WO2007111333A1 - Method of reducing cell injury in nucleic acid transfection - Google Patents

Abstract

[PROBLEMS] To provide a method of reducing cell injury without interfering gene transfection in transferring a nucleic acid into an animal cell. [MEANS FOR SOLVING PROBLEMS] A method of reducing cell injury in transferring a nucleic acid into an animal cell by using a nucleic acid transfection reagent, characterized by comprising adding an acidic polysaccharide component to the medium immediately after the nucleic acid transfection.

Description

 Method for reducing cell damage during nucleic acid introduction

 Technical field

 [0001] The present invention relates to a method for reducing cell damage that does not hinder introduction into a cell when a nucleic acid is introduced. 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.

 Background art

 [0002] Technologies for introducing nucleic acids into cells range from basic fields such as gene product functional analysis, protein expression, gene expression suppression, and transcriptional regulatory region analysis to gene therapy and other application fields. It is the basic technology used.

 [0003] As a method for introducing nucleic acid into cells, a physical method such as an electoporation method, a microindi- tation method, a chemical method using a substance such as calcium phosphate, DEAE-dextran, lipid, ribosome, or polymer, adeno Biological methods using viruses, retroviruses, etc. are known. Since the chemical method is simple, various nucleic acid introduction reagents are now commercially available and widely used. These methods are also considered to be suitable for in vivo use because they are highly safe, can be easily manufactured on a large scale, and can be easily used.

[0004] However, the disadvantages of these techniques include cell damage, cells that are difficult to introduce nucleic acids, and low reproducibility of experiments. Among these, the most prominent are It is a cellular disorder. Cell damage is affected by factors such as the type and amount of 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, and the state of the cells. The introduction rate of nucleic acids into cells is also affected by these factors, and when the above conditions are adjusted to reduce damage to cells, a reduction in introduction rate is often observed.

[0005] 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 it is troublesome. There is a problem with strength. Furthermore, the introduction rate of the nucleic acid into the cells may decrease depending on the timing of the medium exchange.

 [0006] Dmgomir, A. et al. Have reported the only method for reducing the damage to cells that appears at the time of introduction of nucleic acid by adding parin to low molecular weight 匕 within 2 hours after transfection (non- Patent Document 1). In this report, GenePorter2 (manufactured by Gene Therapy Systems) or polyethyleneimine (25 kDa, manufactured by Sigma-Aldrich) was used for CFBE (Cystic fibrosis human bronchial epithelial cells) and CFSME (Cystic fibrosis submucosal epith elial cells) cells. It has been shown that cell damage can be reduced by performing transfection using the medium, and replacing it with a new medium 2 hours after transfection, and adding a non-molecular weight agent to the medium. . However, this method shows a significant decrease in the gene transfer rate. In other words, the two problems of efficient nucleic acid introduction and cell damage reduction have not been achieved at the same time. In addition, this method involves a process of removing the medium after transfection and replacing it with a new medium, which is troublesome.

 Non-patent document 1: Life Sciences 75, 2203-2216 (2004)

 Brief Description of Drawings

 [0007] [Fig.l] Cell viability and gene transfer when COS—7 cells were transfected with polyethyleneimine and the timing of addition of low molecular weight was examined. A figure showing the rate. For example, when the cell survival rate is 60% and the introduction rate is 40%, if 100 cells are initially included, 100 X 0.6 X 0.4 = 24 cells are introduced. Show. The cell viability immediately after the transfer is 100% and the introduction rate is 0%.

 [Fig. 2] A graph showing cell viability and gene transfer rate when examining the timing of addition of parin to lower molecular weight after transfection with Lipofectamine 2000 for COS-7 cells. The view of the figure is the same as in Figure 1.

 Disclosure of the invention

 Problems to be solved by the invention

[0008] Although the conventional technology can reduce cell damage that occurs during introduction of nucleic acid by adding parin to low molecular weight, there is a problem that introduction of nucleic acid into cells is hindered. . Accordingly, an object of the present invention is to provide a method for reducing cell damage that hinders gene transfer when a nucleic acid is introduced into an animal cell.

 Means for solving the problem

 [0009] As a result of various studies, the present inventors have surprisingly contributed to the recovery of cells damaged by transfection by the time until the addition of pallin to the low molecular weight 匕. We found that cell damage can be reduced without lowering the gene transfer rate by conversely extending the time until the addition of parin to the molecular weight reduction from 2 hours.

 The present inventors have also found that cell damage is reduced by using compounds other than heparin having a low molecular weight such as fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, etc. It was.

[0010] That is, the present invention has the following constitutional power.

 Section 1.

 A method for reducing cell damage when a nucleic acid is introduced into an animal cell using a nucleic acid introduction (transfusion) reagent, characterized in that an acidic polysaccharide component is added to the medium after the introduction of the nucleic acid. How to reduce obstacles.

 Section 2.

 2. The method for reducing cell damage according to claim 1, wherein the acidic polysaccharide has a sulfate group.

 Section 3.

 3. The method for reducing cell damage according to claim 2, wherein the acidic polysaccharide has a repeating structure of two or more sugars.

 Section 4.

 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 heno << phosphorus, heparan sulfate, chondroitin sulfate, fucoidan, dextran sulfate, and keratan sulfate.

 Section 5.

5. The cytotoxicity according to claim 4, wherein the acidic polysaccharide has a low molecular weight. Reduction method.

 Section 6.

 2. 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.

 Section 7.

 The method for reducing cell damage according to claim 1, wherein the medium is exchanged when the acidic polysaccharide component is added. Section 8.

 2. The method for reducing cell damage according to claim 1, wherein a substance selected from the group consisting of calcium phosphate, DEAE-dextran, lipid, ribosome and polymer force is used for introduction of nucleic acid into cells.

 Section 9.

 A reagent or kit for reducing cell damage, comprising an acidic polysaccharide used in the method according to claim 1 as a component.

 The invention's effect

 [0011] According to the present invention, it has become possible to reduce cell damage that appears at the time of nucleic acid introduction without hindering introduction of nucleic acid into cells. As a result, nucleic acid can be introduced into animal cells simply, easily and with good reproducibility.

 BEST MODE FOR CARRYING OUT THE INVENTION

 The term “cytotoxicity” as used in the present invention means cell death caused by addition of a nucleic acid introduction reagent, or cell shape change. This cell damage can be evaluated by the MTT method, LDH method, etc. after nucleic acid introduction. In the MTT method, tetrazolium salts such as MTT, XTT, MTS, and 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 addition, the LDH method evaluates cell damage by measuring lactate dehydrogenase released from dead cell force damaged by cell membranes.

[0013] The present invention is a method for reducing cell damage, which comprises adding an acidic polysaccharide component after introduction of a nucleic acid. [0014] The acidic polysaccharide component is preferably dissolved in a koffer and added as an aqueous solution. The buffer for dissolving the acidic polysaccharide component is not particularly limited, and examples thereof include a HEPES buffer and a phosphate buffer.

 In the present invention, the so-called acidic polysaccharide having an acidic residue is used as the polysaccharide added after transfection. 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 hexanelan sulfate.

 [0016] The acidic polysaccharide may be reduced in molecular weight. For example, low molecular weight heparin or low molecular weight fucoidan may be used. The term “reducing molecular weight” as used herein means degrading acidic polysaccharides chemically or enzymatically. The low molecular weight heparin and low molecular weight fucoidan used are preferably those having a molecular weight of 2,000 to 8, OOODa, and more preferably a molecular weight of 4,000 to 6, OOODa.

 [0017] The acidic polysaccharide in the present invention is not limited in the type of force considered to be supplied as various salt compounds. For example, sodium salt, ammonium salt, lithium salt, calcium salt can be used.

 [0018] In the present invention, the time until the addition of acidic polysaccharide (time until the addition of acidic polysaccharide that contributes to the recovery of cells damaged by the transfection) is longer than 2 hours. A more preferred lower limit is 3 hours, more preferably 4 hours, and a more preferred upper limit is 12 hours, more preferably 10 hours. This makes it possible to reduce cell damage without lowering the gene introduction rate.

 In the present invention, the medium may be replaced after the introduction of the nucleic acid, and the nucleic acid introduction reagent may be removed, and then the acidic polysaccharide component may be added. 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 of simply adding an acidic polysaccharide component.

[0020] In the present invention, the method for introducing a nucleic acid into a cell is not particularly limited! How to introduce nucleic acid Examples of the method include a calcium phosphate method, a DEAE-dextran method, and a ribofusion method.

 In this study, as a reagent for introducing nucleic acid, a force using one kind of polymer, polyethyleneimine, and Lipofectamine 2000 mainly composed of lipids. The scope of the present invention is not limited to these. Therefore, it is possible to use a reagent based on other lipids or other polymers, or a misaligned reagent based on calcium phosphate or DEAE-dextran! /.

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 RNA and siRNA introduced by plasmid DNA alone are introduced, cell damage can be reduced by adding acidic polysaccharide.

 [0022] Naturally, the type of cells used is not limited. In this study, we examined the COS-7 cells (derived from the African green monkey kidney) and HeLa cells (derived from human eclampsia cancer). After introducing the nucleic acid into other animal cells, the acidic polysaccharide May be added.

 [0023] The present invention further extends to reagents and kits for reducing cell damage comprising an acidic polysaccharide as a component used in the method of the present invention. This is because it is expected that by supplying a reagent containing the method of the present invention, a number of users can easily perform nucleic acid introduction experiments with good reproducibility.

 [0024] As one embodiment of the present invention, it is possible to consider reagents and kits that reduce cell damage during nucleic acid introduction.

 Specifically, for example, a form in which a parin solution is supplied to 0.1 to: LOmgZmL of heparin solution or a low molecular weight container is conceivable. Use it in combination with a commercially available or separately prepared transfection reagent. For example, a nuclear acid is introduced using a transfection reagent, and after 2 hours (not including 2 hours), a non-heine solution or a low-molecular-weight non-heine solution is added to the medium. In this way, cell damage can be prevented.

Or, for example, set the above low molecular weight hetero solution and transfection reagent What was made into is also contained in the reagent or kit of this invention. Further, for example, in addition to the low molecular weight heparin solution and the transfection reagent, a set of nucleic acids to be introduced into cells is also included in the reagent or kit of the present invention.

 Example

 [0025] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

 [0026] Example 1: Effects of timing of addition of parin to low molecular weight silkworms on cell viability and gene transfer rate

The day before transfection, COS-7 cells were seeded on a 12 well plate at 1.0 X 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. PQBI25 (manufactured by Quantum Biotechnologies) 3. g and polyethyleneimine 3. O ^ g were mixed in serum-free D-MEM and allowed to stand at room temperature for 20 minutes. The medium was replaced with serum-free D-MEM in advance, and the prepared plasmid DNA / polyethyleneimine complex solution was added to each well and incubated in a 37 ° C, 5% CO incubator. Inn

 2

 After 10 hours of Cuprate 2, 4, 6, 8, 10 hours, 10 L of a 1.25 mg ZmL low molecular weight reduced-in weight (4,000,000, 6,000 Da, Sigma) solution dissolved in PBS was added. One day after the addition of parin to low molecular weight, add 1 mL of serum-containing D—MEM, and incubate at 37 ° C and 5% CO.

 2 Incubate for an additional day in a plate.

 When Lipofectamine 2000 was used, transfection was performed according to the following procedure. PQBI25 1. 0; z g and Lipofectamine2000 2.5 ^ L were mixed in Opti-MEM I Reduced Serum Medium (manufactured by Invitrogen) and allowed to stand at room temperature for 20 minutes (see Lipofectamine2000 instruction manual). To each well, add the prepared plasmid DNA and Lipofectamine 2000 complex solution, and incubate at 37 ° C and 5% CO.

 2

 Incubated in. After 2, 4, 6, 8, 10 hours of incubation, add 10 L of 1.25 mg ZmL low molecular weight parin solution dissolved in PBS, and incubate at 37 ° C and 5% CO.

2 Incubate for an additional 2 days in one plate. Cell damage after transfection was evaluated using a commercially available kit containing tetrazolium salt WST-8. Specifically, add 100 L of viable cell count measurement reagent (SF strength tester) to each well, incubate at 37 ° C for 20 minutes, and then use a microplate reader and refer to the absorbance at 650 nm. Absorbance at 450 nm was measured. Based on the obtained absorbance, the survival rate of non-transfected cells was defined 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 samples, and FACSCalibur (Betaton 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. 1 and 2 were obtained. Figures 1 and 2 show the cell viability and gene transfer rate when transfection was performed using polyethyleneimine and Lipofectamine 2000, respectively, and the timing of the addition of parin to low molecular weight was examined. Regardless of which nucleic acid transfection reagent is used, if the low molecular weight is added to the low molecular weight 2 hours after transfection, a sufficient effect of reducing cell damage can be seen! / A drop in the gene transfer rate was also observed. On the other hand, when 4 hours after transfection, the addition of low molecular weight nitrogen does not significantly reduce the rate of gene transfer, and this condition reduces cell damage and increases efficiency. It was found that both nucleic acid introductions were feasible.

Figure 1 focuses on the timing of the addition of parin to low molecular weight 量, and its influence on cell viability and gene transfer rate. Polyethyleneimine used in Non-Patent Document 1 (25 The results of studies on COS-7 cells using kDa, Aldrich) as a nucleic acid transfer reagent are shown.

Addition of low molecular weight phenone 2 hours after transfection resulted in a sufficient reduction in cell damage, but reduced gene transfer rate compared to low molecular weight addition of no parin. It was seen. On the other hand, it was possible to reduce cell damage without lowering the gene transfer rate by extending the time until the addition of parin to the low-molecular-weight protein. [0028] Fig. 2 shows a typical nucleic acid transfer reagent, Lipofectamine 2000 (Invitrogen), and COS-7 cells were subjected to transfection, and the cell viability of the addition of parin to low molecular weight was The results of examining the effect on the gene transfer rate are shown below. Even when Lipofectamine 2000 was used, addition of low molecular weight phenone 2 hours after transfection resulted in a sufficient reduction in cell damage, but compared to low molecular weight addition of no parin. A decrease in the introduction rate was observed. On the other hand, it was possible to reduce cell damage without lowering the gene transfer rate by extending the time until the addition of parin to the low molecular weight matrix.

 [0029] In summary, as a result of examining the effects of the timing of the addition of parin to low molecular weight 匕 on the cell viability and gene transfer rate, the ability to introduce nucleic acid was added to low molecular weight 匕. It was possible to efficiently introduce nucleic acid while maintaining the effect of reducing cell damage by extending the time until the time until the time was increased. Moreover, the nucleic acid introduction reagent to be used is not limited to polyethyleneimine, and even when Lipofectamine 2000 is used, cell damage can be reduced by reducing the molecular weight and adding paris.

[0030] Example 2: Effect of addition of various acidic polysaccharides on cell viability and gene transfer rate On the day before transfection, in the case of COS-7 cells on 12 well plates 1.0 X 10 ⁵ cell s / well In case of HeLa cells, the cells were seeded at 1.5 × 10 ⁵ cells / well. The transformation was performed using Lipofectamine 2000 (Invitrogen) according to the following procedure. pQBI2 5 (manufactured by Quantum Biotechnologies) 1. g and Lipofectamine2000 2.5 μL¾ Opti-MEM I Reduced Serum Medium (manufactured by Invitrogen) were mixed and allowed to stand at room temperature for 20 minutes (see the Lipofectamine2000 instruction manual). To each well, add the prepared plasmid DNA and Lipofectamine 2000 complex solution and add 37 ° C, 5% CO

 2 Incubated in incubator. After 6 hours of incubation, dissolve in PBS. 1.2 Add 5 mg / mL various acidic polysaccharide solutions 10 / z L, in a 37 ° C, 5% CO incubator

 2

 And further incubated for 2 days. Eight types of acidic polysaccharides were used: low molecular weight 匕 henoline, fucoidan, hemolin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, and hyaluronic acid.

For cell damage after transformation, use a commercially available kit containing the tetrazolium salt WST-8. Used and evaluated. Specifically, add 100 L of viable cell count measurement reagent (SF strength tester) to each well, incubate at 37 ° C for 20 minutes, and then use a microplate reader and refer to the absorbance at 650 nm. Absorbance at 450 nm was measured. Based on the obtained absorbance, the survival rate of non-transfected cells was defined 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 samples, and FACSCalibur (Betaton 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 were obtained. Table 1 shows the relationship between the characteristics of the examined acidic polysaccharides and the intensity 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.

 COS—In both 7 cells and HeLa cells, acidic polysaccharides such as low molecular weight heparin, fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B reduce cell damage. Was confirmed. However, even with the same acidic polysaccharide, hyaluronic acid was not able to confirm the reduction of cell damage. In addition, 4 types of low molecular weight 匕 henoline, fucoidan, heparin, and dextran sulfate containing multiple sulfate groups (preferably 1.5 to 4 molecules) per 2 sugar molecules can significantly reduce cell damage. It was possible. Furthermore, in the case of using fucoidan, heparin, and dextran sulfate, it was confirmed that the cell damage reduction effect was higher than that of parin. At this time, no reduction in gene transfer rate was observed with any acidic polysaccharide.

In addition, when the low molecular weight hone was added immediately after gene introduction, the gene was hardly introduced. Even when added 2 hours after gene transfer, the gene transfer rate remained at 25-26%. (Comparative example)

Thus, the present inventors can reduce cell damage by using compounds other than heparin, such as fucoidan, heparin, dextran sulfate, chondroitin sulfate C, alginic acid, chondroitin sulfate B, etc. I found. [0031] [Table 1]

[0032] Example 3 Confirmation of cytopathic effect of low molecular weight heparin in siRNA introduction system

 In Example 1, siRNA was used instead of plasmid DNA, and the effect of parin on lowering the molecular weight was examined.

 We examined the transfer system using Lipofectamine2000 for Neuro 2a cells or HeLa cells. Specifically, Neg. Control siRNA, Fluorescein (Qiagen) 60 pmol and Lipofectamine2000 3. L were mixed in Opti-MEM I Reduced Serum Medium and allowed to stand at room temperature for 20 minutes (see 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 incubator. After 6 hours of incubation, 10 μL of parin solution was added to 1.25 mg ZmL low molecular weight solution dissolved in PBS and incubated in a 37 ° C, 5% CO incubator for another 2 days. 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, it was confirmed that cell damage was reduced by parin to lower molecular weight.

 Industrial applicability

[0033] By 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 preventing efficient introduction of nucleic acid. It can be used in basic fields such as gene product function analysis, protein expression, gene expression suppression, and transcriptional regulatory region analysis, and can be used in gene therapy and other application fields, making a significant contribution to industry. .

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Claims

The scope of the claims  [1] A method for reducing cell damage when a nucleic acid is introduced into animal cells using a nucleic acid introduction (transfusion) reagent, wherein an acidic polysaccharide component is added to the medium after the introduction of the nucleic acid. A method for reducing cell damage.  [2] The method for reducing cell damage according to claim 1, wherein the acidic polysaccharide has a sulfate group.  [3] The method for reducing cell damage according to claim 2, wherein the acidic polysaccharide has a repeating structure of two or more sugars.  [4] The reduction of cell damage according to claim 3, wherein the acidic polysaccharide is one or more selected from the group consisting of heno << phosphorus, heparan sulfate, chondroitin sulfate, fucoidan, dextran sulfate, and keratan sulfate. Method.  5. The method for reducing cell damage according to claim 4, wherein the acidic polysaccharide is reduced in molecular weight!  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 introduction of the nucleic acid. 7. The method for reducing cell damage according to claim 1, wherein the medium is exchanged when the acidic polysaccharide component is added. [8] For introduction of nucleic acid into cells, calcium phosphate, DEAE-dextran, lipid, ribosome 2. The method for reducing cell damage according to claim 1, wherein a substance selected from the group consisting of polymer power is used.  [9] A reagent or kit for reducing cell damage, comprising an acidic polysaccharide used as a component in the method according to any one of claims 1 to 8.