CN101638668A - Application of amphipathy macromolecule cation polymer - Google Patents
Application of amphipathy macromolecule cation polymer Download PDFInfo
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- CN101638668A CN101638668A CN200910067480A CN200910067480A CN101638668A CN 101638668 A CN101638668 A CN 101638668A CN 200910067480 A CN200910067480 A CN 200910067480A CN 200910067480 A CN200910067480 A CN 200910067480A CN 101638668 A CN101638668 A CN 101638668A
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Abstract
The invention relates to application of amphipathy macromolecule cation polymer, wherein the polymer is taken as a siRNA transfection carrier. The polymer is a non virus gene carrier. The gene silentefficiency is high, and cell toxicity is less. The highest gene silent efficiency of CT26 cell for constantly expressing luciferase is 73.4%; and in 6h liquid change experiment after transfection, thehighest gene silent efficiency of 4T1 cell for constantly expressing luciferase is 63.7%. In the Hella cell experiment of transient transfection, compared with commercial PEI-25K and liposome 2000, the amphipathy macromolecule cation polymer has higher gene silent efficiency and less cell toxicity. In the 6h liquid change experiment after transfection, the reduction of the cell toxicity of the amphipathy macromolecule cation polymer is less.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of amphipathy macromolecule cation polymer as siRNA transfection carrier and usage.
Background technology
The RNA interference phenomenon is to utilize the siRNA target to realize the phenomenon of post-transcriptional level gene silencing, and RNAi is the specific efficient inhibition of gene expression approach by the siRNA mediation, by siRNA mediation identification and target cutting homology said target mrna.The siRNA gene silent technology is the most effectual way of treatment range gene relative disease
[1-6]
Studies show that the siRNA with 21～22 nucleotide sequences of synthetic does not have cytotoxicity, and can effectively realize the RNA interference and reach gene therapy purpose
[7-9]However, because siRNA itself can not enter the function that target enters pathological tissues, aspect clinical application, be subjected to great restriction
[10-12]In recent years, non-virus type positively charged ion genophore has been applied in the siRNA transmission system, as PEG-PLL and PEG-PEI
[13-14]
PEI class carrier has been proved to be has good effect in the plasmid transfection system, and also playing a role aspect the siRNA transfection, but a large amount of positive charge that polycation has, when dosage increases, can the intensive effect be arranged and cause necrocytosis with cell surface, therefore, modify on the PEI surface and cover a part of positive charge and can effectively reduce cytotoxicity
[15-18]
Summary of the invention
The objective of the invention is to seek hypotoxicity, transfection efficiency height, the non-virus gene carrier that target and pH susceptibility are strong.
The present invention selects the copolymer p EI-PBLG of hyperbranched polyethyleneimine (the PEI)-poly benzyl glutamate (PBLG) of hyperbranched polyethyleneimine modification for use, choose PEI-25k and Lipofectamine2000 in contrast, mediation luciferase siRNA carries out gene silencing.We find that the relative PEI-25k of PEI-PBLG can effectively improve the reticent efficient of siRNA, and have reduced cytotoxicity.
The invention provides a kind of application of amphipathy macromolecule cation polymer, it is characterized in that, described a kind of amphipathy macromolecule cation polymer is as the siRNA transfection carrier;
Described amphipathy macromolecule cation polymer is that molecular weight is 25000 hyperbranched polyethyleneimine (PEI) and the copolymer p EI-PBLG of poly benzyl glutamate (PBLG), wherein, the mass ratio of described hyperbranched polyethyleneimine and poly benzyl glutamate is 6: 4; Described amphipathy macromolecule cation polymer is of a size of 150-250nm;
Described siRNA is the Luc siRNA of reticent luciferase, its sequence is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 '.Contrast is Rev siRNA, its sequence is 5 '-AGCUUCAUGAGUCGCAUUCdTdT-3 '.
The method for making of described a kind of amphipathy macromolecule cation polymer: the method that provides with reference [19] prepares.
A kind of amphipathy macromolecule cation polymer provided by the invention is as the usage of siRNA transfection carrier, and step and condition are as follows:
(1) with the synthetic siRNA of ordinary method;
(2) preparation of amphipathy macromolecule cation polymer/siRNA composite particles
Get described amphipathy macromolecule cation polymer be dissolved in water to concentration be 1mg/mL, be the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the sterile distilled water compound concentration is the siRNA aqueous solution of 0.1mg/mL, mass ratio according to described amphipathy macromolecule cation polymer: siRNA is 1.25: 1～40: 1, the aqueous solution and the siRNA aqueous solution of described amphipathy macromolecule cation polymer are mixed by above-mentioned solute mass ratio, this mixed aqueous solution is put room temperature to be placed 20 minutes, further promote the formation of amphipathy macromolecule cation polymer and siRNA composite particles, obtain a kind of amphipathy macromolecule cation polymer/siRNA composite particles that contains;
(3) a kind of amphipathy macromolecule cation polymer is as the siRNA transfection carrier
(1) cultivation of cell
The recovery cell is 5% CO in containing the DMEM cell culture fluid that volume fraction is 10% calf serum and containing percent by volume
2, temperature is cultured continuously in 37 ℃ the incubator;
(2) in-vitro transfection
In preceding 24 hours of the transfection, cell is pressed 1 * 10
4Individual cells/well kind places that to contain percent by volume be 5% CO in 96 porocyte culture plates
2, temperature is that cultured continuously to 80 in 37 ℃ the incubator～90% merges; During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after the PBS washed twice, add amphipathy macromolecule cation polymer/siRNA composite particles, and contain the high sugared DMEM nutrient solution that percent by volume is 10%FBS, continue to cultivate 24 hours, adding composite particles 6h changes liquid and tests in contrast.
(3) mensuration of transfection efficiency in vitro
Take out culture plate, inhale and remove nutrient solution, PBS washing 2 times adds 50 μ L cell pyrolysis liquid cracking, and piping and druming evenly back is taken out in 20 μ L to the 1.5mL centrifuge tubes, adds 100 μ L fluorescein substrates then, uses the photometric determination transfection efficiency;
Take out 20 μ L lysates again to new 96 orifice plates, select the U.S. BCA of Pierce company protein detection kit for use, add 200 μ L and detect liquid, 37 ℃ hatch 30 minutes after, microplate reader 562nm wavelength detects.
Beneficial effect: the invention provides a kind of amphipathy macromolecule cation polymer as siRNA transfection carrier and usage.Amphipathy macromolecule cation polymer of the present invention is a kind of non-virus gene carrier.Its gene silencing efficient height, and cytotoxicity is little.The reticent efficient of high gene to the CT26 cell of constant expressing luciferase is 73.4%, is greatly improved with respect to 34.5% of 10.1% and the liposome 2000 of commercial PEI-25k, and has significantly reduced cytotoxicity.6h changes in the liquid experiment after transfection, high transfection efficiency to the 4T1 cell of constant expressing luciferase is 63.7%, also improve a lot with respect to 20.5% of 14.5% and the liposome 2000 of commercial PEI-25k, also demonstrate its hypotoxic characteristics simultaneously.In the Hela of transient transfection cell experiment, this amphipathy macromolecule cation polymer has higher gene silencing efficient and less cytotoxicity with respect to commercial PEI-25k and liposome 2000.6h does not change in the liquid controlled trial after the transfection, and this amphipathy macromolecule cation polymer cytotoxicity reduces less, and commercial PEI-25k and liposome 2000 reduce relatively large.
Description of drawings
Fig. 1 is a liposome 2000, and promptly Lip 2000, and PEI-25k and PEI-PBLG mediate the Luc siRNA of reticent luciferase and Rev siRNA in the intracellular release of the CT26 of constant expressing luciferase, and 6h changes liquid after the transfection.X-coordinate is represented the mass ratio of material and siRNA, and the final concentration of siNRA is 2ng/ μ L.Detect the luciferase expression amount behind the transfection 24h.
Fig. 2 is a liposome 2000, and promptly Lip 2000, and PEI-25k and PEI-PBLG mediate the Luc siRNA of reticent luciferase and Rev siRNA in the intracellular release of the CT26 of constant expressing luciferase, and 6h does not change liquid after the transfection.X-coordinate is represented the mass ratio of material and siRNA, and the final concentration of siNRA is 2ng/ μ L.Detect the luciferase expression amount behind the transfection 24h.
Fig. 3 is a liposome 2000, and promptly Lip 2000, and PEI-25k and PEI-PBLG mediate the Luc siRNA of reticent luciferase and Rev siRNA in the intracellular release of the 4T1 of constant expressing luciferase, and 6h changes liquid after the transfection.X-coordinate is represented the mass ratio of material and siRNA, and the final concentration of siNRA is 2ng/ μ L.Detect the luciferase expression amount behind the transfection 24h.
Fig. 4 is a liposome 2000, and promptly Lip 2000, and PEI-25k and PEI-PBLG mediate the Luc siRNA of reticent luciferase and Rev siRNA in the intracellular release of the 4T1 of constant expressing luciferase, and 6h does not change liquid after the transfection.X-coordinate is represented the mass ratio of material and siRNA, and the final concentration of siNRA is 2ng/ μ L.Detect the luciferase expression amount behind the transfection 24h.
Fig. 5 is a liposome 2000, and promptly Lip 2000, and PEI-25k and PEI-PBLG mediate the Luc siRNA of reticent luciferase and Rev siRNA in the intracellular release of the Hela of transient expression luciferase, and 6h changes liquid after the transfection.X-coordinate is represented the mass ratio of material and siRNA, and the final concentration of siNRA is 2ng/ μ L.Detect the luciferase expression amount behind the transfection 24h.
Fig. 6 is a liposome 2000, and promptly Lip 2000, and PEI-25k and PEI-PBLG mediate the Luc siRNA of reticent luciferase and Rev siRNA in the intracellular release of the Hela of transient expression luciferase, and 6h does not change liquid after the transfection.X-coordinate is represented the mass ratio of material and siRNA, and the final concentration of siNRA is 2ng/ μ L.Detect the luciferase expression amount behind the transfection 24h.
Embodiment
Embodiment 1: utilize amphipathy macromolecule cation polymer to mediate the Luc siRNA of reticent luciferase and the negative control Rev siRNA CT26 cells in vitro transfection to constant expressing luciferase
(1) with the synthetic siRNA of ordinary method;
(2) preparation of amphipathy macromolecule cation polymer/siRNA composite particles
Get described amphipathy macromolecule cation polymer be dissolved in water to concentration be 1mg/mL, be the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the sterile distilled water compound concentration is the siRNA aqueous solution of 0.1mg/mL, mass ratio according to described amphipathy macromolecule cation polymer: siRNA is 1.25: 1,2.5: 1,5: 1,10: 1,20: 1 and 40: 1, the aqueous solution and the siRNA aqueous solution of described amphipathy macromolecule cation polymer are mixed by above-mentioned solute mass ratio, this mixed aqueous solution is put room temperature to be placed 20 minutes, further promote the formation of amphipathy macromolecule cation polymer and siRNA composite particles, obtain a kind of amphipathy macromolecule cation polymer/siRNA composite particles that contains;
(3) a kind of amphipathy macromolecule cation polymer is as the siRNA transfection carrier
(1) cultivation of cell
The CT26 cell of the constant expressing luciferase of recovering is 5% CO in containing the high sugared DMEM cell culture fluid that volume fraction is 10% calf serum and containing percent by volume
2, temperature is cultured continuously in 37 ℃ the incubator;
(2) in-vitro transfection
In preceding 24 hours of the transfection, cell is pressed 1 * 10
4Individual cells/well kind places that to contain percent by volume be 5% CO in 96 porocyte culture plates
2, temperature is that cultured continuously to 80 in 37 ℃ the incubator～90% merges; During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after the PBS washed twice, add amphipathy macromolecule cation polymer/siRNA composite particles, and contain the high sugared DMEM nutrient solution that percent by volume is 10%FBS, continue to cultivate 24 hours, adding composite particles 6h changes liquid and tests in contrast.
(3) mensuration of transfection efficiency in vitro
Take out culture plate, inhale and remove nutrient solution, PBS washing 2 times adds 50 μ L cell pyrolysis liquid cracking, and piping and druming evenly back is taken out in 20 μ L to the 1.5mL centrifuge tubes, adds 100 μ L fluorescein substrates then, uses the photometric determination transfection efficiency;
Take out 20 μ L lysates again to new 96 orifice plates, select the U.S. BCA of Pierce company protein detection kit for use, add 200 μ L and detect liquid, 37 ℃ hatch 30 minutes after, microplate reader 562nm wavelength detects.
Fig. 1,2 have provided amphipathy macromolecule cation polymer/siRNA mixture respectively changes liquid and the gene silencing efficient experimental result of not changing liquid to constant expressing luciferase CT26 cell transfecting after 6 hours
As seen from Figure 1, the active mensuration of luciferase fluorescence intensity depends on Luc siRNA and Rev siRNA, and wherein Luc siRNA can combine with target mRNA specificity, and RevsiRNA can not combine with target mRNA specificity as negative control.The cell of addition polymerization compound and siRNA is not as positive control.The reduction of fluorescence intensity is depended on and has been added LucsiRNA rather than negative control Rev siRNA, has all reduced if add the fluorescence intensity of two kinds of siRNA cells, then is that the toxicity owing to carrier has caused necrocytosis.Do not producing under the cytotoxicity condition, the siRNA gene silencing efficient of simple PEI-25k mediation is not high, the highlyest can only reach 10.1%, only add siRNA and do not produce gene silencing, liposome 2000 mediation siRNA effects are apparent in view, can produce 34.5% reticent efficient nearly, but liposome 2000 cytotoxicities are bigger as can be seen, and the siRNA gene of PEI-PBLG mediation transmits high energy and produces 73.4% gene silencing efficient, and its toxicity is less.
As seen from Figure 2, the toxicity that carrier behind the liquid was not changed in transfection in 6 hours all slightly increases, and liposome 2000 and PEI-25k increase very fast, the PEI-PBLG gene silencing is most effective, can reach 75.2%, and liposome and PEI-25k are respectively 55.5% and 12.6%, only adding siRNA does not have reticent effect.
Embodiment 2: utilize amphipathy macromolecule cation polymer to mediate the Luc siRNA of reticent luciferase and the negative control Rev siRNA 4T1 cells in vitro transfection to constant expressing luciferase
(1) with the synthetic siRNA of ordinary method;
(2) preparation of amphipathy macromolecule cation polymer/siRNA composite particles
Get described amphipathy macromolecule cation polymer be dissolved in water to concentration be 1mg/mL, be the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the sterile distilled water compound concentration is the siRNA aqueous solution of 0.1mg/mL, mass ratio according to described amphipathy macromolecule cation polymer: siRNA is 1.25: 1,2.5: 1,5: 1,10: 1,20: 1 and 40: 1, the aqueous solution and the siRNA aqueous solution of described amphipathy macromolecule cation polymer are mixed by above-mentioned solute mass ratio, this mixed aqueous solution is put room temperature to be placed 20 minutes, further promote the formation of amphipathy macromolecule cation polymer and siRNA composite particles, obtain a kind of amphipathy macromolecule cation polymer/siRNA composite particles that contains;
(3) a kind of amphipathy macromolecule cation polymer is as the siRNA transfection carrier
(1) cultivation of cell
The 4T1 cell of the constant expressing luciferase of recovering is 5% CO in containing the low sugar DMEM cell culture fluid that volume fraction is 10% calf serum and containing percent by volume
2, temperature is cultured continuously in 37 ℃ the incubator;
(2) in-vitro transfection
In preceding 24 hours of the transfection, the 4T1 cell is pressed 1 * 10
4Individual cells/well kind places that to contain percent by volume be 5% CO in 96 porocyte culture plates
2, temperature is that cultured continuously to 80 in 37 ℃ the incubator～90% merges; During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after the PBS washed twice, add amphipathy macromolecule cation polymer/siRNA composite particles, and contain the low sugar DMEM nutrient solution that percent by volume is 10%FBS, continue to cultivate 24 hours, adding composite particles 6h changes liquid and tests in contrast.
(3) mensuration of transfection efficiency in vitro
Take out culture plate, inhale and remove nutrient solution, PBS washing 2 times adds 50 μ L cell pyrolysis liquid cracking, and piping and druming evenly back is taken out in 20 μ L to the 1.5mL centrifuge tubes, adds 100 μ L fluorescein substrates then, uses the photometric determination transfection efficiency.
Take out 20 μ L lysates again to new 96 orifice plates, select the U.S. BCA of Pierce company protein detection kit for use, add 200 μ L and detect liquid, 37 ℃ hatch 30 minutes after, microplate reader 562nm wavelength detects.
Fig. 3,4 have provided amphipathy macromolecule cation polymer/siRNA mixture respectively changes liquid and the gene silencing efficient experimental result of not changing liquid to constant expressing luciferase 4T1 cell transfecting after 6 hours
As seen from Figure 3, the active mensuration of luciferase fluorescence intensity depends on Luc siRNA and Rev siRNA, and wherein Luc siRNA can combine with target mRNA specificity, and RevsiRNA can not combine with target mRNA specificity as negative control.The cell of addition polymerization compound and siRNA is not as positive control.The reduction of fluorescence intensity is depended on and has been added LucsiRNA rather than negative control Rev siRNA, has all reduced if add the fluorescence intensity of two kinds of siRNA cells, then is that the toxicity owing to carrier has caused necrocytosis.Do not producing under the cytotoxicity condition, the siRNA gene silencing efficient of simple lipid body 2000 and PEI-25k mediation is not high, the highlyest can only reach 14.5% and 20.5% respectively, and produce bigger cytotoxicity, only add siRNA and do not produce gene silencing, and the siRNA gene of PEI-PBLG mediation transmits high energy and produces 63.7% gene silencing efficient, and its toxicity is less.
As seen from Figure 4, the toxicity that carrier behind the liquid was not changed in transfection in 6 hours all slightly increases, and liposome 2000 and PEI-25k increase very fast, the PEI-PBLG gene silencing is most effective, can reach 65.2%, liposome and PEI-25k are respectively 9.5% and 11.4%, and only adding siRNA does not have reticent effect.
Luc siRNA and negative control Rev siRNA that embodiment 3 utilizes amphipathy macromolecule cation polymer to mediate reticent luciferase test the in-vitro transfection of Lipofectamine2000 transfection luciferase plasmids pGL3-control transient expression luciferase in the HeLa cell
(1) with the synthetic siRNA of ordinary method;
(2) preparation of amphipathy macromolecule cation polymer/siRNA composite particles
Get described amphipathy macromolecule cation polymer be dissolved in water to concentration be 1mg/mL, be the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the sterile distilled water compound concentration is the siRNA aqueous solution of 0.1mg/mL, mass ratio according to described amphipathy macromolecule cation polymer: siRNA is 1.25: 1,2.5: 1,5: 1,10: 1,20: 1 and 40: 1, the aqueous solution and the siRNA aqueous solution of described amphipathy macromolecule cation polymer are mixed by above-mentioned solute mass ratio, this mixed aqueous solution is put room temperature to be placed 20 minutes, further promote the formation of amphipathy macromolecule cation polymer and siRNA composite particles, obtain a kind of amphipathy macromolecule cation polymer/siRNA composite particles that contains;
(3) a kind of amphipathy macromolecule cation polymer is as the siRNA transfection carrier
(1) cultivation of cell
Recovery Hela cell is 5% CO in containing the high sugared DMEM cell culture fluid that volume fraction is 10% calf serum and containing percent by volume
2, temperature is cultured continuously in 37 ℃ the incubator;
(2) in-vitro transfection
In preceding 24 hours of the transfection, the HeLa cell is pressed 1 * 10
4Individual cells/well kind places that to contain percent by volume be 5% CO in 96 well culture plates
2, temperature is that cultured continuously to 80 in 37 ℃ the incubator～90% merges.During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after the PBS washed twice, add liposome 2000/pGL3-control composite particles transfection 3h, make Hela cell transient expression luciferase, after sopping up the interior liquid of culture plate, the PBS washed twice adds amphipathy macromolecule cation polymer/siRNA composite particles again, and contains the high sugared DMEM nutrient solution that percent by volume is 10%FBS, continue to cultivate 24 hours, adding composite particles 6h changes liquid and tests in contrast.
(3) mensuration of transfection efficiency in vitro
Take out culture plate, inhale and remove nutrient solution, PBS washing 2 times adds 50 μ L cell pyrolysis liquid cracking, and piping and druming evenly back is taken out in 20 μ L to the 1.5mL centrifuge tubes, adds 100 μ L fluorescein substrates then, uses the photometric determination transfection efficiency.
Take out 20 μ L lysates again to new 96 orifice plates, select the U.S. BCA of Pierce company protein detection kit for use, add 200 μ L and detect liquid, 37 ℃ hatch 30 minutes after, microplate reader 562nm wavelength detects.
Fig. 5,6 have provided amphipathy macromolecule cation polymer/siRNA mixture respectively changes liquid and the gene silencing efficient experimental result of not changing liquid to the Hela cell transfecting of transient expression luciferase after 6 hours
As seen from Figure 5, the active mensuration of luciferase fluorescence intensity depends on Luc siRNA and Rev siRNA, and wherein Luc siRNA can combine with target mRNA specificity, and RevsiRNA can not combine with target mRNA specificity as negative control.The cell of addition polymerization compound and siRNA is not as positive control.The reduction of fluorescence intensity is depended on and has been added LucsiRNA rather than negative control Rev siRNA, has all reduced if add the fluorescence intensity of two kinds of siRNA cells, then is that the toxicity owing to carrier has caused necrocytosis.Do not producing under the cytotoxicity condition, the siRNA gene silencing efficient of simple PEI-25k mediation is not high, only add siRNA and do not produce gene silencing, liposome 2000 mediation siRNA effects are apparent in view, but liposome 2000 cytotoxicities are bigger as can be seen, and the siRNA gene of PEI-PBLG mediation transmits high energy and produces higher gene silencing efficient, and its toxicity is less.
As shown in Figure 6, the toxicity that carrier behind the liquid was not changed in transfection in 6 hours all slightly increases, and liposome 2000 and PEI-25k increase very fast, and the PEI-PBLG gene silencing is most effective, and produces minimum cytotoxicity, and only adding siRNA does not have reticent effect.
The reference that the present invention relates to:
[1]M.T.McManus，P.A.Sharp，Gene?silencing?in?mammals?by?small?interferingRNAs，Nat.Rev，Genet?2002；3：737-747.
[2]J.W.Shiver，T.M.Fu，L.Chen，D.R.Casimiro，M.E.Davies，R.K.Evans，etal.Replicationincompetent?adenoviral?vaccine?vector?elicits?effectiveanti-immunodeficiency-virusimmunity，Nature?2002；415：331-335.
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Claims (2)
1. the application of an amphipathy macromolecule cation polymer is characterized in that, described amphipathy macromolecule cation polymer is as the siRNA transfection carrier;
Described amphipathy macromolecule cation polymer is that molecular weight is 25000 the hyperbranched polyethyleneimine and the multipolymer of poly benzyl glutamate: hyperbranched polyethyleneimine-poly benzyl glutamate; The mass ratio of described hyperbranched polyethyleneimine and poly benzyl glutamate is 6: 4; Described amphipathy macromolecule cation polymer is of a size of 150-250nm; Described siRNA is the Luc siRNA of reticent luciferase, its sequence is 5 '-CUUACGCUGAGUACUUCGAdTdT-3 '.
2. a kind of amphipathy macromolecule cation polymer as claimed in claim 1 is as the siRNA transfection carrier, and the step and the condition of its usage are as follows:
(1) with the synthetic siRNA of ordinary method;
(2) preparation of amphipathy macromolecule cation polymer/siRNA composite particles
Get described amphipathy macromolecule cation polymer be dissolved in water to concentration be 1mg/mL, be the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the sterile distilled water compound concentration is the siRNA aqueous solution of 0.1mg/mL, mass ratio according to described amphipathy macromolecule cation polymer: siRNA is 1.25: 1～40: 1, the aqueous solution and the siRNA aqueous solution of described amphipathy macromolecule cation polymer are mixed by above-mentioned solute mass ratio, this mixed aqueous solution is put room temperature to be placed 20 minutes, further promote the formation of amphipathy macromolecule cation polymer and siRNA composite particles, obtain a kind of amphipathy macromolecule cation polymer/siRNA composite particles that contains;
(3) a kind of amphipathy macromolecule cation polymer is as the siRNA transfection carrier
(1) cultivation of cell
The recovery cell is 5% CO in containing the DMEM cell culture fluid that volume fraction is 10% calf serum and containing percent by volume
2, temperature is cultured continuously in 37 ℃ the incubator;
(2) in-vitro transfection
In preceding 24 hours of the transfection, cell is pressed 1 * 10
4Individual cells/well kind places that to contain percent by volume be 5% CO in 96 porocyte culture plates
2, temperature is that cultured continuously to 80 in 37 ℃ the incubator～90% merges; During transfection, the nutrient solution in the Tissue Culture Plate of annotating the day before yesterday is abandoned in suction, after the PBS washed twice, add amphipathy macromolecule cation polymer/siRNA composite particles, and contain the high sugared DMEM nutrient solution that percent by volume is 10%FBS, continue to cultivate 24 hours, adding composite particles 6h changes liquid and tests in contrast;
(3) mensuration of transfection efficiency in vitro
Take out culture plate, inhale and remove nutrient solution, PBS washing 2 times adds 50 μ L cell pyrolysis liquid cracking, and piping and druming evenly back is taken out in 20 μ L to the 1.5mL centrifuge tubes, adds 100 μ L fluorescein substrates then, uses the photometric determination transfection efficiency;
Take out 20 μ L lysates again to new 96 orifice plates, select the U.S. BCA of Pierce company protein detection kit for use, add 200 μ L and detect liquid, 37 ℃ hatch 30 minutes after, microplate reader 562nm wavelength detects.
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