CN1693460A - Regulation and control and use for differentiation by neuro stem cells - Google Patents

Abstract

A gene NM-017084.1 with obviously different expression in the nerve stem cell differentiating procedure is disclosed. It can promote the differentiation of nerve steam cell after its expression is silenced by RNAi.

Description

The regulation and control and the purposes of NM_017084.1 gene pairs cell differentiation of nerve cord

Invention field

The invention belongs to biotechnology and developmental biology, relate to the genetic engineering of cell differentiation of nerve cord development related gene, and utilize technology such as RNA interference, with the target that cell differentiation of nerve cord development related gene and product thereof are grown as the intervention differentiation of stem cells, grow thereby regulate and control cell differentiation of nerve cord.

Background of invention

Neural stem cell is meant to have the ability that is divided into neurone, astroglia cell, oligodendrocyte, and can carry out self, is enough to provide the regenerable cell of brain tissue cell.Grow in mammalian central nervous system, keep the function of central nervous system to have vital role.Cell differentiation of nerve cord is a very complicated process.The factor of the differentiation of stem cells that affects the nerves is a lot, can promote that as CNTF cell differentiation of nerve cord is a spongiocyte, and BDNF then promotes to be divided into neurone.We have found the specifically expressing (seeing patent application) of NM_017084.1 gene in the Embryonic Neural Stem Cells Differentiation.Specificity is intervened the technology of this genetic expression, and for the concrete function of this gene of research, and specificity regulates and control this expression of gene, thereby regulates growth, the differentiation of neural stem cell, and is extremely important.

Because neural stem cell inherent in-vitro separation is cultivated difficulty, it is technical at present its report of doing often to be confined to separation and Culture.

Summary of the invention

The objective of the invention is to confirm that the NM_017084.1 gene is relevant with cell differentiation of nerve cord.Another object of the present invention is to utilize behind reticent this gene of RNAi technology influence to cell differentiation of nerve cord.

The present invention includes the following step: separate and culture of neural stem cells neural; With the Northern technical evaluation NM_017084.1 gene relevant, be synthetic and its homologous RNA of template with this gene, with the differentiation and development of these synthetic nucleic acid fragments intervention neural stem cell, the effect of research regulation and control with cell differentiation of nerve cord.NM_017084.1 genes encoding glycine-N-methyltransgerase utilizes the high cysteine of substrate S-adenosine of this enzyme to suppress glycine-N-methyltransgerase on the other hand, studies its influence to cell differentiation of nerve cord.Concrete comprise following embodiment:

Embodiment

Materials and methods

One, the separation and Culture of neural stem cell and evaluation

1. the neural stem cell cultivation (not breaking up the cultivation of neural stem cell) of going down to posterity

Formerly be commissioned to train that foster (nutrient solution is basic culture solution+20ngmL ^-1EGF and bFGF) after the week, collecting cell group blows and beats into single cell suspension with the rifle head, with pipettor cell suspension is collected the centrifuge tube of 50mL, 1500rpm, 5 minutes again, abandon supernatant, then with fresh culture suspension cell precipitation, in the aseptic culturing bottle with the transfer pipet transfer.

Observations finds in the former process of supporting of being commissioned to train to observe in the 1st day the rounded bright spot of cell; 2nd, as seen several plastidogenetic dumbbell shaped forms were arranged on the 3rd day, most individual cells are adherent, and the part cell has projection to generate; 4th, the suspension ball that can see about tens cells on the 5th day forms, and the minicell regimental commander become medium-sized cell mass in the time of the 7th day cell mass further become big, the neural ball that suspends also obviously increases and can repeatedly go down to posterity.

2. the cultivation of differentiation neural stem cell

Formerly be commissioned to train that foster (nutrient solution is EGF and the bFGF of basic culture solution+20ng/mL) after two weeks, collecting cell group blows and beats into single cell suspension with the rifle head, again with two weeks of base culture base that contain 10% foetal calf serum.In the culturing process of neural stem cell, having added experimental group that foetal calf serum cultivates and the control group that does not add foetal calf serum, can to observe cell preceding 3 days of cultivation rounded.During by the 4th day, most of cell attachment, and the part cell has projection to generate, the rear section cell mass had projection to form in the 7th day, and cell mass and unicellular mutual intersection projection occurs and are woven into netted and tightly are attached on the culturing bottle.

3. identify the indirect immunofluorescence cytochemistry method of neural stem cell

(1) principle

Known antibodies or antigen are checked corresponding antigen or antibody on the cell or tissue sample with the fluorescein thing that makes marks, and under fluorescent microscope, because the antigen and antibody specific combination, the fluorescein of combining site sends bright fluorescence because of the stimulated luminescence irradiation.From position and the quantity that marker exists, can determine location and distribution in histocyte.Common mark fluorescent have isothiocyanic acid (FITC) and tetraethylrhodamine (RB ₂₀₀), the former sends wavelength of fluorescence is 490-619 nanometer (yellow-green fluorescence).Latter's emitting fluorescence wavelength is 540-660 nanometer (fluorescent red-orange).FITC and RB ₂₀₀Be usually used in labelled immune fluorescin Ig.

(2) method

Dye the day before yesterday, with passage, plant on the slide of Poly-L-lysine bag quilt that (the Poly-L-lysine bag is by the processing of slide: slide glass is placed 0.5% Hcl, soaked overnight, flowing water flushing is also cleaned with distilled water, and high-temperature sterilization is immersed in the poly-lysine and was put in the electric heating incubator 2-3 hour, remove unnecessary poly-lysine, and use the 0.01%PBS rinsing).Dye the same day, and took out and treat transfect cell, 40gL is used in PBS rinsing twice ^-1Paraformaldehyde 96 is fixed 15 minutes, absorbs stationary liquid, PBS rinsing twice, and the limit side washing is vibrated gently, uses 10mgL ^-1BSA/3mgL ^-1Triton * 100 were sealed 1 hour, added one again and resisted, hatched under the normal temperature 90 minutes, and PBS rinsing three times, each 10 minutes, remove the antibody of unreacted non-specific adsorption, reduce background dyeing.Add then and be connected with the corresponding two anti-of FITC fluorescein, hatched 30 minutes, abandon two anti-, PBS rinsing three times, each 10 minutes, the glycogelatin mounting, fluorescent microscope is observed down.Nestin albumen is the marker protein of neural stem cell, and dyeing by the single mark of immunofluorescence has confirmed to observe the positive cell of a large amount of Nestin in the control group that adopts serum-free medium, and cell is loose shape or overlap population.

Two, the separation of total RNA extraction, purifying and mRNA

1. be used to extract the cell cultures of total RNA

Get the striatum of the SD rat of E14 under the aseptic condition, mechanical process will be organized and blow and beat into single cell suspension, with 3 * 10 ⁵ML ^-1Cell concn, in the basic culture solution I that has added 20ng/mL EGF and 10ng/mLbFGF, the culturing bottle that nutrient solution will be housed then places 37 ℃, 5%CO with cell seeding ₂Incubator in cultivate.After cultivating a week, collecting cell group, blow and beat into single cell suspension with the rifle head, after repeating aforesaid operations 3 times then, the neural stem cell group that suspends is collected, a part of cell is used to extract total RNA, after another part is is then blown and beaten into single cell suspension, be planted among the basic culture solution I that has added 20ng/mLEGF, 10ng/mL bFGF and 10% foetal calf serum (FCS) with same cell density, in 37 ℃, 5%CO ₂Incubator in cultivated for two weeks after, collect adherent noble cells and extract total RNA.

2. total RNA extracts

Cell counting, the efferent nerve stem cell, add then Trizol (if suspension cell, about 5～10 * 10 ⁷Add 1mL Trizol in the individual cell; If attached cell, about 1mL Trizol/10cm ²), aspirate mixing repeatedly with the rifle head, add glycogen (final concentration 250 μ g/mL) again, thermal agitation or homogenizer homogenate.Deng in the centrifuge tube that after the lysis fully cell pyrolysis liquid is transferred to 1.5mL (1mL in every pipe), add precooling chloroform/primary isoamyl alcohol 200uL, vibrated 30 seconds, 12000g, 4 ℃ centrifugal 5 minutes, take out supernatant in another test tube, add the equal-volume Virahol in supernatant liquor, vibrated 10 seconds, and placed 30 minutes for-20 ℃.12000g, 4 ℃ centrifugal 10 minutes, abandon supernatant (prevent precipitation from losing).Wash (each 12000g, 4 ℃ centrifugal 2 minutes) twice with 70% ethanol, drying adds 0.1%DEPC water, deposits under-70 ℃ standby.

3. the processing (removing a small amount of genomic dna) of total RNA

Handle total RNA with the DNA enzyme that does not contain the RNA enzyme and remove a small amount of genomic dna, concrete grammar is as follows:

In the Eppendorf pipe of a sterilization, add:

10×PCR?buffer 5.0μL

50mmol/L?MgCl ₂ 1.5μL

RNasin 5.0U

DNA enzyme I (RNase-Free) 4.5U

Total RNA 20 μ g

Add DEPC and handled distilled water to 50 μ of institute L → mixing → 37 ℃, 30 minutes → add distilled water to the 50 μ L that DEPC handled → add phenol/chloroform (3: 1) 100 μ L → 4 ℃, 10000g, 20 minutes → transfer supernatant, add equal-volume chloroform → 4 ℃, 10000g, 20 minutes → transfer supernatant, add 15 μ L 2mmol/L sodium acetates, 250 μ L dehydrated alcohols →-20 ℃, 1 hour → 4 ℃, 10000g, 20 minutes → 75% ethanol wash precipitation once → precipitation is dissolved in the distilled water that 20 μ L DEPC handled standby.

4.RNA evaluation and quantitatively

Adopt colorimetry and electrophoretic method to identify the quality of total RNA

1. colorimetry

Above-mentioned RNA solution is obtained the value of A260/A280 through suitably surveying absorbancy (A) in 260nm and 280nm wavelength place after the dilution (50～100 times).

2. electrophoretic method

(1) preparation of gel

Take by weighing agarose 1.2 grams, add 5 * formaldehyde gel electrophoretic buffer 12mL, the distilled water 37.3mL that DEPC handled, heating for dissolving.Treat to add when solution is cooled to 60 ℃ the formaldehyde stock solution 10.7mL of 12.3mol/L, the bromine second pyridine 3.0 μ L of 10mg/mL, mixing left standstill in room temperature about half an hour, and gel is solidified.

(2) preparation of sample and upward sample

In the Eppendorf pipe of a sterilization, add

RNA sample 4.5 μ L

5 * formaldehyde gel electrophoretic buffer, 2.0 μ L

Formaldehyde 3.5 μ l

Methane amide 10.0 μ L

In 65 ℃ of insulations 15 minutes, concentrated on liquid in pipe and manage at the end in centrifugal 5 second behind above-mentioned each composition mixing, places ice abundant then immediately, add 2 μ L sterilization and 10 * formaldehyde gel sample-loading buffer of handling through EDPC, mixing.With gel prerunning 5 minutes (5V/cm), go up sample then.

(3) electrophoresis

In 1 * formaldehyde gel electrophoretic buffer with the field intensity electrophoresis of 4V/cm.Observe electrophoresis result down in ultraviolet lamp after 40 minutes.

5.RNA quantitatively:

After measuring the A260 value of RNA solution, obtain its concentration: the concentration of RNA solution (μ g/mL)=A260 * 40 * extension rate according to following formula

6. the extracting of plasmid

This laboratory previous work shows, Embryonic Neural Stem Cells Differentiation relates to the gene of a differential expression surplus in the of 1400, in order further to study the function that these genes are correlated with in Embryonic Neural Stem Cells Differentiation, select 15 gene clones, carry out plasmid extraction in order to hybridization.

Three, anti-Northern hybridization

1. be the template synthesising probing needle with total RNA

(1) at room temperature, preparation Master Mix in the centrifuge tube of 0.5mL

perr×n 3r×n(+10％)

5×Reaction?Buffer 10μL 33μL

10×Labeling?Mix 5μL 16.5μL

DTT(100mM) 2.5μL 8.2μL

Total?Volume 17.5μL 57.7μL

(2) the PCR instrument is preheating to 70 ℃;

(3) in each reaction, the PCR pipe of every 0.5mL adds following reagent:

50μg RNA

5μL CDS?PrimerMix

Add deionized water then to 30 μ L.

(4) the above-mentioned PCR pipe that slightly vibrates makes to mix, and is centrifugal again;

(5) the PCR pipe 70 ℃ of incubations 10 minutes;

(6) the PCR pipe is put 1 minute on ice then;

(7) PCR manages and the centrifuge tube of Master Mix0.5mL is housed at 48 ℃, incubation 5 minutes, add 2.5 μ L Power script Reverse TraNSCsriptase in the centrifuge tube of each 0.5mL, the vibration mixing respectively adds 20 μ L Master Mix to each PCR pipe then fast;

(8) mixing above-mentioned substance, the PCR pipe was 48 ℃ of incubations 45 minutes;

(9) add 1 μ L0.5M EDTA (pH8.0) termination reaction at last.

2. some film

(1) with 15 genes and a control point on the nylon membrane of 10 * 2 (cm), each sample 3 μ L divides the some film three times, all dries up with hair dryer then at every turn;

(2) DNA on the film is carried out alkaline denaturation, place to be soaked with 0.1mol/L, last 5 minute of filter paper of 1.5mol/LNacl;

(3) repeating step 2.Airing then;

(4) filter membrane is placed be soaked with 0.5mol/L Tris-HCl (pH7.0), last 15 minute of filter paper of 3mol/LNaCl, airing again;

(5) wrap 60-80 ℃ of vacuum-drying 2 hours with clean aluminium platinum paper.

3. prehybridization

(1) get the plastics bag of a suitable size, the nylon membrane with 2 times of SSC solution-wet behind the airing is placed in the band bag, can not be overlapping with this film;

(2) pour in the bag with an amount of prehybridization solution, the configuration of prehybridization solution is as follows: 100-200 μ g/L is denatured calf thymus dna, and the calf thymus DNA 0.2mL that gets 10mg/L concentration boiled 5 minutes in 100, made its sex change;

5 times of Denhardt ' s: get 100 times of Denhardt ' s 0.5mL;

5 times of SSC: get 20 times of SSC solution 2.5mL;

10mmol/L Tris-HCl (pH7.5): get 1mol/LTris-HCl (pH7.5) 0.1mL;

0.1%SDS: get 20%SDS solution 0.05mL;

DH ₂O adds 6.85mL, is settled to 10mL;

(3) bubble in the plastics bag is driven away, on one side the plastics bag opening is sealed with the plastic hot sealer;

(4) plastics bag is placed on (constantly vibration) in 68 ℃ of water-baths, prehybridization 2-4 hour.

4. hybridization

(1) plastics bag that prehybridization is crossed is cut off, and to removing prehybridization solution, and extracts as far as possible;

(2) preparing hybrid solution

5 times of Denhardt ' s: get 100 times of Denhardt ' s 0.5mL

6 * SSC: get 20 times of SSC solution 3.0mL

0.5%SDS: get 20%SDS solution 0.25mL

0.025mol/LHCl: get 5mol/LHCl0.05mL

Add dH ₂O6.2mL is settled to 9mL;

The calf thymus DNA and the biotin labeled dna probe solution 1mL that add sex change again;

(3) an amount of hybridization solution is added in the plastics bag of (prehybridization is crossed) nylon membrane of above-mentioned containing, the bubble in the plastics bag is driven away as far as possible, the heat-sealing mouth;

(4) plastics bag is placed in 60 ℃ the hybrid heater, spends the night.

5. the wash-out of hybridizing the back nylon membrane

(1) wears gloves and cut off plastics bag, take out nylon membrane rapidly, put into another new plastics bag with tweezers;

(2) earlier with 2 * SSC, 60 ℃ of wash-outs twice of 1%SDS solution, each 15 minutes;

(3) use 0.1 * SSC again, 60 ℃ of wash-outs twice of 0.5%SDS solution, each 15 minutes.

6. chemiluminescence detection

(1) cut off plastics bag, outwell elutriant, add 0.5mLGEAblocking Solution Q immediately, incubation is 40 minutes under the room temperature, and constantly rotates in hybrid heater;

(2) discard GEAblocking Solution Q, add the BindingBuffer that 2mL contains AP (dilution in 1: 7500), room temperature incubation 10 minutes, and slight rotation continuously;

(3) with 0.5mL1 * BufferF wash-out four times, each 5 minutes;

(4) with 0.5mLBuffer G wash-out twice, each 5 minutes;

(5) discard Buffer G, add 0.3mL CDP-Star solution room temperature incubation 2-5 minute;

(6) discard CDP-Star solution again, blot debris, the heat-sealing mouth with filter paper;

Radioautograph in (7) 1.5 hours.

7. radioautograph

The X-mating plate is covered on the nylon membrane, step up then in the darkroom, exposed 5-10 minute, flushing X-mating plate.

Technique effect

By above-mentioned technology, we see that 1,3,7,8,10,11,13,14, No. 15 clone gene expresses obviously downward modulation in the process of cell differentiation of nerve cord, 2,4,5,6,9,12, number clone gene expresses obviously rise in atomization.Especially No. 15 clone gene differential expression is particularly remarkable.So this paper studies No. 15 effect of clone gene in cell differentiation of nerve cord emphatically, in Genebank, find its gene number and be NM_017084.1.

Four, synthetic double-stranded RNA (dsRNA)

1. the preparation of linear dna profiling

The plasmid of clone's NM_017084.1 gene is adopted EcoRI and Xhol single endonuclease digestion respectively, and it is as follows that enzyme is cut system:

(1) EcorRI single endonuclease digestion:

Y ⁺/Tanqo ^TMBuffer 2μL

Sample DNA 5 μ L

EcorRI 0.5mL

Water 12.5 μ L

Total?Volume 20μL

(2) Xhol single endonuclease digestion:

Y ⁺/Tanqo ^TMBuffer 2μL

Sample DNA 5 μ L

Xhol 0.5mL

Water 12.5 μ L

Total?Volume 20μL

Above-mentioned enzyme is cut the system mixing be placed on 37 ℃ of incubations 1 hour.

2. gel electrophoresis and band reclaim

Carry out electrophoresis with aforementioned same method, the concentration of sepharose is 1.0%, after enzyme is cut the liquid electrophoresis and confirmed that plasmid has been cut open.Adopt UNIQ-10 pillar DNA glue to reclaim test kit and from sepharose, reclaim DNA.

Recycling step is as follows:

(1) cuts off the DNA agar block that contains the need recovery with clean scalpel, put into the 1.5mL centrifuge tube.The volume of agar block is as far as possible little, but tries not so lose DNA, when judging the position of dna fragmentation, uses long wave ultraviolet light, and the irradiation time under UV-light is short as much as possible;

(2) add Binding Buffer in the ratio that does not have 400 μ L/100mg sepharoses, place 50-60 ℃ of water to bathe 10 minutes, glue is thoroughly melted, when adding thermosol, per two minutes mixings once.The time of heating can extend to 15 minutes, to guarantee that glue all melts;

(3) gelating soln that melts is transferred in the UNIQ-10 post that is mounted in the 2mL collection tube, room temperature was placed 2 minutes, centrifugal 1 minute of 8000rpm room temperature;

(4) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, UNIQ-10 is put into same collection tube, add 500 μ L elutriants, centrifugal 1 minute of 8000rmp room temperature;

(5) repeating step 4 once;

(6) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, UNIQ-10 is put into same collection tube, centrifugal 15 seconds of 12000rmp room temperature;

(7) the UNIQ-10 post is put into the aseptic centrifuge tube of a new 1.5mL, added the went out distilled water of bacterium of 30 μ L, placed 2 minutes for 37 ℃ in man of pillar film central authorities;

(8) the 2000rmp room temperature is centrifugal 1 minute, and the liquid in the centrifuge tube is the dna fragmentation of recovery.

3.RNA synthetic

(1) will be stored in-20 ℃ MEGAscript ^TMT3 and T7 test kit take out, wherein T3 and t7 rna polymerase taken out directly place on ice, four kinds of nucleic acids (ATP, CTP, GTP, UTP) dissolving is placed on ice fully, and 10 * reaction buffer then dissolves and is placed on room temperature, and the experiment back is regained-20 ℃.

(2) following prescription is assembled the reaction system of 20 μ L:

ddH ₂O 4μL

ATP 2μL

CTP 2μL

GTP 2μL

UTP 2μL

10 * reaction buffer, 2 μ L

DNA masterplate 4 μ L

T3 or t7 rna polymerase 2 μ L

Total?Volume 20μL

Note adding each composition the linear dna fragmentation that the corresponding EcoRI single endonuclease digestion of t7 rna polymerase makes, the linear dna fragmentation that the corresponding Xhol single endonuclease digestion of T3 RNA polymerase makes by above-mentioned order

(3) above-mentioned system is assembled after, mixing gently of short durationly centrifugally makes liquid hold-up at the pipe end, then pipe is placed 37 ℃ of following incubations 4 hours.

4.RNA recovery

(1) in each 20 μ L reaction system, adds 25 μ L LiCl;

(2) be put in behind the mixing-20 ℃ freezing 1 hour;

(3) 4 ℃, 14000r/ minute centrifugal 15 minutes, with precipitated rna;

(4) the careful supernatant liquor of removing adds 1mL70% ethanol in 4 ℃ of washing double-stranded RNAs precipitations once, and 4 ℃ then, 14000r/ minute centrifugal 15 minutes, so that farthest remove unconjugated single nucleic acid;

(5) the careful supernatant liquor that eliminates precipitates resuspended and aseptic distilled water with double-stranded RNA, puts-70 ℃ of preservations.

5.RNA renaturation

Two single stranded RNA fragment equivalent of every clock exogenous dna fragment correspondence are placed together, fully behind the mixing, place water-bath, be warming up to 90 ℃ and keep 4 minutes, yet make it gained double-stranded RNA is kept at-70 ℃ then then from being cooled to room temperature (needing 4-5h approximately) slowly.

6. the synthetic result of electrophoresis observation dsRNA

Produce 1.2% sepharose, the glue method is the same, spend the night but in advance electrophoresis chamber and the gel casting platform sealed were immersed among 0.1% the DEPC before glue and place under 37 ℃ of environment, the DEPC that will residue in electrophoresis chamber and glue groove with distilled water again before glue washes away.Observe with conventional RNA electrophoretic method.

Five, electrotransformation

(1) cell suspension is centrifugal, make cell precipitation be resuspended in electricity and transform in the damping fluid, making density is 1 * 10 ⁷ML ^-1Cell suspension;

(2) 8.0 μ g dsRNA are joined to make cumulative volume in the cell suspension be 400 μ L, mixing.Do the blank experiment simultaneously;

(3) cell suspension in the step 2 is moved into the electricity conversion cup that the specification of placing in advance is 400 μ L volumes on ice;

(4) under the room temperature electricity is transformed cup and put on the glass stand of electric conversion system, with 200 volts, 80 microseconds shock by electricity once;

(5) after electric shock finishes, will transform cup and place 10 minutes on ice;

(6) with 10 times of complete culture solution dilutions by cell transformed, and wash electricity with this nutrient solution and transform glass to shift out allly by transformant, place culturing bottle in 37 ℃, 5%CO ₂Middle cultivation, the observation of cell form and the cultivation of going down to posterity.Transforming the back observes neural stem cell.After cultivating 1 day, the cell major part that transforms the control group of experimental group that dsRNA is arranged and unconverted dsRNA all exists with the unicellular of circle, and the cell mass of minority is only arranged; Cultivate after 3 days, the cell of experimental group has pimple to form, and cell becomes the shuttle shape by circle gradually; After the 5th day, cell most of adherent and the differentiation more obvious; The 9th day

The projection of cell is crossed as netted mutually; The 12nd day all cell all breaks up and is closely adherent.And the cell of control group still exists with undifferentiated form.

Six, reverse transcription PCR (RT-PCR)

1. total RNA (total RNA) extracts:

Extract and purifying by the method for narrating previously.

2. reverse transcription

(1) following reaction system is joined in the EP pipe of a no RNase, its reaction system is:

Total RNA 2 μ g behind the purifying

Oligo(dT)18Primer(0.5μg/μL) 1μL

Add DEPC-H ₂O is 12 μ L totally extremely

With this reaction system mixing, cultivated 5 minutes in 70 ℃ of water-baths, cooled on ice is 30 seconds again;

(2) the EP pipe is placed add each reagent, mixing on ice in the following order;

5×reaction?buffer 4μL

RNase?inhibitor(20U/μL) 1μL

dNTP?mix(10mmol/L) 2μL

(3) this EP pipe is placed 42 ℃ of water-baths cultivated 5 minutes;

(4) append AMV reverse traNSCsriptase (20U/ μ L) 1 μ L, in 42 ℃ of water-baths, cultivated 60 minutes;

(5) 10 minutes stopped reaction of heating in 70 ℃ of water-baths place cooled on ice with this EP pipe again, preserve pending PCR reaction in-20 ℃ of refrigerators.

3.PCR reaction

(1) PCR primer design

Full length sequence according to the NM_017084.1 gene, design is corresponding to the PCR primer of NM_017084.1 gene, its primer I sequence is that TTATGCTGGTGGAAGAGGG and primer I I sequence are GCAAGGGAAGGCTGTATTG, respectively is 19bp, and its PCR product size is 400bp.

(2) PCR reaction system

ddH ₂O 18μL

10×PCR?buffer 3μL

dNTPs(10mmol/L) 2μL

Template DNA 3 μ L

Primer I (100ng/ μ L) 1.5 μ L

Primer I I (100ng/ μ L) 1.5 μ L

Taq archaeal dna polymerase (2U/ μ L) 1 μ L

(3) reaction system is placed the PCR instrument, carry out PCR by following pcr amplification condition.

The pcr amplification condition is:

1. 95 ℃ 3 minutes

2. 30 circulations (95 ℃ 45 seconds; 55 ℃ 2 minutes; 72 ℃ 90 seconds)

3. 72 ℃ 10 minutes

(4) after pcr amplification finishes,, get 3 μ L PCR products and carry out agarose gel electrophoresis, make the molecular weight standard thing with PCR Markers simultaneously PCR product centrifugal a moment.

The result shows the gradually reduction of the expression amount of this gene on different time sections mRNA level along with the time.Can observe this genetic expression by experiment in back 12 days to conversion obviously reduces.

Seven, the high cysteine of S-adenosine (SAH) is to the influence of cell differentiation of nerve cord

(1) SAH is made into the mother liquor of 4ng/mL;

(2) former generation neural stem cell go down to posterity twice, transfer to 24 orifice plates then, density is 10 ⁵Individual/mL;

(3) in the prescription of minimum medium, add SAH,, and establish control group with 4ng/mL concentration;

(4) then from the metamorphosis of cellular form observation of cell;

(5) extract the total RNA of stem cell of control group and experimental group respectively;

(6) carry out RT-PCR and electrophoresis and identify its result.

Discover that Gnmt does not have significant differential expression in differentiation with breaking up in the neural stem cell, so we further study this gene on enzyme level, the concentration of product S AH by increasing the Gnmt enzyme influences cell differentiation of nerve cord after inquiring into the expression of downward modulation Gnmt.Neural stem cell is transferred to 24 orifice plates, in basic training after former foster two weeks of being commissioned to train

The SAH that supports adding 4ng/mL in the based sols does not organize to add SAH as experimental group simultaneously in contrast.In the Culture of neural stem cells process, observe, begin 2-3 days experimental group and the equal well-grown of control group; Cultivate after 5 days, the experimental group cell is fusiformis, has all grown projection, and the part cell is linked to be netted; When cultivating the 8th day, cell space is plentiful, and cell is fusiformis, polygon, many projections, and neurocyte increases; Cultivate after 12 days, the good neurocyte of visible growth is linked to be the netted of densification.And control group remains undifferentiated state in culturing process.

After SAH handles 12 days, to the cell while extracted total RNA of experimental group and control group, carry out RT-PCR then, the result shows that the expression amount of mRNA in experimental group of NM_017084.1 gene obviously reduces.

Brief summary:

This patent has been studied the regulating and controlling effect of gene NM_017084.1 to cell differentiation of nerve cord first on genetic expression and enzyme level.The result shows: NM_017084.1 gene mRNA level, enzyme level expression inhibiting, promote the differentiation of neural stem cell, and can be used for the cell differentiation of nerve cord regulation and control and use.

Claims (3)

1. One with the cell differentiation of nerve cord development related gene.
2. 2. the described method of claim 1, the wherein reticent cell differentiation of nerve cord that promotes of the gene NM_017084.1 of regulation and control.
3. 3. the described method of claim 1, the wherein differentiation of the inhibition of Tiao Kong gene NM_017084.1 encoded protein enzyme promotion neural stem cell.