CN1775945A - Method for in vitro inhibiting AF116909 gene induced nerve stem cells from directional differentiating into cholinergic neuron - Google Patents
Method for in vitro inhibiting AF116909 gene induced nerve stem cells from directional differentiating into cholinergic neuron Download PDFInfo
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- CN1775945A CN1775945A CNA2005100258616A CN200510025861A CN1775945A CN 1775945 A CN1775945 A CN 1775945A CN A2005100258616 A CNA2005100258616 A CN A2005100258616A CN 200510025861 A CN200510025861 A CN 200510025861A CN 1775945 A CN1775945 A CN 1775945A
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Abstract
The invention relates to a method to directionally separate the in vitro inducement nerve stem cell into cholinergic neuron that includes nerve stem cell separation, cultivating, and identification. The invention has is easy to operate, well repeatability, and is a new way to gain cholinergic neuron.
Description
Technical field
The present invention relates to the differentiation research of neural stem cell, particularly vitro inhibition AF116909 gene induced nerve stem cells directed differentiation is the method for cholinergic neuron.
Background of invention:
The discovery of neural stem cell is the important breakthrough of neuroscience area research in recent years, has become the focus of recent research, and not only because it has the importance of research nervous system development, main is its potential therapeutic value.Mckay etc. think, the cell that neural stem cell refers to have the self ability and is divided into neurone, astroglia cell and oligodendrocyte ability.Its differentiation, migration, sophisticated process are internal cause and external cause results of interaction, and internal cause is the expression status of cytogene just.Neural stem cell will enter clinical application, and what at first will solve is the directed differentiation problem, because only get the differentiation mechanism of neural stem cell clear, just can make full use of its plasticity-, to realize cell replacement.Cholinergic neuron is a kind of important neurone, can replace because flesh committee contracts or the loss of the motor neuron that Spinal injury causes, and can improve human or animal's abilities such as study, memory, and senile dementia, epilepsy etc. is also had important regulatory role.This invention provides an approach for external acquisition cholinergic neuron, for diseases such as treatment senile dementia, epilepsy provide certain thinking.
Summary of the invention
The objective of the invention is to realize the directed differentiation of neural stem cell to cholinergic neuron by the method that adopts gene regulating.
For achieving the above object, what the present invention adopted is contemplated that: the corresponding A of will encode F116909 gene specific sequence also can form little hairpin structure RNA (small hairpin RNA, shRNA) a pair of 57bp DNA palindromic sequence is inserted in the pSilenCircle carrier that contains poly-and enzyme III (Pol III) the U6 promotor of RNA and is built into the shRNA expression vector, again with this shRNA expression vector transfection in NSCs, observe the differentiation situation of NSC.
According to above-mentioned design, the present invention adopts following technical scheme:
A kind of vitro inhibition AF116909 gene induced nerve stem cells directed differentiation is the method for cholinergic neuron, may further comprise the steps:
(1) separation of neural stem cell, cultivation and evaluation;
(2) structure of shRNA expression vector: according to SilenCircle
TMThe palindromic sequence corresponding to the AF116909 gene of shRNA is transcribed in the design of RNAi Transcription Kit method, two palindromic sequences are synthesized double-stranded sequences 95 ℃ of annealing, be connected with the pSilenCircle carrier of wire again, being transformed into competent DH5 α increases, plasmid extraction, order-checking is identified;
(3) transfection of NSC: transfection same day NSCs is gone down to posterity, NSCs is gone down to posterity, it is 10 that density is made in piping and druming
6-10
8The cell suspension of individual/mL is got the culturing bottle that this suspension of 5mL places 60-mm; To be diluted among the DMEM of 500 μ L antibiotic-frees, serum-free through the expression vector 6.0 μ g-10.0 μ g that order-checking is defined as shRNA; Simultaneously with the Lipofectamine of 20 μ L
TM2000 transfection reagents are diluted among the above-mentioned DMEM of same volume, cultivate under the room temperature after 5 minutes; Then the shRNA expression vector of the dilution transfection reagent with dilution is mixed, incubated at room temperature is after 20 minutes, at last again with this mixture transfection in NSCs;
(4) evaluation of cholinergic neuron.
Above-mentioned neural stem cell is to separate from tire mouse striatum to obtain.
The cultural method of above-mentioned neural stem cell is: in containing F12-DMEM (1: the 1) substratum of 1%B27,20ng/mL EGF, 10ng/mL bFGF, cell suspension is made in piping and druming, in 37 ℃, 50mL/L CO with tire mouse striatum
2Cultivate under the saturated humidity.
Immunofluorescence technique is adopted in the evaluation of above-mentioned neural stem cell.
The time of the transfection of above-mentioned NSC is 11 days.
The RT-PCR way of qualitative analysis is adopted in the evaluation of above-mentioned cholinergic neuron
Adopting vitro inhibition AF116909 gene induced nerve stem cells directed differentiation of the present invention is the method for cholinergic neuron, is increased to (80 ± 3.1) % of the 11st day from (12 ± 1.5) % cholinergic neuron differentiation rate of cultivating the 3rd day.The RT-PCR analytical results shows that the expression amount of AF116909 gene mRNA is compared obvious minimizing with control group.This illustrates that this method is effectively suppressed really to the AF116909 goal gene, shows that simultaneously suppressing this gene has the function of impelling neural stem cell to break up to cholinergic neuron.
Embodiment
The pregnant mouse of laboratory animal: SD (14d) is available from Shanghai The 2nd Army Medical College Animal House.
The separation and Culture of neural stem cell: aseptic condition takes out the striatum of tire mouse down, put into and contain 1%B27,20ng/mL EGF, in the F12-DMEM of 10ng/mL bFGF (1: the 1) substratum, cell suspension is made in piping and druming, in 37 ℃, the conventional cultivation under the 5%CO2 saturated humidity, every one week of cultivation goes down to posterity once.
The evaluation of neural stem cell: cell is attached on the slide glass grows, take out after 2 days.The sucking-off nutrient solution, with PBS flush away residual culture, 4% (m/m) Paraformaldehyde 96 is 20min fixedly.PBS washes 2 times (every 10 minutes 1 time); 10% normal goats serum sealing 1h; PBS washes 3 times; Anti-nestin antibody (v/v 1: 100 dilutes with antibodybuffer) spends the night for 4 ℃, and PBS washes 3 times; Two anti-(v/v 1: 50 is with antibody buffer dilutions) are hatched 1h for 32 ℃, and PBS washes 3 times; 50% (v/v) glycerine mounting is observed fluorescence.
Neural stem cell separation and Culture and evaluation: Nestin albumen is the labelled protein of neural stem cell, it is a kind of intermediate filament protein, be distributed in the tenuigenin, observed green fluorescence with anti-nestin immunofluorescence dyeing detection, assembling agglomerating automatically is the key character of neural stem cell in the vitro culture process.This just further proves the reliability of separation and Culture neural stem cell method in tire mouse striatum.
The structure of shRNA expression vector: according to SilenCircle
TMThe palindromic sequence corresponding to the AF116909 gene (respectively being 57bp) of shRNA is transcribed in the design of RNAi Transcription Kit method, and its sequence is:
sense：
5’ACACCGACCCTCATTCCAGAGAGGGTTTGCTTGAAACCCTCTCTGGAATGAGGGTCT3’
antisense：
5’AAAAAGACCCTCATTCCAGAGAGGGTTTCAAGCAAACCCTCTCTGGAATGAGGGTCG3’
With the synthetic double-stranded sequence of 95 ℃ of annealing of two palindromic sequences, be connected with the pSilenCircle carrier of wire again, be transformed into competent DH5 α and increase, plasmid extraction, order-checking identifies that the sequence after connecting is:
5’ACACCGACCCTCATTCCAGAGAGGGTTTGCTTGAAACCCTCTCTGGAATGAGGGTCT3’
3’GCTGGGAGTAAGGTCTCTCCCAAACGAACTTTGGGAGAGACCTTACTCCCAGAAAAA5’
The transfection of NSC: transfection same day NSCs is gone down to posterity, it is 4 * 10 that density is made in piping and druming
6The cell suspension of individual/mL is got the culturing bottle that this suspension of 5mL places 60-mm, and just the expression vector 8.0 μ g that are defined as shRNA through order-checking are diluted among the DMEM of 500 μ L antibiotic-frees, serum-free, with the Lipofectamine of 20 μ L
TM2000 transfection reagents are diluted among the above-mentioned DMEM of same volume, cultivate after 5 minutes under the room temperature, shRNA expression vector with dilution mixes with the transfection reagent that dilutes again, after the incubated at room temperature 20 minutes, at last again with this mixture transfection in NSCs, design contrast simultaneously, cell is 37 ℃, the conventional cultivation under the 50mL/L CO2 saturated humidity, the differentiation situation of observation experiment group and cellular control unit, and make relevant experimental record.
The evaluation of cholinergic neuron: cultivate after 11 days, collect experimental group and cellular control unit respectively, with Trizol difference extracted total RNA, purifying, respectively getting 2 μ g RNA is that template is carried out cDNA synthetic (its synthetic method is carried out according to AMV FirstStrand cDNA Synthesis Kit).Be that template is carried out PCR respectively to the qualitative analysis of AF116909 gene mRNA expression amount in experimental group and the control group with synthetic cDNA again.Its PCR primer is: 5 ' CTGTGGACTCCTGGTCAG3 ' (Forward) and 5 ' GTCTGTCCAGATGCTTCT3 ' (Reverse), PCR product size is 380bp.
Cellular form is observed after the NSCs transfection: (cell that breaks up in ten visuals field accounts for the per-cent of total cell to the differentiation rate that the control group of untransfected shRNA expression vector and transfection have the experimental group of shRNA expression vector to cultivate respectively and calculate to cultivate different number of days.Found that, cultivate that control group does not have obvious differentiation after 11 days, differentiation rate only is (5 ± 1.2) %, and experimental group just begins differentiation after the 3rd day from cultivating, the quantity showed increased of cytodifferentiation and differentiation rate are (12 ± 1.5) %, along with the increase of cultivating fate, the differentiation rate of cell obviously increases, and the differentiation rate of cell arrives (80 ± 3.1) % after cultivating the 11st day.
Cultivate after 11 days, with Trizol extracted total RNA and the total RNA of purifying, carry out RT-PCR respectively and come the expression amount of the AF116909 gene mRNA in qualitative analysis experimental group (T) and control group (C) cell, the expression amount of AF116909 gene mRNA is obviously greater than the expression amount in the experimental group in the control group as a result, and the expression of goal gene is effectively suppressed in this illustrative experiment group.
Claims (6)
1. a vitro inhibition AF116909 gene induced nerve stem cells directed differentiation is the method for cholinergic neuron, may further comprise the steps:
(1) separation of neural stem cell, cultivation and evaluation;
(2) structure of shRNA expression vector: according to SilenCircle
TMThe palindromic sequence corresponding to the AF116909 gene of shRNA is transcribed in the design of RNAi Transcription Kit method, two palindromic sequences are synthesized double-stranded sequences 95 ℃ of annealing, be connected with the pSilenCircle carrier of wire again, being transformed into competent DH5 α increases, plasmid extraction, order-checking is identified;
(3) transfection of NSC: transfection same day NSCs is gone down to posterity, it is 10 that density is made in piping and druming
6-10
8The cell suspension of individual/mL is got the culturing bottle that this suspension of 5mL places 60-mm; To be diluted among the DMEM of 500 μ L antibiotic-frees, serum-free through the expression vector 6.0 μ g-10.0 μ g that order-checking is defined as shRNA; Simultaneously with the Lipofectamine of 20 μ L
TM2000 transfection reagents are diluted among the above-mentioned DMEM of same volume, cultivate under the room temperature after 5 minutes; Then the shRNA expression vector of the dilution transfection reagent with dilution is mixed, incubated at room temperature is after 20 minutes, at last again with this mixture transfection in NSCs;
(4) evaluation of cholinergic neuron.
2. vitro inhibition AF116909 gene induced nerve stem cells directed differentiation according to claim 1 is the method for cholinergic neuron, it is characterized in that described neural stem cell is to separate from tire mouse striatum to obtain.
3. vitro inhibition AF116909 gene induced nerve stem cells directed differentiation according to claim 1 is the method for cholinergic neuron, the cultural method that it is characterized in that described neural stem cell is: with tire mouse striatum in containing F12-DMEM (1: the 1) substratum of 1%B27,20ng/mL EGF, 10ng/mL bFGF, cell suspension is made in piping and druming, in 37 ℃, 50mL/L CO
2Cultivate under the saturated humidity.
4. vitro inhibition AF116909 gene induced nerve stem cells directed differentiation according to claim 1 is the method for cholinergic neuron, it is characterized in that immunofluorescence technique is adopted in the evaluation of described neural stem cell.
5. vitro inhibition AF116909 gene induced nerve stem cells directed differentiation according to claim 1 is the method for cholinergic neuron, and the time that it is characterized in that the transfection of described NSC is 11 days.
6. the method for claim 1, the RT-PCR way of qualitative analysis is adopted in the evaluation of wherein said cholinergic neuron.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010121465A1 (en) * | 2009-04-23 | 2010-10-28 | 中国科学院广州生物医药与健康研究院 | New serum-free medium for inducing pluripotent stem cells quickly with high efficiency and method using thereof |
| CN101557025B (en) * | 2009-05-20 | 2012-07-18 | 南京赛格微电子科技有限公司 | Compact GSM/CDMA shared combiner for transceiving |
-
2005
- 2005-05-17 CN CNA2005100258616A patent/CN1775945A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010121465A1 (en) * | 2009-04-23 | 2010-10-28 | 中国科学院广州生物医药与健康研究院 | New serum-free medium for inducing pluripotent stem cells quickly with high efficiency and method using thereof |
| CN101557025B (en) * | 2009-05-20 | 2012-07-18 | 南京赛格微电子科技有限公司 | Compact GSM/CDMA shared combiner for transceiving |
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