CN105441389B - Promote mesenchymal stem cell at the highly expressed method of osteanagenesis key gene - Google Patents
Promote mesenchymal stem cell at the highly expressed method of osteanagenesis key gene Download PDFInfo
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- CN105441389B CN105441389B CN201510934333.6A CN201510934333A CN105441389B CN 105441389 B CN105441389 B CN 105441389B CN 201510934333 A CN201510934333 A CN 201510934333A CN 105441389 B CN105441389 B CN 105441389B
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Abstract
The present invention relates to a kind of promotion mesenchymal stem cells into the highly expressed method of osteanagenesis key gene, and steps are as follows：The separation of mesenchymal stem cell：Sterile marrow is obtained, is resuspended and is precipitated with culture solution by pre-treatment, is counted, with 1 × 106/cm2It is seeded in culture bottle and cultivates, a-MEM+10%FBS and a-MEM+5% platelet cracking content+50mg/ml ascorbic acid+10nmol/L glucagon-like-peptide-1 is respectively adopted in culture solution.Bone defect is treated for bone tissue engineer as seed cell using the mesenchymal stem cell of cultivating system culture of the invention, the step of avoiding mesenchymal stem cell external osteogenic induction treats the time that bone defect shortens at least 14 days for clinical application bone tissue engineer.
Description
Technical field
The invention belongs to one of field of biotechnology to promote mesenchymal stem cell high at osteanagenesis key gene
The method of expression.
Background technique
Bone tissue defect is to make some sclerotin of body forfeiture to form larger gap because of factors such as wound, infection, tumours,
This problem is clinical relatively conventional and more intractable.The reparation for how promoting bone tissue defect is always the area research
The emphasis of personnel's research.In recent years, with the development of bone tissue engineer technology, this problem has obtained a degree of solution.
Need its due effect of competence exertion that be combined with each other of timbering material and seed cell in bone tissue engineer, and seed cell
Selection it is particularly important, early stage researcher selects osteocyte and marrow stromal cell, but these seed cells materials are difficult,
It is easy to cause secondary injury to patient.Therefore, researcher is by the target lock-on of seed cell in mescenchymal stem cell.Between fill
Matter stem cell has the characteristic of low immunogenicity, and the potential with Multidirectional Differentiation, can be divided into under certain conditions
The cell of the maturation such as osteocyte, lipoblast and chondroblast.Mesenchymal stem cell (BMSCs) is that mesenchyma is dry thin
One kind of born of the same parents, the potential with Multidirectional Differentiation, and can be into continuing to generate Osteoblastic activity after implanting.It determines in this course
The gene expression for determining the key transcription factor RUNX and Osterix of osteanagenesis is raised and the Typical Representative of osteanagenesis early stage
The factor-osteopontin gene (OPN) and alkaline phosphatase (ALP) can also express up-regulation, but give full play to medulla mesenchyma
The osteogenic ability of stem cell, it could be to osteoblast direction point in the case where needing the induction of osteogenic induction liquid in vitro first
Change, and cell induces this process at least 14 days time of needs.Therefore, how to shorten the osteogenic induction time or avoid skeletonization
The step for induction, aobvious is particularly important, the new cultivating system of one kind that the present invention uses, normal in mesenchymal stem cell
Using cultivating system of the invention when culture, mesenchymal stem cell determines skeletonization again while maintaining its original stemness
The gene expression of raw correlation factor is raised, and is treated bone defect for application bone tissue engineer and is provided necessary seed cell,
Bone defect, which is treated, for bone tissue engineer shortens the time.
Summary of the invention
The purpose of the present invention is to provide a kind of expression of promotion mesenchymal stem cell skeletonization regeneration associated genes to raise
Cultivating system, using cultivating system culture mesenchymal stem cell of the invention, mesenchymal stem cell is not only kept
Higher stemness, and promote in mesenchymal stem cell into osteanagenesis key transcription factor (Osterix, RUNX) and bone again
The gene high expression of raw the early stage Typical Representative factor (OPN, ALP).
The technical solution adopted by the present invention to solve the technical problems is：
A kind of promotion mesenchymal stem cell is at the highly expressed method of osteanagenesis key gene, and steps are as follows：
(1) the separation of mesenchymal stem cell：Sterile marrow is obtained, 1500rpm is centrifuged 5min under aseptic condition；It abandons
Supernatant is removed, using sterile phosphate buffer repeated flushing, 1500rpm is centrifuged 5min, abandons supernatant；The dilution of phosphate buffer equimultiple
Precipitating is mixed, is added slowly in lymph separating liquid, 2000rpm is centrifuged 20min；Tunica albuginea layer monocyte is collected in new sterile
Centrifuge tube in, phosphate buffer cleaning, 1500rpm be centrifuged 5min；Supernatant is abandoned, is resuspended and is precipitated with culture solution, is counted, with 1 ×
106/cm2Be seeded in culture bottle and cultivate, culture solution be respectively adopted a-MEM+10%FBS and a-MEM+5% platelet cracking content+
50mg/ml ascorbic acid+10nmol/L glucagon-like-peptide-1；
(2) the digestion, passage of mesenchymal stem cell, when mesenchymal stem cell it is long to 80% when, using 0.05%
The EDTA of trypsase+0.02% it is digested, with 1 × 104cells/cm2It reaches in new culture bottle.
Moreover, the third generation mesenchymal stem cell of two kinds of cultivating system cultures is taken to carry out streaming identification, two kinds of cultures
The mesenchymal stem cell positive rate of system culture is above 95%, and negative rate is below 5%；
Moreover, taking third generation mesenchymal stem cell, extracts RNA- reverse transcription and obtain cDNA- progress Q-PCR, compare two
The mesenchymal stem cell of kind of cultivating system culture at the relevant transcription factor Osterix of osteanagenesis and RUNX gene and bone again
The expression quantity of raw early stage Typical Representative factor gene OPN and ALP.
At the high expression system of osteanagenesis key gene, a-MEM+5% blood platelet is split for a kind of promotion mesenchymal stem cell
Solve object+50mg/ml ascorbic acid+10nmol/L glucagon-like-peptide-1.
The beneficial effects of the invention are as follows：
(1) the present invention is using a kind of new cultivating system, since mesenchymal stem cell is primary with the cultivating system into
Row culture, compared with traditional cultivating system, in the mesenchymal stem cell that the cultivating system that the present invention uses is turned out
Determine the gene of the regenerated key transcription factor of skeletonization (Osterix, RUNX) and the gene-of osteanagenesis early stage representative factor
Osteopontin (OPN) and the obvious high expression of alkaline phosphatase (ALP).
(2) not only medulla mesenchyma still maintains higher to application cultivating system culture mesenchymal stem cell of the invention
Stemness, and it can promote the regenerated key transcription factor of mesenchymal stem cell skeletonization (Osterix, RUNX) and bone
The gene high expression of the early stage Typical Representative factor (OPN, ALP) is regenerated, this treats bone defect for clinical application bone tissue engineer and mentions
Ideal seed cell is supplied.
(3) bone tissue work is used for as seed cell using the mesenchymal stem cell of cultivating system culture of the invention
The step of journey treats bone defect, avoids mesenchymal stem cell external osteogenic induction, controls for clinical application bone tissue engineer
Treat the time that bone defect shortens at least 14 days.
Detailed description of the invention
Fig. 1 is the mRNA expression (relative intensity of fluorescence) of Osterix；
Fig. 2 is the mRNA expression (relative intensity of fluorescence) of RUNX；
Fig. 3 is the mRNA expression (relative intensity of fluorescence) of OPN；
Fig. 4 is the mRNA expression (relative intensity of fluorescence) of ALP；
Specific embodiment
The invention will be further described with reference to the accompanying drawing and by specific embodiment, and following embodiment is descriptive
, it is not restrictive, this does not limit the scope of protection of the present invention.
A kind of promotion mesenchymal stem cell is at the highly expressed method of osteanagenesis key gene, and steps are as follows：
(1) the separation of mesenchymal stem cell：Sterile marrow is obtained, 1500rpm is centrifuged 5min under aseptic condition；It abandons
Supernatant is removed, using sterile phosphate buffer repeated flushing, 1500rpm is centrifuged 5min, abandons supernatant；The dilution of phosphate buffer equimultiple
Precipitating is mixed, is added slowly in lymph separating liquid, 2000rpm is centrifuged 20min；Tunica albuginea layer monocyte is collected in new sterile
Centrifuge tube in, phosphate buffer cleaning, 1500rpm be centrifuged 5min；Supernatant is abandoned, is resuspended and is precipitated with culture solution, is counted, with 1 ×
106/cm2Be seeded in culture bottle and cultivate, culture solution be respectively adopted a-MEM+10%FBS and a-MEM+5% platelet cracking content+
50mg/ml ascorbic acid+10nmol/L glucagon-like-peptide-1；
(2) the digestion, passage of mesenchymal stem cell, when mesenchymal stem cell it is long to 80% when, using 0.05%
The EDTA of trypsase+0.02% it is digested, with 1 × 104cells/cm2It reaches in new culture bottle；
(3) the third generation mesenchymal stem cell of two kinds of cultivating system cultures is taken to carry out streaming identification, two kinds of cultivating systems
The mesenchymal stem cell positive rate of culture is above 95%, and negative rate is below 5%；
(4) third generation mesenchymal stem cell is taken, RNA- reverse transcription is extracted and obtains cDNA- progress Q-PCR, compare two kinds
The mesenchymal stem cell of cultivating system culture at the relevant transcription factor of osteanagenesis (Osterix, RUNX) gene and bone again
The expression quantity of raw early stage Typical Representative factor gene (OPN, ALP).
Claims (2)
1. at the highly expressed method of osteanagenesis transcription factor in a kind of promotion mesenchymal stem cell, it is characterised in that：Step
It is as follows：
(1) the separation of mesenchymal stem cell：Sterile marrow is obtained, 1500rpm is centrifuged 5min under aseptic condition；It discards
Clear liquid, using sterile phosphate buffer repeated flushing, 1500rpm is centrifuged 5min, abandons supernatant；The dilution of phosphate buffer equimultiple mixes
Precipitating, is added slowly in lymph separating liquid, and 2000rpm is centrifuged 20min；Collect tunica albuginea layer monocyte in it is new it is sterile from
In heart pipe, phosphate buffer cleaning, 1500rpm is centrifuged 5min；Supernatant is abandoned, is resuspended and is precipitated with culture solution, is counted, with 1 × 106/
cm2Be seeded in culture bottle and cultivate, culture solution be respectively adopted a-MEM+10%FBS and a-MEM+5% platelet cracking content+
50mg/ml ascorbic acid+10nmol/L glucagon-like-peptide-1；
(2) the digestion, passage of mesenchymal stem cell, when mesenchymal stem cell it is long to 80% when, using 0.05% pancreas
The EDTA of protease+0.02% digests it, with 1 × 104cells/cm2It reaches in new culture bottle；
Moreover, the third generation mesenchymal stem cell of two kinds of cultivating system cultures is taken to carry out streaming identification, two kinds of cultivating systems
The mesenchymal stem cell positive rate of culture is above 95%, and negative rate is below 5%；
Moreover, taking third generation mesenchymal stem cell, extracts RNA- reverse transcription and obtain cDNA- progress Q-PCR, compare two kinds of trainings
The mesenchymal stem cell for supporting system culture is early at the relevant transcription factor Osterix of osteanagenesis and RUNX gene and osteanagenesis
The expression quantity of phase Typical Representative factor gene OPN and ALP.
2. at the high expression system of osteanagenesis transcription factor in a kind of promotion mesenchymal stem cell, it is characterised in that：a-MEM+
5% platelet cracking content+50mg/ml ascorbic acid+10nmol/L glucagon-like-peptide-1.
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| CN106701671A (en) * | 2016-11-14 | 2017-05-24 | 天津市康婷生物工程有限公司 | Culture system beneficial for mesenchymal stem cells to be applied in bone regeneration |
| CN111534487B (en) * | 2020-06-22 | 2021-05-14 | 孙德强 | Promoter for promoting osteogenic differentiation of mesenchymal stem cells |
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Non-Patent Citations (3)
| Title |
|---|
| GLP-1受体激动剂促进大鼠骨髓间充质干细胞的增殖与成骨分化;王宁等;《中国药学大会暨第十三届中国药师周论文集》;20140609;1-11 * |
| 抗坏血酸、B．甘油磷酸钠和地塞米松联合诱导大鼠骨髓间充质干细胞向成骨细胞的分化;廖庆辉等;《中国组织工程研究与临床康复》;20090101;88-91 * |
| 血小板裂解液对人脐带间充质干细胞体外成骨分化的影响;吕江涛等;《中国组织工程研究》;20121007;7637-7641 * |
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Address after: No.9 Saida South Road, Xiqing Economic and Technological Development Zone, Tianjin 300200 Patentee after: Tianjin Kangting Bioengineering Group Co., Ltd. Address before: No. 126 Youyi South Road, Saida Pharmaceutical Industrial Park, Xiqing Economic and Technological Development Zone, Xiqing District, Tianjin 300200 Patentee before: Tianjin Kangting Biotechnology Co., Ltd. |