CN105441388A - Method for promoting high expression of umbilical cord mesenchymal stem cell ossification regeneration key gene - Google Patents
Method for promoting high expression of umbilical cord mesenchymal stem cell ossification regeneration key gene Download PDFInfo
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- CN105441388A CN105441388A CN201510933962.7A CN201510933962A CN105441388A CN 105441388 A CN105441388 A CN 105441388A CN 201510933962 A CN201510933962 A CN 201510933962A CN 105441388 A CN105441388 A CN 105441388A
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Abstract
The invention relates to a method for promoting high expression of an umbilical cord mesenchymal stem cell ossification regeneration key gene. The method comprises umbilical cord mesenchymal stem cell separation: acquiring sterile marrow, carrying out re-suspension deposition through pretreating nutrient solutions, carrying out counting, inoculating a culture bottle with the stem cells according to a ratio of 1*10<6>/cm<2> and carrying out culture, wherein the nutrient solutions respectively comprise a-MEM+10% FBS and a-MEM+5% FBS+5% blood platelet lysate+50mg/ml ascorbic acid+10nmol/L glucagon-like peptide-1. The umbilical cord mesenchymal stem cells cultured by the culture system can be used as seed cells and be used for bony tissue engineering treatment on bone defects, prevents umbilical cord mesenchymal stem cell in-vitro ossification induction and shortens clinical bone tissue engineering treatment on bone defects by at least 14 days.
Description
Technical field
To the invention belongs in biological technical field a kind of promotes the method that umbilical cord mesenchymal stem cells becomes osteanagenesis key gene high expression level.
Background technology
Osseous tissue defect is because the factors such as wound, infection, tumour make body lose some sclerotin thus form comparatively wide arc gap, and this problem is clinical comparatively common and more difficult.How to promote the reparation of osseous tissue defect, be the emphasis of this area research personnel research always.In recent years, along with the development of bone tissue engineer technology, this problem obtains solution to a certain degree.Seed cell in bone tissue engineering plays a part key.Therefore, the selection of seed cell is most important, and early stage researchist selects osteocyte and marrow stromal cell, and the difficulty but these seed cells are drawn materials, easily causes secondary injury to patient, and these cell expansion ex vivo abilities are more weak.After this, researchist by the target lock-on of seed cell at mescenchymal stem cell.Mescenchymal stem cell has the potential of Multidirectional Differentiation, can be divided into the cell that scleroblast, lipoblast and chondroblast etc. are ripe under certain conditions.Umbilical cord mesenchymal stem cells (UC-MSC) is a kind of mescenchymal stem cell separated from umbilical cord tissue, its tool advantage once: umbilical cord tissue wide material sources, ensures sufficient cell derived; Umbilical cord mesenchymal stem cells has the potential of Multidirectional Differentiation; Umbilical cord mesenchymal stem cells immunogenicity is lower, can not cause the immunological rejection of patient self.Umbilical cord mesenchymal stem cells is as other tissue-derived mescenchymal stem cell, determine after its osteogenic induction that the key transcription factor (Osterix, RUNX) of osteanagenesis and the genetic expression of the early stage Typical Representative factor of osteanagenesis (osteopontin-OPN, alkaline phosphatase-ALP) are all raised, but this process need of osteogenic induction time of at least 14 days.Therefore, how to shorten the osteogenic induction time or avoid the step of osteogenic induction, aobvious is particularly important.
Summary of the invention
The object of the present invention is to provide a kind of culture system that umbilical cord mesenchymal stem cells becomes osteanagenesis key gene up-regulated that promotes, the umbilical cord mesenchymal stem cells adopting culture system of the present invention to cultivate still remains higher dryness; The umbilical cord mesenchymal stem cells adopting culture system of the present invention to cultivate treats Cranial defect as seed cell for bone tissue engineer, avoid the step of the external osteogenic induction of umbilical cord mesenchymal stem cells, for clinical application bone tissue engineer treatment Cranial defect shortens the time of at least 14 days.
The technical solution adopted for the present invention to solve the technical problems is:
Promote that umbilical cord mesenchymal stem cells becomes a method for osteanagenesis key gene high expression level, step is as follows:
(1) the separation of umbilical cord mesenchymal stem cells: obtain aseptic umbilical cord tissue, peels off artery and vein in the adventitia of umbilical cord tissue and umbilical cord tissue under aseptic condition, isolate umbilical cord China's Tong Shi glue and shred to 2-3mm
3, by the magnificent Tong Shi sticker that shreds in culturing bottle bottom surface, in culturing bottle, add appropriate nutrient solution after 1-4h, nutrient solution adopts a-MEM+10%FBS and a-MEM+5% platelet lysates thing+50mg/ml xitix+10nmol/L glucagon-like-peptide-1 respectively;
Umbilical cord mesenchymal stem cells digestion, go down to posterity, when umbilical cord mesenchymal stem cells grows to 80%, adopt the EDTA of trypsinase+0.02% of 0.05% to digest it, with 1 × 10
4cells/cm
2reach in new culturing bottle;
And the third generation umbilical cord mesenchymal stem cells getting two kinds of culture systems cultivations carries out streaming qualification, the umbilical cord mesenchymal stem cells positive rate of two kinds of culture system cultivations is all higher than 95%, and negative rate is all lower than 5%;
And, get third generation umbilical cord mesenchymal stem cells, extract RNA-reverse transcription acquisition cDNA-and carry out Q-PCR, the expression amount of the transcription factor Osterix that the umbilical cord mesenchymal stem cells comparing two kinds of culture systems cultivations becomes osteanagenesis to be correlated with and RUNX gene and the early stage Typical Representative factor gene of osteanagenesis OPN and ALP.
A kind of promotion umbilical cord mesenchymal stem cells becomes osteanagenesis key gene high expression level system, a-MEM+5% platelet lysates thing+50mg/ml xitix+10nmol/L glucagon-like-peptide-1.
The invention has the beneficial effects as follows:
(1) the present invention adopts a kind of new culture system, cultivate with this culture system from umbilical cord mesenchymal stem cells is primary, compared with traditional culture system, in the umbilical cord mesenchymal stem cells that the culture system that the present invention adopts is turned out, determine into the key transcription factor (Osterix, RUNX) of osteanagenesis and the obvious high expression level of gene of the osteanagenesis early stage Typical Representative factor (OPN, ALP).
(2) apply culture system of the present invention and can promote that umbilical cord mesenchymal stem cells becomes the transcription factor of osteanagenesis (Osterix, RUNX) and the osteanagenesis early stage Typical Representative factor (OPN, ALP) high expression level, for bone defect healing provides suitable seed cell.
(3) a kind of new cultural method of the present invention's employing, culture system of the present invention is used when umbilical cord mesenchymal stem cells is normally cultivated, umbilical cord mesenchymal stem cells is while its original dryness of maintenance, the genetic expression determining into the correlation factor of osteanagenesis is raised, for application bone tissue engineer treatment Cranial defect provides necessary seed cell, also for bone tissue engineer treatment Cranial defect shortens the time.
(4) adopt culture system of the present invention to cultivate umbilical cord mesenchymal stem cells, improve into the expression of the gene of osteanagenesis key transcription factor (Osterix, RUNX) and the early stage Typical Representative factor (OPN, ALP) of osteanagenesis;
(5) the umbilical cord mesenchymal stem cells adopting culture system of the present invention to cultivate still remains higher dryness; The umbilical cord mesenchymal stem cells adopting culture system of the present invention to cultivate treats Cranial defect as seed cell for bone tissue engineer, avoid the step of the external osteogenic induction of umbilical cord mesenchymal stem cells, for clinical application bone tissue engineer treatment Cranial defect shortens the time of at least 14 days.
Accompanying drawing explanation
Fig. 1 is the mrna expression level (relative intensity of fluorescence) of Osterix;
Fig. 2 is the mrna expression level (relative intensity of fluorescence) of RUNX;
Fig. 3 is the mrna expression level (relative intensity of fluorescence) of OPN;
Fig. 4 be ALP mrna expression level (relative intensity of fluorescence);
Embodiment
Below in conjunction with accompanying drawing, also by specific embodiment, the invention will be further described, and following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.
Promote that umbilical cord mesenchymal stem cells becomes a method for osteanagenesis key gene high expression level, step is as follows:
(1) the separation of umbilical cord mesenchymal stem cells: obtain aseptic umbilical cord tissue, peels off artery and vein in the adventitia of umbilical cord tissue and umbilical cord tissue under aseptic condition, isolate umbilical cord China's Tong Shi glue and shred to 2-3mm
3, by the magnificent Tong Shi sticker that shreds in culturing bottle bottom surface, in culturing bottle, add appropriate nutrient solution after 1-4h, nutrient solution adopts a-MEM+10%FBS and a-MEM+5% platelet lysates thing+50mg/ml xitix+10nmol/L glucagon-like-peptide-1 respectively;
Umbilical cord mesenchymal stem cells digestion, go down to posterity, when umbilical cord mesenchymal stem cells grows to 80%, adopt the EDTA of trypsinase+0.02% of 0.05% to digest it, with 1 × 10
4cells/cm
2reach in new culturing bottle;
(3) the third generation umbilical cord mesenchymal stem cells getting two kinds of culture systems cultivations carries out streaming qualification, and the umbilical cord mesenchymal stem cells positive rate of two kinds of culture system cultivations is all higher than 95%, and negative rate is all lower than 5%;
(4) get and cultivate third generation umbilical cord mesenchymal stem cells, extract RNA-reverse transcription acquisition cDNA-and carry out Q-PCR, the expression amount of the transcription factor (Osterix, RUNX) that the umbilical cord mesenchymal stem cells comparing two kinds of culture systems cultivations becomes osteanagenesis to be correlated with and the osteanagenesis early stage Typical Representative factor (OPN, ALP) gene.
Claims (4)
1. promote that umbilical cord mesenchymal stem cells becomes a method for osteanagenesis key gene high expression level, it is characterized in that: step is as follows:
(1) the separation of umbilical cord mesenchymal stem cells: obtain aseptic umbilical cord tissue, peels off artery and vein in the adventitia of umbilical cord tissue and umbilical cord tissue under aseptic condition, isolate umbilical cord China's Tong Shi glue and shred to 2-3mm
3, by the magnificent Tong Shi sticker that shreds in culturing bottle bottom surface, in culturing bottle, add appropriate nutrient solution after 1-4h, nutrient solution adopts a-MEM+10%FBS and a-MEM+5% platelet lysates thing+50mg/ml xitix+10nmol/L glucagon-like-peptide-1 respectively;
Umbilical cord mesenchymal stem cells digestion, go down to posterity, when umbilical cord mesenchymal stem cells grows to 80%, adopt the EDTA of trypsinase+0.02% of 0.05% to digest it, with 1 × 10
4cells/cm
2reach in new culturing bottle.
2. promotion umbilical cord mesenchymal stem cells according to claim 1 becomes the method for osteanagenesis key gene high expression level, it is characterized in that: the third generation umbilical cord mesenchymal stem cells getting two kinds of culture systems cultivations carries out streaming qualification, the umbilical cord mesenchymal stem cells positive rate of two kinds of culture system cultivations is all higher than 95%, and negative rate is all lower than 5%.
3. promotion umbilical cord mesenchymal stem cells according to claim 1 becomes the method for osteanagenesis key gene high expression level, it is characterized in that: get third generation umbilical cord mesenchymal stem cells, extract RNA-reverse transcription acquisition cDNA-and carry out Q-PCR, the expression amount of the transcription factor Osterix that the umbilical cord mesenchymal stem cells comparing two kinds of culture systems cultivations becomes osteanagenesis to be correlated with and RUNX gene and the early stage Typical Representative factor gene of osteanagenesis OPN and ALP.
4. promote that umbilical cord mesenchymal stem cells becomes an osteanagenesis key gene high expression level system, is characterized in that: a-MEM+5% platelet lysates thing+50mg/ml xitix+10nmol/L glucagon-like-peptide-1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108570443A (en) * | 2017-12-05 | 2018-09-25 | 皓昇莱生物制药有限公司 | A kind of culture medium for cultivating urine derived cell |
| CN111759865A (en) * | 2020-07-10 | 2020-10-13 | 浙江中医药大学 | Method for promoting umbilical cord stem cell activation and synergistically intervening knee osteoarthritis by using platelet lysate |
-
2015
- 2015-12-15 CN CN201510933962.7A patent/CN105441388A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 吕江涛等: "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", 《中国组织工程研究》 * |
| 廖庆辉等: "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", 《中国组织工程研究与临床康复》 * |
| 王宁等: "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", 《中国药学大会暨第十三届中国药师周论文集》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108570443A (en) * | 2017-12-05 | 2018-09-25 | 皓昇莱生物制药有限公司 | A kind of culture medium for cultivating urine derived cell |
| CN111759865A (en) * | 2020-07-10 | 2020-10-13 | 浙江中医药大学 | Method for promoting umbilical cord stem cell activation and synergistically intervening knee osteoarthritis by using platelet lysate |
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Application publication date: 20160330 |