JP4182449B2 - Method for reducing cell damage during nucleic acid introduction - Google Patents

Description

  The present invention relates to a method for reducing cell damage without interfering with introduction into a cell when introducing a nucleic acid. More specifically, the present invention relates to a method for achieving both efficient introduction of nucleic acid and reduction of cell damage by adding acidic polysaccharide at an appropriate time after introduction of nucleic acid.

  The technology to introduce nucleic acid into cells is widely used in basic fields such as functional analysis of gene products, protein expression, gene expression suppression, transcriptional regulatory region analysis, etc. to application fields such as gene therapy. It has become a technology.

  Methods for introducing nucleic acids into cells include physical methods such as electroporation and microinjection, chemical methods using substances such as calcium phosphate, DEAE-dextran, lipids, liposomes and polymers, adenoviruses, and retroviruses. Biological methods to be used are known. Since the chemical method is simple, various reagents for introducing a nucleic acid are currently commercially available and widely used. These methods are also considered to be suitable for in vivo use because of their high safety, easy production on a large scale, and easy use.

  However, the disadvantages of these methods include cell damage, cells in which introduction of nucleic acid is difficult, and low reproducibility of experiments. Among them, the most serious is cell damage. Cell damage is affected by various factors such as the type and amount of the nucleic acid introduction reagent, the mixing ratio of DNA and nucleic acid introduction reagent, the type of cells, the number of cells at the time of nucleic acid introduction, the state of the cells, and the like. The rate of introduction of nucleic acid into cells is also affected by these factors, and when the above conditions are adjusted to reduce damage to cells, a reduction in the rate of introduction is often observed.

  As a method for reducing cell damage, a method of exchanging a medium after introducing a nucleic acid has been proposed. However, such an effect is often not recognized, and there is a problem that it takes a lot of trouble. Furthermore, the introduction rate of the nucleic acid into the cells may be reduced depending on the timing at which the medium is exchanged.

The only method for reducing the damage to cells that appears at the time of introduction of nucleic acid is Dragomir, A. et al. Have reported a method of adding low molecular weight heparin within 2 hours after transfection (Non-patent Document 1). In this report, CFBE (Cystal fibrosis human epithelial cells) and CFSME (Cystal fibrosis submucosal cells) are used, and GenePorter2 (GeneTer min) It has been shown that cell damage can be reduced by replacing with a new medium and adding low molecular weight heparin to the medium 2 hours after transfection. However, in this method, a remarkable reduction in gene transfer rate is observed. That is, the two problems of efficient nucleic acid introduction and cell damage reduction cannot be achieved at the same time. In addition, this method includes a process of removing the medium after transfection and replacing it with a new medium, which is troublesome.
Life Sciences 75, 2203-2216 (2004)

  In the prior art, the addition of low molecular weight heparin can reduce the cell damage that occurs during the introduction of nucleic acid, but there is a problem that the introduction of nucleic acid into cells is hindered. Accordingly, an object of the present invention is to provide a method for reducing cell damage without interfering with gene introduction when introducing a nucleic acid into an animal cell.

As a result of various studies, the present inventors have surprisingly found that the time from the transfection to the addition of the low molecular weight heparin (from the time until the addition of the low molecular weight heparin that contributes to the recovery of the cells damaged by the transfection). On the contrary, it was found that cell damage can be reduced without further reducing the gene transfer rate by further extending the time) from 2 hours.
The present inventors have also found that cell damage is reduced by using a compound other than low molecular weight heparin such as fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, and the like.

That is, the present invention has the following configuration.
Item 1.
A method for reducing cell damage when nucleic acid is introduced into animal cells using a nucleic acid introduction (transfection) reagent, characterized by adding an acidic polysaccharide component to the medium after introduction of the nucleic acid Reduction method.
Item 2.
The method for reducing cell damage according to claim 1, wherein the acidic polysaccharide has a sulfate group.
Item 3.
The method for reducing cell damage according to claim 2, wherein the acidic polysaccharide has a repeating structure of two or more sugars.
Item 4.
The method for reducing cell damage according to claim 3, wherein the acidic polysaccharide is one or more selected from the group consisting of heparin, heparan sulfate, chondroitin sulfate, fucoidan, dextran sulfate, and keratan sulfate.
Item 5.
The method for reducing cell damage according to claim 4, wherein the acidic polysaccharide has a reduced molecular weight.
Item 6.
The method for reducing cell damage according to claim 1, wherein the acidic polysaccharide is added to the medium after 2 hours (not including 2 hours) after the introduction of the nucleic acid.
Item 7.
The method for reducing cell damage according to claim 1, wherein the medium is exchanged when the acidic polysaccharide component is added.
Item 8.
The method for reducing cell damage according to claim 1, wherein a substance selected from the group consisting of calcium phosphate, DEAE-dextran, lipid, liposome, and polymer is used for introduction of a nucleic acid into a cell.
Item 9.
A reagent or kit for reducing cell damage, comprising an acidic polysaccharide used in the method according to claim 1 as a component.

  According to the present invention, it has become possible to reduce cell damage that appears at the time of nucleic acid introduction without hindering the introduction of nucleic acid into cells. Therefore, the introduction of nucleic acids into animal cells can be carried out simply, easily and with good reproducibility.

  The term “cytotoxicity” as used in the present invention means cell death caused by the addition of a nucleic acid introduction reagent or cell shape change. This cell damage can be evaluated by MTT method, LDH method, etc. after the introduction of nucleic acid. In the MTT method, tetrazolium salts such as MTT, XTT, MTS, WST are used, and these substances are reduced by mitochondrial dehydrogenase in living cells to utilize formazan. In this method, the number of viable cells can be calculated by measuring the absorbance of the generated formazan. In the LDH method, cell damage is evaluated by measuring lactate dehydrogenase released from dead cells whose cell membrane is damaged.

  The present invention is a method for reducing cell damage, wherein an acidic polysaccharide component is added after introduction of a nucleic acid.

  The acidic polysaccharide component is preferably dissolved in a buffer and added as an aqueous solution. Although it does not specifically limit as a buffer which melt | dissolves an acidic polysaccharide component, For example, a HEPES buffer, a phosphate buffer, etc. are mention | raise | lifted.

  In the present invention, as the polysaccharide added after transfection, a so-called acidic polysaccharide having an acidic residue is used. Preferred are those having a sulfate group such as heparin, heparan sulfate, chondroitin sulfate, fucoidan, dextran sulfate, and keratan sulfate. More preferably, it contains a plurality of sulfate groups per two sugar molecules, and is not particularly limited, and examples thereof include heparin, heparan sulfate, fucoidan, and dextran sulfate. More preferably, the acidic polysaccharide is derived from a living body, and examples thereof include heparin and heparan sulfate.

  The acidic polysaccharide may be reduced in molecular weight, and for example, reduced molecular weight heparin or reduced molecular weight fucoidan may be used. The term “reducing molecular weight” as used herein means that the acidic polysaccharide is decomposed chemically or enzymatically. The low molecular weight heparin and low molecular weight fucoidan to be used are preferably those having a molecular weight of 2,000 to 8,000 Da, more preferably a molecular weight of 4,000 to 6,000 Da.

  Moreover, although it is thought that the acidic polysaccharide in this invention is supplied as a compound of various salts, a kind is not limited. For example, a sodium salt, an ammonium salt, a lithium salt, or a calcium salt can be used.

  In the present invention, the time from the transfection to the addition of acidic polysaccharide (the time until the addition of acidic polysaccharide that contributes to the recovery of cells damaged by transfection) is preferably longer than 2 hours. A more preferable lower limit is 3 hours, further preferably 4 hours, and a more preferable upper limit is 12 hours, and further preferably 10 hours. This makes it possible to reduce cell damage without reducing the gene transfer rate.

  In the present invention, the medium may be replaced after the introduction of the nucleic acid, and the acid polysaccharide component may be added after the nucleic acid introduction reagent is removed. The medium replacement timing is preferably immediately before the addition of the acidic polysaccharide component. In this case, a further effect of reducing cell damage may be obtained as compared with the case where the acidic polysaccharide component is simply added.

In the present invention, the method for introducing a nucleic acid into a cell is not particularly limited. Examples of the method for introducing a nucleic acid include a calcium phosphate method, a DEAE-dextran method, and a lipofection method.
In this examination, as a nucleic acid introduction reagent, one kind of polymer imineimine and Lipofectamine 2000 mainly composed of lipids were used. However, the scope of the present invention is not limited to these. In order to achieve this, a reagent based on other lipids or other polymers, or any reagent based on calcium phosphate or DEAE-dextran may be used.

  In the present invention, the type of nucleic acid to be introduced is not limited. Non-Patent Document 1 only discloses the result that cell damage can be reduced when plasmid DNA is introduced. On the other hand, in the present invention, even when not only plasmid DNA but also RNA and siRNA are introduced, the addition of acidic polysaccharide can reduce cell damage.

  Of course, the type of cell used is not limited. In this study, we investigated using COS-7 cells (derived from African green monkey kidney) and HeLa cells (derived from human cervical cancer). After introducing nucleic acids into other animal cells, acidic polysaccharides were introduced. It may be added.

  The present invention further extends to reagents and kits for reducing cell damage comprising an acidic polysaccharide used in the method of the present invention as a component. This is because it is expected that many users will be able to carry out the nucleic acid introduction experiment easily and with good reproducibility by supplying the reagent containing the method of the present invention.

As one embodiment of the present invention, reagents and kits that reduce cell damage during nucleic acid introduction can be considered.
Specifically, for example, a form in which a 0.1 to 10 mg / mL heparin solution or a low molecular weight heparin solution is supplied can be considered. It is used in combination with a commercially available or separately prepared transfection reagent. For example, by introducing a nucleic acid using a transfection reagent and adding a heparin solution or a low molecular weight heparin solution to the medium after 2 hours (not including 2 hours), the cell damage is prevented. be able to.
Alternatively, for example, a set of the above-described reduced molecular weight heparin solution and a transfection reagent is also included in the reagent or kit of the present invention. Furthermore, for example, in addition to the above-mentioned low molecular weight heparin solution and transfection reagent, a set of nucleic acids to be introduced into cells is also included in the reagent or kit of the present invention.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

Example 1: Effect of timing of addition of low molecular weight heparin on cell viability and gene transfer rate On the day before transfection, COS-7 cells were seeded on a 12-well plate at 1.0 × 10 ⁵ cells / well. Transfection was performed using polyethyleneimine (25 kDa, manufactured by Aldrich) or Lipofectamine 2000 (manufactured by Invitrogen).
When polyethyleneimine was used, transfection was performed according to the following procedure. 3.0 μg of pQBI25 (manufactured by Quantum Biotechnologies) and 3.0 μg of polyethyleneimine were mixed in serum-free D-MEM and allowed to stand at room temperature for 20 minutes. The prepared plasmid DNA / polyethyleneimine complex solution was added to each well whose medium had been replaced with serum-free D-MEM in advance, and incubated in a 37 ° C., 5% CO ₂ incubator. After incubation 2, 4, 6, 8, and 10 hours, 10 μL of a 1.25 mg / mL low molecular weight heparin (4,000-6,000 Da, manufactured by Sigma) solution dissolved in PBS was added. One day after the addition of low molecular weight heparin, 1 mL of serum-containing D-MEM was added and incubated for another day in a 37 ° C., 5% CO ₂ incubator.
When Lipofectamine 2000 was used, transfection was performed according to the following procedure. 1.0 μg of pQBI25 and 2.5 μL of Lipofectamine 2000 were mixed in Opti-MEM I Reduced Serum Medium (manufactured by Invitrogen) and allowed to stand at room temperature for 20 minutes (see the Lipofectamine 2000 instruction manual). The prepared plasmid DNA and Lipofectamine 2000 complex solution was added to each well and incubated in a 37 ° C., 5% CO ₂ incubator. After Incubation 2, 4, 6, 8, and 10 hours, 10 μL of a 1.25 mg / mL low molecular weight heparin solution dissolved in PBS was added, and further incubated at 37 ° C. in a 5% CO ₂ incubator.
Cell damage after transfection was evaluated using a commercially available kit containing the tetrazolium salt WST-8. Specifically, after adding 100 μL of the viable cell count measurement reagent SF (manufactured by Nacalai Tesque) to each well and incubating at 37 ° C. for 20 minutes, the absorbance at 450 nm was measured with reference to the absorbance at 650 nm using a microplate reader. It was measured. Based on the obtained absorbance, the survival rate of untransfected cells was taken as 100%, and the cell survival rate after transfection was calculated. Next, the gene transfer rate was measured by FACS (fluorescence activated cell sorting) analysis. That is, cells 2 days after transfection were treated with trypsin, suspended in PBS, used as a sample, and FACSCalibur (manufactured by Becton Dickinson) was used to calculate the gene transfer rate based on the presence or absence of GFP expression.
As a result, the results shown in FIGS. FIGS. 1 and 2 are diagrams showing cell viability and gene transfer rate when the transfection was performed using polyethyleneimine and Lipofectamine 2000, respectively, and the timing of addition of low molecular weight heparin was examined. Regardless of which nucleic acid introduction reagent is used, when low molecular weight heparin is added 2 hours after transfection, a sufficient effect of reducing cell damage is observed, but a decrease in gene introduction rate is also observed. It was. On the other hand, when low molecular weight heparin was added after 4 hours after transfection, there was not much reduction in the gene transfer rate, and both the reduction of cell damage and the efficient introduction of nucleic acids were achieved under these conditions. Was found to be feasible.

FIG. 1 pays attention to the timing of addition of low molecular weight heparin. Regarding its influence on cell viability and gene transfer rate, polyethyleneimine (25 kDa, manufactured by Aldrich) used in Non-Patent Document 1 is used as a reagent for nucleic acid transfer. The result of having examined COS-7 cells as a subject is shown.
When the low molecular weight heparin was added 2 hours after the transfection, a sufficient reduction in cell damage was observed, but a decrease in the gene transfer rate was observed compared to the case where no low molecular weight heparin was added. On the other hand, by extending the time from transfection to the addition of low molecular weight heparin, it was possible to reduce cell damage without reducing the gene transfer rate.

FIG. 2 shows the effect of the addition timing of low molecular weight heparin on the cell viability and gene transfer rate using Lipofectamine 2000 (manufactured by Invitrogen), a typical nucleic acid transfer reagent, and transfection of COS-7 cells. The result of having examined about is shown.
Even when Lipofectamine 2000 was used, when low molecular weight heparin was added 2 hours after transfection, a sufficient reduction in cell damage was observed, but the gene transfer rate decreased compared to the case where low molecular weight heparin was not added. It was seen. On the other hand, by extending the time from transfection to the addition of low molecular weight heparin, it was possible to reduce cell damage without reducing the gene transfer rate.

  In summary, the effects of the timing of addition of low molecular weight heparin on the cell viability and gene transfer rate were examined. By extending the time from introduction of nucleic acid to addition of low molecular weight heparin, It was possible to introduce nucleic acids efficiently while maintaining the reduction effect. In addition, the nucleic acid introduction reagent used is not limited to polyethyleneimine, and even when Lipofectamine 2000 is used, the addition of low molecular weight heparin can reduce cell damage.

Example 2: Effect on cell viability and gene transfer rate by addition of various acidic polysaccharides On the day before transfection, in the case of COS-7 cells in a 12-well plate, 1.0 × 10 ⁵ cells / well, in the case of HeLa cells Seeding was carried out at 1.5 × 10 ⁵ cells / well. Transfection was performed by the following procedure using Lipofectamine 2000 (manufactured by Invitrogen). 1.0 μg of pQBI25 (manufactured by Quantum Biotechnologies) and 2.5 μL of Lipofectamine 2000 were mixed in Opti-MEM I Reduced Serum Medium (manufactured by Invitrogen) and allowed to stand at room temperature for 20 minutes (see the manual for Lipofectamine 2000). The prepared plasmid DNA and Lipofectamine 2000 complex solution was added to each well and incubated in a 37 ° C., 5% CO ₂ incubator. After 6 hours of incubation, 10 μL of 1.25 mg / mL various acidic polysaccharide solutions dissolved in PBS were added, and further incubated at 37 ° C. in a 5% CO ₂ incubator for 2 days. A total of eight types of acidic polysaccharides were used: reduced molecular weight heparin, fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, and hyaluronic acid.
Cell damage after transfection was evaluated using a commercially available kit containing the tetrazolium salt WST-8. Specifically, after adding 100 μL of the viable cell count measurement reagent SF (manufactured by Nacalai Tesque) to each well and incubating at 37 ° C. for 20 minutes, the absorbance at 450 nm was measured using a microplate reader with reference to the absorbance at 650 nm. It was measured. Based on the obtained absorbance, the survival rate of untransfected cells was taken as 100%, and the cell survival rate after transfection was calculated. Next, the gene transfer rate was measured by FACS (fluorescence activated cell sorting) analysis. That is, cells 2 days after transfection were treated with trypsin, suspended in PBS, used as a sample, and FACSCalibur (manufactured by Becton Dickinson) was used to calculate the gene transfer rate based on the presence or absence of GFP expression.
As a result, the results shown in Table 1 could be obtained. Table 1 is a table showing the relationship between the characteristics of the examined acidic polysaccharide and the strength of the effect of reducing cell damage. Table 1 shows the results of studying COS-7 cells and HeLa cells using Lipofectamine 2000 and adding various compounds after 6 hours.
In both COS-7 cells and HeLa cells, reduction of cell damage was confirmed with acidic polysaccharides such as low molecular weight heparin, fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, etc. . However, even with the same acidic polysaccharide, reduction in cell damage could not be confirmed with hyaluronic acid. In addition, four types of low molecular weight heparin, fucoidan, heparin, and dextran sulfate containing a plurality of sulfate groups per two sugar molecules (preferably 1.5 to 4 molecules) can greatly reduce cell damage. . Furthermore, when fucoidan, heparin, and dextran sulfate were used, the effect of reducing cell damage over low molecular weight heparin could be confirmed. At this time, almost no decrease in gene transfer rate was observed in any acidic polysaccharide.
When low molecular weight heparin was added immediately after gene introduction, almost no gene was introduced. Moreover, even when it was added 2 hours after gene transfer, the gene transfer rate remained at 25-26%. (Comparative example)
Thus, the present inventors have found that cell damage is reduced by using compounds other than low molecular weight heparin such as fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B and the like.

Example 3 Confirmation of cytopathic reduction effect of low molecular weight heparin in siRNA introduction system In Example 1, the effect of low molecular weight heparin was examined using siRNA instead of plasmid DNA.
The study was carried out in a system in which Neuro 2a cells or HeLa cells were subjected to transfection using Lipofectamine 2000. Specifically, Neg. 60 pmol of control siRNA, Fluorescein (manufactured by Qiagen) and 3.0 μL of Lipofectamine 2000 were mixed in Opti-MEM I Reduced Serum Medium and allowed to stand at room temperature for 20 minutes (see the Lipofectamine 2000 instruction manual). To each well, the prepared complex solution of siRNA and Lipofectamine 2000 was added and incubated in a 37 ° C., 5% CO ₂ incubator. After 6 hours of incubation, 10 μL of a 1.25 mg / mL low molecular weight heparin solution dissolved in PBS was added and incubated for another 2 days in a 37 ° C., 5% CO ₂ incubator. Thereafter, the cell viability after transfection was calculated using the method in Example 1. As a result, even when siRNA was used instead of plasmid DNA, reduction of cell damage due to low molecular weight heparin was confirmed.

  According to the method for reducing cell damage at the time of introduction of nucleic acid of the present invention, it is possible to reduce damage to cells without hindering efficient introduction of nucleic acid. It can be used from basic fields such as gene product function analysis, protein expression, gene expression suppression, and transcriptional regulatory region analysis to gene therapy and other application fields, which can greatly contribute to industry. .

The figure which shows a cell viability and a gene transfer rate at the time of examining the timing of addition of low molecular weight heparin after implementing a transfection with polyethyleneimine for COS-7 cells. For example, when the cell survival rate is 60% and the introduction rate is 40%, if there are 100 cells initially, 100 × 0.6 × 0.4 = 24 cells are introduced. Show. The cell viability immediately after the transfection is 100% and the introduction rate is 0%. The figure which shows a cell viability and a gene transfer rate at the time of examining the timing of addition of low molecular weight heparin after implementing transfection with Lipofectamine2000 for COS-7 cells. The view of the figure is the same as in FIG.

Claims (3)

A method for reducing cell damage at the time of introducing a nucleic acid into an animal cell using a nucleic acid introduction (transfection) reagent, wherein Lipofectamine 2000 (trademark) is used as a transduction reagent and “6 hours” after transduction A method for reducing cell damage, comprising adding fucoidan or dextran sulfate at the point of time. The method for reducing cell damage according to claim 1, wherein the medium is changed when fucoidan or dextran sulfate is added. Claim 1 containing fucoidan or dextran sulfate for use in the method according to any one of 2 as a component, cytotoxic reducing reagent or kit.

Images