CN112342239B - Use of melatonin in electroporated transfected cells - Google Patents

Abstract

The invention provides an application of melatonin in electroporation transfected cells. The invention discovers that melatonin can reduce apoptosis rate after electroporation transfection, improve electroporation transfection efficiency of fibroblasts, and has a certain promotion effect on cell growth, and can promote formation of fibroblast monoclonal groups.

Description

Use of melatonin in electroporated transfected cells

Technical Field

The invention belongs to the technical field of cell transfection, and relates to application of melatonin in electroporation transfected cells.

Background

Electroporation transfection, as one of the physical methods, not only can DNA, RNA, but also antibodies, enzymes and other biologically active molecules can be transferred into bacteria, yeast, animal cells or plant cells. The method is a high-efficiency and simple gene transfer method, has incomparable advantages as other transfer methods, such as simple and convenient operation, high repeatability, high transfection efficiency, suitability for broad spectrum and the like, and can obtain higher transfection especially for suspension culture cells which are generally considered to be difficult to transfer at present. Electroporation is a technique in which, after an electric field is applied to a cell for several microseconds to several milliseconds, a small hole or opening is temporarily formed in a cell membrane, 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; meanwhile, a small part of plasmids enter the nucleus to integrate with the chromosome, and stable rotation is started; once the DNA diffuses into the cells, the pores of the membrane close.

The low electroporation transfection efficiency can affect the production of in vitro transgenic nuclear donor cells, and the increased apoptosis after electroporation is a main problem of nuclear donor cell production; the production of the reconstructed embryo of the transgenic animal is directly influenced by the nuclear cell culture, and the low production efficiency of the reconstructed embryo of the transgenic animal is one of key factors restricting the development of biotechnology.

Melatonin (MLT) is an indole hormone that is mainly secreted by the pineal gland, and Melatonin is also secreted by other organs such as the retina, gastrointestinal tract, and skin. Melatonin participates in regulating the circadian rhythm of animal organisms, and the synthesis and photoperiod action of melatonin are kept in synchronization for 24 hours. Melatonin and its metabolites can effectively scavenge various Reactive Oxygen Species (ROS), reduce oxidative stress, resist inflammatory reaction, and enhance immunity. Melatonin can activate antioxidant enzyme, increase antioxidant enzyme activity and gene expression, such as catalase, glutathione peroxidase and superoxide dismutase, and inhibit the activity of the enzyme and expression of related genes, such as Nitric Oxide Synthase (NOs), and Xanthine Oxidase (XO) activity and expression level decrease. Melatonin can sequester a variety of metal ions, including iron, copper, aluminum, lead, cadmium, and zinc, and the like, and blocks Fenton reactions (Fenton reactions) from producing ROS.

There is no report on whether melatonin can be applied to electroporation transfection.

Disclosure of Invention

The invention aims to provide an application of melatonin in electroporation transfected cells, and the invention discovers that the melatonin can reduce the apoptosis rate of the cells after electroporation transfection, improve the electroporation transfection efficiency of fibroblasts, and has a certain promotion effect on the growth of the cells, and can promote the formation of fibroblast monoclonal clusters.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides the use of melatonin in electroporated transfected cells.

The cells of the invention are eukaryotic cells, preferably fibroblasts, more preferably mammalian fetal fibroblasts.

In a second aspect, the present invention provides an electrotransfer fluid for electroporating transfected cells, the electrotransfer fluid comprising melatonin.

The invention discovers that the addition of Melatonin (MLT) with a certain concentration into electrotransfer solution can improve electroporation transfection efficiency and has a certain promotion effect on cell growth.

In order to better improve electroporation transfection efficiency and promote cell growth, the concentration of melatonin in the electrotransfection solution is 1×10 ^-11 ～1×10 ^-3 mol/L, e.g. 1X 10 ^-9 mol/L、1×10 ^-7 mol/L、1×10 ^-5 mol/L, etc., preferably 1X 10 ^-7 mol/L。

In a third aspect, the present invention provides a method of increasing the efficiency of electroporation of transfected cells by using melatonin or an electrotransfection solution as described in the second aspect.

The cells of the invention are eukaryotic cells, preferably fibroblasts, more preferably mammalian fetal fibroblasts.

In a fourth aspect, the invention provides a method of electroporating transfected cells, the method comprising: electroporation transfection of cells with the electroporation solution of the second aspect.

Preferably, the cell is a eukaryotic cell, preferably a fibroblast, more preferably a mammalian fetal fibroblast.

As a preferred embodiment of the present invention, the method for electroporating transfected cells according to the present invention comprises the steps of:

(1) Preparing an electrotransport fluid containing melatonin;

(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) And performing electric shock treatment on the electric conversion mixed solution.

The method for electroporation transfection of cells further comprises (4) adding a cell culture solution into the electrotransfer mixed solution after electric shock treatment, and then transferring the cells in the cell culture solution to a culture dish for cell culture.

The molecule to be transfected according to the invention is selected from the group consisting of DNA, RNA, antibodies or enzymes, preferably plasmid DNA.

The preparation method of the molecule to be transfected is a conventional preparation method at present, wherein plasmid DNA containing a green fluorescent protein reporter gene is taken as an example for the following:

adding 250-350mL of sterilized LB liquid medium, 250-300 mu L of colibacillus liquid for converting pIRES-AANA stored at the temperature of between 250 and 80 ℃ and antibiotics carried by 6-7 mu L of 1000X expression vector into a sterile 500mL conical flask, lightly covering a cover, and shaking by a shaking table at 180rpm at the constant temperature of 37 ℃ for 12-16 hours (overnight); then, the plasmids were extracted using a endotoxin-free miniplasmid Kit (Cycle Pure Kit: OMEGA Biotech, USA) according to the Kit instructions, and the extracted plasmids were subjected to concentration measurement by a Nanodrop ultramicro-spectrophotometer (Thermo Scientific, USA) and stored at-20℃for use.

In the actual operation process, the concentration of the molecules to be transfected in the electrotransfer mixed solution is matched with the specific required concentration on the kit.

In order to obtain excellent transfection effect, the concentration of cells in the electrotransfection mixture is 10 ⁶ ～10 ⁸ personal/mL, e.g. 10 ⁷ And each mL.

As a preferred embodiment of the present invention, the method includes:

(1) Preparing the mixture containing (0.5-1.5) x 10 ^-7 Electrotransport fluid of mol/L melatonin;

(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, wherein the adopted electric conversion program is A03;

(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.

Taking sheep fetal fibroblasts as an example here, the isolation and culture process of fibroblasts is enumerated:

(1) Tissue block adherence method for separating and culturing fetal fibroblast

a. The pure-bred dupont sheep is bred, pregnant is carried out for about 35 days, a fetus is taken by aseptic sampling of living operation, the fetus is washed 3 times by PBS containing 3X double antibody, namely penicillin and streptomycin mixed solution, the fetus is soaked in DMEM/F12 containing 2X double antibody, the fetus is sealed by a sealing film, and the fetus is quickly brought back to a laboratory at low temperature; sterilizing the cell chamber clean bench with 75% alcohol surface, and sterilizing with ultraviolet lamp for 15min; PBS, DMEM/F12 cell culture solution and the like are preheated at 37 ℃; b. taking out fetus from DMEM/F12, transferring to 100mm culture dish in super clean bench, sterilizing with 75% alcohol surface for 30-60s, washing fetus with PBS containing 3X double antibody, shearing fetal head, limbs, viscera, PBS, washing with DMEM/F12 for 2 times, shearing the rest with sterilized ophthalmic scissors in 60mm cell culture dish, and pulverizing to about 1mm ³ The method comprises the steps of carrying out a first treatment on the surface of the c. Spreading the tissue fragments uniformly into a 60mm cell culture dish with sterilized forceps, slightly drying, dripping 30% FBS-containing DMEM/F12 culture solution with 1mL pipette, standing at 385 deg.C and 5% CO ₂ Culturing in an incubator for 12h, adding 3mL of DMEM/F12 culture solution containing 10% FBS, continuously culturing, observing the climbing-out condition of cells around the tissue block every 3 days, changing the solution, and performing subculture and freezing preservation when the cells grow to 75% confluence state.

(2) Subculture of fetal fibroblasts

a. When the cells grow to 80% confluence, discarding the old culture solution, and cleaning the cells for 2 times by using DPBS preheated at 37 ℃; b. adding a proper amount of pancreatin (1-2 mL pancreatin is added into a common 60mm cell culture dish), digesting for 3min at 37 ℃, allowing the cells to slide down by tilting the culture dish after 3min of digestion, gently blowing the bottom of the culture dish by using a 1mL pipetting gun, slightly tilting, and blowing all the cells down; note that the time is not too long, otherwise the pancreatin will over digest the cells, the digestion is stopped by adding serum-containing culture medium, and the suspension is then collected into a centrifuge tube; c. balancing the centrifuge tube, centrifuging at 1500rpm for 5min, discarding supernatant, washing with preheated DMEM/F12 culture solution, centrifuging at 1500rpm for 5min, discarding supernatant, and re-suspending cell pellet with DMEM/F12 culture solution containing 10% FBS to adjust cell densityDegree, according to 1X 10 ⁵ Inoculating the cells/mL into a new cell culture dish at 38.5 ℃ and 5% CO ₂ Culturing in an incubator, replacing culture solution every 2 days, and carrying out passage or freezing storage when the cells grow to 80% confluence.

The electric conversion mixed solution and the subsequent electric shock treatment and the like can adopt the conventional methods at present, and are exemplified as follows:

in the electroporation transfection method, the specific steps include: a. soaking the electric rotating cup with 75% ethanol overnight, and air drying before use; b. culturing sheep fetal fibroblasts in a complete culture medium until the sheep fetal fibroblasts reach a logarithmic growth phase, adding pancreatin, digesting for 3min, adding the complete culture medium, stopping digestion, centrifuging at 1500rpm for 5min, discarding the supernatant, adding DPBS for resuspension, centrifuging again, and collecting cells; c. the cells were mixed at 1X 10 ⁷ Resuspension of the density of individual/mL in 100mL of electroporation buffer at 0 ℃, transferring the cell suspension into an electrocuvette, failing to generate bubbles, holding the "windows" on both sides of the electrocuvette, vibrating the bottom thereof to mix the cell suspension; d. and (3) placing the electric rotating cup into the position of the electroporation device for electric shock once by using a set program, taking out the electric rotating cup after OK, adding a proper amount of complete culture medium, sucking out after uniform mixing, centrifugally cleaning cells, transferring the cells into the complete culture medium for culturing, and transferring the cells under a fluorescence microscope for observation for 24-48 hours.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention discovers that melatonin 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 melatonin of the present invention is 1X 10 ^-9 ～1×10 ^-5 mol/L, has remarkable effect on improving electroporation transfection efficiency;

(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.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

Example 1

A method for electroporation transfection of fibroblasts is as follows:

(1) Preparation of melatonin concentrate

23.2mg melatonin was dissolved in 1mL DMSO to give 10 ^-1 M MLT concentrate; diluting 10000 times with complete culture medium to obtain 10 ^-5 M MLT solution; when in use, 1 mu L of 10 is added ^-5 M MLT solution to 99. Mu.L of electrotransfer solution, i.e.MLT working concentration of 10 ^-7 M MLT。

(2) Extraction of plasmid DNA with EGFP reporter Gene:

a. 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; b. shaking: taking a sterile 500mL conical flask, adding 300mL of LB liquid medium, 270 mu L of escherichia coli bacterial liquid stored at the temperature of between 80 ℃ and converted pIRES-AANA and adding 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); c. 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 fetal fibroblasts

a. Dupont sheep are bred, about 35 days of gestation, a dupont sheep fetus is aseptically collected through living operation, the dupont sheep fetus is washed 3 times by PBS containing 3X double antibody, namely penicillin and streptomycin mixed solution, 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; b. 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 ℃; c. remove fetus from DMEM/F12, transfer to 100mm Petri dish in ultra clean bench, sterilize with 75% alcohol surface for 60s, wash with PBS containing 3X diabodyFetal head, limbs, viscera are sheared off by sterilized ophthalmic scissors, PBS is used for cleaning, finally DMEM/F12 is used for cleaning for 2 times, and the rest part is sheared off by sterilized ophthalmic scissors in a 60mm cell culture dish with the size of about 1mm ³ The method comprises the steps of carrying out a first treatment on the surface of the d. Spreading the tissue fragments uniformly into a 60mm cell culture dish with sterilized forceps, slightly drying, dripping 30% FBS-containing DMEM/F12 culture solution with 1mL pipette, standing at 385 deg.C and 5% CO ₂ Culturing in an incubator for 12h, adding 3mL of DMEM/F12 culture solution containing 10% FBS, continuously culturing, observing the climbing-out condition of cells around the tissue block every 3 days, changing the solution, and performing subculture and freezing preservation when the cells grow to 75% confluence state.

(4) Electroporation transfection of fibroblasts

a. 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; b. 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; c. 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; d. preparing a nucleic electric conversion buffer solution with room temperature rewarming, wherein 10 is added ^-7 M melatonin, adding 10 μg of plasmid DNA into 100 μl of electrotransfer buffer, centrifuging, discarding supernatant, and resuspending cells with prepared plasmid-containing electrotransfer buffer; e. transferring the mixed solution into an electrode cup by using a pipetting gun, opening a nucleofector electrotransfer instrument, selecting an electrotransfer program A03 of electrotransfer 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;f. 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.

Examples 2 to 5

The difference from example 1 is that in this example, the concentration of melatonin in the electrotransport fluid is 10 ^-5 M (example 2), 10 ^-9 M (example 3), 10 ^-3 M (example 4), 10 ^-11 M (example 5).

Comparative example 1

The difference from example 1 is that in this comparative example, step (1) was omitted, i.e., melatonin was not added to the electrotransport fluid.

Performance testing

The methods provided in examples 1-6 and comparative example 1 were tested for performance as follows:

(1) Electric conversion efficiency: electrotransformation of plasmid with GFP fluorescence into a sheep fetal fibroblast cell line using electrotransformation procedure A03, and cell electrotransformation efficiency was counted using a flow cytometer within 48 h;

(2) Efficiency of cell monoclonal mass formation after electrotransfection: the working concentration of melatonin is controlled to be the same as that of examples 1-5 and comparative example 1, monoclonal cells are selected by a flow cytometer for culture, liquid is changed every 5 days, cell lines with good growth state are counted after 10 days are counted, and the influence of melatonin with different concentrations on the formation efficiency of cell monoclonal clusters is observed;

(3) Apoptosis results after electrotransfection: screening by using an Annexin V-Alexa flow 647/PI apoptosis detection kit after electrotransfection for 48 hours, and counting experimental results by a flow cytometer;

the test results are shown in Table 1:

TABLE 1

Sample of	Electrotransport efficiency/%	Single cell colony formation rate/%	Apoptosis rate/%
				Example 1	71.01±2.63％ b	19.50±1.31 b	9.67±1.11％ a
Example 2	51.49±6.5％ b	14.00±1.29 a	8.76±1.72％ a
				Example 3	60.77±3.43％ b	15.50±1.12 a	11.19±3.58％ a
Example 4	44.68±1.84％ a	-	13.43±1.02％ b
				Example 5	49.26±3.78％ b	-	15.69±2.11％ b
Comparative example 1	31.57±0.28％ a	13.30±1.05 a	19.28±3.71％b

Note that: the statistical analysis is carried out by adopting SPSS single-factor analysis of variance software, and the data without the upper marks of the same lowercase letters have significant differences; the single cell colony formation rate was not measured in examples 4 to 5.

According to the embodiment and the performance test, the addition of melatonin in the electrotransfer liquid can promote the electrotransfer efficiency, improve the formation efficiency of the fetal fibroblast monoclonal group, reduce the damage to the fetal fibroblast during electroporation transfection and reduce apoptosis.

Meanwhile, as is clear from comparison of example 1 and examples 2 to 5, the melatonin content in the electrotransport fluid was 1X 10 ^-9 ～1×10 ^-5 When the mol/L is in the range, the method has obvious promotion effect on electroporation transfection efficiency and other aspects, and can obviously reduce apoptosis.

(4) Cell viability: melatonin with the same concentration as in examples 1-5 is respectively added into culture solutions of sheep fetal fibroblasts, a group of culture solutions are left without melatonin, cells are collected after a certain period of time and stained with trypan blue, and the cell viability is counted by a visual method on a blood cell counting plate;

the test results are shown in Table 2:

TABLE 2

From the examples and performance tests, it is clear that the addition of melatonin to the culture broth increases the fetal fibroblast viability with significant differences.

(5) Cell proliferation: melatonin with the same concentration as in examples 1-5 is respectively added into culture solutions of sheep fetal fibroblasts, a group of culture solutions are left without melatonin, after a certain time, the cells are collected, and the proliferation capacity of the cells is detected by using a CCK8 kit, and the light absorption value (OD value) is counted;

the test results are shown in Table 3:

TABLE 3 Table 3

From the examples and performance tests, melatonin can promote the proliferation of fetal fibroblasts, and has remarkable promotion effect.

In conclusion, after the fetal fibroblasts are obtained through in vitro separation, melatonin with proper concentration is added into the electrotransfer solution, so that apoptosis after electroporation transfection can be reduced, and electrotransfer efficiency is improved. Therefore, the method provided by the invention is favorable for improving the electroporation transfection efficiency, provides theoretical reference for the application and popularization of melatonin in transgenic animal production and achieves the purpose of improving the electroporation transfection efficiency.

The applicant states that the use of melatonin of the invention in electroporated cells is illustrated by the above examples, 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.

Claims (5)

1. Use of melatonin in electroporated transfected cells, wherein the use comprises one or more of:

(1) Reducing the apoptosis rate after electroporation transfection;

(2) The electroporation transfection efficiency of cells is improved;

wherein the cells are fibroblasts; the application is to carry out cell transfection by using electrotransfer solution added with melatonin, wherein the concentration of the melatonin is 10 ^-9 mol/L～10 ^-5 mol/L。

2. The use according to claim 1, wherein the fibroblast is a mammalian fetal fibroblast.

3. A method for improving the efficiency of electroporation transfected cells, characterized in that the method improves the efficiency of electroporation transfected cells by using an electrotransfection solution to which melatonin is added, the concentration of melatonin being 10 ^-9 mol/L～10 ^-5 mol/L；

Wherein the cells are fibroblasts.

4. The method of claim 3, wherein the fibroblast is a mammalian fetal fibroblast.

5. A method of electroporating a foetus ovis fibroblasts, the method comprising:

(1) Preparing the mixture containing (0.5-1.5) x 10 ^-7 Electrotransport fluid of mol/L melatonin;

(2) Adding plasmid DNA to be transfected into the electrotransfer solution to make its concentration be 0.05-0.15 g/L, then re-suspending sheep fetal fibroblast to make its concentration be (0.5-1.5). Times.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.