CN109082409A - A kind of fat stem cell that tissue repairing ability is strong is separately cultured and screening technique - Google Patents
A kind of fat stem cell that tissue repairing ability is strong is separately cultured and screening technique Download PDFInfo
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Abstract
It is separately cultured the invention discloses a kind of fat stem cell that tissue repairing ability is strong and screening technique.The present invention is on the basis of fat mesenchymal stem cell, its surface antigen is further screened, selected expression CD10 and the cell for not expressing CD200, then secrete the ability of vascular endothelial growth factor to it and screen, obtain a kind of fat stem cell that tissue repairing ability is strong.The present invention solves fat stem cell heterogeneity, filters out the strong fat stem cell of tissue repairing ability, with the wide application prospect in organizational project.
Description
Technical field
The present invention relates to cell engineering field, in particular to a kind of screening technique of fat stem cell.
Background knowledge
Stem cell is that a kind of multipotential cell with the of self-replication capacity can be divided into a variety of under certain condition
Functioning cell plays pivotal player during embryonic development, tissue update and injury repair.Stem cell is regenerative medicine
Basis and soul, it may be said that regenerative medicine is born and development depends on the development of stem-cell research and gos deep into.
Fat stem cell is because have the characteristics that stronger stemness and materials are easy, using wide in Tissue Engineering Study
It is general, while also there is very big potential applicability in clinical practice.However, due to the age of donor, health status, internal microenvironment etc. because
Element, fat stem cell secretion specific cells factor level height is different, and differentiation potential is also inconsistent.
In clinical treatment, it needs that there is lower heterogeneity for the cell of transplanting, to stablize the effect of its repair tissue
Fruit.However it has not yet to see a kind of lower fat stem cell of heterogeneity and is reported.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of fat stem cell that tissue repairing ability is strong and is separately cultured and sieves
Choosing method.
The present invention provides a kind of fat stem cells that tissue repairing ability is strong, it is characterised in that: it is that have following spy
The fat mesenchymal stem cell of sign:
A, when convergence degree is 80%, in culture supernatant, every milligram of total protein medium vascular endothelial growth factor content >=
300pg；
B, antigens c D10 is expressed, does not express antigens c D200.
Aforementioned fat stem cell, it is characterised in that: in the cell, when convergence degree is 80%, in culture supernatant, every milli
Gram total protein medium vascular endothelial growth factor content >=500pg.
The preparation method of cell above-mentioned, it is characterised in that: take fat mesenchymal stem cell, cultivate, detect cell confluency
When degree is 80%, every milligram of total protein medium vascular endothelial growth factor content in culture supernatant, while detecting the antigen of cell
The expression of CD10, CD200 select the cell with feature as claimed in claim 1 or 2.
Method above-mentioned, it is characterised in that: the culture medium is the platelet rich plasma for being 2%-5% containing volume fraction
The DMEM/F12 culture medium of PRP, condition of culture are 37 DEG C, 5%CO2；The platelet rich plasma PRP makes as follows
It is standby: to take blood, sodium citrate is added to 0.05~0.15mol/L, is placed in refrigerated centrifuge and 15min is centrifuged with 4000g, collect
Upper liquid, as platelet rich plasma；Preferably, sodium citrate is added to 0.1mol/L.
Method above-mentioned, it is characterised in that: the fat mesenchymal stem cell is prepared as follows:
(1) adipose tissue is taken, the collagen enzyme solution digestion of 0.05-1% (w/v) is carried out；
(2) postdigestive adipose tissue is taken, is centrifuged, obtains stromal vascular component；
(3) it by isolated vascular stroma component, is added after culture medium is resuspended and is cultivated, obtain fat stem cell；It is described
Culture medium is the DMEM/F12 culture medium for the platelet rich plasma PRP for being 2%-5% containing volume fraction, the rich platelet blood
Slurry PRP is prepared as follows: take blood, sodium citrate be added to 0.1mol/L, be placed in refrigerated centrifuge with 4000g from
Heart 15min collects upper liquid, as platelet rich plasma；
(4) fat stem cell originally culture to convergence degree 80% carries out had digestive transfer culture culture, and it is dry thin to obtain fat mesenchymal
Born of the same parents.
Method above-mentioned, it is characterised in that: in step (1), the digestion be by adipose tissue and digestive juice by volume
1:1 mixing is placed in revolving speed 50-250rpm in 37 DEG C of constant-temperature tables and digests 0.5-4h.
Method above-mentioned, it is characterised in that: in step (2), the centrifugation is 150-2000g centrifugation 5min.
Method above-mentioned, it is characterised in that: in step (3), the culture bottle is T75 culture bottle；The condition of the culture
Are as follows: 37 DEG C, 2-8%CO2, cultivate in saturated humidity incubator, change liquid after 48h for the first time, every 2d is changed the liquid once later.
Method above-mentioned, it is characterised in that: in step (3), passage amplification ratio is 1:4-1:5.
The present invention is by the screenings of Multiple Factors, in addition the restriction to VEGF secretion level, reduces fat stem cell
It is heterogeneous.
In addition, the present invention unexpectedly improves cell VEGE by the cell of the screening CD10 positive and CD200 feminine gender
Secretion level；And VEGF itself can promote angiogenesis, it is highly beneficial to tissue repair.The present invention is reducing fat stem cell
While heterogeneous, the repair ability of fat stem cell is also improved.
The present invention provides a kind of to have been widely used the fat stem cell of prospect in tissue repair.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is morphology of adipose-derived stem cells, 40 × magnification field, (10 times of eyepiece multiple, 4 times of object lens): A1, after inoculation 2 days
It can be seen that attached cell；A2, after adherent 4-5 days it is visible more how long shuttle sample fat stem cell；The fat of A3, convergence degree 80% or so are dry
Cell.
Fig. 2 is that fat stem cell three-dimensional differentiation potency is tried hard to.
Fig. 3 is amplification curve of the fat stem cell passage to P10 generation.
Fig. 4 is fat stem cell colony formation figure.
Fig. 5 is cells and supernatant total protein content figure.AM-MSC: amnion mesenchymal stem cell, N=6；CP-MSC: suede
Trichilemma mescenchymal stem cell, N=5；DP-SMC: decidua mescenchymal stem cell, N=5；UC-MSC: umbilical cord mesenchymal stem cells, N
=13；ASC: fat stem cell, N=22.
Fig. 6 for institute's factor-containing in cells and supernatant protein chip screening results.A:Human XL Cytokine
Array testing result is vascular endothelial growth factor VEGF in red circle；B:Human Adipokine Array testing result, it is red
It is HGF in frame 1, is VEGF in red frame 2, be Fetuin B in red frame 3, be IL-8 in red frame 4, is TIMP-3 in red frame 5.UC-
MSC: umbilical cord mesenchymal stem cells culture supernatant；ASC: fat stem cell culture supernatant；CP-MSC: chorion mescenchymal stem cell
Culture supernatant；AM-MSC: amnion mesenchymal stem cell supernatant.
Fig. 7 is VEGF content in different tissue sources MSC culture supernatant.AM-MSC: amnion mesenchymal stem cell, N=6；
CP-MSC: chorion mescenchymal stem cell, N=5；DP-SMC: decidua mescenchymal stem cell, N=5；UC-MSC: umbilical cord mesenchyma
Stem cell, N=10；ASC: fat stem cell, N=22.
Fig. 8 is the nude mice figure for transplanting Matrixgel, Matrixgel+ fat stem cell compound.
Fig. 9 is to be implanted into 7 days Matrixgel of nude mice.
Figure 10 is the Matrixgel+ fat stem cell compound for being implanted into nude mice 7 days.
Figure 11 is the slice colored graph for being implanted into 7 days Matrixgel of nude mice.
Figure 12 be implanted into nude mice 7 days Matrixgel+ fat stem cell compound slice colored graph.
Figure 13 is full cortex defect SD rat modeling figure.A, SD rat back shaving；B, left and right is each after skin degerming draws one
The circle of a diameter 2cm；The full thickness dermal of a diameter 2cm is respectively cut in C and D, left and right.
Figure 14 is that defect of skin repairs outside drawing.HASCs, human adipose-derived stem cell.
Figure 15 is defect of skin wound repairing healing rate statistical chart.HASCs, human adipose-derived stem cell.
Figure 16 is defect of skin wound repairing H&E colored graph.
Specific embodiment
It will further illustrate by way of examples below.
A kind of fat stem cell that tissue repairing ability is strong of embodiment is separately cultured and screens
One, the acquisition of fat mesenchymal stem cell
(1) separation method
1. cell obtains
The adipose tissue for taking liposuction to obtain is cleaned 3-5 times with injection stage physiological saline, it is preferable that raw using injection stage
Salt water is managed to clean 5 times；By the adipose tissue after cleaning, 1:1 is mixed by volume with digestive juice, is placed in 37 DEG C of constant-temperature table transfers
Fast 150rpm digests 2h.The digestive juice is the collagen enzyme solution of 0.2% (w/v).
2. obtaining vascular stroma component (SVF)
Fat tissue fragments postdigestive in step 1 are centrifuged 5-10min with 900g, lower sediment is taken to obtain SVF, wherein
Contain fat stem cell.
Culture is resuspended in 3.SVF
Vascular stroma component SVF after being centrifuged in step 2 is added in culture medium and is resuspended, with every 5mL liposuction product digestion
The density that obtained SVF is inoculated with 1 bottle of T75 culture bottle is inoculated in culture bottle, is placed in 37 DEG C, 5%CO2, saturated humidity incubator
Middle culture changes liquid after 48h for the first time, and every 2d is changed the liquid once later.The culture medium is DMEM/F12 culture medium, wherein containing body
Fraction is people's platelet rich plasma PRP of 2-5%.
The acquisition methods of platelet rich plasma PRP are as follows: take human body new blood that sodium citrate 0.1mol/L is added, by the blood
Liquid, which is placed in refrigerated centrifuge, is centrifuged 5-20min with 3000-10000g, and collecting upper liquid is platelet rich plasma.
(2) identification method
1. separating the fat stem cell energy adherent growth obtained, spindle shape is presented in cellular morphology, and effect is shown in Fig. 1.
2. the streaming of cell surface marker is identified.
When cell culture convergence degree reaches 80% or so, with the trypsin solution digestion containing 0.1%, centrifugation.It is thin with streaming
Born of the same parents' instrument evaluation and screening expresses CD73/CD90/CD105, and positive rate >=95% does not express CD14/CD34/CD45/CD79 α/HLA-
The cell (table 1) of DR, positive rate < 2%.
1 fat stem cell surface marker flow cytometer detection result of table
3. fat stem cell three-dimensional differentiation capability is identified
Obtained fat stem cell should also have skeletonization, at rouge and at the differentiation capability of cartilage.
Oil red O, alizarin red and safranin O dyeing are carried out to cell, there is the dyeing effect of similar Fig. 2.Conclusion: rouge has been obtained
Fat mescenchymal stem cell.
Two, it screens
(1) screening technique
1. the identification of fat stem cell CD10 and CD200
Streaming examines CD10 and CD200, and gained fat stem cell expresses antigens c D10, and positive rate >=95% is not expressed anti-
Former CD200, positive rate < 2%.
2. fat stem cell VEGF secretion capacity screens
When cultured cell line convergence degree reaches 80% or so, its supernatant is taken, VEGF content is detected by ELISA, often
VEGF content >=500pg in milligram total protein, corresponding cell is then required cell.
(2) it identifies
1. long-term proliferative capacity
Continuous passage has been carried out to three different biological samples, reach P10 generation cell-proliferation activity is not observed
It reduces (Fig. 3).Passage in every 3-4 days is primary, and per generation expands 6.4 ± 2.3 times.
2. fat stem cell clonality
HASCs is inoculated with by the every 100mm ware of 100 and 50 cells, after culture 14 days, respectively obtain 48.0 ±
7.7 and 28.0 ± 5.2 clones (Fig. 4), it was demonstrated that the hASCs that screening obtains has powerful clonality.
Technical solution of the present invention is described further in a manner of experimental example below.
The screening and identification of the specific cytokines of 1 fat stem cell of experimental example secretion
Before determining the specific cytokines that VEGF is fat stem cell secretion, this experimental example is for determining special secretion
The factor.
1. experimental method
(1) CD10 of convergence degree about 80% is collected+/CD200-Fat stem cell and people's amnion (AM) MSC, human chorionic
(CP) MSC, people's decidua (DP) MSC, people's umbilical cord (UC) MSC supernatant first measure P3 generation with Bio-Rad DC protein determination kit
Total protein content in cell conditioned medium.
(2) by two kinds of protein chips of R&D company, (Human XL Cytokine Array, detects 105 kinds of solubilities
Cell factor；Human Adipokine Array detects 58 kinds of Adipocyte Factors) to hASCs (fat stem cell)/UC-MSC (navel
Band mescenchymal stem cell) egg in/CP-MSC (chorion mescenchymal stem cell)/AM-MSC (amnion mesenchymal stem cell) supernatant
Bai Yinzi is detected, and the more important factor for selecting differential expression carries out ELISA detection, determine hASCs it is specifically expressed because
Son, as the standard screened later.
2. experimental result
(1) total protein content in supernatant
The supernatant total protein content of various cells is on close level, and is 2-3mg/mL (Fig. 5).
(2) in supernatant institute's factor-containing protein chip the selection result
As shown in fig. 6, compared with umbilical cord MSC, chorion MSC, amnion MSC culture supernatant, in human adipose-derived stem cell culture
Cell factor contained in clear only has VEGF (in Fig. 6 shown in red circle) relatively higher.And HGF, Fetuin B, IL-8, TIMP-3
The cytokine profiles such as (in Fig. 6 shown in red frame) and IL-6, IL-11, GDF-15, uPAR (not marking) are than umbilical cord MSC, chorion
MSC, amnion MSC secretion are low.
(3) VEGF content ELISA testing result in supernatant
As shown in fig. 7, VEGF content mescenchymal stem cell more tissue-derived than other in fat stem cell culture supernatant
It is much higher in supernatant, the amount of rear contained VEGF is standardized in supernatant with every milligram of total protein generally in 300pg or more,
Most of reachable 500pg or more, highest reachable 1785.23pg.
Then, the specific cytokines that final choice VEGF is secreted as fat stem cell, preferably every milligram in supernatant
Expression quantity >=500pg in total protein.
2 fat stem cell of experimental example subcutaneously fills experiment
1. method
P3 fat subsitutes stem cell after 100 μ L Matrixgel, 1,000,000 screenings is injected (according to implementation to experimental group nude mice
Example 1 method screening cell) and 100 μ L PBS mixture, nude mice of control group injection 100 μ L Matrixgel and 100 μ L
The mixture (Fig. 8) of PBS puts to death mouse after 7 days respectively, takes out mixture observation, and do H&E dyeing.
2. result
(1) there is calcification phenomenon (Fig. 9) in control group Matrixgel gel, and experimental group has no obvious calcification (Figure 10).
(2) it is sliced from H&E and dyes (the figure it is observed that fat stem cell of experimental group can survive in Matrixgel
11, Figure 12).
3 mouse full thickness dermal reparative experiment of experimental example
1. method
(1) mouse full thickness dermal model construction
12 week old cleaning grades or SPF grades of SD rats are selected, weight 200g or so raises one week adaptation ring after quarantine is qualified
Then border is raised 3 weeks using high lipid food.The injection streptozotocin STZ of 30mg/kg weight, injection are pressed from rat tail vein
It is latter week rat tail vein blood sampling with blood glucose meter can be detected rat blood sugar increase, using blood glucose be higher than 16.7mmol/L as modeling at
Function is eliminated not at mould rat.Blood sampling detects a blood glucose weekly later, confirms rat diabetes model stability.It is held after STZ injection
Continuous progress uses the successful Diabetes Mellitus SD Rats of the aforementioned modeling of chloral hydrate anesthesia after High fat diet 12 weeks, picks on Sterile surgery platform
Except rat back hair exposure operative site is mould on a rat back left side using the disk of diameter 2cm with iodophor disinfection operative site
The circle that right two sides are 2cm with two internal diameters of oiliness stroke.Rat back full thickness skin is cut i.e. along circle inside with operating scissors
Modeling full thickness skin damages successfully (Figure 13).
(2) injury repair
Defect of skin model is randomly divided into experimental group and control group.Multi-point injection around the surface of a wound of experimental mice is contained
Have 106100 μ L phosphate buffers of fat stem cell (cell screened according to the method for embodiment 1), control group after a screening
100 μ L phosphate buffer of multi-point injection around the surface of a wound of mouse observes its skin after 3,7,14,21 and 28 days after injection respectively
Healing effect, and the surface of a wound is taken to carry out slice H&E dyeing.
2. result
(1) skin healing gross examination of skeletal muscle
The skin healing speed of experimental group is higher than control group (Figure 14), and especially at first 7 days, Wound healing rate has significant poor
Different (Figure 15).
(2) surface of a wound coloration result
As shown in figure 16, PBS control group 28 regeneration for having not yet to see skin accessory organ after surgery；Fat stem cell injection group
It is initially observed a small amount of neoplastic skin accessory organ within 21 days after surgery, more newborn accessory organ was observed by postoperative 28 days.Figure
Middle circle frame is the accessory organ at neoplastic skin position.
The results show, the tool that the present invention screens, can be in nude mices and diabetes rat body there are two types of the cell of characteristic
Interior survival, and skin and its regeneration of accessory organ can be promoted well.Cell of the invention has stronger tissue repair
Ability has great application prospect in field of tissue engineering technology.
Claims (9)
1. a kind of fat stem cell that tissue repairing ability is strong, it is characterised in that: it is fat mesenchymal with the following characteristics
Stem cell:
A, when convergence degree is 80%, in culture supernatant, every milligram of total protein medium vascular endothelial growth factor content >=300pg；
B, antigens c D10 is expressed, does not express antigens c D200.
2. fat stem cell according to claim 1, it is characterised in that: in the cell, when convergence degree is 80%, in culture
In clear liquid, every milligram of total protein medium vascular endothelial growth factor content >=500pg.
3. the preparation method of cell of any of claims 1 or 2, it is characterised in that: fat mesenchymal stem cell is taken, is cultivated, inspection
When survey cell confluency degree is 80%, every milligram of total protein medium vascular endothelial growth factor content in culture supernatant detects simultaneously
The expression of antigens c D10, CD200 of cell selects the cell with feature as claimed in claim 1 or 2.
4. according to the method described in claim 3, it is characterized by: it is 2%-5%'s that the culture medium, which is containing volume fraction,
The DMEM/F12 culture medium of platelet rich plasma PRP, condition of culture are 37 DEG C, 5%CO2；The platelet rich plasma PRP according to
Following method preparation: taking blood, and sodium citrate is added to 0.05~0.15mol/L, is placed in refrigerated centrifuge and is centrifuged with 4000g
15min collects upper liquid, as platelet rich plasma；Preferably, sodium citrate is added to 0.1mol/L.
5. according to the method described in claim 4, it is characterized by: the fat mesenchymal stem cell is made as follows
It is standby:
(1) adipose tissue is taken, the collagen enzyme solution digestion of 0.05-1% (w/v) is carried out；
(2) postdigestive adipose tissue is taken, is centrifuged, obtains stromal vascular component；
(3) it by isolated vascular stroma component, is added after culture medium is resuspended and is cultivated, obtain fat stem cell；The culture
Base is the DMEM/F12 culture medium for the platelet rich plasma PRP for being 2%-5% containing volume fraction, the platelet rich plasma
PRP is prepared as follows: taking blood, sodium citrate is added to 0.1mol/L, is placed in refrigerated centrifuge and is centrifuged with 4000g
15min collects upper liquid, as platelet rich plasma；
(4) fat stem cell originally culture to convergence degree 80% carries out had digestive transfer culture culture, obtains fat mesenchymal stem cell.
6. according to the method described in claim 5, it is characterized by: the digestion is by adipose tissue and digestion in step (1)
1:1 is mixed liquid by volume, is placed in revolving speed 50-250rpm in 37 DEG C of constant-temperature tables and is digested 0.5-4h.
7. according to the method described in claim 5, it is characterized by: the centrifugation is 150-2000g centrifugation in step (2)
5min。
8. according to the method described in claim 5, it is characterized by: the culture bottle is T75 culture bottle in step (3)；It is described
The condition of culture are as follows: 37 DEG C, 2-8%CO2, cultivate in saturated humidity incubator, change liquid after 48h for the first time, every 2d changes liquid later
Once.
9. according to the method described in claim 3, it is characterized by: passage amplification ratio is 1:4-1:5 in step (3).
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| CN113521103A (en) * | 2021-07-27 | 2021-10-22 | 吴志新 | Application of mesenchymal stem cells in combination with PRP (platelet-rich plasma) in preparation of medicine for treating vulvar atrophy |
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Application publication date: 20181225 |