JP2019170213A - Methods for producing immortalized human tracheal epithelial cells, immortalized human tracheal epithelial cells obtainable by the methods, and kits for viral separation or kits for screening of viral inhibitors provided with the immortalized human tracheal epithelial cells - Google Patents

Abstract

To provide cells that solve the problems of primary culture cells derived from epithelia of respiratory mucosa such as quick aging and functional decline.SOLUTION: Disclosed herein is a method for producing an immortalized human respiratory epithelial cell comprising introduction to a human respiratory epithelial cell the following genes: (1) CDK4R24C gene or BMI1 gene; and (2) TERT gene.SELECTED DRAWING: None

Description

  The present invention relates to immortalization of cells and uses of immortalized cells.

  In order to measure the functional changes of human tracheal epithelial cells and human nasal mucosal epithelial cells due to respiratory virus infection, the ability of the cells to proliferate the virus, and the activity of antiviral drugs, they were excised by tracheal tissue and surgery provided by pathological anatomy Primary culture cells were used to culture cells collected from the nasal mucosa.

  Primary cultured cells are useful for analyzing functional changes in airway epithelial cells related to respiratory virus isolation and exacerbations such as bronchial asthma and chronic obstructive pulmonary disease. is there. Human trachea is very difficult to obtain due to the procedure provided by pathological anatomy. In addition, it was necessary to collect and culture cells repeatedly from the trachea and nasal mucosa, which required a lot of labor. Moreover, since the virus growth ability between individual differences is different, it is difficult to obtain reproducible data for antiviral drug evaluation.

JP2004-254509

  An object of the present invention is to provide a cell that solves the problems of primary cultured cells derived from airway epithelium that are aged in a short period of time and have reduced function.

Under such circumstances, the present inventors have conducted intensive research and found that a specific gene is introduced into human airway epithelial cells to become immortal and can be used to solve such problems. Accordingly, the present invention provides the following sections:
Item 1. The following genes (1) and (2) (1) CDK4R24C gene or BMI1 gene
(2) A method for producing immortalized human airway epithelial cells, comprising a step of introducing a TERT gene into human airway epithelial cells.

  Item 2. Item 2. The method according to Item 1, wherein the gene of (1) is a CDK4R24C gene, and (3) a cyclin D1 gene is further introduced.

  Item 3. Item 3. An immortalized human airway epithelial cell obtained by the method according to item 1 or 2.

  Item 4. A virus isolation kit comprising the immortalized human airway epithelial cell according to Item 3.

  Item 5. A kit for screening a virus inhibitor, comprising the immortalized human airway epithelial cell according to Item 3.

  Since the cells obtained by the method of the present invention are immortalized by suppressing the decrease in the proliferation ability of the cells due to passage, it is possible to reduce the labor of cell preparation. In addition, the cells have the ability to infect viruses that cause respiratory infections (influenza viruses, rhinoviruses, respiratory syncytial viruses, etc.). Uniform lots of virus-separating host cells can be used, and the time and frequency of preparation of primary cultured cells can be reduced, so that virus isolation experiments and evaluation of the inhibitory effect of antiviral drugs can be performed at any time. . In addition, measures of airway inflammation such as inflammatory substances and active oxygen synthesis for viral infection in the cells are measured, and functional changes in the respiratory tract are related to pathological conditions such as bronchial asthma and chronic obstructive pulmonary disease due to viral infection. Can be analyzed.

In the Examples of the present application, the results of the “virus growth in immortalized human tracheal epithelial cells” test are shown. In the Examples of the present application, a micrograph is shown in the “virus growth in immortalized human tracheal epithelial cells” test. In the Examples of the present application, the results of the “virus growth in immortalized human nasal mucosal epithelial cells” test are shown. In the Examples of the present application, the results of the “virus growth in immortalized human nasal mucosal epithelial cells” test are shown. In the Examples of the present application, photomicrographs in the “virus growth in immortalized human tracheal epithelial cells” test are shown (magnification: x100). Immortalized tracheal epithelial cells proliferate in wells not treated with collagen as in the case of treated wells. The nucleic acid sequence of human CDK4R24C used in the Examples of the present application is shown. The nucleic acid sequence of human BMI1 used in the examples of the present application is shown. The nucleic acid sequence of human TERT used in the Examples of the present application is shown. The nucleic acid sequence of human TERT used in the Examples of the present application is shown. 1 shows the nucleic acid sequence of human cyclin D1 used in Examples of the present application.

  The present invention relates to a method for introducing a gene, which is a nucleic acid molecule, into a cell. In the present invention, unless otherwise specified, the term “gene” refers not only to a structural gene defining a primary structure such as a protein, tRNA, or rRNA, but also to a nucleic acid having a specific regulatory function such as a promoter or an operator. Regions are also included. Therefore, in the present invention, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified. The “structural gene” also includes silent DNA in which the original DNA sequence has been subjected to silent mutation. In the present invention, nucleic acid molecules such as siRNA that interfere with gene expression are also included in the “gene”.

  In the present specification, “nucleic acid” is synonymous with nucleotide, oligonucleotide, and polynucleotide, and may be any of DNA, RNA, and DNA-RNA hybrids. Further, these may be double-stranded or single-stranded, and in the case of a nucleic acid molecule having a certain sequence, a nucleic acid molecule having a complementary sequence (or nucleotide, oligonucleotide) unless otherwise specified. And polynucleotide) are also meant to be inclusive. These nucleic acid molecules may be circular or linear, and may be synthetic or biological.

Method for producing immortalized human airway epithelial cell and immortalized cell obtained by the method The present invention includes the following genes (1) and (2): (1) CDK4R24C gene or BMI1 gene
(2) Provided is a method for producing immortalized human airway epithelial cells, comprising the step of introducing a TERT gene into human airway epithelial cells.

  In the present invention, the airway includes nasal cavity, nasopharynx, pharynx, larynx, trachea, bronchi, bronchiole, and lung. In the present invention, trachea, nasal mucosal epithelial cells and the like are preferable.

  The method of the present invention is characterized by introducing the genes (1) and (2) above. CDK4R24C is a mutant cyclin-dependent kinase 4. A typical example is human CDK4R24C. Examples of the nucleic acid sequence of CDK4R24C include Genbank accession No. Examples include NM — 000075 (CDK4) codon 24 (CGT = Arg) introduced with mutation (C to T) so as to be (TGT = Cys) (SEQ ID NO: 1, FIG. 6). BMI1 is a kind of polycomb group protein cloned from a cDNA library of human erythroleukemia cell line (K562). A typical example is human BMI1. Examples of the nucleic acid sequence of BMI1 include Genbank accession No. Examples thereof include L13689 (SEQ ID NO: 2, FIG. 7). TERT (telomerase reverse transcriptase) is a protein having a molecular weight of about 100 kD, and is known to comprise a telomerase-specific domain at the N-terminal side, a reverse transcriptase domain at the center, and a C-terminal domain. A typical example is human TERT. As a nucleic acid sequence of TERT, for example, Genbank accession No. Examples thereof include those represented by AB085628 (SEQ ID NO: 3, FIGS. 8 and 9).

  In the present invention, the gene of (1) is a CDK4R24C gene, and (3) the cyclin D1 gene is further introduced. Therefore, (1) the CDK4R24C gene (2), the TERT gene and (3 ) It is preferable to introduce a cyclin D1 gene. Cyclin D1 is a protein known as a target gene for chromosomal translocation. A typical example is human cyclin D1. Examples of the nucleic acid sequence of cyclin D1 include Genbank accession No. Examples thereof include those represented by M64349 (SEQ ID NO: 4, FIG. 10).

  The method for introducing a gene is not particularly limited, and examples thereof include a physical method (electroporation, microinjection, gene gun, etc.), a chemical method (lipofection, etc.), a method using a viral vector, and the like. In the present invention, a method using a viral vector is preferred. Although it does not specifically limit as a viral vector, For example, a lentiviral vector, a retroviral vector, etc. are mentioned. A specific method for gene transfer can be performed according to a method known per se, except that human airway epithelial cells are used as target cells and the specific gene is introduced. For example, as a method using a viral vector, a method comprising a step of cultivating a target cell in the presence of a viral vector containing the target gene to infect the target cell with a virus can be mentioned.

  The medium used for the culturing step is not particularly limited as long as it is a medium suitable for the cell culture and virus infection. Examples of such a medium include Dulbecco's Modified Eagle Medium (DMEM) medium, Alpha Modified Eagle Minimum Essential Medium (αMEM), and the like. The culture medium may contain additives that are included when culturing ordinary somatic cells as necessary. Specific examples of the additive include fetal bovine serum, amino acids (eg, L-glutamine), antibiotics (eg, penicillin, streptomycin, etc.), and the like. Commercially available products such as DMEM / F12 (manufactured by Life Technologies), to which necessary additives are added in advance, can also be used. Culturing can be performed in a suitable vessel. The culture temperature is not particularly limited, and may be about 30 to 40 ° C. The period for culturing is not particularly limited as long as the effect of the present invention is not impaired, but can be, for example, about 12 hours to 3 days.

Human airway epithelial cells expressing these genes obtained by introducing CDK4R24C gene or BMI1 gene and TERT gene by the method of the present invention acquire immortalization, that is, infinite proliferation ability. Accordingly, in a preferred embodiment, the present invention provides immortalized human airway epithelial cells obtained by the above method. In a preferred embodiment of the present invention, human immortalized airway epithelial cells of the present invention such as tracheal epithelial cells and nasal mucosal epithelial cells do not have a coating step of pretreating growth support such as collagen in a culture container such as a well. Is also useful because it can also grow. The present invention relates to the following genes (1) and (2) (1) CDK4R24C gene or BMI1 gene
(2) A method for immortalizing epithelial cells of human airway mucosa, including the step of introducing a TERT gene into epithelial cells of human airway mucosa is also provided. The details of each step in the embodiment are the same as the method for producing immortalized human airway mucosal epithelial cells of the present invention.

Kits Immortalized cells of the present invention are useful for isolation experiments because they have the ability to infect viruses that cause respiratory infections. Accordingly, in a preferred embodiment, the present invention provides a virus isolation kit comprising the aforementioned immortalized human airway epithelial cells.
In addition, the kit of the present invention can contain an article other than the cells as necessary. Examples of other articles include, but are not limited to, tubes for cell culture and virus infection, cell culture plates; media or materials thereof. The kit of the present invention can also include a document in which a procedure for performing the virus separation method is written.

  The virus separation kit according to the present invention can be used to separate unknown or known viruses in a sample. The kit of the present invention was obtained, for example, by contacting a sample derived from a subject (eg, sputum, saliva, blood, etc.) with the immortalized cell of the present invention, culturing it, and infecting the virus contained in the sample derived from the subject. It can be used by detecting the virus in which the cells are infected. According to the kit of the present invention, a virus suspected to be infected by a subject can be separated and detected from a sample derived from the subject. Since the immortalized human airway epithelial cells of the present invention require less labor and frequency of adjustment than primary cultured cells and can be prepared immediately if necessary, the present invention is particularly suitable for such applications that require examination in a short time. Useful.

  The present invention also provides a virus inhibitor screening kit comprising the aforementioned immortalized human airway epithelial cells. Also in this embodiment, the kit of the present invention can contain articles other than the cells as necessary. Examples of other articles include tubes for cell culture and virus infection, cell culture plates; virus samples targeted by virus inhibitors; reagents for use in virus detection; media or materials thereof, and the like. However, it is not limited to this. The kit of this invention can also contain the document etc. which wrote down the procedure for performing the virus inhibitor screening method. The kit of the present invention comprises, for example, infecting the immortalized cell of the present invention with a target virus, contacting the cell with a candidate drug for a virus inhibitor, culturing, and measuring and evaluating the virus inhibitory effect of the candidate drug. Can be performed. Since the immortalized cells of the present invention can be prepared in large numbers with the same properties such as virus growth ability, the present invention can take reproducible data and is useful.

Cell establishment method 1) Preparation method of primary cultured cell used for establishment of immortalized cell 1-1
1-1-1: When cells are collected from human tracheal epithelial tissue, the mucosal epithelial tissue is detached from the human trachea provided by pathological dissection with Kochel.
1-1-2: As a human nasal mucosa epithelial tissue, a surgical specimen is used as it is.
1-2: The peeled tracheal mucosa epithelial tissue and nasal mucosa tissue are washed with a phosphate buffer to remove mucus and wrinkles.
1-3: The mucosal epithelial tissue is allowed to stand at 4 ° C. overnight in a protease (0.5 mg / mL) solution dissolved in a phosphate buffer.
1-4: On the next day, shake the plastic tube containing the mucosal epithelial tissue and protease (0.5 mg / mL) solution to isolate the cells.
1-5: The isolated cells are cultured in a flask, 24-well plate or 6-well plate. The bottom of the flask or well bottom is coated with human placental collagen to promote cell adhesion and proliferation. For culture, a culture solution (Dulbecco's MEM + Ham's F12 + 2% ultroser G) supplemented with 2% ultroser G is used. It is used to establish immortalized cells as primary cultured cells at the stage of proliferation.

2) Establishment method of immortalized cells 2-1: Lentiviral vectors or retroviral vectors are used as primary cultured cells of proliferated human tracheal epithelial cells and human nasal mucosal epithelial cells, and genes are introduced and immortalized in the following combinations. Cells were established. The nucleic acid sequences of CDK4R24C, BMI1, TERT and cyclin D1 are shown in FIGS. Non-gene-transfected cells, GFP-transfected cells, etc. stopped growing by passage number 5 under the following culture conditions.
1. CDK4R24C + cyclin D1 + TERT
2. CDK4R24C + TERT
3. BMI1 + TERT
4. CDK4R24C + cyclin D1
5. CDK4R24C
6. BMI1
7. TERT
8. GFP
0. mock
Lentiviral vector, retroviral vector used
CSII-CMV-TERT
CSII-CMV-CDK4R24C
CSII-CMV-cyclin D1
CSII (ins) -CMV-BMI1
CSII-CMV-EGFP
pCLXSH-TERT (only retrovirus vector)

2-2: Cells proliferated after gene introduction by the above combination were collected as immortalized cells. In addition to 2% Ultroser G, the culture medium contains Y-27632 (ROCK inhibitor) (Rho-associated coiled-coil forming kinase / Rho-binding kinase inhibitor) (5-10 μM) and A83-01 (TGF-βRI inhibitor) (5 μM) was added. Among the combinations of the above genes 1 to 8, the immortalized cells are 1 (CDK4R24C + cyclin D1 + TERT), 2 (CDK4R24C + TERT) and 3. (BMI1 + TERT). In the following “method of virus infection using cells”, cells into which the combination of 1 (CDK4R24C + cyclin D1 + TERT) was introduced were used.

Virus infection method using cells 1) Cell culture operation method Cells are cultured on the bottom of a 24-well plate (influenza virus) or on the side wall of a plastic tube (rhinovirus, respiratory syncytial virus (RS virus)). Collagen gel is made on the filter surface and cells are cultured (influenza virus, rhinovirus, respiratory syncytial virus (RS virus)). Place the filter in a 24-well plate. The medium used for cell culture is a mixture of DMEM (Dulbecco's Modified Eagle Medium) and F-12 (Nutrient Mixture F-12) in a 1: 1 ratio. Add A83-01.

2) Virus infection operation method 2-1: Infect a virus when cells grow on the well, plastic tube, or filter surface.
2-2: The culture solution on the cells is removed, and the culture solution containing the virus is added to the wells, plastic tubes, or cells grown on the filter surface to cause infection. At the time of infection, a culture solution containing virus is appropriately diluted with a culture solution not containing virus, and a virus dose of usually 10 ^{3 to} 10 ⁶ median tissue culture infectious dose (TCID) ₅₀ / mL is used.
2-3: After adding the culture solution containing the virus to the cells, the cells are cultured at 33-34 ° C. (rhinovirus, RS virus) or 37 ° C. (influenza virus) for 60 minutes to establish infection.
2-4: Thereafter, the culture medium containing the virus is removed, and the phosphate buffer is put into a 24-well plate, a plastic tube or a filter container, and the cells are washed twice. By this operation, the virus not adsorbed on the cells is removed.
2-5: After virus infection and washing, the cells cultured in plastic tubes are rotationally cultured at 33 ° C-34 ° C. Cells cultured in 24-well plates or filters are cultured at 33 ° C-34 ° C (rhinovirus, RS virus) or 37 ° C (influenza virus).
2-6: After infection, the culture medium, cells, or RNA is collected as appropriate, and virus replication or release is measured. The medium used for cell culture was a mixture of DMEM (Dulbecco's Modified Eagle Medium) and F-12 (Nutrient Mixture F-12) in a 1: 1 ratio. Add A83-01.

Test results 1) Virus growth in immortalized human tracheal epithelial cells 1-1: Influenza A virus (A / H1N1 2009) 10 ³ TCID) After infection with ₅₀ / mL, the amount of influenza virus released into the culture medium is The infection increased to 1 day and 3 days, and was detected continuously even after 5 days. Viral load decreased in cells to which the protease inhibitor camostat (10 μg / mL) was added.
1-2: After infection with rhinovirus 10 ⁵ TCID ₅₀ / mL, the amount of influenza virus released into the culture increased to 1 day and 3 days of infection (FIG. 1).
1-3: After infection with RS virus 10 ⁵ TCID ₅₀ / mL, the amount of influenza virus released into the culture increased to 1 day and 3 days of infection.

2) Ion transport in immortalized human tracheal epithelial cells On the cultured cell surface, a dome formed by water that migrated with the absorption of sodium was observed. It was shown that this cell forms a tight junction as an airway epithelial cell and performs ion transport and water absorption (arrow in FIG. 2; x100).

3) Virus growth in immortalized human nasal mucosal epithelial cells 3-1: Influenza A virus (A / H1N1 2009) 10 ³ TCID) After ₅₀ / mL infection, the amount of influenza virus released into the culture is It increased to 1 day and 3 days and was detected continuously (FIG. 3). Viral load decreased in cells to which the protease inhibitor camostat (10 μg / mL) was added.
3-2: After infection with rhinovirus 14 type 10 ⁵ TCID ₅₀ / mL, the amount of influenza virus released into the culture increased to 1 day and 3 days of infection, and was detected continuously (right side of FIG. 3). ).
3-3: After infection with RS virus 10 ⁵ TCID ₅₀ / mL, the amount of influenza virus released into the culture increased to 1 day and 3 days of infection, and was detected continuously (FIG. 4 below).
4) Immortalized cells proliferate without a coating process that pre-treats growth support such as collagen in a culture container such as a well (confirmed in immortalized tracheal epithelial cells and immortalized nasal mucosal epithelial cells) (immortalized) (The growth of tracheal epithelial cells is shown in FIG. 5). This eliminates the need to use expensive collagen for culturing, and saves the labor of coating. This makes the scientists reduce costs and labor, which is a great benefit.

Claims (5)

The following genes (1) and (2) (1) CDK4R24C gene or BMI1 gene
(2) A method for producing immortalized human airway epithelial cells, comprising a step of introducing a TERT gene into human airway epithelial cells. The method according to claim 1, wherein the gene of (1) is a CDK4R24C gene, and (3) a cyclin D1 gene is further introduced. Immortalized human airway epithelial cells obtained by the method according to claim 1 or 2. A virus isolation kit comprising the immortalized human airway epithelial cell according to claim 3. A kit for screening a virus inhibitor, comprising the immortalized human airway epithelial cell according to claim 3.