CN110438162A - A kind of modified form non-liposomal transfection reagent box and its application method - Google Patents
A kind of modified form non-liposomal transfection reagent box and its application method Download PDFInfo
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- CN110438162A CN110438162A CN201910776365.6A CN201910776365A CN110438162A CN 110438162 A CN110438162 A CN 110438162A CN 201910776365 A CN201910776365 A CN 201910776365A CN 110438162 A CN110438162 A CN 110438162A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
Abstract
The present invention discloses a kind of modified form non-liposomal transfection reagent box and its application method, wherein modified form non-liposomal transfection reagent box of the present invention includes: the star-shaped cationic polymer for preparing transfection reagent；And sodium chloride, 4- hydroxyethyl piperazineethanesulfonic acid and citric acid-sodium citrate buffer solution for preparing Contrast agent.The good biocompatibility of each ingredient in modified form non-liposomal transfection reagent box provided by the invention, immunogenicity is low, toxicity is low, safety is good, it is easy to operate, repeatable strong when for gene transfection, and transfection efficiency and cell survival rate are high, use scope is wide, and the gene for being applicable to a variety of attached cell systems and suspension cell line transfects.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of modified form non-liposomal transfection reagent box and its application side
Method.
Background technique
Gene therapy, which refers to, imports target cell for external source normal gene to correct or compensate because gene defect and exception cause
Disease, reach therapeutic purposes.Gene therapy is that have currently used for one kind of cancer and innate immune system disease treatment
Efficacious prescriptions method, the key implemented is the suitable genophore of selection and method of gene introduction, and then enables gene in cell
Obtain safe and efficient, controllable and stable expression.
Now widely used genophore is broadly divided into viral vectors and non-virus carrier two major classes.
Virus is the carrier being modified earliest as gene therapy, and most study in clinical trial, closest at present
The genophore of actual product, but there is a series of safety problems and application problem always in viral vectors, such as potential carcinogenicity,
Autoantigenic, target gene capacity is small, targeting specific is poor etc., to limit its answering in clinical gene therapy
With.Non-virus carrier is a kind of novel gene vector developed rapidly after viral vectors, non-viral gene vector one
As be divided into cationic-liposome and cationic polymer two major classes, wherein cationic-liposome (such as Invitrogen
LipofectamineTM2000) there is very high efficiency in gene transfection in vitro, be widely used in outer-gene transfection assay.
But cationic-liposome induces strong inflammatory reaction, causes there is also that can be removed from serum rapidly and be assembled in lung
The defects of high-caliber toxicity.Due to the limitation of cationic-liposome, cationic polymer transfection reagent is paid more and more attention.
Chinese patent CN102604114B discloses a kind of star-shaped cationic polymer of primitive of polylysine containing dendroid
And preparation method thereof, this contains the star-shaped cationic polymer good biocompatibility, biodegradable of dendroid polylysine primitive
And its metabolite small toxicity, but its be used for gene transfection when, transfection efficiency is lower, and transfection efficiency is to be improved.
Summary of the invention
In order to solve defect existing in the prior art, the purpose of the present invention is to provide a kind of modified form non-liposomal to turn
Transfection reagent box.Modified form non-liposomal transfection reagent box of the present invention using star-shaped cationic polymer as transfection reagent it is main at
Point, by addition Contrast agent, the transfection efficiency of foreign gene is significantly improved, meanwhile, modified form non-liposomal of the present invention turns
The safety of transfection reagent box is good, and when for gene transfection, cell survival rate is high, is suitble to a plurality of types of cell lines, can be widely used for
Transient transfection and stable transfection.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of modified form non-liposomal transfection reagent box, comprising:
For preparing the star-shaped cationic polymer of transfection reagent；And
For preparing sodium chloride, 4- hydroxyethyl piperazineethanesulfonic acid and the citric acid-sodium citrate buffer solution of Contrast agent.
Star-shaped cationic polymer of the present invention is Chinese patent CN102604114B disclosed containing the poly- bad ammonia of dendroid
The star-shaped cationic polymer of acidic group member.
In the present invention, star-shaped cationic polymer, sodium chloride, 4- hydroxyethyl piperazineethanesulfonic acid and citric acid-sodium citrate
Buffer can respective independent packaging, be sub-packed in kit, be configured to transfection reagent and Contrast agent when needing again for gene
Transfection；After can also being configured to transfection reagent and Contrast agent, transfection reagent and Contrast agent are subjected to independent packaging respectively, dispensed
In kit, transfection reagent and Contrast agent directly can be used for gene transfection when needing.Wherein, each independent packet in kit
The packing specification of the ingredient of dress can be arranged on demand.
The present invention is using the star-shaped cationic polymer of the primitive of polylysine containing dendroid as genophore, biocompatibility
Well, immunogenicity is low, toxicity is low, is used cooperatively with Contrast agent, is remarkably improved efficiency gene transfection, and cell survival rate is high,
And be applicable to the transfection of a variety of attached cells system and suspension cell line, as HEK293 cell, A549 cell, LNCAP cell and
HepG2 cell etc., use scope is wide.
Preferably, the preparation method of transfection reagent may include steps of: star-shaped cationic polymer is dissolved in nothing
In bacterium water, pH value is adjusted to get transfection reagent.It is further preferred that in transfection reagent, the concentration of star-shaped cationic polymer is
1mg/mL, the pH of transfection reagent are 7.0.
Preferably, the preparation method of Contrast agent includes the following steps: sodium chloride and 4- hydroxyethyl piperazineethanesulfonic acid is molten
Solution adjusts pH value in citric acid-sodium citrate buffer solution to get Contrast agent.It is further preferred that in Contrast agent, lemon
Lemon acid-sodium citrate buffer solution concentration is 10mmol/L, the final concentration of 150mmol/L of sodium chloride, 4- hydroxyethyl piperazine second
The final concentration of 10mmol/L of sulfonic acid, the pH value of Contrast agent are 7.2~7.4.
Correspondingly, the present invention also provides the sides that the above-mentioned modified form non-liposomal transfection reagent box of application carries out gene transfection
Method includes the following steps:
(1) star-shaped cationic polymer is dissolved in sterile water, adjusting pH value is 7.0 to get transfection reagent；
(2) sodium chloride and 4- hydroxyethyl piperazineethanesulfonic acid are dissolved in citric acid-sodium citrate buffer solution, adjust pH value
For 7.2~7.4 to get Contrast agent, wherein the concentration of citric acid-sodium citrate buffer solution is 10mmol/L；
(3) plasmid is added in Contrast agent, mixes, obtains mixture；
(4) transfection reagent is added in the mixture, 10~30min is stood after mixing, obtains Transfection solution；
(5) Transfection solution is added in cell culture fluid, cell is placed in incubator after mixing and is cultivated.
When kit of the present invention is transfected for gene, Contrast agent facilitates the formation of plasmid and transfection reagent compound,
So as to effectively improve transfection efficiency.
Preferably, in step (1), the concentration of star-shaped cationic polymer is 1mg/mL.
Preferably, in step (2), the final concentration of 150mmol/L of sodium chloride, the final concentration of 4- hydroxyethyl piperazineethanesulfonic acid
For 10mmol/L.
Preferably, the mass ratio of plasmid and star-shaped cationic polymer is 1:(2~4).
Preferably, the volume ratio of Contrast agent and cell culture fluid is 1:(5~15).
The present invention has the advantages that
(1) for the present invention using the star-shaped cationic polymer of the primitive of polylysine containing dendroid as genophore, when use, is molten
It is configured to transfection reagent in sterile water, is aided with sodium chloride, 4- hydroxyethyl piperazineethanesulfonic acid and citric acid-sodium citrate buffer solution and matches
Manufactured Contrast agent is transfected for gene, is remarkably improved the transfection efficiency of gene；
(2) good biocompatibility of each ingredient, immunogenicity are low, malicious in modified form non-liposomal transfection reagent box of the present invention
Property it is low, good for gene transfection safety, cell survival rate is high, gene can be enable to obtain in cell safe and efficient, steady
Fixed expression；
(3) reagent stability that application modified form non-liposomal transfection reagent box of the present invention is prepared is strong；
(4) easy to operate, repeatable strong when modified form non-liposomal transfection reagent box of the present invention is transfected for gene, and
The transfection that present invention can be suitably applied to a variety of attached cell systems and suspension cell line, as HEK293 cell, A549 cell, LNCAP are thin
Born of the same parents and HepG2 cell etc., use scope is wide.
Detailed description of the invention
Fig. 1 is modified form non-liposomal transfection reagent box of the present invention, PEI-25 transfection reagent, star-shaped cationic polymer turn
Transfection reagent and Lip2000 transfection reagent transfect the fluorescent effect figure of the cell after pEGFP；
Fig. 2 be with modified form non-liposomal transfection reagent box of the present invention preparation transfection reagent and Contrast agent mixed liquor,
The result of variations figure of cell survival rate when PEI-25 transfection reagent and Lip2000 transfection reagent and cell co-culture.
Specific embodiment
The present invention is described in further detail for attached drawing combined with specific embodiments below.
The modified form non-liposomal transfection reagent box of the present invention of embodiment 1 and its application method
Modified form non-liposomal transfection reagent box of the present invention includes: the star-shaped cationic polymer of respective independent packaging, chlorine
Change sodium, 4- hydroxyethyl piperazineethanesulfonic acid and citric acid-sodium citrate buffer solution.Wherein, star-shaped cationic polymer, sodium chloride,
The packing specification of 4- hydroxyethyl piperazineethanesulfonic acid and citric acid-sodium citrate buffer solution can be arranged on demand, such as:, can in kit
Comprising star-shaped cationic polymer 10mg, sodium chloride 1.5g, 4- hydroxyethyl piperazineethanesulfonic acid 0.5g and citric acid-sodium citrate are slow
Fliud flushing 100mL, wherein the concentration of citric acid-sodium citrate buffer solution is not less than 10mmol/L.
Include the following steps: using the method that modified form non-liposomal transfection reagent box of the present invention carries out gene transfection
(1) star-shaped cationic polymer is dissolved in sterile water, adjusting pH value is 7.0 to get transfection reagent；Wherein,
The concentration of star-shaped cationic polymer is 1mg/mL；
(2) sodium chloride and 4- hydroxyethyl piperazineethanesulfonic acid are dissolved in citric acid-sodium citrate buffer solution, adjust pH value
For 7.2~7.4 to get Contrast agent；Wherein, the concentration of citric acid-sodium citrate buffer solution is 10mmol/L, the end of sodium chloride
Concentration is 150mmol/L, the final concentration of 10mmol/L of 4- hydroxyethyl piperazineethanesulfonic acid；
(3) plasmid is added in Contrast agent, mixes, obtains mixture；
(4) transfection reagent is added in the mixture, 10~30min is stood after mixing, obtains Transfection solution；Wherein, plasmid and
The mass ratio of star-shaped cationic polymer is 1:(2~4)；The dosage of Contrast agent is mainly according to the culture body of cell to be transfected
Long-pending or used culture vessel determines, specifically may is that the volume ratio of Contrast agent and cell culture fluid is 1:(5~15), it is excellent
It is selected as 1:10；
(5) Transfection solution is added in cell culture fluid, cell is placed in incubator after mixing and is cultivated.Ordinary circumstance
Under, recombinant protein can be detected after transfection 36~48 hours, maximum expression quantity usually can be observed after 72~96 hours in transfection.
The application modified form non-liposomal transfection reagent box of the present invention of embodiment 2 carries out gene transfection to attached cell
Using the method that modified form non-liposomal transfection reagent box of the present invention carries out gene transfection to attached cell, including such as
Lower step:
(1) in the single hole of 6 orifice plates after inoculating cell, culture 18~for 24 hours, when cell confluency reaches 60~80%,
It is transfected, before transfection, the fresh culture with 3mL containing 2% serum changes liquid, cultivates 1~2h；High concentration serum will affect transfection
Efficiency, the low serum fresh culture using serum content no more than 5% change liquid and transfection efficiency can be improved；
(2) star-shaped cationic polymer is dissolved in sterile water, adjusting pH value is 7.0 to get transfection reagent；Wherein,
The concentration of star-shaped cationic polymer is 1mg/mL；
(3) sodium chloride and 4- hydroxyethyl piperazineethanesulfonic acid are dissolved in citric acid-sodium citrate buffer solution, adjust pH value
For 7.2 up to Contrast agent；Wherein, the concentration of citric acid-sodium citrate buffer solution be 10mmol/L, sodium chloride it is final concentration of
The final concentration of 10mmol/L of 150mmol/L, 4- hydroxyethyl piperazineethanesulfonic acid；
(4) 300 μ L Contrast agents, 2 μ g plasmids are added in EP (eppendorf) pipe, concussion mixes or vortex mixes, and obtains
Mixture；
(5) 8 μ L transfection reagents are added in the mixture, vortex 5 seconds, mixes, obtains Transfection solution；
(6) Transfection solution stands 20 minutes in room temperature environment, forms transfection reagent-DNA compound, is gently mixed molten
Liquid is drawn 3 times up and down；
(7) will through step (6), treated that Transfection solution is added in 6 orifice plates (single hole), 6 orifice plates are then put into culture
Case (5%CO2, 37 DEG C) and it inner cultivates；Under normal circumstances, recombinant protein, transfection 72~96 can be detected after transfecting 36~48 hours
Maximum expression quantity usually can be observed after hour.
The application modified form non-liposomal transfection reagent box of the present invention of embodiment 3 carries out gene transfection to suspension cell
Using the method that modified form non-liposomal transfection reagent box of the present invention carries out gene transfection to suspension cell, including such as
Lower step:
(1) 1.0 × 10 are added in 250mL shaking flask6The cell of/mL concentration contains the fresh culture of 2% serum with 25mL
Cultivate 2~3h；
(2) star-shaped cationic polymer is dissolved in sterile water, adjusting pH value is 7.0 to get transfection reagent；Wherein,
The concentration of star-shaped cationic polymer is 1mg/mL；
(3) sodium chloride and 4- hydroxyethyl piperazineethanesulfonic acid are dissolved in citric acid-sodium citrate buffer solution, adjust pH value
For 7.2 to get Contrast agent；Wherein, the concentration of citric acid-sodium citrate buffer solution is 10mmol/L, the final concentration of sodium chloride
For 150mmol/L, the final concentration of 10mmol/L of 4- hydroxyethyl piperazineethanesulfonic acid；
(4) 2.5mL Contrast agent, 50 μ g plasmids are added in EP pipe, concussion mixes or vortex mixes, and obtains mixture；
(5) 200 μ L transfection reagents are added in the mixture, vortex 5 seconds, mixes, obtains Transfection solution；
(6) Transfection solution stands 20 minutes in room temperature environment, forms transfection reagent-DNA compound, is gently mixed molten
Liquid is drawn 3 times up and down；
(7) will through step (6), treated, and Transfection solution is added in the cell suspending liquid of 25mL, shake up 2~3h, be added
25mL contains the fresh culture of 2% serum, is then placed in incubator and cultivates；It under normal circumstances, can after transfecting 36~48 hours
Detect recombinant protein, maximum expression quantity usually can be observed after 72~96 hours in transfection.
The transfection efficiency of the different transfection reagents of embodiment 4 compares
4 kinds of different cells (HEK293 cell, A549 cell, LNCAP cell and HepG2 cell) grow in 24 orifice plates
When cell confluency reaches 70%, modified form non-liposomal transfection reagent box of the present invention, PEI-25 transfection reagent (article No. are used respectively
23966, Polysciences, Inc), star-shaped cationic polymer transfection reagent (take modified form non-liposomal of the present invention transfection examination
Star-shaped cationic polymer in agent box is dissolved in sterile water, and adjusting pH value is 7.0 to get star-shaped cationic polymer transfection
Reagent；Wherein, the final concentration of 1mg/mL of star-shaped cationic polymer) and Lip2000 transfection reagent (article No. 11668019,
Thermo Fisher) transfection pEGFP (article No. VT1110, excellent precious biology), every 0.5 μ g pEGFP of hole cell transfecting transfects 72h
It with fluorescence microscope green EGFP fluorescence and takes pictures afterwards, as a result as shown in figure 1 and table 1.All data (mean ± standards
Difference) it indicates, it is handled using 22.0 statistical software of SPSS.More comparison among groups one-way analysis of variances.P < 0.05 indicates statistics
Difference is learned, P < 0.01 indicates significant statistical difference.
1 cell transfecting efficiency of table compares
| HEK293 | A549 | LNCAP | HepG2 | |
| The present invention | 85.06±0.02 | 55.023±0.0152 | 60.023±0.0058 | 65.567±0.1528 |
| Star-shaped cationic polymer | 50.633±0.1102** | 34.913±0.0208** | 36.920±0.02** | 40.850±0.0361** |
| PEI-25 | 84.913±0.0814 | 54.907±0.0379** | 55.163±0.0252** | 60.263±0.0306** |
| Lip2000 | 84.976±0.0153 | 54.973±0.015 | 59.987±0.0152* | 65.197±0.0603** |
Note: compared with the present invention,*Indicate P < 0.05；**Indicate P < 0.01.
By the result of Fig. 1 and table 1 it is found that modified form non-liposomal transfection reagent box of the present invention is in HEK293 cell, A549
All have very high transfection efficiency on cell, LNCAP cell and HepG2 cell, wherein A549 cell, LNCAP cell and
The transfection efficiency of cationic polymer PEI-25 transfection reagent is all remarkably higher than on HepG2 cell, in LNCAP cell and HepG2
Transfection efficiency on cell is also significantly greater than the transfection efficiency of cationic-liposome Lip2000 transfection reagent；In addition, with not adding
The transfection efficiency of the star-shaped cationic polymer transfection reagent of Contrast agent is compared, modified form non-liposomal transfection reagent of the present invention
The transfection efficiency of box significantly improves, and illustrates that the Contrast agent in modified form non-liposomal transfection reagent box of the present invention is remarkably improved
The transfection efficiency of star-shaped cationic polymer.
Influence of the different transfection reagents of embodiment 5 to cell survival rate
When 293 cells grow to cell confluency in 24 orifice plates and reach 70%, it is separately added into non-with modified form of the present invention
(transfection reagent and Contrast agent volume ratio are the same as implementation for the transfection reagent and Contrast agent mixed liquor that lipofectamine box is prepared
Example 2 after being incubated for different time altogether for 2:75), PEI-25 transfection reagent and Lip2000 transfection reagent, measures cell using mtt assay
Survival rate.In total incubation 6h, 12h, for 24 hours and after 48h, 10 μ L CCK8 solution (article No. CK04, DOJINDO) of every hole addition, 37 DEG C
It is incubated for 1~4h, microplate reader measures light absorption value at 450nm, cell survival rate is calculated, as a result as shown in Fig. 2 and table 2.All data
It is indicated with (mean ± standard deviation), is handled using 22.0 statistical software of SPSS.More comparison among groups one-way analysis of variances.P<
0.05 indicates statistical difference, and P < 0.01 indicates significant statistical difference.
2 cell survival rate of table compares
| 6h | 12h | 24h | 48h | |
| The present invention | 99.033±0.3055 | 97.0667±0.1528 | 94.2533±0.1626 | 91.2±0.2646 |
| PEI-25 | 98.927±0.3215 | 96.8467±0.0493* | 93.92±0.0529** | 87.233±0.3786** |
| Lip2000 | 99.067±0.1528 | 96.9±0.0458 | 94.02±0.01* | 88.067±0.2517** |
Note: compared with the present invention,*Indicate P < 0.05；**Indicate P < 0.01.
By the result of Fig. 2 and table 2 it is found that the cell survival rate of product of the present invention is significant when incubation time is not less than 12h altogether
Higher than PEI-25 transfection reagent, and altogether incubation time not less than for 24 hours when, the cell survival rate of product of the present invention is also significantly greater than
Lip2000 transfection reagent illustrates that modified form non-liposomal transfection reagent box good biocompatibility of the present invention, immunogenicity are low, malicious
Property it is low, for gene transfection safety it is better than common PEI-25 transfection reagent and Lip2000 transfection reagent, cell survival rate
It is high.
Above-described is only some embodiments of the present invention.For those of ordinary skill in the art, not
Under the premise of being detached from the invention design, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.
Claims (10)
1. a kind of modified form non-liposomal transfection reagent box characterized by comprising
For preparing the star-shaped cationic polymer of transfection reagent；And
For preparing sodium chloride, 4- hydroxyethyl piperazineethanesulfonic acid and the citric acid-sodium citrate buffer solution of Contrast agent.
2. modified form non-liposomal transfection reagent box according to claim 1, which is characterized in that the transfection reagent is matched
Method processed includes the following steps: for star-shaped cationic polymer to be dissolved in sterile water, adjusts pH value to get transfection reagent.
3. modified form non-liposomal transfection reagent box according to claim 2, which is characterized in that in the transfection reagent,
The concentration of star-shaped cationic polymer is 1mg/mL, and the pH of the transfection reagent is 7.0.
4. described in any item modified form non-liposomal transfection reagent boxes according to claim 1~3, which is characterized in that the increasing
The preparation method of strong reagent includes the following steps: sodium chloride and 4- hydroxyethyl piperazineethanesulfonic acid being dissolved in citric acid-citric acid
In sodium buffer, pH value is adjusted to get Contrast agent.
5. modified form non-liposomal transfection reagent box according to claim 4, which is characterized in that in the Contrast agent,
The concentration of citric acid-sodium citrate buffer solution is 10mmol/L, the final concentration of 150mmol/L of sodium chloride, 4- hydroxyethyl piperazine
The final concentration of 10mmol/L of ethanesulfonic acid, the pH value of the Contrast agent are 7.2~7.4.
6. the method that the described in any item modified form non-liposomal transfection reagent boxes of application Claims 1 to 5 carry out gene transfection,
It is characterized by comprising the following steps:
(1) star-shaped cationic polymer is dissolved in sterile water, adjusting pH value is 7.0 to get transfection reagent；
(2) sodium chloride and 4- hydroxyethyl piperazineethanesulfonic acid are dissolved in citric acid-sodium citrate buffer solution, adjusting pH value is
7.2~7.4 to get Contrast agent, wherein the concentration of citric acid-sodium citrate buffer solution is 10mmol/L；
(3) plasmid is added in Contrast agent, mixes, obtains mixture；
(4) transfection reagent is added in the mixture, 10~30min is stood after mixing, obtains Transfection solution；
(5) Transfection solution is added in cell culture fluid, cell is placed in incubator after mixing and is cultivated.
7. the method according to claim 6 for carrying out gene transfection using modified form non-liposomal transfection reagent box, special
Sign is, in the step (1), the concentration of star-shaped cationic polymer is 1mg/mL.
8. the method according to claim 6 for carrying out gene transfection using modified form non-liposomal transfection reagent box, special
Sign is, in the step (2), the final concentration of 150mmol/L of sodium chloride, 4- hydroxyethyl piperazineethanesulfonic acid it is final concentration of
10mmol/L。
9. the method according to claim 6 for carrying out gene transfection using modified form non-liposomal transfection reagent box, special
Sign is that the mass ratio of plasmid and star-shaped cationic polymer is 1:(2~4).
10. the method according to claim 6 for carrying out gene transfection using modified form non-liposomal transfection reagent box, special
Sign is that the volume ratio of Contrast agent and cell culture fluid is 1:(5~15).
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