JP5311885B2 - Conjunctival epithelial cell line - Google Patents

Description

  The present invention relates to a conjunctival epithelial cell line and a method for producing the same.

  The conjunctiva is a mucosal tissue for keeping the corneal surface moist with tears, and is partially divided into a conjunctiva, a bulbous conjunctiva, and a conical conjunctiva. The conjunctiva covers the posterior surface of the eyelid, the bulbar conjunctiva covers the surface of the eyeball, and the conical conjunctiva exists between the upper and lower conjunctiva and the bulbar conjunctiva. The conjunctiva and bulbar conjunctiva are covered by squamous epithelium, and the conjunctiva of the cap is covered by columnar epithelium. Histologically, it consists of epithelial layer, basement membrane and lamina propria.

  Conjunctival epithelial cells are cells contained in the conjunctiva, and play an important role in protecting the eyeball as a physical / biological barrier by preventing the entry of foreign substances and bacteria from the outside. Culture of conjunctival epithelial cells in vitro and analysis of their functions are important for elucidating the pathological conditions of various eye diseases and promoting the development of therapeutic agents for the diseases.

  However, the conjunctival epithelial cells rapidly decrease in proliferative ability in the culture system, and it is impossible to subculture for a long period of time, or even if possible, stable culture is extremely difficult. In addition, conjunctival epithelial cells obtained from newborns are relatively easy to cultivate, but still have limited proliferation ability, and there is a possibility that cell enlargement and degeneration occur during long-term passage. Therefore, it is desired to develop a conjunctival epithelial cell line capable of long-term passage that can be used for molecular cell biological research while maintaining its original function as a conjunctival epithelial cell.

  On the other hand, a human lens epithelial cell line was prepared by introducing a large T antigen gene of simian virus 40 (SV40) known as an immortalizing gene into human lens epithelial cells [Japanese Patent Laid-Open No. 10-52272: Patent Document 1]. Andley UP et al., Invest. Ophthalmol. Vis. Sci., 35: 3094-3102 (1994): Non-Patent Document 1]. In addition, it has been reported that immortalized human foreskin cell lines were prepared by introducing E6 and E7 genes of papillomavirus [J. Virol., Vol.65, p.473-478 (1991): non-patent Reference 2].

  In WO 2007/094301 [Patent Document 2] and WO 2007/094302 [Patent Document 3], a vitreous cell line was prepared by introducing an SV40 large T antigen gene, and papillomavirus E6 was obtained. It has been reported that a scleral cell line was prepared by introducing the E7 gene.

  As for conjunctival epithelial cells, it has been reported that an immortalized rat conjunctival epithelial cell line was prepared by introducing SV40 large T antigen gene into rat conjunctival epithelial cells [Exp. Eye Res., 84 (2 ), p323-331 (2007): Non-Patent Document 3]. However, there has been no report that a human-derived conjunctival epithelial cell line has been produced by introducing an exogenous immortalizing gene.

JP-A-10-52272 WO 2007/094301 WO 2007/094302 Invest. Ophthalmol. Vis. Sci., 35: 3094-3102 (1994) J. Virol., Vol.65, p.473-478 (1991) Exp. Eye Res., 84 (2), p323-331 (2007)

  Therefore, the object of the present invention is useful as a research means for developing physiological and biochemical studies of the conjunctiva and for the prevention and treatment of eye diseases such as conjunctivitis, and for the regeneration of the conjunctiva into the living body. To provide a conjunctival epithelial cell line, particularly a human conjunctival epithelial cell line. Another object of the present invention is to provide a method for producing such a conjunctival epithelial cell line.

  The present inventors confer infinite proliferation ability in vitro to conjunctival epithelial cells by infecting human conjunctival epithelial cells with a retroviral vector functionally incorporating an immortalizing gene encoding human papillomavirus E6 and E7. The present invention was completed by successfully establishing a clonal human conjunctival epithelial cell line that can be subcultured semipermanently.

That is, the present invention relates to the following.
[1] A conjunctival epithelial cell line capable of expressing an exogenous immortalizing gene encoding human papillomavirus E6 and E7.
[2] The cell line according to [1] above, which is derived from human.
[3] The cell line according to [1], which has the following properties (1) to (3):
(1) No decrease in growth rate is seen;
(2) No substantial enlargement / degeneration of cells;
(3) Keratin 13 is expressed.
[4] The cell line according to [3] above, wherein the properties of (1) to (3) are maintained for 10 passages or more.
[5] Established by transfecting conjunctival epithelial cells with an expression vector functionally carrying exogenous immortalizing genes encoding human papillomavirus E6 and E7, and subculturing the cells in a medium. The cell line according to [1] above.
[6] Conjunctival epithelium comprising transfecting conjunctival epithelial cells with an expression vector functionally carrying an exogenous immortalizing gene encoding human papillomavirus E6 and E7, and subculturing the cells in a medium Cell line production method.

  Since the conjunctival epithelial cell line of the present invention can obtain a sufficient number of cells and has a certain continuous proliferation ability, elucidation of the etiology of eye diseases such as conjunctivitis, prevention or / and treatment of the disease It can be used advantageously for development. Furthermore, the cell line is not only very useful for biochemical and physiological studies of the conjunctiva, but also for the study of cell differentiation mechanisms, and may be used as a biomaterial for artificial conjunctiva.

  The conjunctival epithelial cell line of the present invention is a cell group that contains exogenous immortalizing genes encoding human papillomavirus E6 and E7, and has acquired in vitro infinite proliferation ability as a result of expression of the immortalizing gene.

  The conjunctival epithelial cell line of the present invention is derived from a mammal. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs, and laboratory animals such as rabbits, hoofs such as pigs, cows, goats, and sheep, odd hoofs such as horses, dogs, cats, etc. Primates such as pets, humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees and the like. The conjunctival epithelial cell line of the present invention is preferably derived from a human.

  Here, the “immortalizing gene” means a gene that immortalizes cells and acquires infinite proliferation ability. Further, in the present invention, the “exogenous immortalizing gene” means an immortalizing gene newly introduced from the outside of the conjunctival epithelial cell that is the origin of the conjunctival epithelial cell line of the present invention. . As the exogenous immortalizing gene of the present invention, preferably, E6 and E7 genes of human papillomavirus are used. By using the E6 and E7 genes of human papillomavirus, conjunctival epithelial cells can be immortalized with higher efficiency than when the SV40 large T antigen gene or the like is used.

  The conjunctival epithelial cell line of the present invention is preferably an expression vector that functionally carries the above-mentioned exogenous immortalizing gene in a conjunctival epithelial cell isolated from a tissue, that is, a form that can be expressed in a target cell. And can be established by subculturing the cells in an appropriate medium.

The conjunctival epithelial cells are obtained by, for example, isolating the conjunctival tissue from the extracted eye tissue, further separating the conjunctival epithelial cells from the isolated conjunctival tissue, and finely cutting (about 1 mm square) with scissors or the like. Epithelial fragments can be obtained by digesting with type IV collagenase. Conjunctival epithelial cells are suspended in a suitable liquid medium containing fetal bovine serum or calf serum 10-20%, such as Eagle MEM medium, Dulbecco's modified Eagle MEM medium, Ham medium F12, Katsuta medium DM-160, etc. _The cells are cultured for about 2 days in ₂ incubators and are used for transfection.

  Examples of expression vectors used in the present invention include plasmid vectors and virus vectors. When a viral genome is used as a vector, it is more preferable that a part of the gene is deleted or mutated so that at least complete virus particles are not produced in the cell into which the vector is introduced.

  As a method for constructing an expression vector functionally carrying an exogenous immortalizing gene, for example, when the human papillomavirus E6 and E7 genes are used as the exogenous immortalizing gene, the following method is exemplified. A DNA fragment containing an immortalizing gene (ie, E6 and E7 genes) present in the early region of human papillomavirus genomic DNA is excised using an appropriate restriction enzyme and contains a promoter capable of expressing the gene in conjunctival epithelial cells. Into the expression vector. Examples of the promoter include SV40 early promoter, Rous sarcoma virus (RSV) promoter, MuMLV LTR and the like.

  Examples of plasmid vectors include pBR322 and pGEM. Furthermore, a transcription termination signal, a specific sequence that enhances the expression of an exogenous immortalizing gene, and the like can be placed at an appropriate position of the vector. The exogenous immortalizing gene expression vector thus constructed is synthesized in a large amount in an appropriate host, for example, Escherichia coli, yeast, Bacillus subtilis, etc., recovered and purified by a conventional method, For example, it is introduced into conjunctival epithelial cells by calcium phosphate coprecipitation method, microinjection method, polyethylene glycol method, electroporation method and the like.

  Examples of viral vectors include retroviruses such as Moloney murine leukemia virus and lentivirus, adenovirus, herpes virus, adeno-associated virus, parvovirus, Semliki forest virus, vaccinia virus, Sendai virus and the like. Gene transfer by retrovirus is preferable because the gene is introduced so that the gene is integrated into the chromosome. Lentiviruses can also infect both dividing and non-dividing cells and introduce genes. By culturing conjunctival epithelial cells in a culture solution containing virus particles, an exogenous immortalizing gene is introduced into the conjunctival epithelial cells. In this case, the introduction efficiency can be increased by using retronectin, fibronectin, polybrene or the like as a gene introduction reagent.

The transfected cells are cultured in a liquid medium such as Eagle MEM medium, Dulbecco's modified Eagle MEM medium, Ham medium F12, Katsuta medium DM-160, and the like. As conditions such as culture temperature, medium pH, and CO ₂ concentration, general conditions usually used in animal cell culture can be appropriately employed. Replace with fresh medium every 2-3 days and passage when cell growth reaches saturation point. Once the cell growth has stopped, the culture is continued, and the cell group that has begun to re-grow is classified as a single clone cell group. The clone can be separated as follows. A small-diameter filter paper soaked with trypsin and EDTA solution is allowed to stand on the target clonal cell group, and the cells are detached from the culture vessel and attached to the filter paper. The clonal cell group adhering to the filter paper piece is transferred together with the filter paper to another culture container. In addition, clones can be separated by a method of picking up colonies, a limiting dilution method, or a method using a cell sorter. Because clonal cell lines have uniform cell characteristics, physiological and biochemical studies of conjunctiva, which have been performed using heterogeneous conjunctival epithelial cells, elucidation of the etiology of eye diseases such as conjunctivitis and It can be a useful model cell for the development of prophylactic / therapeutic drugs.

The conjunctival epithelial cell line obtained as described above can be subcultured by a general animal cell culture technique. As an index of cell proliferation ability, a cell population doubling level (PDL) calculated by the following equation is used.
PDL = log ₁₀ (N _i / N ₀ ) / log ₁₀ 2
N _i = number of cells at the end of the _i -th subculture N ₀ = number of cells at the start of the first subculture The PDL of the cell line of the present invention is usually about 1.1 to 2.2. Increase by 0, preferably by 1.4 to 1.5.

The cell line of the present invention may have the following properties (1) to (3):
(1) No decrease in growth rate is seen;
(2) substantially no cell enlargement / degeneration; and (3) keratin 13 is expressed.

  Here, “substantially no enlargement / denaturation of cells” means that even though some cells may be seen by microscopic observation of cells, the majority (for example, about 95%) The above means that it shows the morphological characteristics of the original conjunctival epithelial cells. “Expression of a gene” means expression of mRNA encoding the gene or the gene product (protein). The expression of keratin (cytokeratin) 13 is an indicator that the cell line of the present invention has characteristics as conjunctival epithelial cells. Keratin 13 is one of the constituent components of the intermediate filament of epithelial cells. The cell lines of the present invention provide a powerful tool for studying various eye diseases involving conjunctival epithelial cells.

  The conjunctival epithelial cell line of the present invention stably maintains the above properties for at least 10 passages or more, preferably 15 passages or more, more preferably 20 passages or more.

  The cell line of the present invention can be a useful model cell for physiological and biochemical studies of the conjunctiva, elucidation of the etiology of conjunctival diseases, development of preventive / therapeutic drugs, and safety testing of pharmaceuticals for ophthalmic diseases. In addition, since the cells can be stably supplied, they can be useful materials for transplantation for the purpose of conjunctival regeneration (for example, biomaterials for artificial conjunctiva such as sheets of conjunctival epithelial cells). In addition, a cell line into which a papillomavirus antigen gene has been introduced can be used for cell biology experiments.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

(Preparation of human conjunctival epithelial cells)
Sample No. 53: Conjunctival tissue was isolated from a 13-year-old man suffering from endoblastoma at the time of excision of the affected area.
Sample No. 63: A conjunctival tissue was isolated from a 66-year-old man suffering from conjunctival squamous cell carcinoma at the time of excision of the affected area.
The conjunctival epithelial tissue separated from the conjunctival tissue was cut into 1 mm squares with scissors. The resulting conjunctival epithelial tissue fragment was digested in 0.05% type IV collagenase / DMEM at 37 ° C. for 16 hours. Conjunctival epithelial cells collected from the digested product were suspended in DMEM / F-12 (Invitrogen) (15% FBS (Nichirei)) and cultured at 37 ° C. and 5% CO ₂ . This study was approved by the Yamagata University School of Medicine Ethics Committee. Prior to the trial, a license was obtained from the above patient.

(Preparation of packaging cell PA317 incorporating human papillomavirus type 16 E6 and E7 and preparation of virus stock)
PA317 (mouse packaging cell: ATCC CRL-2203) into which the pLXSN vector into which human papillomavirus type 16 E6 and E7 were inserted was introduced into DMEM (10% FBS, high glucose) at 37 ° C. and 5% CO ₂ . And cultured. The cells were cultured to 90% confluence, and the culture supernatant was collected as a virus stock and stored at −80 ° C. until use.

(RetroNectin coating)
1 mg / ml of retronectin (Wako # T100A) was diluted to 25 μg / ml with PBS (pH 7.2), and 2 ml each was added to a 35 mm culture dish. The dish was allowed to stand at room temperature for 2 hours in a clean bench to perform retronectin coating. Thereafter, blocking was performed by adding 2 ml of 2% BSA / PBS, and the blocking solution was washed with 2 ml PBS, and then PBS was removed.

(Retroviral infection of conjunctival epithelial cells)
To a 35 mm culture dish coated with retronectin, 2.5 ml of the virus stock solution was added and left at 37 ° C., 5% CO _{2 for} 4 to 6 hours to bind the retronectin and the retroviral vector. Thereafter, the virus stock solution was removed, the dish was washed with PBS, and conjunctival epithelial cells were collected at a concentration of 0.5 to 1.0 × 10 ⁵ cells / 2 ml DMEM / F-12 (15% FBS) / 35 mm dish. In addition, the cells were cultured at 37 ° C. and 5% CO _{2 for} 2 days.

(Isolation of conjunctival epithelial cell line)
Conjunctival epithelial cells infected with retrovirus were passaged to 10 mm dishes and further passaged for 5 days. The formed colony was picked up, transferred to a 24-well plate, and the colony was cultured to establish a monoclonal human conjunctival epithelial cell line. The cell lines established for each of samples No. 53 and No. 63 (No. 53-1, 2 and No. 63-1, 2) are cell lines cultured from a single colony, respectively.

(Cell passage)
The isolated colonies were continuously cultured under the same conditions until the 17th passage for sample No. 53 and until the 20th passage for sample No. 63. The doubling times for No. 53 and No. 63 were 26 hours and 44 hours, respectively. Throughout the culture, the cells could continue to be passaged at a constant ratio (1: 3), and no decrease in the cell growth rate was observed.
Cell morphology was observed under a microscope. The appearance of the cells was photographed with a cooled CCD camera DP-70 (Olympus) connected to a DMIRBE microscope (Leica). The photographing conditions are as follows: objective lens 5 × / eyepiece 10 × / light quantity 9.0V. The cells consisted of a single layer and had a fibroblast-like morphology (in Fig. 1, No.53-1,2 was taken at passage 7 and No.63-1,2 was taken at passage 4) is there). No cell enlargement or degeneration was observed.

(Confirmation of expression of human papillomavirus type 16 E6 and E7)
Total RNA was isolated from the cells, cDNA was synthesized, and mRNA expression of the immortalized gene human papillomavirus type 16 E6 and E7 was confirmed by RT-PCR method.

(Analysis of phenotype of conjunctival epithelial cell line)
The expression of keratin 13 in four established conjunctival epithelial cell lines (clone No. 53-1, 2 and No. 63-1, 2) was examined. The expression was observed by RT-PCR.
That is, when the conjunctival epithelial cell line was 60-70% confluent, the medium of the cell was replaced with DMEM containing 1% bovine serum and cultured for 24 hours. After culturing for 24 hours, the cells were collected, extracted with total RNA and synthesized with cDNA, and RT-PCR was performed using primers specific for keratin 13 (FIG. 2). PCR was performed on a Gene Amp PCR System 9700 (Applied Biosystems) using the Roche FastStart High Fidelity PCR System. PCR conditions are as follows:
95 ° C for 2 minutes → (95 ° C for 30 seconds → X ° C for 30 seconds → 72 ° C for 30 seconds) x Y cycle → 72 ° C for 7 minutes X = 55, Y = 33 ... Keratin 13
X = 60, Y = 20 ... beta-Actin

  As a result, expression of keratin 13 was observed in all established cell lines. From the above, it was shown that the obtained conjunctival epithelial cell line retains characteristics as conjunctival epithelial cells.

(Comparative example)
(Introduction of immortalized gene SV40 large T antigen)
The vector (RIKEN Taq-LTR-pUC18 sense # 4) in which the SV40 large T antigen sequence was incorporated into the conjunctival epithelial cell of sample No. 53 obtained in the above example was subjected to the calcium phosphate method (BD calphos Mammalian Transfection Kit). Introduced.
At 5 days after gene transfer, isolation of the cell population was attempted. Cells forming colonies were selected, the cells were detached with 0.25% trypsin / EDTA, and each colony was cultured in a 12-well plate. When it became about 80% confluent, it was subcultured into a 6-well plate and cultured. However, when the cells were subsequently passaged 3 times at a 1: 3 dilution, cell division was reduced.

  Since the conjunctival epithelial cell line of the present invention can obtain a sufficient number of cells and has a certain continuous proliferation ability, elucidation of the etiology of an eye disease such as conjunctivitis, and a prophylactic or / and therapeutic agent for the eye disease Can be advantageously used for the development of Furthermore, the cell line is not only very useful for biochemical and physiological studies of the conjunctiva, but also for the study of cell differentiation mechanisms, and may be used as a biomaterial for artificial conjunctiva.

It is a photograph showing the morphology of the conjunctival epithelial cell line (bar: 200 μm). It is a figure which shows the expression of keratin 13 in a conjunctival epithelial cell line.

Claims (5)

Conjunctival epithelial cell lines capable of expressing exogenous immortalizing genes encoding human papillomavirus E6 and E7 having the following properties (1) to (3) :
(1) No decrease in growth rate is seen;
(2) No substantial enlargement / degeneration of cells;
(3) Keratin 13 is expressed .   The cell line according to claim 1, which is derived from human. The cell line according to claim 1 or 2, wherein the properties (1) to (3) are maintained for 10 passages or more.   2. Established by transfecting conjunctival epithelial cells with an expression vector functionally carrying exogenous immortalizing genes encoding human papillomavirus E6 and E7, and subculturing the cells in medium. The cell line described.   A conjunctival epithelial cell line comprising: transfecting a conjunctival epithelial cell with an expression vector functionally carrying an exogenous immortalizing gene encoding human papillomavirus E6 and E7; and subculturing the cell in a medium. Production method.

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