in an aseptic environment liquid is frozen into solid-state, vacuumize the dry aseptic powder of water sublimed, the preparation method of lyophilized powder is the technology of this area comparative maturity, the present invention adopts the preparation method of any one 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 is the special feature for above-mentioned human adipose mesenchymal stem cells extract also, and have developed the preparation method of human adipose mesenchymal stem cells extract freeze-drying powder, this preparation method comprises the steps:
In human adipose mesenchymal stem cells extract, add vehicle and water, filtration sterilization after mixing, then carries out frozen dried, then prepares required human adipose mesenchymal stem cells extract freeze-drying powder.
In the preparation method of above-mentioned lyophilized powder, described vehicle adopt in N.F,USP MANNITOL, trehalose or sucrose any one.
In the preparation method of above-mentioned lyophilized powder, the consumption of described vehicle: vehicle accounts for 5 ~ 10% of human adipose mesenchymal stem cells extract gross weight.
In the preparation method of above-mentioned lyophilized powder, described hydromining ultrapure water.
In the preparation method of above-mentioned lyophilized powder, adding ultrapure water to final volume is 100ml.
In the preparation method of above-mentioned lyophilized powder, described frozen dried refers to that freeze temperature is-20 ~-35 DEG C, vacuum tightness is 50 ~ 200Pa, freeze-drying 48 hours.
Human adipose mesenchymal stem cells extract freeze-drying powder of the present invention has various biological activity equally, can be used for biomedical product such as preparation wound healing medicine or cytothesis medicine etc., 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 FCM analysis figure of human adipose mesenchymal stem cells;
Fig. 2 is that human adipose mesenchymal stem cells extract freeze-drying powder affects result histogram to HDF Growth of Cells;
Fig. 3 is that human adipose mesenchymal stem cells extract freeze-drying powder affects result histogram to SOD enzyme activity in cell;
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml group; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is negative control group; Wherein: * * P≤0.01, * P≤0.05;
Fig. 4 is the detected result histogram of SOD enzyme activity in the mice plasma of 5 experimental group;
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml group; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is negative control group; Wherein: * * P≤0.01, * P≤0.05;
Fig. 5 is the detected result histogram of GPx activity in the mice plasma of 5 experimental group;
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml group; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is negative control group; Wherein: * * P≤0.01, * P≤0.05;
Fig. 6 is the detected result histogram of MDA content in the mice plasma of 5 experimental group.
In figure, 1 is human adipose mesenchymal stem cells extract freeze-drying powder 20 μ g/ml group; 2 is human adipose mesenchymal stem cells extract freeze-drying powder 10 μ g/ml group; 3 is model group; 4 is positive controls; 5 is negative control group; Wherein: * * P≤0.01, * P≤0.05.
Embodiment
Below in conjunction with specific embodiment the present invention done and describe further, but specific embodiment does not do any restriction to the present invention.
the primary separation of embodiment 1 human adipose mesenchymal stem cells, cultivation and qualification
1, the primary separation of human adipose mesenchymal stem cells and cultivation
By centrifugal for the human abdomen's subcutaneus adipose tissue extract obtained by liposuction (preoperative oneself gets rid of endocrinopathy and transmissible disease) 800g, use PBS wash buffer, soak, then 800g × 4min washs 3 times; Reject macroscopic blood vessel and knot hoof tissue, put into 50ml centrifuge tube after it fully being shredded with eye scissors, add 2 times of volume 0.2% collagenases, 1 times of volume 1%BSA and dual anti-, mixing, sealing, puts into shaking table 37 DEG C and digests 45min, to pasty state;
Add isopyknic containing volume fraction be 10% foetal calf serum low sugar DMEM stop digestion, the centrifugal 10min of 1800r/min, is divided into 3 layers after centrifugal, upper strata is grease and does not digest fatty tissue completely, middle level is supernatant liquor, and lower floor is the precipitation of the cell mixing such as fat stem cell and red corpuscle;
Abandon upper strata and middle level, lower floor adds perfect medium re-suspended cell precipitation, 70 μm of cell screen filtrations, is 5 × 10 by the filtrate adjustment cell concn after filtering
5monocyte/mL is inoculated in T-25cm
2in culturing bottle, 37 DEG C, 5%CO
2, saturated humidity cultivate, change liquid after 48h, add fat stem cell perfect medium continue cultivate.
2, the qualification of human adipose mesenchymal stem cells
Flow cytometer is adopted to carry out the qualification 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.
embodiment 2 human adipose mesenchymal stem cells extract
The human adipose mesenchymal stem cells extract of the present embodiment, this extract prepares by the following method and obtains:
Step 1.
By the P3 ~ P30 of the Secondary Culture through qualification for human adipose mesenchymal stem cells, cell growth medium (described cell growth medium be add final concentration in commercially available basic DMEM/F12 nutrient solution be that the foetal calf serum of 10% and the Streptomycin sulphate preparation of final concentration to be the penicillin of 200 U/ml and final concentration be 250 U/ml obtain) is utilized to be cultured to cell 80% when merging, discard nutrient solution, first use the phosphate buffered saline buffer PBS solution of 7.0 ~ 7.2 (the described phosphate buffered saline buffer to be pH value be) washed cell three times, then add commercially available serum-free medium and continue cultivation after 72 hours, collect serum-free medium that P3 ~ P30 obtains respectively for each generation human adipose mesenchymal stem cells and merge, and the cell be not collected carries out subsequent disposal,
Step 2.
Cell step 1 be not collected utilizes Digestive system, and (described Digestive system is 0.25% trypsinase-0.02%EDTA mixture slaking liquid, and this mixture slaking liquid is degerming through the membrane filtration of 0.22 μm) digestion after, the cell growth medium adding step 1 stops digestion, 4 DEG C, 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; Become after clarification until liquid, 4 DEG C, 17000g, centrifugal 30min collect supernatant, the supernatant that P3 ~ P30 obtains respectively for each generation human adipose mesenchymal stem cells is collected and merged;
Step 3,
The supernatant that serum-free medium step 1 collected and step 2 are collected is with after the mixing of the volume ratio of 1:1, utilize 0.45 μm of frit solution, collect filtered liquid and this filtered liquid is concentrated into 1/10th of original volume, gained concentrated solution is degerming with aseptic 0.22 μm of membrane filtration, then prepare the human adipose mesenchymal stem cells extract solution of the present embodiment.
embodiment 3 human adipose mesenchymal stem cells extract freeze-drying powder
BCA method is adopted to carry out determination of protein concentration to human adipose mesenchymal stem cells extract solution prepared by embodiment 2, microplate reader measures the absorbancy of A570, obtain the protein concentration of human adipose mesenchymal stem cells extract solution prepared by embodiment 2, then according to this protein concentration, according to the ultrapure water mixing needing the final quality of Solutions in Freeze-drying to add vehicle N.F,USP MANNITOL (N.F,USP MANNITOL accounts for 5% of human adipose mesenchymal stem cells extract gross weight) and 50 ~ 60 DEG C, 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 DEG C, vacuum tightness is 50 ~ 200Pa, freeze-drying time 48 hours, then obtained required human adipose mesenchymal stem cells extract freeze-drying powder, lyophilized powder according to 1ml measure/prop up packing.
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.Be clarification, transparent incarnadine solution after dissolving by labelled amount 1ml ultrapure water.
(2) mensuration of pH value
Accurate pH measurement is adopted to determine the pH value of human adipose mesenchymal stem cells extract freeze-drying powder.First utilize reference liquid to correct pH meter according to process specifications, then the stem cell extract freeze-drying powder aqueous solution is measured.The pH value of the human adipose mesenchymal stem cells extract freeze-drying powder aqueous solution is between 6.5 ~ 7.5.
embodiment 4 human adipose mesenchymal stem cells extract freeze-drying powder In Vitro Anti light aging test
(1) mtt assay measures the impact that human adipose mesenchymal stem cells extract freeze-drying powder grows fibroblasts of adult human dermis
Detect sample: human adipose mesenchymal stem cells extract freeze-drying powder (protein content be 50.0 ± 0.5 μ g /) prepared by embodiment 3.
Biological subject body: HDF cell.
Concrete steps are as follows:
(1) 10%(v/v is utilized) the DMEM/F12 substratum (penicillin 200 U/ml, Streptomycin sulphate 250 U/ml, pH7.2) of FBS cultivates HDF cell, when cell density reaches 100% fusion, utilizes cell dissociation buffer peptic cell, and adjustment cell concn is for being 1 × 10
5cell/ml, is inoculated in 96 orifice plates, every hole 100 μ l, sets 4 multiple holes, 37 DEG C, 5%CO2, saturated humidity cell culture incubator in continue to cultivate;
(2) after 24h, discard substratum, diluting stem cell extract to final concentration with the DMEM/F1 substratum 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, control group adds the DMEM/F12 substratum of 1%FBS.37 DEG C, 5%CO2, saturated humidity cell culture incubator in continue 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 wavelength be 570nm, reference wavelength is 630nm place mensuration absorbancy, and calculates cell survival rate.
Cell survival rate (%)=(experimental group mean absorbance values/control group mean absorbance values) × 100%
Experimental result as shown in Figure 2, during human adipose mesenchymal stem cells extract freeze-drying powder Nong Du≤400 μ g/ml to HDF cell without obvious restraining effect, Microscopic observation cell is also without the obvious symptom such as dead.Experimental group is compared with control group, and its cell survival rate does not have obvious difference, illustrates and does not occur significant necrocytosis, and can promote the growth of HDF cell as human adipose mesenchymal stem cells extract freeze-drying powder Nong Du≤100 μ g/ml.
(2) human adipose mesenchymal stem cells extract freeze-drying powder is to the provide protection of ultraviolet damage fibroblasts of adult human dermis
Detect sample: human adipose mesenchymal stem cells extract freeze-drying powder (protein content be 50.0 ± 0.5 μ g /) prepared by embodiment 3.
Biological subject body: HDF cell.
Concrete steps are as follows:
(1) when fibroblasts of adult human dermis grows to 80% fusion, with UVA uviolizing cell, irradiate 2 hours every day, concurrent irradiation 4 days, negative control group tinfoil encases Tissue Culture Flask.Examine under a microscope the form of cell, find that cell starts to occur shrinkage, respective cells is in the medium floating; 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) human adipose mesenchymal stem cells extract freeze-drying powder is on the impact of SOD enzyme activity in cell: after trysinization, and cell is centrifugal 10min under 1500g, 4 DEG C of conditions.Cell precipitation is suspended in (Tris – HCl, the 5 mM EDTA of 50mM and 1 mM DTT) in the cold damping fluid of chance and carries out ultrasonic, and lysate is centrifugal 10min under 10000g, 4 DEG C 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 improve the activity of SOD enzyme in cell to some extent; thus reduce because the oxyradical of ultraviolet radiation induction generation is to the attack of cell and infringement; reduce the mortality ratio of cell; thus play the effect of Cell protection, reach effect of anti-light aging.
embodiment 5 human adipose mesenchymal stem cells extract freeze-drying powder causes the provide protection of Mice Photoaging to ultraviolet
One, experimental program
1, sample is detected: human adipose mesenchymal stem cells extract freeze-drying powder (protein content be 50.0 ± 0.5 μ g /) prepared by embodiment 3.
2, test method:
2.1 animal rearings: in SPF level female KM mouse in 4 week age (body weight 20 ± 2g), Guangdong Province's Medical experimental center provides, after adaptability raises one week, reject mouse back hair, baring skin cotton knot is about 2cm
2, test.Normal feedwater and feed in process of the test.
2.2 experiment grouping and drug treating: mouse is divided into five groups, often organizes 5, proceeds as follows respectively:
1st group: with physiological saline solution human adipose mesenchymal stem cells extract freeze-drying powder, prepare the solution that human adipose mesenchymal stem cells extract freeze-drying powder concentration is 20 μ g/ml; The solution being 20 μ g/ml by aforementioned human adipose mesenchymal stem cells extract freeze-drying powder concentration is applied on mouse, and applying amount is lml/, 1 times/day of (pre-irradiation), then uv irradiating;
2nd group: with physiological saline solution human adipose mesenchymal stem cells extract freeze-drying powder, prepare the solution that human adipose mesenchymal stem cells extract freeze-drying powder concentration is 10 μ g/ml; The solution being 10 μ g/ml by aforementioned human adipose mesenchymal stem cells extract freeze-drying powder concentration is applied on mouse, and applying amount is lml/, 1 times/day of (pre-irradiation), then uv irradiating;
3rd group: model group, the mouse of this group does not smear human adipose mesenchymal stem cells extract freeze-drying powder, uv irradiating;
4th group: positive controls, prepare the solution that vitamine concentration is 20 μ g/ml, be applied on mouse by this solution with physiological saline solution vitamins C (VitC), applying amount is lml/, 1 times/day of (pre-irradiation), then uv irradiating;
5th group: negative control group, the mouse of this group is not smeared whatever, also not uv irradiating.
The concrete operations of uviolizing: after medicine is absorbed, uv irradiating is carried out to mouse administration 1h, first week every day irradiated 1h, and second week irradiates 2h every day, and the 3rd week every day irradiated 3h, and from the 4th week, every day irradiated 4h, total concurrent irradiation 42 days; Ultraviolet intensity is: UVA:0.25mJ/cm
2/ s, UVB:0.02mJ/cm
2/ s; To be illuminated complete after, by mouse put to death, carry out the detection of indices; Carry out bloodletting to mouse, serum is centrifugal 4min under 2500g, 4 DEG C of conditions, gets the detection that supernatant carries out indices.
3, test item:
The mensuration of SOD enzyme, Selenoperoxidase (GPx) and mda (MDA) content in blood plasma: bloodletting is carried out to mouse, centrifugal 4min under 2500g, 4 DEG C 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 the specification sheets of supplier.
Skin slice HE dyes, and observe the change of skin texture: the skin getting mouse back outshot wool, the formaldehyde solution of 10% is fixed, and routine paraffin wax is cut into slices, and HE dyes observation.
4, experimental result
Fig. 4 is the detected result histogram of SOD enzyme activity in the mice plasma of 5 experimental group, as seen from Figure 4: the SOD enzymic activity 7.26 ± 2.24U/ml in model group Mice Body, and SOD enzymic activity is 53.3 ± 5.99U/ml in the 1st group of Mice Body, in the 2nd group of Mice Body, SOD enzyme activity is 37.26 ± 2.24U/ml; Although SOD enzymic activity does not return to levels found in normal controls in the Mice Body of stem cell extract protection; but compare with model group and positive drug VitC group; equal tool is greatly improved; illustrate that the objectionable impuritiess such as the oxyradical in animal body in antioxidant system purged body can more effectively be removed or assist to stem cell extract, thus make anti-oxidant equilibrium system keep stable.
Fig. 5 is the detected result histogram of GPx activity in the mice plasma of 5 experimental group, as can be seen from Figure 5, the GPx activity of the 1st group of mouse and the 2nd group of mouse all than model group and positive drug VitC group good, thus protect mouse skin; Wherein in the 1st group and the 2nd group of test mice blood plasma, GPx enzymic activity is respectively: 1125.33 ± 104.47U/ml, 811.27 ± 125.88U/ml; In model group mice plasma, GPx enzymic activity is 591.55 ± 176.47U/ml.
Fig. 6 is the detected result histogram of MDA content in the mice plasma of 5 experimental group, 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 significantly reduces the content of MDA, maintains anti-oxidant equilibrium system in body.
The detected result of the mice skin tissue of 5 experimental group is as follows:
1st group: skin texture is complete and clear, and normal skin tissue does not have obvious difference, epidermis and skin corium are showed no and obviously thicken, and have no the phenomenons such as inflammatory cell infiltration;
2nd group: intradermal arrangement of collagen fibers is comparatively neat, and the appendages of skin have a little hyperplasia;
3rd group (model group): mouse skin thickness obviously increases, and dermis thickness sharply reduces, collegen filament abnormal growth and arrangement disorder, also there is obvious hyperplasia in the appendages of skin, presents skin initial stage aging feature;
4th group (positive controls): epidermis has certain hyperplasia phenomenon, and deep skin arrangement of collagen fibers is comparatively loose;
5th group (negative control group): skin is normal, does not have obvious slight crack and hyperplasia phenomenon, does not have the feature that the initial stage is aging.
Due to the medicine with good sun-proof result that vitamins C is known; therefore as the positive drug of this experiment; and net 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 to 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 powder
The toxicity assessment of the human adipose mesenchymal stem cells extract freeze-drying powder of the present embodiment is divided into the chmice acute Oral toxicity of human adipose mesenchymal stem cells extract freeze-drying powder to test (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 test of human adipose mesenchymal stem cells extract freeze-drying powder.
One, chmice acute Oral toxicity test (method of once limiting the quantity) of human adipose mesenchymal stem cells extract freeze-drying powder
Sample: human adipose mesenchymal stem cells extract freeze-drying powder prepared by embodiment 3.
Biological subject body: kunming mice.
Test method:
The result of preliminary in vitro experiments shows, human adipose mesenchymal stem cells extract freeze-drying powder is substantially nontoxic, therefore method of once limiting the quantity is adopted to confirm its security to organism, namely once maximum given low adopts its 10 times of actual effects to indicate the tested material of dose (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, then 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, does not also have animal dead.
Two, the rat acute percutaneous toxicity test (method of once limiting the quantity) of human adipose mesenchymal stem cells extract freeze-drying powder
Sample: human adipose mesenchymal stem cells extract freeze-drying powder prepared by embodiment 3.
Biological subject body: SD rat (Guangdong Province's animal experimental center).
Test method:
The result of preliminary in vitro experiments shows, human adipose mesenchymal stem cells extract freeze-drying powder is substantially nontoxic, therefore method of once limiting the quantity is adopted to confirm its security to organism further, namely 10 animal (male and female half and half are used, body weight is 149.1 ± 12.4g), the stem cell solution of extract of dermal application 2000mg/kg body weight dose, when not causing animal dead, then no longer carries out the acute dermal toxicity test of multiple dosage.
Observe the time limit: 14 days.
Observations: not observing experimental animal has any untoward reaction, does not also have animal dead.
Three, the rabbit skin phototoxicity test of human adipose mesenchymal stem cells extract freeze-drying powder
Sample: human adipose mesenchymal stem cells extract freeze-drying powder prepared by embodiment 3.
Biological subject body: white rabbit (Guangdong Province's animal experimental center) 50 of growing up, female half and half.
UV light source: wavelength is the UVA of 320nm ~ 400nm.
Irradiation time: 1.5h.
Testing sequence:
Carry out the front 18 ~ 24h of formal light poison test, by rabbit backbone both sides skin unhairing, test area skin needs intact, can't harm and injures exception; Standby 4 pieces of unhairing districts, every block 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, applies 1ml respectively in animal unhairing district 1 and 2 containing 10 μ g/ml, the 20 μ g/ml tested material aqueous solution; After 30min, left side (unhairing district 1 and 3) is topped with aluminium foil, and adhesive tape is fixed, right side UVA irradiates, observe skin reaction respectively at 1,24,48 and 72h after end, judge whether there is irritant reaction to skin according to skin wound repair grade form, result is as shown in table 2.
The test arrangement in table 1 animal unhairing district
| Unhairing district numbers | Test process |
| 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 |
Reaction grade form is commented in table 2 skin irritation
Test-results shows: 60 tested rabbit after treatment 1h, 24h, 48h and 72h all do not observe skin wound repair, 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 lotion
Hand lotion composition divides into groups by the present embodiment, as shown in table 3, prepares the hand lotion of the human adipose mesenchymal stem cells extract freeze-drying powder containing embodiment 3 preparation of 3 kinds of formulas.
Table 3 hand lotion formula table
The preparation technology of the present embodiment hand lotion comprises the steps:
After component A being dissolved in 70 ~ 80 DEG C, add B component homogeneous (negative pressure, homogeneous 1 ~ 2 time, each 15 ~-30 minutes) emulsification, C and D component is added after cooling to 37 ~ 50 DEG C, stir, vacuum outgas, obtains the human adipose mesenchymal stem cells extract freeze-drying powder hand lotion of the present embodiment.
embodiment 8 human adipose mesenchymal stem cells extract freeze-drying powder skin cream
Skin cream composition divides into groups by the present embodiment, as shown in table 4, prepares the skin cream of the human adipose mesenchymal stem cells extract freeze-drying powder containing embodiment 3 preparation of 3 kinds of formulas:
Table 4 skin lotion formula table
The preparation technology of the present embodiment skin cream comprises the steps:
By component A after 70 ~ 80 DEG C of water-baths are dissolved, after xanthan gum and disodium ethylene diamine tetraacetate are uniformly dispersed by B component glycerine, water-bath is dissolved, A is poured in B, homogeneous (negative pressure, homogeneous 1 ~ 2 time, each 15 ~-30 minutes) emulsification, add component C and D component after cooling to 37 ~ 50 DEG C, homogeneous uniform, then prepare the human adipose mesenchymal stem cells extract freeze-drying powder skin cream of the present embodiment.
embodiment 9 human adipose mesenchymal stem cells extract freeze-drying powder eye cream
Eye cream composition divides into groups by the present embodiment, as shown in table 5, prepares the eye cream of the human adipose mesenchymal stem cells extract freeze-drying powder containing embodiment 3 preparation of 3 kinds of formulas.
Table 5 eye cream formula table
The preparation technology of the present embodiment eye cream comprises the steps:
By B component portions of de-ionized water heating for dissolving, stir, component A is in 70 ~ 80 DEG C of dissolvings, component A is poured in B component, stir, component C and D component is added after being cooled to 37 ~ 50 DEG C, stir, then prepare the human adipose mesenchymal stem cells extract freeze-drying powder eye cream of the present embodiment.
Claims (1)
1. a preparation method for human adipose mesenchymal stem cells extract, is characterized in that the method comprises:
Step 1.
By human adipose mesenchymal stem cells after primary separation and Culture, carry out Secondary Culture, get the P3 ~ P30 of Secondary Culture for human adipose mesenchymal stem cells, cell growth medium is utilized to be cultured to cell 80% when merging, discard nutrient solution, first use phosphate buffer wash cell three times, then add serum-free medium and continue cultivation after 72 hours, collect serum-free medium, and the cell be not collected carries out subsequent disposal;
Step 2.
After cell step 1 be not collected utilizes Digestive system to digest, Ultrasonic Cell Disruptor smudge cells, collected after centrifugation supernatant; The condition of Ultrasonic Cell Disruptor smudge cells is 4 DEG C, 400W, ultrasonic 3s, interval 6s, 99 circulations;
Step 3.
After the supernatant mixing that serum-free medium step 1 collected and step 2 are collected, filter, collect filtered liquid and it is concentrated, after gained concentrated solution carries out bacteria removing again, then prepare required human adipose mesenchymal stem cells extract;
In above-mentioned steps 1, cell growth medium refers to the DMEM/F12 nutrient solution containing 10% foetal calf serum, the Streptomycin sulphate also containing final concentration to be the penicillin of 200 U/ml and final concentration be 250 U/ml in this nutrient solution;
In above-mentioned steps 2, Digestive system is through 0.25% degerming trypsinase-0.02%EDTA mixture slaking liquid of the membrane filtration of 0.22 μm;
In above-mentioned steps 3, the blending ratio of the serum-free medium that step 1 is collected and the supernatant that step 2 is collected is 1:1, volume ratio, filtration adopts aperture to be the membrane filtration of 0.45 μm, concentrated be adopt molecular weight cut-off be the filtering membrane of 3KD be concentrated into original volume 1/10, bacteria removing adopts 0.22 μm of aseptic membrane filtration degerming.
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