CN102978164B - Channelrhodopsin-2 (ChR2)-green fluorescence protein (GFP) gene engineered nerve stem cell line and construction method thereof - Google Patents
Channelrhodopsin-2 (ChR2)-green fluorescence protein (GFP) gene engineered nerve stem cell line and construction method thereof Download PDFInfo
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- CN102978164B CN102978164B CN201210459119.6A CN201210459119A CN102978164B CN 102978164 B CN102978164 B CN 102978164B CN 201210459119 A CN201210459119 A CN 201210459119A CN 102978164 B CN102978164 B CN 102978164B
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Abstract
The invention discloses a channelrhodopsin-2 (ChR2)-green fluorescence protein (GFP) gene engineered nerve stem cell line and a construction method of the channelrhodopsin-2-green fluorescence protein (GFP) gene engineered nerve stem cell line. The cell line is the recombined nerve stem cell line C 17.2 of a channelrhodopsin-2 (ChR2) which is capable of stably expressing and a green fluorescence protein (GFP). The recombined ChR2-GFP gene engineered nerve stem cell line C 17.2 is obtained by constructing recombined slow virus, transducing C 17.2 nerve stem cell line and sifting. The channelrhodopsin-2 (ChR2)-green fluorescence protein (GFP) gene engineered nerve stem cell line can be used for in vivo and in vitro studies on the functional integration of a nerve neuron of a transplanted stem cell differentiation source and a nervous system of a host and provides a favorable platform for the study on the functional integration of a nerve neuron of a transplanted stem cell differentiation and a nervous system of a host. Especially with the utilization of a filter paper digestion method, the stem cell recombining method improves the rate of GFP positive cells, is beneficial to streaming Fluorescence Activated Cell Sorting (FACS) separation, and greatly improves the efficiency of an experiment.
Description
Technical field
The present invention relates to biomedicine field, particularly the genetically engineered neural stem cell strain of a kind of ChR2-GFP and construction process thereof.
Background technology
Nervous system degeneration disease is a class disease, comprises the disease such as Parkinson, Alzheimer, there is no at present effective, specific treatment, and stem cell transplantation alternative medicine has good prospect.Be implanted into source of human stem cell in body neurone must and the neurone that retains of host's neural system between foundation have the synaptic contact of function, the transmission that could recover cynapse information.Current multiple research just provides circumstantial evidence aspect morphology, and not yet in function, especially the cynapse aspect of transmission of information is illustrated this problem.The development of light genetics technology can address this problem.Utilize genetics technology to make cell expressing photaesthesia albumen, can realize the optics control of cellular function, professor Deisseroth of Stanford University waits the technology of this combination optics and genetics combination is called to light genetics technology.Photaesthesia albumen is by a class membranin of photoactivation, to be distributed widely in the vision system of prokaryotic organism, plant and animal, at present in light-operated neurone research widespread use be photaesthesia albumen (Channelrhodopsin-2 is called for short ChR2).ChR2 is that a kind of photosensitive film of separating from unicell green alga is expressed cationic channel protein, can express on neuronic cytolemma by Gene transfer techniques, and under irradiating in low-yield 470nm blue light moment (5-15ms), ChR2 passage is opened, Na
+, Ca
2+entering and in born of the same parents, cause that thereby cell depolarization produces action potential Deng positively charged ion, and first biological activity that can not affect the nerves, is therefore the most frequently used photaesthesia albumen at present.
By gene transfection, make stem cell Simultaneous Stabilization express ChR2-GFP, wherein GFP(green fluorescent protein, English is green fluorescent protein, is called for short GFP) for tracking stem cell, ChR2 is for irritation cell; After being implanted in body, neurone stably express ChR2 and the GFP of source of human stem cell, by its position of GFP spike, 470nm laser light stimulates the differentiation of stem cells source neuronal cell of expressing ChR2 simultaneously, record the postsynaptic currents of host neuron by patch clamp technique simultaneously, the neurone of source of human stem cell and the Function Integration Mechanism of host neuron cell are transplanted in research, for clinical cellular replacement therapy degenerative disease provides theoretical foundation.
But, rapid build, screening recombinant cell strain are not nothing the matters, the structure screening efficiency that improves the strain of recombination tissue-engineered nerve stem cell is also one of important content of restructuring stem cell experiment always, good structure, screening method can be saved a large amount of human and material resources, and accelerate the paces of scientific research.
Summary of the invention
Given this, the object of this invention is to provide the genetically engineered neural stem cell strain of a kind of ChR2-GFP, described cell strain is energy stably express photaesthesia albumen (channelrhodopsin-2, and the recombinant C 17.2 neural stem cell strains of green fluorescent protein (green fluorescent protein, GFP) ChR2).
Another object of the present invention is to provide the method for the genetically engineered neural stem cell strain of a kind of ChR2-GFP of structure, comprises the steps:
The first step, the structure of recombinant slow virus and screening:
Inoculation 293FT cell is in being added with 24 orifice plates of 10%FBS+DMEM substratum, and every porocyte inoculating cell number is 1 × 10
5individual; then use 293FT cell described in slow virus packaging plasmid mixture and ChR2-GFP slow virus expression plasmid cotransfection; within 24 hours, be changed to the nutrient solution containing 1%FBS DMEM; within 48 hours, the centrifugal 5min of 5000rpm collects nutrient solution supernatant, and the pvdf membrane of 0.45 μ m filters described supernatant and obtains recombinant slow virus liquid.
With standard virus liquid 1 × 10
8cfu/L is contrast, recombinant slow virus liquid and standard virus liquid are established respectively to 5 gradients by infection multiplicity value: 1,3,5,10 and 20, infect 293FT cell and also after 24 hours, determine virus titer according to the intensity of green fluorescence cell and quantity, when expressing, the cell of GFP exceedes 95%, slow virus drop degree reaches 1.0 × 10
8the recombinant slow virus liquid of cfu/L is for next step experiment.
Second step, structure and the screening of recombinant C 17.2 neural stem cell strains:
C17.2 neural stem cell strain 10%FBS(foetal calf serum)+5% horse serum+DMEM culture medium culturing, at 37 DEG C, 5%CO
2when 6 orifice plate Tissue Culture Plate culturing cell to 50% fusion, the new 10%FBS(foetal calf serum of 1ml is changed in every hole)+5% horse serum+DMEM substratum, add recombinant slow virus liquid described in 1ul, add virus infection auxiliary Polybrene to make final concentration is 8 μ g/ml simultaneously, after transfection 6 hours, be changed to DMEM culture medium culturing when adherent to cell 80%-90% with density 2 × 10
4/ ml is passaged in 10cm Tissue Culture Dish.
Under fluorescent microscope at the cell mass of the bottom of described 10cm Tissue Culture Dish marker expression GFP, PBS cleans after 3 times, 0.05% pancreas enzyme-EDTA is dropped on the aseptic filter paper sheet that is cut into diameter 3mm, the filter paper that contains pancreatin is attached to above the cell mass of mark GFP, after 37 DEG C of 3min, filter paper is added in the new 10cm plate that contains 10%FBS DMEM substratum, stop digestion, piping and druming gently, the cell that is attached on filter paper is dispersed in plate, after 4 days, sub-elect the cell of expressing GFP 0.5% ratio by FACS, and reach in 96 orifice plates with the density of 1 cells/well.Detect with full cell light reflex patch clamp qualification and obtain the genetically engineered neural stem cell strain of recombinant C hR2-GFP by the two marks of immunofluorescence again.
In the present invention, the genetically engineered neural stem cell strain of ChR2-GFP can Simultaneous Stabilization be expressed ChR2-GFP, and can stablize and go down to posterity, and 470nm blue light stimulates and can cause that cell produces inward electric current.The inventive method builds restructuring stem cell, especially utilizes filter paper digestion method to improve the ratio of GFP positive cell, is conducive to streaming (FACS) sorting, has improved conventional efficient.The neurone that cell strain of the present invention can be used for transplanting differentiation of stem cells source and host's nervous function integrate at body and vitro study, for the neurone in differentiation source after stem cell transplantation and the research of host's nervous function integration provide a good platform.
Brief description of the drawings
Fig. 1 is the opticmicroscope figure of the genetically engineered neural stem cell strain of ChR2-GFP of the present invention;
Fig. 2 is the fluorogram that the two marks of immunofluorescence of the present invention detect the genetically engineered neural stem cell strain expression ChR2-GFP albumen of ChR2-GFP and neural stem cell marker Nestin albumen;
Fig. 3 is the map of current to 470nm blue light irritant reaction ability of the genetically engineered neural stem cell strain of the full cell light reflex of the present invention patch clamp qualification ChR2-GFP;
Embodiment
Describe the present invention in detail below in conjunction with embodiment and accompanying drawing, these embodiment and accompanying drawing only play illustrative effect, are not limited to range of application of the present invention.The invention is not restricted to following embodiment or embodiment, all amendments of making without prejudice to spirit of the present invention and distortion, within all should being included in the scope of the invention.
The present invention builds the genetically engineered neural stem cell strain of ChR2-GFP experimental example:
The first step, the structure of recombinant slow virus and screening:
Inoculation 293FT cell is in being added with 24 orifice plates of 10%FBS+DMEM substratum, and every porocyte inoculating cell number is 1 × 10
5individual, then use slow virus packaging plasmid mixture and ChR2-GFP slow virus expression plasmid (buying the company in Addgene) cotransfection 293FT cell; Within 24 hours, be changed to the nutrient solution containing 1%FBS DMEM; 48 hours collecting cells, the centrifugal 5min of 5000rpm, the pvdf membrane of 0.45 μ m is used as virus liquid to be measured (recombinant slow virus liquid) after filtering supernatant; With standard virus liquid 1 × 10
8cfu/L is contrast, virus liquid to be measured and standard virus liquid are established respectively to 5 gradients by infection multiplicity value: 1,3,5,10 and 20, infect 293T cell and observed the intensity of green fluorescence cell in 24 hours and quantity to determine virus titer, the cell of expressing GFP is exceeded to 95%, and virus liquid titre reaches 1.0 × 10
8the recombinant slow virus liquid of cfu/L is for next step experiment.
Second step, the strain of transduction C17.2 neural stem cell, fluorescent activation cell sorting system (fluorescence activated cell sorting is called for short FACS) screening stable cell strain:
C17.2 neural stem cell strain 10%FBS(foetal calf serum)+5% horse serum+DMEM culture medium culturing, at 37 DEG C, 5%CO
2merge 6 orifice plate Tissue Culture Plate culturing cell to 50% left and right, is first changed to 1ml10%FBS(foetal calf serum)+5% horse serum+DMEM substratum, then get 1ul(1.0 × 10
8cfu/L) slow virus that contains ChR2-GFP gene adds in nutrient solution, add virus infection auxiliary Polybrene(final concentration 8 μ g/ml) simultaneously, after transfection 6 hours, be changed to normal DMEM culture medium culturing cell, after cell covers with, be passaged in 10cm Tissue Culture Dish (2 × 10
4/ ml), simultaneously under fluorescent microscope at the cell mass of the bottom of 10cm plate marker expression GFP, then PBS cleans 3 times, then 0.05% pancreas enzyme-EDTA (Gibco) is dropped on the aseptic filter paper sheet that is cut into diameter 3mm, the filter paper that contains pancreatin is attached to above the cell mass of mark GFP, after 37 DEG C of 3min, filter paper is added in the new 10cm plate that contains 10%FBS DMEM substratum, stop digestion, piping and druming gently, the cell that is attached on filter paper is dispersed in plate, after 4 days, sub-elect the cell of expressing GFP 0.5% ratio by FACS, and reach in 96 orifice plates with the density of 1 cells/well.The opticmicroscope figure of the genetically engineered neural stem cell strain of ChR2-GFP that FACS sub-elects as shown in Figure 1, is mono-clonal sample and grows up.
The cell of sorting is carried out to the expression of the two mark detection of immunofluorescence ChR2-GFP albumen, experimental technique is
The genetically engineered stem cell of a.ChR2-GFP is with 1 × 10
5the density of/ml is inoculated in 24 orifice plates of putting in advance the Circular glass creep plate that the diameter of sterilizing is 12mm, fixes with stationary liquid 4% paraformaldehyde solution, then abandons stationary liquid, and PBS shakes and washes 3 × 5min.
B. every hole adds the 0.1%triton of 4 DEG C of preservations of 200 μ l, incubated at room 10min, and PBS shakes and washes 3 × 5min.
C. to add 200 μ l concentration be 10% normal goats serum working fluid in every hole, incubated at room 30min.
D. suck unnecessary liquid, add 300 μ l and contain 1:200 Rabbit-anti-GFP primary antibodie (green skies biotechnology research institute) and 1:300 Mouse-anti-Nestin (Abcam) primary antibodie, hatch after 1h for 37 DEG C, 4 DEG C are spent the night.
E. next day, PBS shakes and washes 3 × 5min.
F. add the goat anti-rabbit igg of FITC-mark and the goat-anti mouse of Cy3-mark two of 300 μ l 1:200 anti-, hatch 1h for 37 DEG C.
G.PBS shakes and washes 3 × 5min.
After h.DAPI dyeing, PBS shakes and washes 3 × 5min.
The water-soluble mountant mounting of i.GVA, Olympus DP71 digital micrograph photographic camera is taken a picture.
As shown in Figure 2, blue-fluorescence is nucleus to the fluorogram of the expression of the two mark detection of immunofluorescence ChR2-GFP albumen, and green fluorescence is the expression that GFP albumen illustrated ChR2-GFP albumen, and red fluorescence is the expression of neural stem cell marker Nestin albumen.Illustrate thus and successfully obtain the genetically engineered neural stem cell strain of ChR2-GFP.
The cell of sorting is carried out to full cell light reflex patch clamp qualification light reflex ability, and experimental technique for to be placed in cell climbing sheet perfusion groove, with oxygen-saturated extracellular fluid perfusion (1-2ml/min) under room temperature in darkroom.Selection any surface finish, the cell that refractivity is strong carry out the experiment of full cell patch pincers, and glass microelectrode draws and charge liquid (Potassium Gluconate 140mM, MgCl in appropriate electrode
22mM, K
2aTP2mM, EGTA 1.1mM, HEPES 10mM), regulate narishige, make eletrode tip approach cell, utilize specific test square wave electric pulse to form sealing-in, Giga Europe (10
9Ω) sealing-in stable 1min after completing, inhales broken cell film gently, forms full cell patch pincers pattern, after cytotostatic 2min, and its resting membrane electric potential of immediate record (RMP), membrane capacitance (Cm) and membrane impedance.Under voltage clamp pattern, membrane potential is clamped down at-25mV, give 470nm blue light stimulates simultaneously, records cell light reflex electric current.To the map of current of 470nm blue light irritant reaction ability as shown in Figure 3, photoresponse electric current maximum can be 150pA to cell, also illustrates and successfully obtains the genetically engineered neural stem cell strain of ChR2-GFP.
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (1)
1. a method that builds the genetically engineered neural stem cell strain of ChR2-GFP, is characterized in that: comprise the steps:
The first step, the structure of recombinant slow virus and screening:
Inoculation 293FT cell is in being added with 24 orifice plates of 10%FBS+DMEM substratum, and every porocyte inoculating cell number is 1 × 10
5individual, then use 293FT cell described in slow virus packaging plasmid mixture and ChR2-GFP slow virus expression plasmid cotransfection, within 24 hours, be changed to the nutrient solution containing 1%FBS DMEM, within 48 hours, the centrifugal 5min of 5000rpm collects nutrient solution supernatant, and the pvdf membrane of 0.45 μ m filters described supernatant and obtains recombinant slow virus liquid;
With standard virus liquid 1 × 10
8cfu/L is contrast, described recombinant slow virus liquid and standard virus liquid are established respectively to 5 gradients by infection multiplicity value: 1,3,5,10 and 20, infect 293FT cell and also after 24 hours, determine virus titer according to the intensity of green fluorescence cell and quantity, when expressing, the cell of GFP exceedes 95%, slow virus drop degree reaches 1.0 × 10
8the recombinant slow virus liquid of cfu/L is for next step experiment;
Second step, structure and the screening of recombinant C 17.2 neural stem cell strains:
10%FBS+5% horse serum+DMEM culture medium culturing for the strain of C17.2 neural stem cell, at 37 DEG C, 5%CO
2when 6 orifice plate Tissue Culture Plate culturing cell to 50% fusion, the new 10%FBS+5% horse serum+DMEM substratum of 1ml is changed in every hole, add recombinant slow virus liquid described in 1ul, add virus infection auxiliary Polybrene to make final concentration is 8 μ g/ml simultaneously, after transfection 6 hours, be changed to DMEM culture medium culturing when adherent to cell 80%-90% with density 2 × 10
4/ ml is passaged in 10cm Tissue Culture Dish;
Under fluorescent microscope at the cell mass of the bottom of described 10cm Tissue Culture Dish marker expression GFP, PBS cleans after 3 times, 0.05% pancreas enzyme-EDTA is dropped on the aseptic filter paper sheet that is cut into diameter 3mm, the filter paper that contains pancreatin is attached to above the cell mass of mark GFP, after 37 DEG C of 3min, filter paper is added in the new 10cm plate that contains 10%FBS DMEM substratum, stop digestion, piping and druming gently, the cell that is attached on filter paper is dispersed in plate, after 4 days, sub-elect the cell of 0.5% ratio by FACS, the cell of described 0.5% ratio can be expressed GFP, and reach in 96 orifice plates with the density of 1 cells/well, detect with full cell light reflex patch clamp qualification and obtain the genetically engineered neural stem cell strain of recombinant C hR2-GFP by the two marks of immunofluorescence again.
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