CN103952444B - A kind of plasma nanometer gold dimer is for the method for genes within cells expression regulation - Google Patents
A kind of plasma nanometer gold dimer is for the method for genes within cells expression regulation Download PDFInfo
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Abstract
A kind of plasma nanometer gold dimer, for the method for genes within cells expression regulation, belongs to material chemistry technical field.The present invention includes that golden nanometer particle modifies the coupling of cell-penetrating peptide, golden nanometer particle and regulating and controlling sequence, and dual-functional nanometer gold dimer assembles, the regulation and control to genes within cells of the nanometer gold dimer, the sign of genes within cells regulation and control.The present invention selects golden nanometer particle as excimer, modify having the antisense nucleotide that can regulate and control survivin gene on single golden nanometer particle, again it is assembled with being connected the particle having cell-penetrating peptide so that it is can the most quickly play the effect of genes within cells regulation and control.The invention provides the nanometer gold dimer assemble method with genes within cells regulating and controlling effect, obtain structure homogeneous, the nanometer gold dimeric structure of superior performance.
Description
Technical field
The present invention relates to a kind of plasma nanometer gold dimer for the method for genes within cells expression regulation, belong to material
Technical field of chemistry.
Background technology
Gene expression is prevalent in each life process of cell, is that cell is converted into the information in hereditary material
There is the process of the protein of certain function.Gene expression regulation is mainly being transcribed, is being translated and profit on mRNA level of processing
With relevant DNA regulation sequence, enzyme and regulation albumen, vital movement rule is regulated and controled in order.Utilize the anti-of synthetic
Justice nucleotide, is incorporated on specific target gene by base complementrity principle, it is possible to the expression of Effective Regulation target gene.
On the other hand, gold nano-material has good biocompatibility because of it, is widely used in biological medicine carrying, and disease treatment etc. is received
Rice medical domain.By the way of self assembly, the particle assembling that two kinds have difference in functionality is become dimeric structure, so that two
Dimeric structure shows the effect of efficient genes within cells regulation and control.
Summary of the invention
In place of it is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of plasma nanometer gold dimer is for thin
The method of intracellular gene expression regulation.
Technical scheme: a kind of plasma nanometer gold dimer is used for the method for genes within cells expression regulation,
Step is as follows:
(1) golden nanometer particle modifies cell-penetrating peptide: by golden nanometer particle and the mercapto-polyglycol of the synthesis of citric acid reducing process
PEG 5000, cell-penetrating peptide TAT and DNA1 mixing, must arrive surface and be modified with the Au-TAT-DNA1 complex of cell-penetrating peptide after 24h；
(2) golden nanometer particle and the coupling of regulating and controlling sequence: by the antisense nucleotide of golden nanometer particle Yu survivin gene
Anti-DNA and DNA2 coupling, must arrive surface and be modified with the Au-anti-DNA-DNA2 complex of regulating and controlling sequence；
(3) dual-functional nanometer gold dimer assembles: Au-TAT-DNA1 complex step (1) obtained and step (2) obtain
The Au-anti-DNA-DNA2 complex mixing arrived, obtains dual-functional nanometer gold dimer package assembly；
(4) the nanometer gold dimer regulation and control to genes within cells: the dual-functional nanometer gold dimer that will obtain in step (3)
After package assembly and cell hatch 3h jointly, with trypsin digestion and cell, obtain the cell after the regulation and control of nanometer gold dimer and hang
Liquid；
(5) sign of genes within cells regulation and control: the Total RNAs extraction in the cell suspension obtain step (4) out, then is incited somebody to action
Its reverse transcription is cDNA, with fluorescence quantitative PCR detection survivin gene expression in vivo, and with reference gene β-
Actin compares, and calculates its relative expression quantity, relative expression quantity=survivin gene expression amount/β-actin gene expression amount.
Described plasma nanometer gold dimer, for the method for genes within cells expression regulation, concretely comprises the following steps:
(1) golden nanometer particle modifies cell-penetrating peptide: take the golden nanometer particle speed with 8000rpm of 100mL, 2nmol/L, 20nm
The centrifugal 10min of degree, and it is resuspended in the phosphate buffer PBS of 10mL 0.01M pH7.4 to obtain aurosol；By golden nanometer particle
PEG TAT DNA1 is molar concentration rate addition PEG, TAT, DNA1 in 5mL aurosol simultaneously of 1 1,000 100 5；Room temperature
After concussion 24h, 8000rpm is centrifuged 10min, removes supernatant, and precipitation is resuspended in pure water, is centrifuged repeatedly resuspended 3 times, obtains
Au-TAT-DNA1 complex；
(2) golden nanometer particle and the coupling of regulating and controlling sequence: separately take the above-mentioned aurosol of 5mL, and press golden nanometer particle anti-
DNA DNA2 is 1 100 5 molar concentration rates, adds anti-DNA and DNA2 in 5mL aurosol；After room temperature concussion 12h, to
Solution adds NaCl to final concentration of 80mM, after continuing concussion 24h, is centrifuged and is resuspended in pure water, obtain Au-anti-DNA-
DNA2 complex；
(3) dual-functional nanometer gold dimer assembles: Au-TAT-DNA1 and Au-anti-step (1) and (2) obtained
DNA-DNA2 complex, by volume 11 mixing, 60 DEG C hatch 20min after be cooled to 4 DEG C immediately, stand after 24h,
To dual-functional nanometer gold dimer；
(4) the nanometer gold dimer regulation and control to genes within cells: the nanometer gold dimer 8000rpm that step (3) is obtained
Centrifugal 10min, precipitation is resuspended in cell culture fluid, makes the dimeric final concentration of 5nM of nanometer gold；Cell is inoculated in 24 holes
In culture plate, the cell quantity in every hole is made to be about 104Individual, remove culture fluid after cultivating 24h, take 7 holes and be separately added into above-mentioned
Containing each 300 μ L of the dimeric cell culture fluid of nanometer gold；After each hole transfects 0h, 1h, 2h, 3h, 4h, 5h, 10h respectively, go
Except culture fluid, with cell 5 times in each hole of PBS cyclic washing, then add without the dimeric fresh training of nanometer gold in cell
Nutrient solution, after continuing to cultivate 48h, collects cell, obtains the cell suspension that different transfection time processes；
(5) sign of genes within cells regulation and control: extract test kit with RNA and extract in step (4) each cell suspension
Total serum IgE, then becomes cDNA the total serum IgE reverse transcription extracted respectively with Reverse Transcription box；In this, as template DNA fluorescence
The expression of intracellular survivin gene after the different transfection time of quantitative PCR method mensuration, simultaneously with intracellular reference gene
β-actin compares, and calculates its relative expression；Relative expression quantity=survivin gene expression amount/β-actin base
Because of expression.
(6) Electronic Speculum characterizes: the cell suspension using biological transmission electron microscope to obtain step (4) characterizes.
Described DNA1 sequence as shown in SEQ ID NO.1, DNA2 sequence as shown in SEQ ID NO.2, Anti-DNA sequence
As shown in SEQ ID NO.3, TAT peptide sequence is as shown in SEQ ID NO.4.
Table 1 DNA and the numbering of polypeptide, sequence and length
Above material is purchased from Sangon Biotech (Shanghai) Co., Ltd..
Beneficial effects of the present invention: the invention provides the dimeric preparation side of nanometer gold with genes within cells regulation and control
Method, takes full advantage of the dimeric structural advantage of nanometer gold, has regulation and control by having the particle efficiently entering cell ability with coupling
The particle assembling of sequence, has obtained structure homogeneous, stable in properties, preferable at intracellular dispersibility, and has the most intracellular
The nanometer gold dimer of survivin gene deregulation effect.
Accompanying drawing explanation
Fig. 1 nanometer gold dimer biological transmission electron microscope picture in cell.
Fig. 2 is the relative expression quantity of intracellular survivin gene after different transfection time.
Detailed description of the invention
Embodiment 1
All of glass apparatus all soaks 24h with chloroazotic acid, and cleans with distilled water, dries standby.The water used in experiment
It is the Milli-Q ultra-pure water of 18.2 M Ω.
(1) golden nanometer particle modifies cell-penetrating peptide: take 100 mL(2nmol/L) golden nanometer particle 8000 rpm of 20 nm from
Heart 10min, and be resuspended in the phosphate buffer (PBS) of 10mL 0.01M pH7.4, obtain aurosol.Take 5mL, by nanoparticle
Sub-PEG TAT DNA1 is molar concentration rate addition PEG, TAT, DNA1 in aurosol simultaneously of 1 1,000 100 5.Room temperature
After concussion 24h, 8000 rpm are centrifuged 10min, go supernatant, precipitation to be resuspended in pure water.It is centrifuged repeatedly resuspended 3 times, obtains Au-
TAT-DNA1 complex.
(2) golden nanometer particle and the coupling of regulating and controlling sequence: separately take the above-mentioned aurosol of 5mL, and press golden nanometer particle anti-
DNA DNA2 is 1 100 5 molar concentration rates, adds anti-DNA and DNA2 in aurosol.After room temperature concussion 12h, to solution
Middle addition Nacl to final concentration of 80mM, after continuing concussion 24h, is centrifuged and is resuspended in pure water, obtain Au-anti-DNA-DNA2
Complex.
(3) dual-functional nanometer gold dimer assembles: Au-TAT-DNA1 and Au-anti-DNA-DNA2 obtained above is multiple
Zoarium, by volume 11 mixing, 60 DEG C hatch 20min after be cooled to 4 DEG C immediately, stand after 24h, i.e. can get difunctional receiving
Rice gold dimer.
(4) the nanometer gold dimer regulation and control to genes within cells: by nanometer gold dimer 8000 rpm obtained above from
Heart 10min, precipitation is resuspended in the RPMI-1640 cell culture medium containing 10% hyclone, makes nanometer gold dense for dimeric end
Degree is 5nM.HeLa cell is inoculated in 24 well culture plates, makes the cell quantity in every hole be about 104Individual, go after cultivating 24h
Fall culture fluid, take 7 holes and be separately added into above-mentioned containing each 300 μ L of the dimeric cell culture fluid of nanometer gold.Each hole turns respectively
After dye 0h, 1 h, 2h, 3 h, 4h, 5 h, 10h, remove culture fluid, with cell 5 times in each hole of PBS cyclic washing.Again to cell
Middle addition does not contains the dimeric fresh medium of nanometer gold, after continuing to cultivate 48h, collects cell, obtains at different transfection time
The cell suspension of reason.
(5) sign of genes within cells regulation and control: with RNA extract that test kit extracts in each cell suspension above-mentioned total
RNA, then becomes cDNA the total serum IgE reverse transcription extracted respectively with Reverse Transcription box.Fixed with fluorescence in this, as template DNA
Amount PCR method measures the expression of intracellular survivin gene after different transfection time, simultaneously with intracellular reference gene β-
Actin compares, and calculates its relative expression, relative expression quantity=survivin gene expression amount/β-actin gene
Expression.
Electronic Speculum characterizes: the cell suspension glutaraldehyde taking above-mentioned different incubation time section respectively is fixed, osmic acid is fixed, acetone
Embed, dry and cut into slices, dyeing.Transmission electron microscope uses the Electronic Speculum of JEOL JEM-2100 model, and its accelerating potential is 80 kV.
SEQ ID NO.1
DNA1:
TTAACGTTGA AATGTCCGTA AGAACAGGAC ATCACCAATA GCCCTTGGAT AAAAAAAAAA-SH
SEQ ID NO.2
DNA2
ATCCAAGGGC TATTGGTGAT GTCCTGTTCT TACGGACATT TCAACGTTAA AAAAAAAAAA-SH
SEQ ID NO.3
Anti-DNA
SH-TTTTTCCCAG CCTTCCAGCT CCTTG
SEQ ID NO.4
TAT(RNA peptide sequence)
YGRKKRRQRR RC
Claims (2)
1. the method that a plasma nanometer gold dimer is used for genes within cells expression regulation, it is characterised in that step is as follows:
(1) golden nanometer particle modifies cell-penetrating peptide: by golden nanometer particle and the mercapto-polyglycol PEG of the synthesis of citric acid reducing process
5000, cell-penetrating peptide TAT and DNA1 mixing, must arrive surface and be modified with the Au-TAT-DNA1 complex of cell-penetrating peptide after 24h；
(2) golden nanometer particle and the coupling of regulating and controlling sequence: by antisense nucleotide anti-of golden nanometer particle Yu survivin gene
DNA and DNA2 coupling, must arrive surface and be modified with the Au-anti-DNA-DNA2 complex of regulating and controlling sequence；
Described DNA1 sequence as shown in SEQ ID NO.1, DNA2 sequence as shown in SEQ ID NO.2, Anti-DNA sequence such as SEQ
Shown in ID NO.3, TAT peptide sequence is as shown in SEQ ID NO.4；
(3) dual-functional nanometer gold dimer assembles: Au-TAT-DNA1 complex step (1) obtained and step (2) obtain
Au-anti-DNA-DNA2 complex mixes, and obtains dual-functional nanometer gold dimer package assembly；
(4) the nanometer gold dimer regulation and control to genes within cells: the dual-functional nanometer gold dimer obtained in step (3) is assembled
After structure and cell hatch 3h jointly, with trypsin digestion and cell, obtain the cell suspension after the regulation and control of nanometer gold dimer；
(5) sign of genes within cells regulation and control: the Total RNAs extraction in the cell suspension obtain step (4) is out, more anti-by it
Be transcribed into cDNA, with fluorescence quantitative PCR detection survivin gene expression in vivo, and with reference gene β-actin phase
Relatively, its relative expression quantity, relative expression quantity=survivin gene expression amount/β-actin gene expression amount are calculated.
The most according to claim 1, plasma nanometer gold dimer is for the method for genes within cells expression regulation, its feature
It is to concretely comprise the following steps:
(1) golden nanometer particle modifies cell-penetrating peptide: take the golden nanometer particle of 100mL, 2nmol/L, 20nm with the speed of 8000rpm from
Heart 10min, and it is resuspended in the phosphate buffer PBS of 10mL 0.01M pH7.4 to obtain aurosol；By golden nanometer particle: PEG:
TAT: DNA1 is molar concentration rate addition PEG, TAT, DNA1 in 5mL aurosol simultaneously of 1: 1000: 100: 5；Room temperature is shaken
After 24h, 8000rpm is centrifuged 10min, removes supernatant, and precipitation is resuspended in pure water, is centrifuged repeatedly resuspended 3 times, obtains Au-
TAT-DNA1 complex；
(2) golden nanometer particle and the coupling of regulating and controlling sequence: separately take the above-mentioned aurosol of 5mL, and press golden nanometer particle: anti-DNA:
DNA2 is 1: 100: 5 molar concentration rate, adds anti-DNA and DNA2 in 5mL aurosol；After room temperature concussion 12h, to solution
Middle addition NaCl to final concentration of 80mM, after continuing concussion 24h, is centrifuged and is resuspended in pure water, obtain Au-anti-DNA-DNA2
Complex；
(3) dual-functional nanometer gold dimer assembles: Au-TAT-DNA1 and Au-anti-DNA-step (1) and (2) obtained
DNA2 complex, by volume 11 mixing, 60 DEG C hatch 20min after be cooled to 4 DEG C immediately, stand after 24h, i.e. obtain double merit
Can nanometer gold dimer；
(4) the nanometer gold dimer regulation and control to genes within cells: nanometer gold dimer 8000rpm step (3) obtained is centrifuged
10min, precipitation is resuspended in cell culture fluid, makes the dimeric final concentration of 5nM of nanometer gold；Cell is inoculated in 24 holes cultivate
In plate, making the cell quantity in every hole is 104Individual, remove culture fluid after cultivating 24h, take 7 holes and be separately added into above-mentioned containing receiving
The rice each 300 μ L of golden dimeric cell culture fluid；After each hole transfects 0h, 1h, 2h, 3h, 4h, 5h, 10h respectively, remove and cultivate
Liquid, with cell 5 times in each hole of PBS cyclic washing, then adds without the dimeric fresh medium of nanometer gold in cell, continues
After continuous cultivation 48h, collect cell, obtain the cell suspension that different transfection time processes；
(5) sign of genes within cells regulation and control: with RNA extract that test kit extracts in step (4) each cell suspension total
RNA, then becomes cDNA the total serum IgE reverse transcription extracted respectively with Reverse Transcription box；Fixed with fluorescence in this, as template DNA
Amount PCR method measures the expression of intracellular survivin gene after different transfection time, simultaneously with intracellular reference gene β-
Actin compares, and calculates its relative expression；Relative expression quantity=survivin gene expression amount/β-actin gene
Expression；
(6) Electronic Speculum characterizes: the cell suspension using biological transmission electron microscope to obtain step (4) characterizes.
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Address after: 214016 Jiangsu city of Wuxi Province District Liangxi No. 898 South Road 7 layer beam Creek area of food science and Technology Park Patentee after: Jiangnan University Address before: Food College of Jiangnan University No. 1800 Li Lake Avenue 214122 in Jiangsu province Wuxi City Binhu District Patentee before: Jiangnan University |