CN105779382A - Culture medium and method for inducing adipose-derived stem cells to differentiate into tendon cells - Google Patents
Culture medium and method for inducing adipose-derived stem cells to differentiate into tendon cells Download PDFInfo
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- CN105779382A CN105779382A CN201610338965.0A CN201610338965A CN105779382A CN 105779382 A CN105779382 A CN 105779382A CN 201610338965 A CN201610338965 A CN 201610338965A CN 105779382 A CN105779382 A CN 105779382A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/066—Tenocytes; Tendons, Ligaments
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/76—Undefined extracts from plants
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/19—Growth and differentiation factors [GDF]
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
- C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
- C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells
Abstract
The invention relates to the technical field of cell culture, and in particular relates to a culture medium and a method for inducing adipose-derived stem cells to differentiate into tendon cells. The culture medium comprises GDF-7, prepared rehmannia root aqueous extract and a serum-free culture medium. The culture medium provided by the invention can remarkably improve the ability of differentiating the adipose-derived stem cells into tendon cells; the prepared rehmannia root aqueous extract is utilized to culture the adipose-derived stem cells, so that the phenotype and the characteristics of the adipose-derived stem cells further can be kept while cell proliferation is promoted; a growth differentiating factor GDF-7 is used as an inductor, so that the effect is single, and the complex components of blood plasma are greatly reduced, and therefore, the adipose-derived stem cells are differentiated into the tendon cells in a directional manner, and the risk and the immunogenicity of animal serum application can be reduced.
Description
Technical field
The present invention relates to technical field of cell culture, be divided into flesh particularly to a kind of induced lipolysis stem cell
The culture medium of tendon cell and method thereof.
Background technology
Injury of muscle and tendon (muscle and tendon, injury of) is common motor system damage.
Rupture or portion fractures completely in muscle or the belly of muscle portion of dividing a word with a hyphen at the end of a line that direct violence causes, the myorrhexis of meaning.Outward
The muscle that power causes contraction the most with sudden force, can cause the tear wholly or in part of tendon terminal, call it
Tendon rupture.If the prolonged and repeated microtrauma that stands of tendon, or tendon itself has chronic abrasion, causes tendon
Fibre modification, attenuates, and the most slightly sprains and can cause tendon rupture, the spontaneous rupture of tendon of this meaning.
There is a great strain on the muscles, muscle acute sprain malpractice or bad attitude and the muscle balance caused by deformity are lost
Adjust, the chronic muscular strain of meaning.The general symptom of injury of muscle and tendon is local pain, swelling, tenderness,
Occasionally there is subcutaneous hemorrhage.If muscle or tendon rupture, then the function at this will weaken or lose.
At present, tendon rupture, damage or the most idealless Therapeutic Method of disease of the motor system such as tear,
The method of clinical treatment tendon injury includes directly sewing up, autologous or heteroplastic transplantation, synthetic material
Repair.But poor due to self healing ability interior after Xiu Fuing, easily occur that calcification, tendon slide
Limited；Autologous or heteroplastic transplantation may cause tendon again to rupture, transplant rejection and ethical issues.
Fat stem cell (ADSC) is that zuk in 2002 et al. separates in fatty tissue, and it comes
Source is enriched, convenience of drawing materials, cell amount to obtain are big, is the mescenchymal stem cell with multi-lineage potential,
Can be described as one of organizational project preferable seed cell source.Having research worker to utilize goes the tendon of cell to make
For support, add fat stem cell and form complex, successfully repair in mouse Tendon Defection model
The tendon of defect.Also there is research using the co-glycolic acid of nanostructured as skeleton, load fat dry
Cell and PDGF, for the reparation of tendon serious defect.
It is applied to Tendon Tissue Engineering as seed cell, it is necessary first to sufficient amount of using fat stem cell
Cell and by its field planting on biological support.The Tenocyte cell of In vitro culture adds blood plasma or plasma clot,
Tenocyte cell propagation and secretion collagen can be stimulated；But plasma fraction is complicated, the fat cultivated in vitro is dry thin
Born of the same parents add platelet rich plasma be difficult to control and induce its orientation Tenocyte cell differentiation.And existing induction
In cultivating system, to be divided into the ability of Tenocyte cell more weak for fat stem cell.Therefore, a set of not containing is set up
Animal serum, may use clinic, safety and definite effect fat stem cell and is divided into Tenocyte cell
Method for inducing and cultivating, have important practical significance.
Summary of the invention
In view of this, the invention provides a kind of induced lipolysis stem cell and be divided into the culture medium of Tenocyte cell
And method.This culture medium is remarkably improved fat stem cell and is divided into the ability of Tenocyte cell.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides a kind of induced lipolysis stem cell and be divided into the culture medium of Tenocyte cell, including
GDF-7, Radix Rehmanniae Preparata water extract and serum-free medium.
The cultivating system that GDF-7, Radix Rehmanniae Preparata water extract combine with serum-free medium can be shown by the present invention
Write raising fat stem cell and be divided into the ability of Tenocyte cell.
As preferably, in culture medium, each amounts of components is:
GDF-7:15～40ng/mL；
Radix Rehmanniae Preparata water extract: 0.1～1mg/mL；
Serum-free medium: supply.
In some embodiments that the present invention provides, in culture medium, each amounts of components is:
GDF-7:15ng/mL；
Radix Rehmanniae Preparata water extract: 0.1mg/mL；
Serum-free medium: supply.
In other embodiments that the present invention provides, in culture medium, each amounts of components is:
GDF-7:30ng/mL；
Radix Rehmanniae Preparata water extract: 0.5mg/mL；
Serum-free medium: supply.
In other embodiments that the present invention provides, in culture medium, each amounts of components is:
GDF-7:40ng/mL；
Radix Rehmanniae Preparata water extract: 1mg/mL；
Serum-free medium: supply.
As preferably, serum-free medium is DMEM/F12 culture medium.
In the present invention, the preparation method of Radix Rehmanniae Preparata water extract is: take Radix Rehmanniae Preparata, the long-pending and Radix Rehmanniae Preparata xanthin by liquid
Amount ratio 6:1 soak by water, filtering and concentrating is to containing crude drug 1g/mL.
Present invention also offers a kind of method that induced lipolysis stem cell is divided into Tenocyte cell, use right
Require culture medium inducing culture fat stem cell any one of 1 to 6, it is thus achieved that Tenocyte cell.
As preferably, fat stem cell is the 3rd generation～the 5th fat subsitutes stem cell.
As preferably, the condition of inducing culture is 37 DEG C, 5%CO2。
As preferably, the time of inducing culture is 15～20 days, within every 3 days, changes a not good liquor.
The invention provides a kind of induced lipolysis stem cell and be divided into culture medium and the method thereof of Tenocyte cell.
This culture medium includes GDF-7, Radix Rehmanniae Preparata water extract and serum-free medium.The present invention at least has the most excellent
One of gesture:
1, this culture medium is remarkably improved fat stem cell and is divided into the ability of Tenocyte cell；
2, the present invention utilizes Radix Rehmanniae Preparata water extract to cultivate fat stem cell, while promoting cell proliferation,
Also can maintain phenotype and the characteristic of fat stem cell；Utilize growth and differentiation factor GDF-7 as inducer, make
With single, greatly reduce the complicated ingredient of blood plasma so that fat stem cell directed differentiation is Tenocyte cell；
3, culture medium of the present invention can reduce risk and the immunogenicity of animal serum application.
Accompanying drawing explanation
Fig. 1 shows P3 fat subsitutes stem cell morphology figure, and wherein 1-1 is the cellular morphology figure of amplification 40 times, and 1-2 is
Amplify the cellular morphology figure of 100 times；
Fig. 2 shows the streaming qualification result of fat stem cell, and wherein Fig. 2-1,2-2,2-3 are matched group streaming mirror
Determining result, 2-1 shows the expression that blank, 2-2 show CD73, HLA-DR, and 2-3 shows CD90, CD45
Expression；Fig. 2-4,2-5,2-6 are sample sets streaming qualification result, and 2-4 shows blank, 2-5
Showing the expression of CD73, HLA-DR, 2-6 shows the expression of CD90, CD45；
Fig. 3 shows the expression change of beta-actin (internal reference), type i collagen and tenomod μ Lin albumen, its
In, 3-1 shows the westernblotting testing result of beta-actin, and 3-2 shows I-type collagen
Westernblotting testing result, 3-3 shows the westernblotting testing result of tenomod μ Lin albumen；
In Fig. 3-1,3-2 and 3-3 swimming lane 1～6 respectively represent induction before, matched group 1, matched group 2, experimental group 1,
The protein expression situation of experimental group 2 and experimental group 3.
Detailed description of the invention
The invention discloses a kind of induced lipolysis stem cell and be divided into culture medium and the method thereof of Tenocyte cell,
Those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes.Of particular note
, all similar replacements and change apparent to those skilled in the art, they are all
It is deemed to be included in the present invention.Method and the application of the present invention are described by preferred embodiment,
Related personnel substantially can in without departing from present invention, spirit and scope to method described herein and should
With being modified or suitably changing and combine, realize and apply the technology of the present invention.
The induced lipolysis stem cell that the present invention provides is divided in the culture medium of Tenocyte cell and method thereof used
Biomaterial or reagent all can be buied by market.
In the present invention, the preparation method of Radix Rehmanniae Preparata water extract is: take Radix Rehmanniae Preparata, the long-pending and Radix Rehmanniae Preparata xanthin by liquid
Amount ratio 6:1 soak by water, filtering and concentrating is to containing crude drug 1g/mL, and 4 DEG C of Refrigerator stores are standby.
Below in conjunction with embodiment, the present invention it is expanded on further:
Embodiment 1
(1) the primary separation of fat stem cell
Treat fatty tissue and Tumescent fluid natural layering Hou Xiqi lower floor liquid, add isopyknic normal saline
Clean fatty tissue 2 times, inhale and abandon lower section solution；By in fatty tissue subpackage to 50mL centrifuge tube, often manage
20mL, often adds isopyknic 0.5% NTx enzyme (final concentration of 0.25%), fully mixes in pipe,
Sealing, be transferred in Tempeerature-constant air shaking table, 37 DEG C, 200R digest 50min；It is centrifuged after having digested,
1500rpm is centrifuged 10min；Discarding the fat after being centrifuged and fatty oil, often pipe adds 40mL normal saline weight
Outstanding cell, 11500rpm is centrifuged 5min, and supernatant discarded, with ordinary culture medium (DMEM/F12+10%FBS)
Re-suspended cell, and be seeded in culture dish, when cell confluency degree reaches 80%-90%, carry out passing on place
Reason.Fig. 1 is shown in by fat stem cell form picture.
(2) qualification of human adipose-derived stem cell (ADSCs) surface markers
Take the logarithm the cell in trophophase the 3rd generation, after sucking-off culture medium, add 0.25% trypsin
The Digestive system of+0.02%EDTA digests, and terminates digestion 1000rpm with appropriate serum afterwards and is centrifuged
10min, abandons supernatant, after meeting cold PBS washed cell 2 times with 4 DEG C resuspended uniformly, adjust cell concentration about
It is 105-106Individual/mL.Taking 2 loading pipes, often pipe adds the single cell suspension of 500 μ L, centrifugal rear No. 1 pipe
It is designated as standard control, No. 2 each addition 2 μ L FITC or the mouse anti human cell surface molecule CD59 of PE labelling,
CD45, CD34, CD105 antibody working solution.Room temperature, lucifuge, hatch 20min；PBS washes twice,
To remove unconjugated antibody, after 500 μ L 1640 culture medium are resuspended, flow cytometer identifies surface markers.
Streaming identifies that Fig. 2 is shown in by picture.
Understand as shown in Figure 2, ADSCs CD73, CD90 positive expression in the 3rd generation of amplification, and
HLA-DR, CD45 are negative expression, meet the characteristic of stem cell, may be used for follow-up induction differentiation real
Test.
(3) fat stem cell is to the inducing culture of Tenocyte cell
1, experiment packet
Test packet situation is shown in Table 1:
Table 1 test packet
| Group | Culture medium forms |
| Matched group 1 | DMEM/F12+10%FBS |
| Matched group 2 | Commercialization tendon inducing culture is basis set |
| Experimental group 1 | DMEM/F12+15ng/mL GDF-7 |
| Experimental group 2 | DMEM/F12+0.1mg/mL Radix Rehmanniae Preparata water extract |
| Experimental group 3 | DMEM/F12+15ng/mL GDF-7+0.1mg/mL Radix Rehmanniae Preparata water extract |
2, GDF-7 and Radix Rehmanniae Preparata water extract common induced lipolysis stem cell are to Tenocyte cell differentiation and proliferation
Take the ADSCs in the 3rd generation, after sucking-off culture medium, add 0.25% trypsin+0.02%EDTA's
Digestive system digests, and terminates digestion with appropriate serum afterwards.1200rpm is centrifuged 5min, fat stem cell
Complete medium is resuspended, and adjusting density is 1 × 104/ mL, is inoculated in 6 orifice plates, every hole 2mL.
(1) quantitative fluorescent PCR
When cell reaches 40%-50% fusion, culture medium shown in application experiment packet, lure under same environment
Lead cultivation, within every 3 days, change inducing culture, cultivate 15 days rear section cells for extracting RNA, reverse transcription
Become cDNA, with this cDNA as template, with forward primer: CTGGTACGGCGAGAGCAT, under
Trip primer: CAGGCTCCGGTGTGACTC (type i collagen)；With forward primer:
AAGACTCTCCCCTTAATG, downstream primer: CAAACAAATCACTGCCAC
(tenomoduLin) being that primer carries out immunofluorescence quantitative PCR, wherein beta-actin is as internal reference, inspection
Survey Tenocyte cell marker protein type i collagen, the gene expression amount of tenomoduLin.Finally according to the C obtained
T () value, in the front cell of induction, the expression of gene is as reference, genes of interest in cell after calculating induction
Relative to the expression before induction.Quantitative fluorescent PCR analysis result is as follows:
Table 2 quantitative fluorescent PCR analysis result
| Group | Type i collagen relative expression quantity | TenomoduLin relative expression quantity |
| Before induction | 1 | 1 |
| Matched group 1 | 1.0 | 1.1 |
| Matched group 2 | 7.2** | 3.6** |
| Experimental group 1 | 7.8** | 3.8** |
| Experimental group 2 | 6.9** | 3.4** |
| Experimental group 3 | 15.2** | 8.6** |
Wherein: *: compare with before induction, p < 0.05；*: compare with before induction, p < 0.01
From result of the test in table 2, matched group 1 compares type i collagen and tenomoduLin gene before induction
Express without significant change, and the type i collagen of cell in matched group 2, experimental group 1, experimental group 2 and experimental group 3
All raise with tenomoduLin gene expression amount, illustrate that fat stem cell success is broken up to Tenocyte cell；And I
The order that Collagen Type VI and tenomoduLin gene expression amount raise is respectively as follows: experimental group 3, experimental group 1, right
According to group 2, experimental group 2, illustrate and commodity induction liquid phase compares, addition GDF-7 and the luring of Radix Rehmanniae Preparata water extract
Drain is more easy to make cell induction break up, but the induction differentiation capability being individually added into Radix Rehmanniae Preparata water extract is more weak,
Both are used in combination and are both greatly improved induction differentiation capability, replace again animal serum, decrease its application
Risk and immunogenicity.
(2)westernblotting
Cultivate the extraction that rear section cell is used for total protein 15 days, and use westernblotting experiment detection I
The expression change of Collagen Type VI and tenomoduLin albumen, using beta-actin as internal reference.
Westernblotting experimental result is shown in Fig. 3.
From westernblotting experimental result, beta-actin is as the protein expression of each sample of internal reference
Amount is just as, it is ensured that the accuracy of experiment；Type i collagen before induction and TenomoduLin are almost
Do not express；The expressing quantity of matched group 1 is lower than other several groups, the albumen table of matched group 2 and experimental group 2
The amount of reaching is similar, but experimental group 3 is higher than other expressing quantity of several groups, illustrates to add in culture medium
Enter growth and differentiation factor GDF-7 as inducer, act on single, greatly reduce the complicated ingredient of blood plasma,
Orientation Tenocyte cell differentiation, consistent with fluorescence quantitative PCR detection result.
Embodiment 2
1, experiment packet
Test packet situation is shown in Table 3:
Table 3 test packet
| Group | Culture medium forms |
| Matched group 1 | DMEM/F12+10%FBS |
| Matched group 2 | Commercialization tendon inducing culture is basis set |
| Experimental group 1 | DMEM/F12+30ng/mL GDF-7 |
| Experimental group 2 | DMEM/F12+0.5mg/mL Radix Rehmanniae Preparata water extract |
| Experimental group 3 | DMEM/F12+30ng/mL GDF-7+0.5mg/mL Radix Rehmanniae Preparata water extract |
2, GDF-7 and Radix Rehmanniae Preparata water extract common induced lipolysis stem cell are to Tenocyte cell differentiation and proliferation
Take the ADSCs in the 4th generation, after sucking-off culture medium, add 0.25% trypsin+0.02%EDTA's
Digestive system digests, and terminates digestion with appropriate serum afterwards.1200rpm is centrifuged 5min, fat stem cell
Complete medium is resuspended, and adjusting density is 1 × 104/ mL, is inoculated in 6 orifice plates, every hole 2mL.
(1) quantitative fluorescent PCR
When cell reaches 40%-50% fusion, culture medium shown in application experiment packet, lure under same environment
Lead cultivation, within every 3 days, change inducing culture, cultivate 15 days rear section cells for extracting RNA, reverse transcription
Become cDNA, with this cDNA as template, with forward primer: CTGGTACGGCGAGAGCAT, under
Trip primer: CAGGCTCCGGTGTGACTC (type i collagen)；With forward primer:
AAGACTCTCCCCTTAATG, downstream primer: CAAACAAATCACTGCCAC
(tenomoduLin) being that primer carries out immunofluorescence quantitative PCR, wherein beta-actin is as internal reference, inspection
Survey Tenocyte cell marker protein type i collagen, the gene expression amount of tenomoduLin.Finally according to the C obtained
T () value, in the front cell of induction, the expression of gene is as reference, genes of interest in cell after calculating induction
Relative to the expression before induction.Quantitative fluorescent PCR analysis result is as follows:
Table 4 quantitative fluorescent PCR analysis result
Wherein: *: compare with before induction, p < 0.05；*: compare with before induction, p < 0.01
From result of the test in table 4, matched group 1 compares type i collagen and tenomoduLin gene before induction
Express without significant change, and the type i collagen of cell in matched group 2, experimental group 1, experimental group 2 and experimental group 3
All raise with tenomoduLin gene expression amount, illustrate that fat stem cell success is broken up to Tenocyte cell；And I
The order that Collagen Type VI and tenomoduLin gene expression amount raise is respectively as follows: experimental group 3, experimental group 1, right
According to group 2, experimental group 2, illustrate and commodity induction liquid phase compares, addition GDF-7 and the luring of Radix Rehmanniae Preparata water extract
Drain is more easy to make cell induction break up, but the induction differentiation capability being individually added into Radix Rehmanniae Preparata water extract is more weak,
Both are used in combination and are both greatly improved induction differentiation capability, replace again animal serum, decrease its application
Risk and immunogenicity.
(2)westernblotting
Cultivate the extraction that rear section cell is used for total protein 15 days, and use westernblotting experiment detection I
The expression change of Collagen Type VI and tenomoduLin albumen, using beta-actin as internal reference.
Westernblotting experimental result is close with Fig. 3.
From westernblotting experimental result, beta-actin is as the protein expression of each sample of internal reference
Amount is just as, it is ensured that the accuracy of experiment；Type i collagen before induction and TenomoduLin are almost
Do not express；The expressing quantity of matched group 1 is lower than other several groups, the albumen table of matched group 2 and experimental group 2
The amount of reaching is similar, but experimental group 3 is higher than other expressing quantity of several groups, illustrates to add in culture medium
Enter growth and differentiation factor GDF-7 as inducer, act on single, greatly reduce the complicated ingredient of blood plasma,
Orientation Tenocyte cell differentiation, consistent with fluorescence quantitative PCR detection result.
Embodiment 3
1, experiment packet
Test packet situation is shown in Table 5:
Table 5 test packet
| Group | Culture medium forms |
| Matched group 1 | DMEM/F12+10%FBS |
| Matched group 2 | Commercialization tendon inducing culture is basis set |
| Experimental group 1 | DMEM/F12+40ng/mL GDF-7 |
| Experimental group 2 | DMEM/F12+1mg/mL Radix Rehmanniae Preparata water extract |
| Experimental group 3 | DMEM/F12+40ng/mL GDF-7+1mg/mL Radix Rehmanniae Preparata water extract |
2, GDF-7 and Radix Rehmanniae Preparata water extract common induced lipolysis stem cell are to Tenocyte cell differentiation and proliferation
Take the ADSCs in the 5th generation, after sucking-off culture medium, add 0.25% trypsin+0.02%EDTA's
Digestive system digests, and terminates digestion with appropriate serum afterwards.1200rpm is centrifuged 5min, fat stem cell
Complete medium is resuspended, and adjusting density is 1 × 104/ mL, is inoculated in 6 orifice plates, every hole 2mL.
(1) quantitative fluorescent PCR
When cell reaches 40%-50% fusion, culture medium shown in application experiment packet, lure under same environment
Lead cultivation, within every 3 days, change inducing culture, cultivate 15 days rear section cells for extracting RNA, reverse transcription
Become cDNA, with this cDNA as template, with forward primer: CTGGTACGGCGAGAGCAT, under
Trip primer: CAGGCTCCGGTGTGACTC (type i collagen)；With forward primer:
AAGACTCTCCCCTTAATG, downstream primer: CAAACAAATCACTGCCAC
(tenomoduLin) being that primer carries out immunofluorescence quantitative PCR, wherein beta-actin is as internal reference, inspection
Survey Tenocyte cell marker protein type i collagen, the gene expression amount of tenomoduLin.Finally according to the C obtained
T () value, in the front cell of induction, the expression of gene is as reference, genes of interest in cell after calculating induction
Relative to the expression before induction.Quantitative fluorescent PCR analysis result is as follows:
Table 6 quantitative fluorescent PCR analysis result
Wherein: *: compare with before induction, p < 0.05；*: compare with before induction, p < 0.01
From result of the test in table 6, matched group 1 compares type i collagen and tenomoduLin gene before induction
Express without significant change, and the type i collagen of cell in matched group 2, experimental group 1, experimental group 2 and experimental group 3
All raise with tenomoduLin gene expression amount, illustrate that fat stem cell success is broken up to Tenocyte cell；And I
The order that Collagen Type VI and tenomoduLin gene expression amount raise is respectively as follows: experimental group 3, experimental group 1, right
According to group 2, experimental group 2, illustrate and commodity induction liquid phase compares, addition GDF-7 and the luring of Radix Rehmanniae Preparata water extract
Drain is more easy to make cell induction break up, but the induction differentiation capability being individually added into Radix Rehmanniae Preparata water extract is more weak,
Both are used in combination and are both greatly improved induction differentiation capability, replace again animal serum, decrease its application
Risk and immunogenicity.
(2)westernblotting
Cultivate the extraction that rear section cell is used for total protein 15 days, and use westernblotting experiment detection I
The expression change of Collagen Type VI and tenomoduLin albumen, using beta-actin as internal reference.
Westernblotting experimental result is close with Fig. 3.
From westernblotting experimental result, beta-actin is as the protein expression of each sample of internal reference
Amount is just as, it is ensured that the accuracy of experiment；Type i collagen before induction and TenomoduLin are almost
Do not express；The expressing quantity of matched group 1 is lower than other several groups, the albumen table of matched group 2 and experimental group 2
The amount of reaching is similar, but experimental group 3 is higher than other expressing quantity of several groups, illustrates to add in culture medium
Enter growth and differentiation factor GDF-7 as inducer, act on single, greatly reduce the complicated ingredient of blood plasma,
Orientation Tenocyte cell differentiation, consistent with fluorescence quantitative PCR detection result.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. an induced lipolysis stem cell is divided into the culture medium of Tenocyte cell, it is characterised in that include
GDF-7, Radix Rehmanniae Preparata water extract and serum-free medium.
Culture medium the most according to claim 1, it is characterised in that in described culture medium, each component is used
Amount is:
GDF-7:15～40ng/mL；
Radix Rehmanniae Preparata water extract: 0.1～1mg/mL；
Serum-free medium: supply.
Culture medium the most according to claim 2, it is characterised in that in described culture medium, each component is used
Amount is:
GDF-7:15ng/mL；
Radix Rehmanniae Preparata water extract: 0.1mg/mL；
Serum-free medium: supply.
Culture medium the most according to claim 2, it is characterised in that in described culture medium, each component is used
Amount is:
GDF-7:30ng/mL；
Radix Rehmanniae Preparata water extract: 0.5mg/mL；
Serum-free medium: supply.
Culture medium the most according to claim 2, it is characterised in that in described culture medium, each component is used
Amount is:
GDF-7:40ng/mL；
Radix Rehmanniae Preparata water extract: 1mg/mL；
Serum-free medium: supply.
Culture medium the most according to claim 1, it is characterised in that described serum-free medium is
DMEM/F12 culture medium.
7. the method that an induced lipolysis stem cell is divided into Tenocyte cell, it is characterised in that use right
Require culture medium inducing culture fat stem cell according to any one of 1 to 6, it is thus achieved that Tenocyte cell.
Method the most according to claim 7, it is characterised in that described fat stem cell was the 3rd generation
～the 5th fat subsitutes stem cell.
Method the most according to claim 7, it is characterised in that the condition of described inducing culture is 37 DEG C,
5%CO2。
Method the most according to claim 7, it is characterised in that the time of described inducing culture is
15～20 days, within every 3 days, change a not good liquor.
Priority Applications (1)
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| CN104805051A (en) * | 2015-02-10 | 2015-07-29 | 广州赛莱拉干细胞科技股份有限公司 | Method used for inducing differentiation of adipose-derived stem cells into fibroblast |
| JP2018123130A (en) * | 2017-02-01 | 2018-08-09 | ロート製薬株式会社 | Cosmetic composition and mitochondrial transfer promoting agent |
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| CN104805051A (en) * | 2015-02-10 | 2015-07-29 | 广州赛莱拉干细胞科技股份有限公司 | Method used for inducing differentiation of adipose-derived stem cells into fibroblast |
| JP2018123130A (en) * | 2017-02-01 | 2018-08-09 | ロート製薬株式会社 | Cosmetic composition and mitochondrial transfer promoting agent |
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| 倪明等: "生长分化因子7体外促进BMSCs向肌腱细胞分化的研究", 《中国修复重建外科杂志》 * |
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