CN106609260A - Method for improving efficiency of differentiation of umbilical cord mesenchymal stem cells into vascular endothelial cells - Google Patents
Method for improving efficiency of differentiation of umbilical cord mesenchymal stem cells into vascular endothelial cells Download PDFInfo
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- CN106609260A CN106609260A CN201510724439.3A CN201510724439A CN106609260A CN 106609260 A CN106609260 A CN 106609260A CN 201510724439 A CN201510724439 A CN 201510724439A CN 106609260 A CN106609260 A CN 106609260A
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Abstract
The invention belongs to the technical field of biological cells, and in particular, relates to a method for improving in-vitro directional induced differentiation of umbilical cord mesenchymal stem cells into vascular endothelial cells. A basic fibroblast growth factor with the concentration of 20 ng/mL and an epidermal growth factor with the concentration of 10 ng/ml are added into a culture liquid (containing an M199 culture medium, fetal bovine serum and a vascular endothelial growth factor) for directional induced differentiation of the umbilical cord mesenchymal stem cells into the vascular endothelial cells, and about 50-70% of the vascular endothelial cells are obtained; compared with pure vascular endothelial growth factor induction (with the differentiation efficiency of about 30%), the differentiation efficiency is improved obviously; the nature and functions of the cells are identified, the obtained cells are confirmed to be the vascular endothelial cells having mature endothelial cell functions, and important experimental basis data are provided for studies of repair of tissues and organs and studies of tissue engineering graft revascularization.
Description
Technical field：The invention belongs to biological cell technical field, and in particular to one kind improves umbilical cord mesenchymal stem cells directional induction in vitro and is divided into
The method of vascular endothelial cell.
Background technology：Endotheliocyte has very important effect to histoorgan reparation, angiogenesiss, and is tissue-engineering graft constructed blood vessel again
The necessary cell component of change.But mature endothelial cell in-vitro multiplication is limited in one's ability, it is difficult to meet the needs of external revascularization.Umbilical cord mesenchyma is done
Cell proliferation in vitro ability is strong, and with multi-lineage potential, and these cell separation are convenient, and baby and mother are not affected, can with it is frozen with
Just use in the future, so, umbilical cord mesenchymal stem cells will have very big in histoorgan reparation and cardiovascular organization engineering revascularization research field
Prospect.We adopt enzyme digestion, successfully stem cell (Wu Kaihong, Mo Xuming, Liu Yinglong, Lu Shihong, Han Zhongchao are isolated from umbilical cord tissue.
Enzyme digestion separates umbilical cord stem cells.Chinese pediatric surgery magazine.2008；29(3)：159-162).We have discovered that, in derivant
In the presence of skin cell growth factor (50ng/ml), umbilical cord mesenchymal stem cells can be divided into vascular endothelial cell, but differentiation efficiency is relatively low,
Only about 30% umbilical cord mesenchymal stem cells can be divided into vascular endothelial cell (Wu Kaihong, Mo Xuming, Lu Shihong, Han Zhongchao.Fill between umbilical cord
Matter stem cell is divided into the experimentation of endotheliocyte.Chinese pediatric surgery magazine 2010；31(12)：954-6).Our Jing experimental studies have found that
Basic fibroblast growth factor and epidermal growth factor are added in derivant, umbilical cord mesenchymal stem cells can be significantly improved and be divided into endotheliocyte
Efficiency, differentiation efficiency brings up to 50-70% by 30%, is histoorgan repairing research and tissue-engineering graft constructed revascularization research, further improves
Stem cell is divided into the efficiency of interior blood vessel chrotoplast, there is provided important experimental basis data.
The content of the invention：Using collagenase digestion separating funicle mesenchyme stem cell.The 5th generation umbilical cord mesenchymal stem cells of culture of isolated are taken, is inoculated with
In 24 well culture plates, culture plate is coated with Matrigel, and liquid culture medium containing M199,2% hyclone and 50ng/ml are broken up in vascular endothelial cell induction
VEGF.After 2 weeks, endothelium-specific antibodies CD34 detections and intake are carried out to the umbilical cord mesenchymal stem cells after induction differentiation
DiI-Ac-LDL detections, observe positive cell ratio.Add basic fibroblast growth factor and epidermal growth in skin induction broth in intravascular
The factor, using flow cytomery cell differentiation efficiency.Induction divergaence time is determined simultaneously, is the concentration in practical application and time to provide reference
Basis.It is determined that umbilical cord can be significantly improved with the basic fibroblast growth factor of 20ng/ml concentration and 10ng/ml epidermal growth factors
Derived from Mesenchymal Stem Cells is the efficiency of vascular endothelial cell.
The present invention is histoorgan repairing research and tissue-engineering graft constructed revascularization research, there is provided important experimental basis data.In of the invention
Hold to be not disclosed and deliver, the person skilled of this area obtain the present invention as do not spent creative work may not infer according to existing technology
Method for inducing and cultivating.
Description of the drawings：
Fig. 1：The umbilical cord mesenchymal stem cells of separation and Culture, ability of cell proliferation is vigorous, in swirl shape growth.
Fig. 2：After endotheliocyte directional induction 1 week, umbilical cord mesenchymal stem cells form grid spline structure.
Fig. 3：After 2 weeks, immunofluorescence dyeing shows the umbilical cord mesenchymal stem cells expression endothelium-specific antibodies CD34 after differentiation, induces differentiation efficiency 30%.
Fig. 4：After adding basic fibroblast growth factor and epidermal growth factor, flow cytomery endothelium-specific antibodies CD144 shows Ink vessel transfusing
Epithelial cell differentiation efficiency is 50-70%.
Specific embodiment：
1. umbilical cord stem cells are separated using enzyme digestion and identified.Flow cytometry umbilical cord stem cells expression CD13, CD29, CD44, CD90,
CD105, CD166 and MHC-I, and CD31, CD34 and CD106 are not expressed, MHC-II.Induction before umbilical cord stem cells form into typically into
After fibroblast-like cell form, induction 2 weeks, the form of cell constantly changes the volume increase of some cells, and cavity sample changes.Take culture of isolated
The 5th generation umbilical cord mesenchymal stem cells be inoculated in 24 well culture plates, culture plate is coated with Matrigel, and umbilical cord mesenchymal stem cells are inoculated with per hole
5×103It is individual.Endothelium induction breaks up liquid for M199 nutrient chemicals, 2% hyclone, 50ng/mlVEGF, 100U/ml penicillin and streptomycin.Per 2 days more
A culture fluid is changed, D-Hank ' s is taken when changing liquid and is gently rinsed cell, remove the cell that death comes off, successive induction 2 weeks, experiment finds induction
Umbilical cord mesenchymal stem cells after differentiation are expressed endothelium-specific antibodies CD34 and absorb positive (the intake DiI-Ac-LDL of DiI-Ac-LDL detections
It is function that mature endothelial cell has), observation positive cell ratio is about 30%.
2. basic fibroblast growth factor and epidermal growth factor are added in intravascular in skin induction broth, and cultural method is with method 1, experiment
It was found that the umbilical cord mesenchymal stem cells expression endothelium-specific antibodies CD34 after induction differentiation, with intake DiI-Ac-LDL functions, while streaming is thin
The detection of born of the same parents' instrument finds that the efficiency of vascular endothelial cell differentiation brings up to 50-70%.Meanwhile, it is determined that can be with the basic fibroblast of 20ng/ml concentration
Somatomedin and 10ng/ml epidermal growth factors are improving the efficiency that umbilical cord mesenchymal stem cells are divided into vascular endothelial cell.
Claims (4)
1. patent of the present invention is characterized in that further improving the efficiency that umbilical cord mesenchymal stem cells Differentiation Induction in vitro is vascular endothelial cell through the following steps：
1) separate, cultivate umbilical cord mesenchymal stem cells and identified.
2) vascular endothelial cell directional induction is carried out to umbilical cord mesenchymal stem cells using 50ng/ml VEGFs and is identified.
3) basic fibroblast growth factor and epidermal growth factor are added in chrotoplast directional induction culture fluid in intravascular, using flow cytometer pair
Endothelial cell differentiation efficiency is analyzed, and analysis shows molecule CD34 and CD144 using blood vessel endothelium specificity.
2. the method for pressing claim 1, after inducing 1 week, the umbilical cord mesenchymal stem cells of directional induction form grid spline structure, after 2 weeks, express endothelium
Specific antibody CD34, the function with intake DiI-Ac-LDL, endothelial cell differentiation efficiency is 30%.
3. the method for pressing claim 1, the optium concentration of basic fibroblast growth factor is 20ng/ml, and the optium concentration of epidermal growth factor is
10ng/ml。
4. the method for pressing claim 1, after adding basic fibroblast growth factor and epidermal growth factor, flow cytomery is divided into mature blood
The umbilical cord mesenchymal stem cells of endothelial cell substantially increase, and are 50-70%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105748523A (en) * | 2016-02-28 | 2016-07-13 | 深圳爱生再生医学科技有限公司 | Stem cell preparation for treating hepatic failure as well as preparation method and application thereof |
| CN110042125A (en) * | 2018-01-16 | 2019-07-23 | 深圳市伊思科生物科技有限公司 | Fat mesenchymal stem cell transdifferentiation prepares method, vascular cell and its application of vascular cell |
| CN113897331A (en) * | 2021-09-29 | 2022-01-07 | 苏州大学 | Differentiation method for inducing human artery endothelial cells |
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| WO2010059828A1 (en) * | 2008-11-19 | 2010-05-27 | Anthrogenesis Corporation | Amnion derived adherent cells |
| CN102776150A (en) * | 2011-05-13 | 2012-11-14 | 上海交通大学医学院附属第九人民医院 | Method for inducing umbilical cord mesenchymal stem cells to differentiate into endothelial cells |
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- 2015-10-27 CN CN201510724439.3A patent/CN106609260A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010059828A1 (en) * | 2008-11-19 | 2010-05-27 | Anthrogenesis Corporation | Amnion derived adherent cells |
| CN102776150A (en) * | 2011-05-13 | 2012-11-14 | 上海交通大学医学院附属第九人民医院 | Method for inducing umbilical cord mesenchymal stem cells to differentiate into endothelial cells |
Non-Patent Citations (2)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105748523A (en) * | 2016-02-28 | 2016-07-13 | 深圳爱生再生医学科技有限公司 | Stem cell preparation for treating hepatic failure as well as preparation method and application thereof |
| CN110042125A (en) * | 2018-01-16 | 2019-07-23 | 深圳市伊思科生物科技有限公司 | Fat mesenchymal stem cell transdifferentiation prepares method, vascular cell and its application of vascular cell |
| CN113897331A (en) * | 2021-09-29 | 2022-01-07 | 苏州大学 | Differentiation method for inducing human artery endothelial cells |
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Address after: 210008 Guangzhou Road, Nanjing, Jiangsu 72 Applicant after: Children's Hospital Affiliated to Nanjing Medical University Address before: 210008 Guangzhou Road, Nanjing, Jiangsu 72 Applicant before: Nanjing Children's Hospital of Nanjing Medical University |
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Application publication date: 20170503 |