CN112725377B - Application of resveratrol in electroporation transfected cells and electrotransfection liquid - Google Patents
Application of resveratrol in electroporation transfected cells and electrotransfection liquid Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly discloses application of resveratrol in electroporation transfected cells, and simultaneously discloses an electrotransfection solution containing resveratrol and a method for electroporation transfected cells. According to the invention, resveratrol is added into electroporation transfected cells, and experiments prove that the electroporation transfection efficiency of the fibroblasts can be improved, and the aim of improving the electrotransfection efficiency without affecting the growth of the cells can be fulfilled. Resveratrol is natural and harmless, is safe to cells, and provides a guarantee for the application of resveratrol in transgenic animal production.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to a cell electroporation transfection technology, and particularly relates to application of resveratrol in electroporation transfected cells, an electrotransfection solution and a method for electroporation transfected cells by adopting the electrotransfection solution.
Background
Electroporation transfection (electroporation for short) is a highly efficient and simple gene transfer technique, and is commonly used to obtain transgenic cells in vitro. Electroporation is a technique in which a small hole or opening is formed in a cell membrane for several microseconds to several milliseconds by an electric field acting on a cell, and a macromolecule such as DNA is introduced into the cell and finally into the nucleus. The process is briefly described as follows: firstly, in the electric shock process, perforation appears on a cell membrane, a plasmid is contacted with the cell membrane under the action of electrophoresis force, and a transferable complex is formed in the electroporation area on the cell membrane; after the electric shock again, the plasmid breaks away from the complex and diffuses into the cytoplasm to start transient rotation; at the same time, a small part of plasmid enters the nucleus to integrate with chromosome, and stable rotation is started. Once the DNA diffuses into the cells, the pores of the membrane close. Electroporation transfection is used as a physical method, and not only can DNA, RNA, but also can antibody, enzyme and other bioactive molecules be transferred into bacteria, yeast, animal cells and plant cells, and has incomparable superiority to other transfer methods, such as simple and convenient operation, rapidness, strong repeatability, high transfection efficiency, suitability for broad spectrum and the like.
However, since electric shock damages cells and generates a large amount of ROS (reactive oxygen species), apoptosis of cells after transfection is unavoidable. The electric shock damages most cells and causes apoptosis, which is also a major cause of the inefficiency of electric transduction.
Transgenic animal models are an important role in life sciences, the construction of the models consumes a great deal of cost, and the damage of ROS to cells severely reduces the application of biotechnology in production. The low production efficiency of the reconstructed embryo of the transgenic animal is one of key factors restricting the development of biotechnology in China. Wherein, the low electroporation transfection efficiency affects the production of in vitro transgenic nuclear supply cells, and the increased apoptosis after electroporation is a main difficult problem of the production of the transgenic nuclear supply cells.
Therefore, improving electroporation transfection efficiency and reducing apoptosis rate of cells after electroporation is a technical problem which needs to be solved urgently at present.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide the application of resveratrol in electroporation transfected cells, and the application of resveratrol in the electroporation transfected cells can promote the electrotransfection efficiency and reduce the apoptosis.
To achieve the purpose, the invention adopts the following technical scheme.
In a first aspect, the invention provides the use of resveratrol in electroporated transfected cells.
As a preferred embodiment of the invention, the cell is a eukaryotic cell. Further preferred are fibroblasts.
More preferably, the cell is a mammalian fibroblast, such as a sheep fetal fibroblast.
As a preferred embodiment of the invention, the resveratrol is used as an electrotransfection fluid additive in electroporated transfected cells.
As a preferred embodiment of the present invention, the resveratrol has a concentration of 10 in the electrotransport fluid -9 ～10 - 3 M is more preferably 10 -7 ～10 -5 M. Wherein M represents the volume molar ratio concentration in mol/L.
More preferably, the concentration of resveratrol in electrotransport fluid is 10 -7 M、10 -6 M or 10 -5 M。
In a second aspect, the invention provides an electrotransport fluid, which contains resveratrol.
As a preferred embodiment of the present invention, the concentration of resveratrol in the electrotransport fluid is 10 -9 ～10 -3 M。
Further preferably, the concentration of resveratrol in the electrotransport fluid is 10 -7 ～10 -5 M, more preferably 10 -7 M、10 -6 M or 10 -5 M。
As a preferred embodiment of the invention, the electrotransport buffer is also included in the electrotransport solution.
Further preferably, the electrotransport buffer is a nucleofector electrotransport buffer.
In a third aspect, the invention provides the use of an electrotransfer fluid according to the invention in electroporating transfected cells. Preferably, the cell is a eukaryotic cell.
Further preferred, the electrotransfection solution of the present invention is used in electroporation transfection of fibroblasts.
More preferably, the electrotransfection solution of the present invention is used in electroporation transfection of mammalian fibroblasts. For example, it can be used for electrotransfection of sheep fetal fibroblasts.
In a fourth aspect, the present invention provides a method for electroporating cells, using resveratrol or an electrotransfection solution of the present invention to electroporate cells, comprising the steps of: cells are electrotransformed in the electrotransformation solution.
As a preferred embodiment of the present invention, the cells are eukaryotic cells, more preferably fibroblasts, even more preferably mammalian fibroblasts.
As a preferred embodiment of the present invention, the method for electroporating transfected cells provided by the present invention comprises the steps of:
(1) Preparing an electrotransport liquid containing resveratrol;
(2) Adding a molecule to be transfected into the electrotransfer solution, and then re-suspending cells in the molecule to obtain an electrotransfer mixed solution;
(3) Performing electric shock treatment on the electric conversion mixed solution;
preferably, the method further comprises (4) adding a cell culture solution to the electrotransfer mixed solution after the electric shock treatment, and then transferring the cells in the cell culture solution to a culture dish for cell culture.
As a preferred embodiment of the invention, the molecule to be transfected is selected from the group consisting of DNA, RNA, antibodies or enzymes, preferably plasmid DNA.
As a preferred embodiment of the present invention, the concentration of cells in the electrotransport mixture is 10 6 ～10 8 And each mL.
As a more preferred embodiment of the present invention, the present invention provides a method for electroporating transfected cells comprising:
(1) Preparing an electrotransport liquid containing resveratrol, wherein the concentration of resveratrol in the electrotransport liquid is 10 -7 ～10 -5 M；
(2) Plasmid DNA to be transfected is added into the electrotransfer solution to make the concentration of the plasmid DNA be 0.05-0.15 g/L, and then the sheep fetal fibroblast is resuspended in the concentration of (0.5-1.5) x 10 7 Obtaining electric conversion mixed solution by adopting the volume per mL;
(3) Performing electric shock treatment on the electric conversion mixed solution;
(4) And (3) adding a cell culture solution into the electrotransfer mixed solution after the electric shock treatment, and transferring cells in the cell culture solution into a culture dish for cell culture.
The electrotransport treatment operation and the cultivation after electrotransport of the present invention can be performed by conventional methods.
The invention adopts resveratrol, in particular to an electrotransfection liquid containing resveratrol with proper concentration to improve the electroporation transfection efficiency of fibroblasts, can achieve the purpose of improving the electrotransfection efficiency without affecting the cell growth, and has low apoptosis rate after electrotransfection.
Resveratrol (Resveratrol) is a natural plant-based glucose derivative that is found in a variety of plants and has a greater antioxidant capacity than Vitamins (Vitamins) E and C. Resveratrol acts at the initiation and propagation stages of the oxidation reaction, increasing the fluidity of the cell membrane by acting on the cell membrane where peroxidation occurs. Resveratrol has various biological activities, and resveratrol treatment can induce cell cycle arrest in the G1 phase by affecting cyclin expression. In the process of researching the influence of resveratrol on the growth of somatic cells, the resveratrol with proper concentration has a promoting effect on the growth of cells, and can obviously improve the cell electrotransformation efficiency of fibroblasts, such as the fibroblasts of sheep fetuses, so that the reagent for enhancing the cell electrotransfection can be prepared by utilizing the characteristic of the resveratrol.
Experiments prove that the adoption of resveratrol with proper concentration can improve the electroporation transfection efficiency of the fibroblast and can achieve the aim of improving the electroporation transfection efficiency without affecting the cell growth. In the invention, sheep is taken as an example, and the result shows that resveratrol can improve the electrotransformation efficiency of the sheep fetal fibroblasts and reduce the damage of electric shock to the cells. In particular using resveratrol at a concentration of 10 -5 、10 -6 Or 10 -7 The electrotransfection liquid of M can obviously improve the electroporation transfection efficiency of the fibroblast and has obvious effect. And resveratrol is natural and harmless, is safe to cells, and provides a guarantee for the application of resveratrol in transgenic animal production.
The method for electrotransformation of the invention is used for detecting the cells after electrotransformation, and the method for electrotransformation of the invention can promote proliferation of fetal fibroblasts, and has obvious promotion effect; it can also enhance the mitochondrial level of fibroblast and reduce ROS in fibroblast, and has remarkable protecting effect on cell.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention discovers that resveratrol can improve electroporation transfection efficiency of fibroblasts;
(2) The electrotransfection liquid provided by the invention can reduce the damage to fibroblasts during electroporation transfection, reduce apoptosis and further improve electroporation transfection efficiency;
(3) The preferred concentration of resveratrol in the invention is 10 -7 ～×10 -5 M, the effect on improving electroporation transfection efficiency is remarkable;
(4) The electrotransfection liquid provided by the invention is safe and harmless, and can achieve the purpose of improving electrotransfection efficiency under the condition of not damaging cells.
Resveratrol is suitable for electroporation transfection of fibroblast lines, provides a safe and effective method for cell electrotransformation, and has wide market prospect. The invention can promote the in vitro production of the transgenic cells when applied to the production practice of transgenic animals, greatly improve the production efficiency of transgenic cloned animals and promote the development of life science research. Transgenic animals are important scientific research models, and efficient production of transgenic animals has important significance for scientific research and popularization of transgenic animal products. Meanwhile, the transgenic animal research consumes a great amount of financial resources, and the method of the invention improves the production efficiency of the transgenic animal and has considerable direct economic benefit.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It should be apparent to those skilled in the art that the detailed description is merely provided to aid in understanding the invention and should not be taken as limiting the invention in any way. Unless otherwise indicated, the technical means used in the examples below are conventional means well known to those skilled in the art, and the raw materials used are commercially available conventional products.
Cell electrotransformation tests are carried out by adopting electrotransformation liquid containing resveratrol, and the test method comprises the following steps:
(1) Preparing resveratrol for storage;
(2) Extracting plasmid DNA with green fluorescent protein (EGFP) reporter gene;
(3) Isolation and culture of sheep fetal fibroblasts;
(4) Cell electrotransfection.
Meanwhile, a comparative test was performed using a blank electrotransport fluid (i.e., without resveratrol).
Specific experiments are described in the following examples and comparative examples.
Example 1
The embodiment provides a method for electroporation transfection of fibroblasts by using an electrotransfer solution containing resveratrol, comprising the following steps:
(1) Preparation of resveratrol concentrate
22.8mg resveratrol was dissolved in 1mL DMSO to give 10 -1 A concentrated solution of M resveratrol; diluting 1000 times with complete culture medium to obtain 10 -4 M resveratrol solution; when in use, 1 mu L of 10 is added -4 The working concentration of the resveratrol is 10 when the M resveratrol solution is added into 99 mu L of electrotransfer solution -6 M；
(2) Extraction of plasmid DNA with EGFP reporter Gene
(1) Preparing an LB liquid culture medium: weighing 10g of Tryptone (Tryptone), 5g of Yeast Extract (Yeast Extract), 10g of sodium chloride (NaCl) and 1L of ultrapure water, and autoclaving for later use;
(2) shaking: taking a sterile 500mL conical flask, adding 300mL of LB liquid medium, 270 mu L of pIRES-AANAT transformed escherichia coli bacterial liquid stored at the temperature of 80 ℃ and 6 mu L of antibiotics carried by 1000X expression vectors, lightly covering a cover, and shaking at 180rpm by a constant temperature shaking table at 37 ℃ for 16 hours (overnight);
(3) extracting plasmid: extracting plasmid with endotoxin-free small plasmid Kit (Cycle Pure Kit: OMEGA Biotech, USA), measuring concentration of the extracted plasmid with Nanodrop ultramicro-spectrophotometer (Thermo Scientific, USA), and preserving at-20deg.C;
(3) Isolation and culture of sheep fetal fibroblasts
(1) Dupont sheep are bred, pregnant is carried out for about 35 days, live operation is carried out aseptically, a dupont sheep fetus is adopted, PBS containing 3X double antibody, namely penicillin and streptomycin mixed solution is used for washing 3 times, the dupont sheep is soaked in DMEM/F12 containing 2X double antibody, and the dupont sheep fetus is sealed by a sealing film and placed at low temperature;
(2) sterilizing the cell chamber clean bench with 75% alcohol surface, and sterilizing with ultraviolet lamp for 30min; PBS and DMEM/F12 cell culture solution are preheated at 37 ℃;
(3) taking out fetus from DMEM/F12, transferring to 100mm culture dish in super clean bench, sterilizing with 75% alcohol surface for 60s, washing fetus with PBS containing 3X double antibody, shearing fetal head, limbs, viscera, PBS, washing with DMEM/F12 for 2 times, shearing the rest in 60mm cell culture dish with sterilized ophthalmic scissors, and cutting into pieces of about 1mm 3 ；
(4) Uniformly spreading the tissue fragments into a 60mm cell culture dish by using a sterilizing forceps, slightly drying, uniformly dripping DMEM/F12 culture solution containing 30% FBS into the culture dish by using a 1mL pipetting gun until the tissue fragments adhere to the culture dish, and about 1mL; then placed at 38.5 ℃ and 5% CO 2 Culturing in an incubator for 12 hours, and then supplementing 3mL of DMEM/F12 culture solution containing 10% FBS for continuous culture; observing the climbing-out condition of cells around the tissue block every 3 days, changing the liquid, and when the cells grow to a confluent state with the confluence degree of 75%, carrying out subculture and freezing preservation for later use;
(4) Electroporation transfection of fibroblasts
(1) Taking sheep fetal fibroblasts in a logarithmic growth phase, culturing to 80% confluence, discarding old culture solution, washing with preheated DPBS for 2 times, discarding washing solution, adding a proper amount of pancreatin (2 mL pancreatin is added into a common 60mm cell culture dish), then digesting for 3min at 37 ℃, and preparing a centrifuge tube on the other side;
(2) after digestion for 3min, the cells slide down in the inclined culture dish, the bottom of the culture dish is gently blown by a 1mL pipetting gun, all the cells are blown down, the time is not too long, or pancreatin excessively digests the cells, the digestion is stopped by adding a culture solution containing serum, and then the suspension is collected into a centrifuge tube;
(3) placing the balanced centrifuge tube into a centrifuge, centrifuging at 1500rpm for 5min, discarding the supernatant, washing with preheated DPBS again, and centrifuging at 1500rpm for 5min;
(4) preparation of room temperature rewriter electrotransport buffer 99. Mu.L, 1. Mu.L of 10 was added thereto -4 M resveratrol solution, i.e. resveratrol with working concentration of 10 -6 M; adding 10 mug of the plasmid DNA with the EGFP reporter gene obtained by extraction in the step (2) into the 100 mug of electrotransport buffer solution;
(5) the supernatant was discarded from the centrifuged cell pellet, and the cells were resuspended in the plasmid-containing electrotransfer buffer prepared in step (4) at a cell concentration of 1X 10 7 Obtaining mixed solution per ml;
(6) transferring the mixed solution in the step (5) into an electrode cup by using a pipetting gun, opening a nucleofector electrotransfer instrument, selecting an electrotransfer program A03 for electrotransferring fetal fibroblasts, placing the electrotransfer cup into an electric shock tank, taking out the electrotransfer cup after electric shock is finished, adding 300 mu L of DMEM/F12+10% FBS culture solution into the electrode cup, and transferring the cells out of the electrotransfer cup by using a special straw attached to the nucleofector kit;
(7) cells after electrotransfection were used in 1X 10 5 Is inoculated in 60mm dishes at 38.5℃with DMEM/F12+10% FBS medium and 5% CO 2 Cells were cultured in an incubator.
Example 2
The method for electroporation transfection of fibroblasts with an electrotransfer solution containing resveratrol provided in this example differs from that in example 1 only in that in step (4) of step (4), the working concentration of resveratrol is 10 -5 M。
Example 3
The method for electroporation transfection of fibroblasts with an electrotransfer solution containing resveratrol provided in this example differs from that in example 1 only in that in step (4) of step (4), the working concentration of resveratrol is 10 -7 M。
Comparative example 1
This comparative example was a test of electroporating fibroblasts using a blank electrotransfection solution (i.e., a resveratrol-free nucleic acid electrotransfection buffer), in which extraction of plasmid DNA with EGFP reporter gene, isolation and culture of sheep fetal fibroblasts were performed as in example 1, differing from example 1 only in that in the case of electroporating fibroblasts, a resveratrol-free nucleic acid electrotransfection buffer was used in step (4) of step (4).
Example 4
The cells after electrotransfection prepared in examples 1-3 and comparative example 1 were subjected to performance tests, mainly to examine the electrotransfection efficiency and apoptosis after electrotransfection. The test method comprises the following steps:
(1) Electric conversion efficiency: electrotransformation of the plasmid with EGFP fluorescence into a sheep fetal fibroblast cell line using electrotransformation procedure A03, and counting the electrotransformation efficiency of the cells using a flow cytometer within 48 hours;
(2) Apoptosis results after electrotransfection: after 48h of electrotransfection, screening was performed using an Annexin V-Alexa flow 647/PI apoptosis detection kit, and experimental results were counted by flow cytometry.
The test results are shown in Table 1.
TABLE 1 electroporation efficiency and apoptosis after electroporation
| Test sample | Electrotransport efficiency/% | Apoptosis rate/% |
| Example 1 | 63.02±3.52 b | 11.31±1.28 a |
| Example 2 | 50.13±2.78 b | 12.77±7.56 a |
| Example 3 | 57.52±8.06 b | 16.23±0.66 a |
| Comparative example 1 | 31.57±0.28 a | 20.50±4.96 a |
Note that: statistical analysis was performed using SPSS single factor analysis of variance software, with no significant differences between the data with the same lowercase superscript.
As shown by the performance test results of the table, the electric conversion efficiency can be promoted after the resveratrol is added into the electric conversion liquid, and compared with the electric conversion efficiency of the comparative example, the electric conversion efficiency is obviously improved; and the damage to fetal fibroblast during electroporation transfection can be reduced after the resveratrol is added, so that the apoptosis rate is reduced.
As can be seen from Table 1, resveratrol content in electrotransport fluid was 1X 10 -7 ～1×10 -5 When the mol/L is in the range, the method has a remarkable promoting effect on electroporation transfection efficiency, and can reduce apoptosis.
Example 5
This example examined the effect of resveratrol on cell proliferation. The cells cultured in step (4) and step (7) of examples 1 to 3 and comparative example 1 were collected after culturing for 48 hours, and the proliferation capacity of the cells was measured with CCK8 kit, and the absorbance (OD value) was counted.
The test results are shown in Table 2.
TABLE 2
| Test sample | Absorbance value |
| Example 1 | 1.15±0.08 b |
| Example 2 | 1.12±0.09 b |
| Example 3 | 1.00±0.06 a |
| Comparative example 1 | 0.71±0.12 a |
From the test results in table 2, it is clear that the addition of resveratrol can promote proliferation of fetal fibroblasts, and has a remarkable promoting effect.
Example 6
This example examined the effect of resveratrol on intracellular mitochondrial levels. The cells cultured in step (4) and step (7) of examples 1 to 3 and comparative example 1, respectively, were collected after culturing for 48 hours, and were subjected to detection of the cellular mitochondrial level using a MitoTracker Red CMXRos mitochondrial red fluorescent probe detection kit.
The test results are shown in Table 3.
TABLE 3 cell mitochondrial levels after electrotransformation
| Test sample | Fluorescence value |
| Example 1 | 90.35±9.56 b |
| Example 2 | 82.62±3.36 a |
| Example 3 | 87.51±7.58 a |
| Comparative example 1 | 63.04±3.62 a |
From table 3, it is clear that resveratrol can enhance the mitochondrial level of fibroblasts and has a remarkable promoting effect.
Example 7
This example examined the effect of resveratrol on intracellular ROS levels. The cells cultured in step (7) of step (4) of examples 1 to 3 and comparative example 1, respectively, were collected after culturing for 48 hours, and were subjected to cellular ROS (reactive oxygen species) level detection using a fluorescent probe DCFH-DA using a reactive oxygen species detection kit (Reactive Oxygen Species Assay Kit).
The test results are shown in Table 4.
TABLE 4 cellular ROS levels following electrotransformation
| Test sample | Fluorescence intensity (10X 6) |
| Example 1 | 1.24±0.04 b |
| Example 2 | 2.29±0.17 a |
| Example 3 | 1.35±0.14 b |
| Comparative example 1 | 2.99±0.27 a |
From the results in table 4, resveratrol can reduce ROS in fibroblasts and has a remarkable protective effect.
In conclusion, after the fetal fibroblasts are obtained through in vitro separation, resveratrol with proper concentration is added into the electrotransfer liquid, so that apoptosis after electroporation transfection can be reduced, and electrotransfer efficiency is improved.
Therefore, the scheme of the invention is beneficial to improving the electroporation transfection efficiency, provides technical support for the application and popularization of resveratrol in transgenic animal production, and provides a new solution for achieving the purpose of improving the electroporation transfection efficiency.
The applicant states that the invention is illustrated by the above examples as applied to electroporation transfected cells, but the invention is not limited to the detailed methods described above, i.e. it is not meant that the invention must be practiced in dependence on the detailed methods described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (2)
1. Use of resveratrol for reducing apoptosis in electroporated transfected cells, wherein the cells are foetus ovis fibroblasts,
addition in electroporation transfected cells by using resveratrol as an electrotransfection fluidCell transfection with agents; the concentration of resveratrol in electrotransfer liquid is 10 -6 M。
2. A method of electroporating a foetus ovis fibroblasts, the method comprising the steps of:
(1) Preparing an electrotransport liquid containing resveratrol, wherein the concentration of resveratrol in the electrotransport liquid is 10 -6 M；
(2) Plasmid DNA to be transfected is added into the electrotransfer solution to make the concentration of the plasmid DNA be 0.05-0.15 g/L, and then the sheep fetal fibroblast is resuspended in the concentration of (0.5-1.5) x 10 7 Obtaining electric conversion mixed solution by adopting the volume per mL;
(3) Performing electric shock treatment on the electric conversion mixed solution;
(4) And (3) adding a cell culture solution into the electrotransfer mixed solution after the electric shock treatment, and transferring cells in the cell culture solution into a culture dish for cell culture.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011568176.9A CN112725377B (en) | 2020-12-25 | 2020-12-25 | Application of resveratrol in electroporation transfected cells and electrotransfection liquid |
| PCT/CN2021/141333 WO2022135584A1 (en) | 2020-12-25 | 2021-12-24 | Use of resveratrol in cell electrotransfection and electrotransfection solution |
| ZA2023/07267A ZA202307267B (en) | 2020-12-25 | 2023-07-20 | Use of resveratrol in cell electrotransfection and electrotransfection solution |
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| GB201521101D0 (en) * | 2015-11-30 | 2016-01-13 | Koninklijke Nederlandse Akademie Van Wetenschappen | Transduction buffer |
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| Title |
|---|
| Resveratrol Enhances mRNA and siRNA Lipid Nanoparticles Primary CLL Cell Transfection;Edo Kon等;《Pharmaceutics》;20200607;第12卷(第520期);第3.3、3.4节，第4节最后1段 * |
| 白藜芦醇对LPS诱导的SH-SY5Y细胞损伤的保护作用;葛金芳等;《中药药理与临床》;20130815(第04期);摘要 * |
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