CN101591642A - Two olfactory cells are trained the method for inducing Olfactory stem cell propagation and differentiation altogether - Google Patents
Two olfactory cells are trained the method for inducing Olfactory stem cell propagation and differentiation altogether Download PDFInfo
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- CN101591642A CN101591642A CNA200810110780XA CN200810110780A CN101591642A CN 101591642 A CN101591642 A CN 101591642A CN A200810110780X A CNA200810110780X A CN A200810110780XA CN 200810110780 A CN200810110780 A CN 200810110780A CN 101591642 A CN101591642 A CN 101591642A
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Abstract
The method of the co-culturing, inducing Olfactory stem cell of a kind of Olfactory essheathing cell and Olfactory stem cell propagation and differentiation is collected the no obvious stunted fetus of Obstetric and Gynecologic Department miscarriage and induced labor, separate respectively, obtain Olfactory essheathing cell and former generation the olifactory nerve stem cell.The Transplanted cells clinical treatment that makes people's embryo Olfactory stem cell be applied to central nervous system injury after external evoked differentiation becomes possibility.
Description
Affiliated technical field
Patent of the present invention relates to a kind of Olfactory essheathing cell cultivation and unites the dried method of inducing the Olfactory stem cell differentiation of cultivation with Olfactory stem cell, obtains the ripe neurocyte after Olfactory stem cell breaks up, and is used for the Transplanted cells clinical treatment of central nervous system injury.
Background technology
Existing report has carried out the different dry cells in vitro and has induced differentiation research, attempt carrying out Transplanted cells after external evoked differentiation, but rarely report is studied still in the differentiation of inducing of Olfactory stem cell.
Summary of the invention
For the difficult problem on the cellular transplantation therapy that overcomes central nervous system injury, patent of the present invention provides a kind of Olfactory essheathing cell cultivation to unite to cultivate with Olfactory stem cell and has induced the Olfactory stem cell differentiation method.After external evoked differentiation, obtain the ripe neurocyte after Olfactory stem cell breaks up, the Transplanted cells clinical treatment that is used in central nervous system injury becomes possibility.
Patent of the present invention solves the technical scheme that its technical problem adopts: in-vitro separation cultivator embryo Olfactory essheathing cell, set up the extracorporeal culturing method of people's embryo olfactory mucosa neural stem cell, obtain behind the stable Olfactory stem cell that goes down to posterity of foot itself and Olfactory essheathing cell co-cultivation are set stem cell media group, serum-free group simultaneously.Fixed cell after five days is done the immunocyte fluorescent dye with nidogen (Nestin), tubulin (Tubulin), microtubule-associated protein (MAP2), the multiple antibody of neuroglial acidic protein (GFAP), positive cell is carried out morphology identify.
The beneficial effect of patent of the present invention is, is the induction method of olifactory nerve stem cell, and the Transplanted cells clinical treatment that makes people's embryo Olfactory stem cell be applied to central nervous system injury after external evoked differentiation becomes possibility.
Embodiment
(1) collect the no obvious stunted fetus of Obstetric and Gynecologic Department miscarriage and induced labor, separate respectively, obtain Olfactory essheathing cell and former generation the olifactory nerve stem cell.
(2) reagent culture medium (DMEM/F12), cytokine (B27), Urogastron (EGF), fibroblast growth factor (FGF), glutamine, Collagenase.
(3) along opening cranium on the dead fetus geisoma, remove cerebral tissue, basis cranii before exposing, take off olfactory bulb and see sieve plate, cockscomb, in sieve plate periphery cut the basis cranii hone lamella to the nasal cavity direction cut the both sides lateral wall of nasal cavity on, middle concha and corresponding nasal septum middle and upper part branch, taking off sample puts into substratum and cleans several all over the blood on the flush away olfactory mucosa, shred the back and add a spot of basic medium, the tissue that will shred with the elbow suction pipe moves in 15 milliliters of centrifuge tubes, add 5 milliliters of basic mediums, blow and beat up and down with thick elbow suction pipe and to make it to become tissue suspension, centrifugal 5min 1000 changes and abandons supernatant; So repeat 2 times, adding Collagenase 0.5ug/ml blows even with suction pipe, put into 37 ℃ of water-baths, blow and beat gently with suction pipe behind the digestion 15min and leave standstill 1min sucking-off supernatant, add in the centrifuge tube of (containing 10% foetal calf serum) substratum, so repeating 3 times can be single cell suspension with tissue digestion substantially, and the suspension after digestion is stopped is collected centrifugal together, D/F12 cleans 3 times, goes down to posterity with the conditioned medium that includes Urogastron and cultivates more than at least 20 times.
(4) the Olfactory essheathing cell substratum that will repeatedly collect carries out replacing after the 0.22 μ m cellulose membrane filtration sterilization olifactory nerve stem cell media to be observed to the 5th day and carries out immuning fluorescent dyeing analysis.
(5) under the light microscopic first three day observation of cell and control group do not have considerable change, the 4th day and the 5th day visible cell projection increases, proliferation slows.Immunofluorescence dyeing is observed GFAP stained positive cell proportion obviously to be increased, and nidogen, tubulin positive cell reduce.
Claims (3)
1. the method for the co-culturing, inducing Olfactory stem cell of Olfactory essheathing cell and Olfactory stem cell propagation and differentiation.It is characterized in that: observe the Olfactory essheathing cell cultivation and unite the inducing action of cultivation, obtain the ripe neurocyte after Olfactory stem cell breaks up, be used for the Transplanted cells clinical treatment of central nervous system injury Olfactory stem cell with Olfactory stem cell.
2. the co-culturing, inducing Olfactory stem cell propagation of Olfactory essheathing cell according to claim 1 and Olfactory stem cell and the method for differentiation.It is characterized in that: in-vitro separation cultivator embryo Olfactory essheathing cell, set up the extracorporeal culturing method of people's embryo olfactory mucosa neural stem cell, obtain behind the stable Olfactory stem cell that goes down to posterity of foot itself and Olfactory essheathing cell co-cultivation are set stem cell media group, serum-free group simultaneously.Fixed cell after five days is done the immunocyte fluorescent dye with nidogen (Nestin), tubulin (Tubulin), microtubule-associated protein (MAP2), the multiple antibody of neuroglial acidic protein (GFAP), positive cell is carried out morphology identify.
3. the co-culturing, inducing Olfactory stem cell propagation of Olfactory essheathing cell according to claim 1 and Olfactory stem cell and the method for differentiation.It is characterized in that: present method is the induction method of olifactory nerve stem cell, and the transplanting clinical treatment that makes people's embryo Olfactory stem cell be applied to central nervous system injury after external evoked differentiation becomes possibility.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CNA200810110780XA CN101591642A (en) | 2008-05-30 | 2008-05-30 | Two olfactory cells are trained the method for inducing Olfactory stem cell propagation and differentiation altogether |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA200810110780XA CN101591642A (en) | 2008-05-30 | 2008-05-30 | Two olfactory cells are trained the method for inducing Olfactory stem cell propagation and differentiation altogether |
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| CN101591642A true CN101591642A (en) | 2009-12-02 |
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| CNA200810110780XA Pending CN101591642A (en) | 2008-05-30 | 2008-05-30 | Two olfactory cells are trained the method for inducing Olfactory stem cell propagation and differentiation altogether |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012164137A1 (en) | 2011-05-30 | 2012-12-06 | Fundación Investigación En Regeneración Del Sistema Nervioso | Stem cells and neural crest cells derived from olfactory ensheathing glia, and uses thereof |
| CN105349491A (en) * | 2015-12-15 | 2016-02-24 | 黄红云 | Separation and preparation technology derived from olfactory mucosa neuronal cells |
| WO2018120437A1 (en) * | 2016-12-30 | 2018-07-05 | 中国医药大学 | Adult pluripotent olfactory stem cell and separation method therefor and use thereof |
| CN109735493A (en) * | 2019-03-11 | 2019-05-10 | 湖南师范大学 | A method of induction olfactory mucosa mescenchymal stem cell directed differentiation is dopaminergic neuron |
-
2008
- 2008-05-30 CN CNA200810110780XA patent/CN101591642A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012164137A1 (en) | 2011-05-30 | 2012-12-06 | Fundación Investigación En Regeneración Del Sistema Nervioso | Stem cells and neural crest cells derived from olfactory ensheathing glia, and uses thereof |
| CN105349491A (en) * | 2015-12-15 | 2016-02-24 | 黄红云 | Separation and preparation technology derived from olfactory mucosa neuronal cells |
| CN105349491B (en) * | 2015-12-15 | 2019-09-24 | 北京市虹天济神经科学研究院 | From olfactory mucosa neuronal cell separation and preparation technology |
| WO2018120437A1 (en) * | 2016-12-30 | 2018-07-05 | 中国医药大学 | Adult pluripotent olfactory stem cell and separation method therefor and use thereof |
| CN109735493A (en) * | 2019-03-11 | 2019-05-10 | 湖南师范大学 | A method of induction olfactory mucosa mescenchymal stem cell directed differentiation is dopaminergic neuron |
| CN109735493B (en) * | 2019-03-11 | 2021-09-14 | 湖南师范大学 | Method for inducing olfactory mucosa mesenchymal stem cells to directionally differentiate into dopaminergic neurons |
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Open date: 20091202 |