CN108267432A - A kind of method of the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model - Google Patents
A kind of method of the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model Download PDFInfo
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- CN108267432A CN108267432A CN201711370927.4A CN201711370927A CN108267432A CN 108267432 A CN108267432 A CN 108267432A CN 201711370927 A CN201711370927 A CN 201711370927A CN 108267432 A CN108267432 A CN 108267432A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- 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
- C12N5/0618—Cells of the nervous system
- C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a kind of methods of the Olfactory essheathing cell of tracer in spinal cord injury mouse model transplanting, and quantum dot is diluted in serum free medium, is incubated in cell incubator；Serum-free medium containing quantum dot is washed out, then phosphate buffer cleaning is replaced with the culture medium containing fetal calf serum.The beneficial effects of the invention are as follows can simply and rapidly complete label of the tracer for stem cell.
Description
Technical field
The invention belongs to medicine technology fields, are related to a kind of Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model
Method.
Background technology
In recent years, Treatment of spinal cord injury with cellular transplantation becomes the hot spot of research, and achieves great successes.But while
A large amount of clinical experimental study confirms that olfactory ensheathing cell transplantation for spinal cord injury achieves the effect of preferable, and still someone holds this
Suspect attitude.The clinical treatment of Olfactory essheathing cell is still in the presence of dispute, lacks substantive evidence.And by Olfactory essheathing cell
Before clinical treatment, still there are many practical problems to need to solve, such as Olfactory essheathing cell come source problem, therapic opportunity, whether
Inflammatory reaction can be adjusted and be implanted in intravital curative effect etc..If in vivo directly it dynamically can observe transplanting
Survival, proliferation, differentiation, the migration of Olfactory essheathing cell, then many knotty problems can all be readily solved.Currently used Olfactory essheathing cell
Tracer includes Superparamagnetic Iron Oxide, radionuclide and fluorescent reporter gene such as green fluorescent protein etc..Although these
Tracer is studied in terms of Olfactory essheathing cell label and is made some progress, but there is also many problems such as, cytotoxicity
It is high, markers step is cumbersome etc..Its most important problem be the above method can not tracer in a long time be implanted into it is internal
Stem cell.The problem of stem cell tracer is primarily present at present is that markers step is cumbersome, time-consuming, and shorter, the nothing of tracer failure
Method realizes that tracer in a long time is implanted into the biological behaviours such as the migration of internal stem cell, distribution.
Invention content
The purpose of the present invention is to provide a kind of method of the Olfactory essheathing cell of tracer in spinal cord injury mouse model transplanting, solutions
Current stem cell tracer markers step of having determined is cumbersome and tracer failure is shorter, can not realize that tracer in a long time moves
The problem of migration of the stem cell to implant, distribution.
The technical solution adopted in the present invention is to follow the steps below：
Step 1：Quantum dot is diluted in serum free medium, is incubated in cell incubator；
Step 2：Serum-free medium containing quantum dot is washed out, then phosphate buffer cleaning is replaced with containing tire
The culture medium of cow's serum.
Further, quantum dot is indium phosphide/zinc sulphide fluorescence quantum.
Further, quantum dot is pressed in the concentration dilution to serum free medium of 10nM in step 1；37 in cell incubator
DEG C, 5%CO2, incubation time 2 hours.
Further, phosphate buffer cleans 3 times in step 2；The culture medium of replacement contains 10% fetal calf serum.
The beneficial effects of the invention are as follows can simply and rapidly complete label of the tracer for stem cell.
Description of the drawings
Fig. 1 is the endocytosis schematic diagram that quantum dot-albumen crown compound prevents cell for quantum dot.
In figure, 1. quantum dots, 2. cells, the protein ingredient in 3. serum, 4. quantum dots-albumen crown compound.
Specific embodiment
The present invention is described in detail With reference to embodiment.
The method of the present invention step is as follows：
Step 1：Indium phosphide/zinc sulphide fluorescent quantum is pressed in the concentration dilution to serum free medium of 10nM, thin
Born of the same parents' incubator (37 DEG C of 5%CO2) in be incubated 2h.
Step 2：It will be washed out containing the serum-free medium of indium phosphide/zinc sulphide fluorescence quantum, phosphate buffer
(PBS) it cleans 3 times, is replaced with the culture medium containing 10% fetal calf serum (FBS).
InP/ZnS-MPA is the negatively charged quantum dot in surface, if as shown in Figure 1, in the culture medium containing serum with
Cell co-cultures, and quantum dot 1 can form quantum dot-albumen crown compound 4 with the protein ingredient 3 in serum first and be attached to quantum
1 surface of point is so as to prevent endocytosis of the cell 2 for quantum dot 1；On the contrary in the culture systems of serum-free, quantum dot 1 can be in 2h
Interior completion labeling process.The present invention can complete cell marking process using quantum dot-labeled Olfactory essheathing cell in 2 hours,
In experiment in vivo, the Olfactory essheathing cell of label by way of tail vein injection, is implanted into the spinal cord injury model of rat by us
In, it can be in the distribution situation of period tracer Olfactory essheathing cell in vivo more than 28 days.Finally, it our experiment demonstrates that, measures
Son point is a kind of efficiently, a kind of Cellular tracking agent that cytotoxicity is low and timeliness is long.The technology of the present invention can simplify tracer
It is the step of labeled stem cells, easy to operate, easy.It, can be when relatively long compared to other Olfactory essheathing cell Cellular tracking agent
Interior (＞ 28d) tracer is implanted into internal cell.
The above is only the better embodiment to the present invention, not makees limit in any form to the present invention
System, any simple modification that every technical spirit according to the present invention makes embodiment of above, equivalent variations and modification,
Belong in the range of technical solution of the present invention.
Claims (4)
1. a kind of method of the Olfactory essheathing cell of the tracer in spinal cord injury mouse model transplanting, it is characterised in that according to following steps into
Row：
Step 1：Quantum dot is diluted in serum free medium, is incubated in cell incubator；
Step 2：Serum-free medium containing quantum dot is washed out, then phosphate buffer cleaning is replaced with ox blood containing tire
Clear culture medium.
2. according to the method for the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model a kind of described in claim 1, feature
It is：The quantum dot is indium phosphide/zinc sulphide fluorescence quantum.
3. according to the method for the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model a kind of described in claim 1, feature
It is：Quantum dot is by the concentration dilution to serum free medium of 10nM in the step 1；37 DEG C in cell incubator, 5%
CO2, incubation time 2 hours.
4. according to the method for the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model a kind of described in claim 1, feature
It is：Phosphate buffer cleans 3 times in the step 2；The culture medium of replacement contains 10% fetal calf serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711370927.4A CN108267432A (en) | 2017-12-19 | 2017-12-19 | A kind of method of the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711370927.4A CN108267432A (en) | 2017-12-19 | 2017-12-19 | A kind of method of the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model |
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| Publication Number | Publication Date |
|---|---|
| CN108267432A true CN108267432A (en) | 2018-07-10 |
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|---|---|---|---|
| CN201711370927.4A Pending CN108267432A (en) | 2017-12-19 | 2017-12-19 | A kind of method of the Olfactory essheathing cell of the transplanting of the tracer in spinal cord injury mouse model |
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Citations (6)
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|---|---|---|---|---|
| CN101531993A (en) * | 2008-03-12 | 2009-09-16 | 中国医学科学院肿瘤研究所 | Method and kit for labeling karyon stably by fluorescence |
| CN102453747A (en) * | 2010-10-18 | 2012-05-16 | 中国科学院上海生命科学研究院 | Tracing analysis method in cell of deoxyribonucleic acid (DNA) |
| CN102643894A (en) * | 2011-02-17 | 2012-08-22 | 中国科学院上海生命科学研究院 | Exogenous gene tracing system and application thereof |
| CN103048298A (en) * | 2012-12-21 | 2013-04-17 | 中国人民解放军第三军医大学第一附属医院 | Method of using glycine modified quantum dot probes to mark living cell |
| CN105018082A (en) * | 2015-07-10 | 2015-11-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing carbon quantum dot labeled probe for silk fibroin extracted cell development |
| CN105368774A (en) * | 2014-08-21 | 2016-03-02 | 南京大学医学院附属鼓楼医院 | Kit for tracing uterus implanting stem cell prognosis, and preparation method thereof |
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2017
- 2017-12-19 CN CN201711370927.4A patent/CN108267432A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101531993A (en) * | 2008-03-12 | 2009-09-16 | 中国医学科学院肿瘤研究所 | Method and kit for labeling karyon stably by fluorescence |
| CN102453747A (en) * | 2010-10-18 | 2012-05-16 | 中国科学院上海生命科学研究院 | Tracing analysis method in cell of deoxyribonucleic acid (DNA) |
| CN102643894A (en) * | 2011-02-17 | 2012-08-22 | 中国科学院上海生命科学研究院 | Exogenous gene tracing system and application thereof |
| CN103048298A (en) * | 2012-12-21 | 2013-04-17 | 中国人民解放军第三军医大学第一附属医院 | Method of using glycine modified quantum dot probes to mark living cell |
| CN105368774A (en) * | 2014-08-21 | 2016-03-02 | 南京大学医学院附属鼓楼医院 | Kit for tracing uterus implanting stem cell prognosis, and preparation method thereof |
| CN105018082A (en) * | 2015-07-10 | 2015-11-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing carbon quantum dot labeled probe for silk fibroin extracted cell development |
Non-Patent Citations (3)
| Title |
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| ER-QUN SONG 等: "Tumor Cell Targeting Using Folate-Conjugated Fluorescent Quantum Dots and Receptor-Mediated Endocytosis", 《CLINICAL CHEMISTRY》 * |
| HICHAM CHIBLI 等: "Cytotoxicity of InP/ZnS quantum dots related to reactive oxygen species generation", 《NANOSCALE》 * |
| 陈文新: "基于生物质甘蔗渣的荧光碳量子点制备", 《暨南大学学报( 自然科学与医学版)》 * |
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Application publication date: 20180710 |