CN108624649A - A kind of culture medium of the secretion containing amniotic fluid stem cell and its application - Google Patents
A kind of culture medium of the secretion containing amniotic fluid stem cell and its application Download PDFInfo
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- CN108624649A CN108624649A CN201810310573.2A CN201810310573A CN108624649A CN 108624649 A CN108624649 A CN 108624649A CN 201810310573 A CN201810310573 A CN 201810310573A CN 108624649 A CN108624649 A CN 108624649A
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Abstract
The invention discloses a kind of culture mediums of secretion containing amniotic fluid stem cell, by 4.9 × 105Amniotic fluid stem cell and basal medium of a/ml through inoculation processing form.It will be applied to cell therapy containing amniotic fluid stem cell the present invention overcomes traditional existing matching and tests the unhandy various drawbacks of process, it will contain amniotic fluid stem cell secretion and will be combined with culture medium, realize preservation it is convenient, can in batches using, without now matching, avoiding Liquid nitrogen storage to need recovery etc. in due course temporarily, and can effectively reduce contaminated possibility when cell during experiment shifts.The present invention can also disclose application of the culture medium in cancer cell detection, have the function of promoting cancer cell-apoptosis, lay a good foundation for Clinical practice in the future.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of culture medium.
Background technology
Cell in amniotic fluid is the cell mass that a group is in embryonic development early stage, the amniotic fluid stem cell therefrom detached
(Amniotic Fluid-derived Stem Cells, hAFSCs) is between embryonic stem cell (Embryonic Stem
Cells, ES) cell type between adult stem cell, the mark of ES cells and adult stem cell is expressed, is had similar more
To differentiation potential, and with the ability for the different cells for being divided into three germinal layer sources.Unlike embryonic stem cell,
Not the problems such as AFSCs does not form tumour or teratoma in vivo, and histocompatbility and immunological rejection is not present in AFSCs.In future
It, will be advantageously using AFSCs in treatment use, regenerative medicine and research.
Use of the cell therapy in clinic at present is more and more, but in cell clinical treatment in use, cell consumption
Amount is very big, and some researches show that there is no survive for a long time in patient's body for the cell of heteroinoculation.With the dead of inoculating cell
It dies, the disease of patient can obtain different degrees for the treatment of, this means that the substance of cell secretion can play in disease treatment
Certain effect.The secretory substance of MSCs is to kinds of experiments sexual organ and tissue injury model such as myocardial infarction, acute tubular
Damage, cerebral ischemia and skin injury have the preferable therapeutic effect for promoting wound healing and functional rehabilitation, mechanism of action main
Be inhibit cell death, promote cell differentiation, proliferation and realize the reparations of damaged tissues.Amniotic fluid stem cell is drawn in the prior art
Enter and has obtained certain effect into cell therapy, however because cell therapy is very big to cell consumption amount, and amniotic fluid stem cell is big
It is easy the problems such as there are unknown pathogen body infection risks when amount culture, and toxigenic capacity is very high, therefore it can not still be promoted and be answered
With.
Invention content
It is a kind of containing amniotic fluid stem cell secretion it is an object of the invention in view of the above shortcomings of the prior art, provide
Culture medium, while application of the culture medium on detection cancer cell being provided.
The technical solution used in the present invention is：A kind of culture medium of the secretion containing amniotic fluid stem cell, by 4.9 × 105
Amniotic fluid stem cell and basal medium of a/ml through inoculation processing form.
As being further improved for said program, the basal medium by mass percentage by 5% human seralbumin
Protein injection liquid and 95% DMEM composition.
As being further improved for said program, the amniotic fluid stem cell is selected from the amniotic fluid stem cell of the third generation.
As being further improved for said program, the inoculation processing is coated for the amniotic fluid stem cell is placed in gelatin
Tissue Culture Dish discards supernatant liquid after carrying out the adherent processing of 8h cells, then is washed three times with PBS.
As being further improved for said program, the Tissue Culture Dish is the Tissue Culture Dish of 100mm.
A kind of application of the culture medium of the secretion containing amniotic fluid stem cell as described above in cancer cell detection.
The beneficial effects of the invention are as follows：The present invention overcomes traditional existing matching to be applied to cell therapy containing amniotic fluid stem cell
The unhandy various drawbacks of experiment process, will contain amniotic fluid stem cell secretion and be combined with culture medium, and realize preservation side
Just, can in batches using, without now matching, avoiding Liquid nitrogen storage to need recovery etc. in due course temporarily, and during can effectively reducing experiment
Cell contaminated possibility when shifting.
The present invention, to the detection of cancer cell, has the function of promotion cancer cell-apoptosis suitable for cell therapy procedures,
It lays a good foundation for Clinical practice in the future.
Description of the drawings
Fig. 1 is the growth curve chart of amniotic fluid stem cell of the present invention；
Fig. 2 is the identified by immunofluorescence result figure of amniotic fluid stem cell surface marker of the present invention；
Fig. 3 is amniotic fluid stem cell of the present invention into fat Osteoblast Differentiation result figure；
Fig. 4 is the apoptogene detection figure of cancer cell during amniotic fluid stem cell culture of the present invention is co-cultured with cancer cell.
(it is amniotic fluid stem cell secretion that Fig. 4 A, which are with the Hela Apoptosis detection of amniotic fluid stem cell co-cultivation three days, Fig. 4 B,
The Hela Apoptosis detection of object culture solution culture three days, Fig. 4 C are the hela Apoptosis inspections after ordinary culture medium culture three days
The control group picture of survey)
Specific implementation mode
The present invention is specifically described with reference to embodiment, in order to technical field personnel to the present invention
Understand.It is necessary to it is emphasized that embodiment is only intended to, the present invention will be further described herein, should not be understood as to this
The limitation of invention protection domain, fields person skilled in the art, the non-intrinsically safe that the present invention is made according to foregoing invention content
The modifications and adaptations of property, should still fall within protection scope of the present invention.Mentioned raw materials following simultaneously are unspecified, are
Commercial product；The processing step or preparation method not referred in detail be processing step known to a person skilled in the art or
Preparation method.
The serum free medium is purchased from LONZA companies；
Pancreatin, Adipogenesis induction culture medium (ADM) and the Osteoblast Differentiation inducing culture (ODM) is purchased from
Gibco companies；
The DAPI is purchased from Invitrogen companies；
The oil red O, Alizarin red staining liquid are purchased from Solarbio companies；
The AnexinV-IFTC apoptosis detection kits are purchased from BD Pharmingen companies；
Dual anti-, the DMEM is purchased from Hylone companies；
The serum is purchased from BI companies；
The Transwell plates are purchased from Corning companies.
One, amniotic fluid stem cell is separately cultured
(1) original cuiture of amniotic fluid stem cell：Amniotic fluid samples carry out 1 with the PBS added with EDTA:1 dilution；1200r/min
Centrifuge 5min；Supernatant is abandoned, amniotic fluid special culture media is added and blows and beats mixing repeatedly；It is seeded in the coated culture dish of gelatin；With 37 DEG C,
5%CO2Environmental condition culture 4~5 days amniotic fluid stem cell growth can be observed.
(2) secondary culture of amniotic fluid stem cell：When primary amniotic fluid stem cell, which reaches 80~90%, to be converged, cell training is discarded
Nutrient solution；It is washed three times with PBS；Cell is set to be dissociated with the 0.25% trypsase covering cell containing 0.02%EDTA；It waits for
When cell retraction is rounded and is started shedding off from culture dish digestion is terminated with the complete medium containing 10% fetal calf serum；It will mix
Liquid is closed to be blown and beaten repeatedly until attached cell falls off to be formed under cell suspension 1200rpm from culture dish and centrifuges 5min；Discard liquid
Afterwards be added culture medium softly blow it is even so that cell is scattered, to primary amniotic fluid stem cell carry out secondary culture.
Two, the drafting of amniotic fluid stem cell growth curve
It takes the third generation, the amniotic fluid stem cell in the 6th generation to be counted, and growth curve is drawn according to the data obtained, with 1 ×
104It is seeded in 24 orifice plates, at interval of taking three holes for 24 hours, and writes down average.It is bent to obtain amniotic fluid stem cell growth shown in FIG. 1
Line chart.
Three, the identification of amniotic fluid stem cell
(1) identified by immunofluorescence
The amniotic fluid stem cell stem cell for taking the third generation is seeded on coated 24 orifice plate of gelatin, it is to be achieved 70% fusion when,
Culture solution is discarded, is washed twice with PBS；3.7% paraformaldehyde solution is added in 24 orifice plates and fixes 30min, is used in combination containing 5%
The PBS washing cells of FBS (wash 10min) every time three times；Be added in 24 orifice plates permeabilization agent (0.1%TritonX-100,
PBS it discards, is washed three times with PBS, each 5min after) being incubated at room temperature 15min；Confining liquid (10%FBS is added in 24 orifice plates：
PBS it) is discarded after 37 DEG C of closing 1h, PBS is washed three times, each 5min；
At 4 DEG C and for CD44 and CD90 (1:200, Santa Cruz, CA) primary antibody be incubated overnight, remove primary antibody
Afterwards, it washs cell with the PBS containing 5%FBS and (washs 10min every time) three times；At room temperature in the dark, it is added and is marked with FITC
Secondary antibody (1：500, Santa Cruz, CA) 1h, is washed three times, each 5min with PBS；Finally sample is used under the conditions of being protected from light
DAPI is incubated 5min, is washed three times with PBS, each 5min；
Identified by immunofluorescence result figure shown in Fig. 2 is analyzed to obtain under fluorescence microscope after anti-fluorescence quenching is added.
(2) the Multidirectional Differentiation identification of AFSCs
It is seeded in wherein three holes of six orifice plates with equal densities when third time is passed on, label 1,2,3, wherein 1,2
Holes is experimental group, and 3 holes are control, and when cell growth to 80% is when converging, then removal culture medium washs cell with PBS
Three times.Adipogenesis induction culture medium is added in 1 hole；Osteoblast Differentiation inducing culture is added in 2 holes；It will train completely
It supports and is added in 3 hole cellular control units in base.
Culture medium is replaced for every 3~4 days completely.After induction 14 days, observe logical when apparent a large amount of calcium tubercles occurs in 1 hole
It crosses Alizarin red staining and measures osteogenic ability, commented by oil red O stain when generating a large amount of greases in observing 2 holes after inducing 17 days
Valence fat generative capacity.It observes and takes pictures under an optical microscope, obtain differentiated result figure as shown in Figure 3.
Four, the preparation of the culture medium of the secretion of the invention containing amniotic fluid stem cell
Take the amniotic fluid stem cell of the third generation with 4.9 × 105A/ml is inoculated in the Tissue Culture Dish of the coated 100mm of gelatin,
After 8h cells are adherent, supernatant liquid is discarded, PBS is washed three times；7ml base culture base 48h, the basal medium is added
It is made of 5% Human Seroalbumin and 95% DMEM, after 0.22 μm of filtering, obtains containing sheep by mass percentage
The culture medium of water stem cell secretion object.
Five, it is applied to cancer cell to detect
(1) culture of Hela cells
Hela cells are purchased in Cell Bank of Chinese Academy of Sciences (cell strain number 101460), and secondary culture is dry with above-mentioned amniotic fluid
Cell, culture medium is dual anti-by 1%, 10% serum, 89%DMEM are formed.
(2) co-cultivation of Hela cells and amniotic fluid stem cell
By amniotic fluid stem cell 1 × 106A/ml is placed in the small interior of 0.4 μm of aperture Transwell plate, and each cell is added
70 μ l are added 4 × 105500 μ l are added per hole in the lower layer for co-culturing plate for a/ml Hela cells, after co-culturing 72h, discard
Supernatant liquid, PBS are washed three times；0.25%% trypsase covering cell makes cell be dissociated, and 5min is centrifuged under 1000rpm
It is spare that cell is collected afterwards.
(3) Hela cells and the culture medium of the secretion containing amniotic fluid stem cell co-culture
By the Hela cells of collection, it is resuspended with 6ml ordinary culture mediums and is seeded to the coated 100mm cell culture of gelatin
Plate, the cell for controlling each plate are 1.0 × 106It is a, after cell is adherent, it is changed to dividing containing amniotic fluid stem cell for the 6ml present invention
The culture medium of secretion, collection cell is spare after cultivating 72h.
The apoptosis of the Hela cells of (4) three kinds of training method cultures detects
The Hela cells for the three groups of difference training methods that suspended with 1 × Binding Buffer of 400 μ l, concentration is about 1
×106A/ml；5 μ l Annexin V-FITC are added cell suspensions, careful mixing, after 5 DEG C or so avoid light place 15min,
Cell suspension, careful mixing, 5 DEG C or so avoid light place 5min are added in 10 μ l PI (Propidine Iodide, PI).After 1h
With flow cytomery natural death of cerebral cells rate, testing result is as shown in Figure 4.
Above-described embodiment is the preferred embodiment of the present invention, it is all with similar technique of the invention and made by equivalence changes,
The protection category of the present invention should all be belonged to.
Claims (6)
1. a kind of culture medium of secretion containing amniotic fluid stem cell, it is characterised in that：By 4.9 × 105Sheep of a/ml through inoculation processing
Water stem cell and basal medium composition.
2. a kind of culture medium of secretion containing amniotic fluid stem cell according to claim 1, it is characterised in that：The basis training
Foster base is made of 5% Human Seroalbumin and 95% DMEM by mass percentage.
3. a kind of culture medium of secretion containing amniotic fluid stem cell according to claim 1, it is characterised in that：The amniotic fluid is dry
Cell is selected from the amniotic fluid stem cell of the third generation.
4. a kind of culture medium of secretion containing amniotic fluid stem cell according to claim 1, it is characterised in that：At the inoculation
Reason discards supernatant liquid for the amniotic fluid stem cell to be placed in after the coated Tissue Culture Dish of gelatin carries out the adherent processing of 8h cells,
It is washed three times with PBS again.
5. a kind of culture medium of secretion containing amniotic fluid stem cell according to claim 1, it is characterised in that：The cell training
Support the Tissue Culture Dish that ware is 100mm.
6. a kind of as right will go the culture medium of 1~5 any one of them secretion containing amniotic fluid stem cell in cancer cell detection
Application.
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