WO2015015655A1 - Lignées cellulaires immortalisées de dystrophie de l'endothélium cornéen de fuchs et leur procédé de préparation - Google Patents

Lignées cellulaires immortalisées de dystrophie de l'endothélium cornéen de fuchs et leur procédé de préparation Download PDF

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WO2015015655A1
WO2015015655A1 PCT/JP2013/071096 JP2013071096W WO2015015655A1 WO 2015015655 A1 WO2015015655 A1 WO 2015015655A1 JP 2013071096 W JP2013071096 W JP 2013071096W WO 2015015655 A1 WO2015015655 A1 WO 2015015655A1
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corneal endothelial
cells
collagen
extracellular matrix
fuchs
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Japanese (ja)
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範子 小泉
直毅 奥村
木下　茂
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京都府公立大学法人
学校法人 同志社
千寿製薬株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

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  • the present invention relates to a model system of Fuchs corneal endothelial dystrophy, and techniques, methods and the like for treating or preventing the disease, or a disorder or condition thereof.
  • Visual information is transmitted from the cornea, the transparent tissue in the foreground of the eyeball, to reach the retina and excite the neurons of the retina, and the generated electrical signals are transmitted to the visual cortex via the optic nerve.
  • the cornea needs to be transparent.
  • the transparency of the cornea is maintained by keeping the water content constant by the pump function and the barrier function of corneal endothelial cells.
  • Human corneal endothelial cells are present at a density of about 3000 per square millimeter at birth, but have no ability to regenerate once damaged.
  • Fuchs corneal endothelial dystrophy is a disease in which endothelial cells inside the cornea become abnormal and cause corneal edema, and the details of the cause are unknown.
  • Fuchs corneal endothelial dystrophy an extracellular matrix such as collagen is deposited on a part of the posterior surface of the Descemet's membrane at the back of the cornea, forming a gutta (corneal guttae) or thickening of the Descemet's membrane. It becomes.
  • Fuchs corneal endothelial dystrophy has no effective treatment other than corneal transplantation, but the cornea donation in Japan is insufficient, and it is conducted in Japan annually for about 2600 patients waiting for corneal transplantation. The number of corneal transplants is about 1700.
  • animals have been mainly used for research and studies on the safety and efficacy of pharmaceuticals, but instead of using animals from the viewpoint of animal welfare, the efficacy and safety of pharmaceuticals in vitro using cultured cells, etc.
  • Research is being conducted at a practical level of technology for testing and researching. For example, an animal test is performed after testing in advance by a method using a primary cultured cell collected from a living tissue or an immortalized cell (established cell) system that grows indefinitely.
  • primary cells proliferate well at the initial stage, but gradually stop growing (cell aging) or undergo transformation with subculture.
  • primary cells change with passage in addition to the problem of heterogeneity that their characteristics differ each time they are collected from living tissue.
  • Non-patent Documents 1 and 3 there are cultures of corneal endothelial cells derived from patients with Fuchs cornea (Non-patent Documents 1 and 3) and reports of immortalization (Non-patent Document 2). Since there are no reports of cells suitable for screening for therapeutic drugs and prophylactic drugs that maintain the characteristics of the disease, there is a limit to the development of such therapeutic drugs, and there are currently no therapeutic drugs in clinical use. I have to rely on corneal transplantation.
  • the present inventors introduced SV40 large T antigen and hTERT into corneal endothelial cells derived from patients with Fuchs corneal endothelial dystrophy, in particular, (1) thickening of extracellular matrix layer, ( 2) Overproduction of at least one extracellular matrix protein selected from the group consisting of collagen I, collagen IV, collagen VIII and fibronectin, (3) At least one epithelial-mesenchymal transition selected from the group consisting of ZEB1 and Snail1 (EMT) related marker expression enhancement, (4) non-fibroblast morphology and (5) normal cell function (for example, Na + / K + -ATPase and ZO-1 are expressed and / or function to the same extent as normal cells) At least one, preferably all stored well We found that we could supply immortalized cells and succeeded in providing a model system for Fuchs corneal endothelial dystrophy.
  • the present invention provides the following inventions.
  • the cells are (1) thickening of the extracellular matrix layer, (2) overproduction of at least one extracellular matrix protein selected from the group consisting of collagen I, collagen IV, collagen VIII and fibronectin, (3 )
  • EMT epithelial-mesenchymal transition
  • the cells are (1) thickening of the extracellular matrix layer, (2) overproduction of at least one extracellular matrix protein selected from the group consisting of collagen I, collagen IV, collagen VIII and fibronectin; ) All the features selected from the group consisting of at least one epithelial-mesenchymal transition (EMT) related marker expression selected from the group consisting of ZEB1 and Snail1, (4) non-fibroblast morphology and (5) normal cell function 3.
  • EMT epithelial-mesenchymal transition
  • a method for producing immortalized cells of Fuchs corneal endothelial dystrophy comprising introducing SV40 large T antigen and hTERT into corneal endothelial cells derived from a patient with Fux corneal endothelial dystrophy.
  • a corneal endothelial cell derived from a patient with Fuchs' corneal endothelial dystrophy is treated with 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazole-2. -Il] benzamide and 4- [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine).
  • it is intended that the one or more features described above can be provided in further combinations in addition to the specified combinations. Still further embodiments and advantages of the invention will be recognized by those of ordinary skill in the art upon reading and understanding the following detailed description as needed.
  • a model system of Fuchs corneal endothelial dystrophy is provided, and a technique that can be used in industries (cell culture industry, pharmaceuticals, etc.) relating to techniques such as screening for Fuchs corneal endothelial dystrophy treatment and prevention drugs is provided. .
  • FIG. 1 shows phase contrast micrographs of immortalized corneal endothelial cells established from healthy subjects and Fuchs corneal endothelial dystrophy patients.
  • FIG. 1 shows the results of preparing immortalized cell lines by introducing SV40 and hTERT genes into lentivirus in Fuchs corneal endothelial dystrophy patient-derived corneal endothelial cells.
  • Corneal endothelial cells were obtained from three patients who had bullous keratopathy due to clinical diagnosis of Fuchs corneal endothelial dystrophy and who had undergone corneal endothelial transplantation (DMEK) with written consent and approval of the ethical committee.
  • DMEK corneal endothelial transplantation
  • the cells were peeled together with the mechanically pathological cells in the cornea and the Descemet's membrane, which is the basement membrane, and immersed in Optisol-GS (Bochrom), which is a corneal preservation solution. Thereafter, collagenase treatment was performed, and corneal endothelial cells were enzymatically collected and cultured in SB203580 + SB431542 + 3T3 conditioned medium (see Examples).
  • the cultured corneal endothelial cells derived from Fuchs corneal endothelial dystrophy were immortalized by introducing SV40 and hTERT genes with lentivirus.
  • FIGS. 1a, 1b, c and FIGS. 1d, e, f Phase contrast microscopic images of an immortalized corneal endothelial cell line (iFECD) derived from a patient with Fuchs corneal endothelial dystrophy are shown for each donor in FIGS.
  • iFECD immortalized corneal endothelial cell line
  • FIGS. 1a, 1b, c and FIGS. 1d, e, f all have one more polygonal shape as normal corneal endothelial cells.
  • FIG. 2 shows the expression of iHCEC and iFCED function-related proteins.
  • FIG. 2 shows the results of testing whether the prepared immortalized cell line maintains normal function. Immunostaining with Na + / K + -ATPase and ZO-1 was performed as markers for the pump function and the barrier function, which are functions of corneal endothelial cells. The left side shows Na + / K + -ATPase, and the right side shows ZO-1. The upper side shows iHCEC and the lower side shows iFECD.
  • FIG. 3 shows transmission electron microscope images of iHCEC and iFCED.
  • FIG. 3 shows morphological observation images of iHCEC and iFECD using a transmission electron microscope.
  • iHCEC left
  • iFECD right
  • FIG. 4 shows enhanced protein production in iFECD.
  • FIG. 4 shows the results of immunostaining and real-time PCR of the extracellular matrix of corneal endothelial cells of patients with Fuchs corneal endothelial dystrophy.
  • Corneal endothelial cells of patients with Fuchs corneal endothelial dystrophy are known to overproduce extracellular matrix resulting in the formation of guttae and thickening of the Descemet's membrane. Therefore, iHCEC and iFECD were cultured in a culture dish and immunostained for expression of type I collagen, type IV collagen, and fibronectin, which are proteins constituting the extracellular matrix.
  • the upper panel is a photograph of immunostaining, and the lower graph shows the gene expression level.
  • the upper panel in the upper panel shows iHCEC, and the lower panel shows iFECD. And I type collagen is shown from the right side. The middle shows type IV collagen and the right shows fibronectin. It was shown that the expression of type I collagen, type IV collagen and fibronectin is increased in iFECD as compared with iHCEC. Further, when the gene expression levels of cultured iHCEC and iFECD were examined by real-time PCR, type I collagen and fibronectin significantly increased expression levels, and type IV collagen tended to increase expression levels.
  • FIG. 5 shows enhanced extracellular matrix production of iHCEC and iFCED.
  • iHCEC and iFECD were cultured on Transwell in serum-free with DMEM, and after 1 week, fixed in a confluent state and stained with HE.
  • a picture of HE staining is shown on the left. In the photograph, the upper part is iHCEC, and the lower part is iFECD.
  • the right graph is a display of thickness. The left bar is iHCEC and the right bar is iFCEC. * Indicates statistical significance at p ⁇ 0.01.
  • FIG. 6 shows that expression of Snail1 and ZEB1 is increased in corneal endothelial cells derived from Fuchs corneal endothelial dystrophy.
  • FIG. 6 shows the results of analysis of the expression level using real-time PCR for genes involved in epithelial-mesenchymal transition (EMT) involved in extracellular matrix production.
  • EMT epithelial-mesenchymal transition
  • the cultured corneal endothelial cells derived from Fuchs corneal endothelial dystrophy were introduced with SV40 and hTERT genes by lentivirus to prepare an immortal cell line.
  • corneal endothelial cells cultured from a research cornea imported from Seattle Eye Bank were immortalized by the same method to prepare an immortal cell line (iHCEC).
  • iHCEC and iFECD were maintained in culture using DMEM + 10% FBS.
  • Real-time PCR significantly increased the expression of Snail1 and ZEB1 in iFECD compared to iHCEC.
  • FIG. 7 shows that TGF ⁇ increases the expression of Snail1, ZEB1 and in vitro matrix constituent proteins.
  • TGF ⁇ which is known to promote the expression of Snail1 and ZEB1, in order to confirm whether the increased expression of Snail1 and ZEB1 is involved in the production of extracellular matrix.
  • TGF ⁇ TGF ⁇
  • optical cell is used in a broad sense and means any cell present in the eye, and any cell present in the eyelid, sclera, cornea, uvea, lens, vitreous, retina, optic nerve. Cells are included.
  • cornea endothelial cell is used in the usual meaning used in the art.
  • the cornea is one of the layered tissues constituting the eye, is transparent, and is located in the portion closest to the outside world.
  • the cornea is said to be composed of five layers in order from the outside (body surface) in humans, and is composed of the corneal epithelium, Bowman's membrane (outer boundary line), eigenlayer, Descemet's membrane (inner boundary line), and corneal endothelium from the outside. Is done. Unless otherwise specified, portions other than the epithelium and endothelium are sometimes collectively referred to as “corneal stroma” and are referred to as such in this specification. As used herein, “iFECD” (immobilized Fuchs' endothelial cortical dystrophy) is an abbreviation for immortalized cells of Fuchs corneal endothelial dystrophy.
  • HCEC human Corneal Endothelial cells
  • IHCEC immortalized human corneal endothelial cells
  • corneal tissue is used in a normal sense and refers to the tissue itself constituting the cornea.
  • undifferentiated cell refers to any cell capable of differentiation.
  • undifferentiated cells include stem cells as well as cells that have differentiated to a certain extent but can still differentiate.
  • immortalized cell refers to a cell line (strain) that has an apparently infinite life in culture (has the ability to proliferate through an infinite number of divisions).
  • Such immortalized cells can acquire the ability to proliferate indefinitely by avoiding cellular senescence in the course of repeated primary cultures, or immortalized by introducing specific genes into the target cells. Abilities can also be granted.
  • many of such conventional immortalized cells are said to lose some or all of the forms and functions that the cells originally had in the living body.
  • examples in which the form and function originally possessed by Fuchs's corneal endothelial dystrophy are maintained have not been reported by conventional methods, including techniques such as Non-Patent Documents 1-3.
  • the technique of the present invention could provide immortalized cells that maintain the desired characteristics of Fuchs corneal endothelial dystrophy.
  • “established” or “established” cells maintain certain properties (eg, pluripotency) and the cells continue to grow stably under culture conditions. State. Usually, the immortalized cells often have the desired characteristics and thus often correspond to established cells.
  • transformation (cell) refers to spontaneous conversion (cell) to a growth state that cannot be controlled. Transformed cells acquire the ability to grow through an infinite number of divisions in an incubator.
  • Transformed cells may be referred to using terms such as neoplastic, undifferentiated and / or hyperplastic with respect to their lack of growth control.
  • isolated means that the substances naturally associated with it in a normal environment are at least reduced, preferably substantially free.
  • an isolated cell, tissue, etc. refers to a cell that is substantially free of other materials (eg, other cells, proteins, nucleic acids, etc.) that accompany it in its natural environment.
  • thickening of the extracellular matrix layer refers to having an extracellular matrix layer thicker than the extracellular matrix layer in the normal form of the cell when referring to a certain cell.
  • extracellular matrix layer is thicker than about 3 ⁇ m, and preferably the thickness is about 5 ⁇ m or more. Preferably, it may be about 6 ⁇ m or more.
  • extracellular matrix ECM
  • extracellular matrix is also called “extracellular matrix” and refers to a substance existing between somatic cells regardless of whether they are epithelial cells or non-epithelial cells.
  • the extracellular matrix is usually produced by cells and is therefore one of the biological materials.
  • the extracellular matrix is responsible not only for tissue support, but also for the construction of the internal environment necessary for the survival of all somatic cells.
  • the extracellular matrix is generally produced from connective tissue cells, but some are also secreted from the cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells.
  • the fiber component is roughly classified into a fiber component and a matrix filling the space, and the fiber component includes collagen fiber and elastic fiber.
  • the basic component of the substrate is glycosaminoglycan (acid mucopolysaccharide), most of which binds to non-collagenous protein to form a polymer of proteoglycan (acid mucopolysaccharide-protein complex).
  • laminin of the basement membrane, microfibril around the elastic fiber, fiber, and glycoprotein such as fibronectin on the cell surface are included in the substrate.
  • the basic structure is the same in specially differentiated tissues, for example, hyaline cartilage produces chondrocytes that contain a large amount of proteoglycan, characteristically by chondroblasts, and bone produces bone matrix that causes calcification by osteoblasts. .
  • extracellular matrix protein refers to a protein constituting the extracellular matrix.
  • Such proteins include, but are not limited to, collagen I, collagen IV, collagen VIII, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, and thrombospondin.
  • the properties of Fuchs corneal endothelial dystrophy are maintained, particularly using at least one, preferably 2, more preferably 3, and still more preferably 4 of collagen I, collagen IV, collagen VIII and fibronectin as an index. It can be determined whether or not.
  • extracellular matrix protein refers to the production of extracellular matrix proteins such as collagen I, collagen IV, collagen VIII, and fibronectin in the extracellular matrix in a normal form. It means that it is produced more than the amount that has been.
  • the production status includes not only stimulation but also that the expression level is increased by a response to transforming growth factor (TGF) ⁇ as necessary.
  • TGF transforming growth factor
  • the amount of extracellular matrix in normal is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times. Times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, or about 2.0 times or more.
  • the difference from normal is preferably significant, but not necessarily significant.
  • epithelial-mesenchymal transition (EMT) -related marker refers to a marker serving as an index in epithelial-mesenchymal transition (EMT), and examples thereof include ZEB1 and Snail1.
  • at least one epithelial-mesenchymal transition (EMT) -related marker expression enhancement selected from the group consisting of ZEB1 and Snail1 refers to a cell that is produced in ZEB1 or Snail1 in a normal form when referring to a certain cell. It means that it is produced more than the amount it has. The production status includes not only stimulation but also that the expression level is increased by a response to transforming growth factor (TGF) ⁇ as necessary.
  • TGF transforming growth factor
  • the amount of extracellular matrix in normal is about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times. Times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, or about 2.0 times or more.
  • the difference from normal is preferably significant, but not necessarily significant.
  • the “non-fibroblast form” refers to the form of a cell and means that it does not have fibroblasts.
  • the “normal cell function” of a cell refers to a function inherent to the cell when referring to a specific cell such as a corneal endothelial cell.
  • a corneal endothelial cell such functions include ZO-1 and Na.
  • ZO-1 and Na + / K + -ATPase can be evaluated by observing expression at the nucleic acid level such as immunological means or RT-PCR.
  • Na + / K + -By confirming that ATPase and ZO-1 are expressed and / or functioning to the same extent as normal cells, it is possible to confirm whether the cells of interest have normal functions.
  • the adaptability to corneal transplantation can be carried out by transplanting cultured cells by mechanically scraping the corneal endothelium as a bullous keratosis model even in laboratory animals such as rabbits.
  • the “subject” refers to a subject (for example, a living organism such as a human or an organ (eye) or a cell taken out of the living organism) as a subject of diagnosis or detection of the present invention.
  • sample refers to any substance obtained from a subject or the like, and includes, for example, ocular cells.
  • the gene (nucleic acid sequence) of the large T antigen (SV40 large T antigen) is used in transformation.
  • detection, diagnosis, preliminary detection, prediction or pre-diagnosis for a certain condition is a drug, agent, factor or means specific for the marker associated with the condition, or It can be realized by using a composition, kit or system containing them.
  • a certain condition eg, a disease such as differentiation disorder
  • gene product refers to a protein or mRNA encoded by a gene.
  • a gene product ie, a molecule such as CD98
  • telomerase reverse transcriptase (TERT) is a kind of telomerase reverse enzyme, a catalytic subunit of telomerase enzyme, and a telomerase RNA component (TERC) together with telomerase RNA component (TERC). It constitutes an important component of the complex.
  • the IDs are Entrez Gene ID: 7015, NM_001193376 (human mRNA), NP_001180305 (human protein).
  • hTERT includes not only a protein having an amino acid sequence described in a specific accession number (and a SEQ ID NO as specifically described herein) (or a nucleic acid encoding it), but also functionally active. A derivative thereof, or a functionally active fragment thereof, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. , Understood to mean.
  • “Snail1” is a protein of the C2H2 type zinc finger family that regulates transcription, and is one of the markers associated with epithelial-mesenchymal transition (EMT).
  • Snail1 is not only a protein having an amino acid sequence described in a specific accession number (and a sequence number as specifically described herein) (or a nucleic acid encoding it), but also functionally active. A derivative thereof, or a functionally active fragment thereof, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. , Understood to mean.
  • ZEB1 refers to zinc finger E-box-binding homebox 1 and is one of epithelial-mesenchymal transition (EMT) -related markers.
  • EMT epithelial-mesenchymal transition
  • BZP BZP
  • DELTAEF1 FECD6
  • NIL2A NIL2A
  • PPCD3 PPCD3
  • TCF8 ZFHEP
  • ZFHX1A The ID is Entrez Gene ID: 6035, which are NM_001128128 (human mRNA) and NP_001121600 (human protein).
  • ZEB1 is not only a protein having an amino acid sequence described in a specific accession number (and a sequence number as specifically described herein) (or a nucleic acid encoding the same), but also functionally active.
  • a “derivative” or “analog” or “variant” preferably includes, but is not intended to be limited to, a molecule comprising a region that is substantially homologous to a component protein, Such molecules may, in various embodiments, be at least 30%, 40% when compared to sequences that are aligned over amino acid sequences of the same size or aligned by computer homology programs known in the art.
  • This is a product of a naturally occurring protein modified by amino acid substitutions, deletions and additions, respectively, and derivatives thereof that do not necessarily exhibit the same degree of biological function of the naturally occurring protein. means.
  • the biological function of such proteins can be examined by suitable and available in vitro assays described herein or known in the art. In the present invention, humans are mainly discussed, but it is understood that this applies to other species such as those of other species within primates or other species of animals, It is understood that it is within the scope of the invention.
  • “functionally active” as used herein refers to a protein, such as a biological activity such as telomerase activity, according to the embodiment to which the polypeptide of the invention, ie, a fragment or derivative, relates.
  • a polypeptide, ie, a fragment or derivative having a structural function, a regulatory function, or a biochemical function.
  • polynucleotide hybridizing under stringent conditions refers to well-known conditions commonly used in the art.
  • Such a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then a 0.1 to 2-fold concentration was obtained. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C.
  • SSC serum-sodium citrate
  • composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate.
  • Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe.
  • sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions.
  • a polypeptide (for example, transthyretin) used in the present invention is a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule encoding a polypeptide particularly described in the present invention.
  • the polypeptide encoded by is also encompassed. These low stringency conditions are: 35% formamide, 5 ⁇ SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% polyvinylpyrrolidone (PVP), 0.02% BSA, 100 ⁇ g / ml denatured salmon sperm DNA , And 10% (weight / volume) dextran sulfate buffered at 40 ° C.
  • a “corresponding” gene eg, a polynucleotide sequence or molecule
  • a gene for example, a polynucleotide sequence or a molecule
  • a gene corresponding to a gene can be an ortholog of that gene. Therefore, molecules such as mouse and rat TERT can each find a corresponding molecule such as TERT in humans.
  • Such corresponding genes can be identified using techniques well known in the art.
  • a corresponding gene in an animal eg, mouse, rat
  • a sequence found in the gene accession number as a reference gene for the corresponding gene (eg, human) as a query sequence. It can be found by searching an animal sequence database.
  • expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form.
  • a gene, polynucleotide or the like is transcribed and translated into a polypeptide form.
  • transcription and production of mRNA can also be an aspect of expression.
  • polypeptide forms may be post-translationally processed (derivatives as referred to herein).
  • a functional equivalent of a molecule such as TERT used in the present invention can be found by searching a database or the like.
  • search refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods.
  • BLAST Altschul et al., J. Mol. Biol. 215: 403-410 (1990)
  • FASTA Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-). 2448 (1988)
  • the Smith and Waterman method Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)
  • Needleman and Wunsch method Needleman and Wunsch, J. Mol. Biol. 48:44:44). -453 (1970)) and the like.
  • Bio searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR and in situ hybridization, etc. It is not limited to. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search. (Description of Preferred Embodiment) The description of the preferred embodiment is described below, but it should be understood that this embodiment is an illustration of the present invention and that the scope of the present invention is not limited to such a preferred embodiment. It should be understood that those skilled in the art can easily make modifications, changes and the like within the scope of the present invention with reference to the following preferred embodiments.
  • the present invention provides immortalized cells of Fuchs corneal endothelial dystrophy.
  • the immortalized cell of Fuchs corneal endothelial dystrophy of the present invention comprises (1) thickening of the extracellular matrix layer, (2) at least one extracellular matrix protein selected from the group consisting of collagen I, collagen IV, collagen VIII and fibronectin.
  • EMT epithelial-mesenchymal transition
  • normal cell function eg, Na + / K + -ATPase and ZO-1 are expressed and / or functioning to the same extent as normal cells
  • TGF transforming growth factor
  • said normal cell function is Na.
  • Fuchs corneal endothelial dystrophy dystrophy cells have characteristics that could not be achieved in the past, and suitable cells are provided for screening drugs for the treatment or prevention of Fuchs corneal endothelial dystrophy. is there.
  • Each feature can be evaluated as follows: (1) Thickening of the extracellular matrix layer Confirm that it is thicker than normal cells (usually 2-4 ⁇ m). It is possible to determine whether the target cells are immortalized cells of Fuchs corneal endothelial dystrophy by observing that the thickness is preferably at least 1.5 times that of normal cells, more preferably at least 2 times that of normal cells. .
  • the expression level is preferably at least 1.5 times that of normal cells, more preferably at least 2 times, it is possible to determine whether the target cells are immortalized cells of Fuchs corneal endothelial dystrophy. .
  • whether or not the expression of the EMT-related marker is increased in response to TGF ⁇ can be used as a basis for judgment.
  • Non-fibroblast morphology Judging from the shape of the cells. It can be determined by maintaining a polygonal morphology as in normal cells and confirming that it exhibits a single epithelial cell-like morphology without significant stratification.
  • Normal cell function Na + / K + -Expression of ATPase and ZO-1 can be confirmed by immunostaining etc.
  • the present invention provides a method for screening an agent for treating or preventing Fuchs corneal endothelial dystrophy using Fuchs corneal endothelial dystrophy immortalized cells of the present invention. (Method for preparing immortalized cells of Fuchs corneal endothelial dystrophy)
  • the present invention provides a method for producing immortalized cells of Fuchs corneal endothelial dystrophy comprising introducing SV40 large T antigen and hTERT into corneal endothelial cells from a patient with Fux corneal endothelial dystrophy.
  • Such immortalized cells can acquire the ability to proliferate indefinitely by avoiding cellular senescence in the course of repeated primary cultures, or introduce a specific gene into a target cell. You can also grant immortality.
  • many of such immortalized cells lose part or all of the forms and functions originally possessed in the living body, in the test using such an immortalized cell line, It is said that it is difficult to accurately reflect the original characteristics of the tissue from which the cell line is derived.
  • examples in which the form and function originally possessed by Fuchs's corneal endothelial dystrophy are maintained have not been reported by conventional methods, including techniques such as Non-Patent Documents 1-3. It has been difficult to provide immortalized cells that maintain the desired characteristics.
  • the present invention maintains the desired characteristics, that is, the characteristics of Fuchs corneal endothelial dystrophy by using the above-described specific gene transfer technology, and retains the same function as that of normal cells (that is, has been transformed). Not) cells could be provided.
  • immortalized cells that have acquired the ability to proliferate indefinitely by avoiding cell aging through repeated passages of primary culture, they will have stable and uniform characteristics, but many of these immortalized cells are In many cases, some or all of the forms and functions that the cells originally have in the living body are lost. Therefore, in tests using such an immortalized cell line, it has been difficult to accurately reflect the original characteristics of the tissue from which the cell line is derived.
  • Fuchs corneal endothelial dystrophy is difficult to maintain as normal cells in the first place, while maintaining normal cells in corneal endothelium is more difficult to maintain as cultured cells. It seemed impossible. Therefore, in the present invention, in addition to using SV40 large T antigen, by introducing telomerase reverse transcriptase (TERT) as a transgene, immortalization is maintained while maintaining the characteristics of Fuchs corneal endothelial dystrophy. Succeeded in doing.
  • TERT telomerase reverse transcriptase
  • the method for introducing SV40 large T antigen and hTERT in the present invention may be a gene (nucleic acid molecule) introduction technique (transformation, transduction, transfection, etc.) or a gene product (protein) introduction method known in the art. Any technique can be used, and introduction of these genes can usually be realized by introduction of nucleic acid molecules.
  • gene transfer refers to introducing a foreign gene, preferably a functional gene, into a chromosome genome or the like, for example, by an appropriate transfer technique.
  • an exogenous functional gene can be introduced by replacing the endogenous functional gene.
  • the exogenous functional gene is a gene that does not originally exist in the chromosome genome of the organism, and may be a gene derived from another species, a synthetic gene prepared by PCR, or the like.
  • a transfection technique is an example of such a gene introduction technique.
  • the target gene in the present invention, SV40 large T antigen, hTERT gene, etc.
  • an appropriate viral vector for example, but not limited to, a lentiviral vector.
  • an appropriate cell for example, 293T cell
  • a helper plasmid as necessary (for example, pLP1, pLP2, pLP / VSVG, etc.).
  • the culture supernatant containing the virus is recovered after infection, and the culture is added to the culture solution of the target cells (in the present invention, cultured corneal endothelial cells derived from Fuchs corneal endothelial dystrophy patients).
  • Kiyo can be added with an appropriate reagent (here, but not limited to polybrene) to produce an immortalized corneal endothelial cell line (iFECD) derived from a patient with Fuchs corneal endothelial dystrophy.
  • iFECD immortalized corneal endothelial cell line
  • exogenous DNA containing DNA encoding a transforming gene can be introduced into a cell of interest using techniques for transfection into retroviruses (eg, lentiviral vectors).
  • retroviruses eg, lentiviral vectors
  • recombinant retroviruses are produced in packaging cell lines and have high titer virus particles (generally 10 5 ⁇ 10 6 (but not limited to pfu / ml) produce a culture supernatant.
  • a recombinant virus particle is a culture medium containing a target cell culture containing a virus culture and a reagent such as polybrene at an appropriate concentration (for example, 5 to 10 ⁇ g / ml but not limited thereto).
  • the cells are used for infection by incubating for an appropriate time (for example, about 1 to 12 hours, but not limited thereto).
  • the cells are rinsed and cultured in standard medium.
  • the infected cells are then analyzed for foreign DNA uptake and expression.
  • the cells may be subjected to selective conditions that select for cells that have taken up and expressed the selectable marker gene.
  • exogenous DNA may be introduced using calcium phosphate mediated transfection techniques. Calcium phosphate precipitates containing DNA encoding the gene of interest, eg, a transforming gene, are described in Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76: 1373-1376. After transfection, the cells are analyzed for foreign DNA uptake and expression.
  • the cells may be subjected to selective conditions that select for cells that have taken up and expressed the selectable marker gene.
  • Such techniques are exemplified in JP 2013-509179 A and JP 2004-254509 A in addition to the examples specifically disclosed in the embodiments, but are not limited thereto.
  • the immortalized cell line of the present invention is cultured in the presence of a test substance, and the degree of increase or decrease in the thickness of the extracellular matrix (ECM) in the cell can be measured and evaluated.
  • ECM extracellular matrix
  • the presence / absence, increase / decrease, etc. of apoptosis can be measured and evaluated, or the degree of increase / decrease of other markers can be measured / evaluated.
  • the screening of useful substances in the above-mentioned immortalized cells involves culturing immortal cell lines in the presence of various concentrations of test substances, and after culturing for a certain period of time, the number of cells, cell morphology, amount of marker protein expressed, etc. Is detected and measured, and is compared and evaluated with a control sample cultured in the absence of the test substance.
  • Useful substances in immortalized cells obtained by these screening methods are useful because they may be used as therapeutic agents, prophylactic and / or symptom improving agents used to treat patients with Fuchs corneal endothelial dystrophy.
  • additional detailed useful conditions can include the following.
  • the corneal endothelial cells from a patient with Fuchs corneal endothelial dystrophy are SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide) and SB203580 (4- [4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -1H-imidazol-5-yl] pyridine)
  • Preferred cells can be obtained by culturing in the above.
  • This medium may preferably be SB203580 + SB431542 + 3T3 conditioned medium used in the examples, but is not limited thereto.
  • Fuchs corneal endothelial dystrophy causes cell death of corneal endothelial cells, and if the remaining corneal endothelial cells cannot compensate for the pump function and the barrier function, the transparency of the cornea cannot be maintained, resulting in blindness due to corneal opacity. It is also known that corneal endothelial cells of patients with Fuchs corneal endothelial dystrophy produce excessive extracellular matrix, resulting in the formation of guttae and thickening of the Descemet's membrane. The formation of the guts and the thickening of the Descemet's membrane cause light scattering and the like, which significantly damages the patient's QOL, which is not responsible for vision loss, photophobia, and fog vision.
  • An immortalized corneal endothelial cell line (iFECD) derived from a patient with Fuchs corneal endothelial dystrophy was prepared.
  • Example 1 Production of immortalized corneal endothelial cell line (iFECD) derived from Fuchs corneal endothelial dystrophy patient
  • an immortalized corneal endothelial cell line (iFECD) was prepared from corneal endothelial cells derived from a patient with Fuchs corneal endothelial dystrophy.
  • Corneal endothelial cells were mechanically detached together with the basement membrane from the research cornea purchased from Seattle Eye Bank, peeled off from the basement membrane using collagenase, and then subjected to primary culture.
  • the medium is Opti-MEM I Reduced-Serum Medium, Liquid (INVITROGEN catalog number: 31985-070), 8% FBS (BIOWEST, catalog number: S1820-500), 200 mg / ml CaCl 2 .2H 2 O (SIGMA catalog number).
  • SB431542 (1 ⁇ mol / l) and SB203580 (4- (4-fluorophenyl) -2- (4-methylsulfonylphenyl) -5 (4-pyridyl) imidazole ⁇ 4- [4- (4-fluoro) were added to the basic medium.
  • collagenase treatment was performed to enzymatically collect corneal endothelial cells, which were then cultured in the order of SB203580 + SB431542 + 3T3.
  • the cultured corneal endothelial cells derived from Fuchs corneal endothelial dystrophy patients were amplified by SV40 large T antigen and hTERT gene by PCR and introduced into a lentiviral vector (pLenti6.3_V5-TOPO; Life Technologies Inc).
  • the lentiviral vector was transformed into 293T cells (RCB2202; Riken) using three types of helper plasmids (pLP1, pLP2, pLP / VSVG; Life Technologies Inc.) and a transfection reagent (Fugene HD; Promega Corp., Madison, WI). Bioresource Center, Ibaraki, Japan).
  • the culture supernatant containing the virus was collected and added to a culture of corneal endothelial cells derived from a patient with Fuchs corneal endothelial dystrophy using 5 ⁇ g / ml of polybrene to obtain SV40 large T antigen and The hTERT gene was introduced.
  • FIGS. 1d, 1e, and 1f Phase contrast microscopic images of an immortalized corneal endothelial cell line (iFECD) derived from a patient with Fuchs corneal endothelial dystrophy are shown in FIGS. 1d, 1e, and 1f for each donor.
  • iHCEC normal corneal endothelial cells
  • FIGS. 1a, 1b, and 1c Phase contrast microscopic images of an immortalized corneal endothelial cell line (iHCEC) derived from a healthy donor.
  • iHCEC and iFECD were maintained in culture using DMEM + 10% FBS.
  • Example 2 Confirmation of normal function of immortalized corneal endothelial cell line (iFECD)
  • iFECD immortalized corneal endothelial cell line
  • ZO-1 Zymed Laboratories, Inc., South San Francisco, Calif.
  • ZO-1 and Na + / K + -ATPase were used as markers related to cell function.
  • iHCEC and iFECD were cultured in Dulbecco's modified Eagle medium (DMEM) (Nacalai Tesque, Cat no .: serum-free in 08456-94) on Transwell Permeable Supports: 0.4 ⁇ m, 6 well plates (Costar, Cat no .: 3450). One week later, the cells were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in a confluent state.
  • DMEM Dulbecco's modified Eagle medium
  • FIG. 3 shows morphological observation images of iHCEC and iFECD using a transmission electron microscope.
  • iHCEC and iFECD are cultured on Transwell in DMEM without serum and fixed in a confluent state one week later, the morphology is observed with a transmission electron microscope. It has been shown.
  • Example 3 Extracellular matrix production of immortalized corneal endothelial cell line (iFECD)
  • iFECD extracellular matrix production of immortalized corneal endothelial cell line
  • iHCEC and iFECD were cultured in a culture dish and immunostained for expression of type I collagen, type IV collagen, and fibronectin, which are proteins constituting the extracellular matrix.
  • type I collagen, type IV collagen and fibronectin is increased in iFECD as compared with iHCEC. Further, when the gene expression levels of cultured iHCEC and iFECD were examined by real-time PCR, type I collagen and fibronectin significantly increased expression levels, and type IV collagen tended to increase expression levels. (Real-time PCR) Further, PCR was performed by Taqman method for type I collagen, type IV collagen, and fibronectin by the following method. Taqman probe was purchased from INVITROGEN. The amount of mRNA of type I collagen, type IV collagen, and fibronectin was examined by real-time PCR.
  • RNEasy (QIAGEN, catalog number: 74106) was used for extraction of total RNA from the cells.
  • the extracted RNA was subjected to reverse transcription reaction (42 ° C., 60 minutes) using RiverTra Ace (TOYOBO, catalog number: TRT-101), and type I collagen using GAPDH as an internal standard using the reaction reagent TaqMan Fast Advanced mastermix (Applied Biosystems).
  • Type IV collagen and fibronectin were amplified.
  • the PCR reaction was performed by StepOne TM (Applied Biosystems) real-time PCR system using the probes shown below.
  • iFECD immortalized corneal endothelial cell line
  • iHCEC and iFECD were cultured in Dulbecco's modified Eagle medium (DMEM) (Nacalai Tesque, Cat no .: serum-free in 08456-94) on Transwell Permeable Supports: 0.4 ⁇ m, 6 well plates (Costar, Cat no .: 3450).
  • DMEM Dulbecco's modified Eagle medium
  • HE staining was performed by fixing in a confluent state. The procedure is as follows. Deparaffinization (for example, with pure ethanol) and water washing were performed as necessary, and the sample was soaked with omni hematoxylin for 10 minutes. Thereafter, it was washed with running water and colored with ammonia water for 30 seconds.
  • Example 4 Gene involved in epithelial-mesenchymal transition (EMT) as a further cell marker of immortalized corneal endothelial cell line (iFECD)
  • EMT epithelial-mesenchymal transition
  • iFECD immortalized corneal endothelial cell line
  • RNEasy (QIAGEN, catalog number: 74106) was used for extraction of total RNA from the cells.
  • the extracted RNA was subjected to reverse transcription reaction (42 ° C., 60 minutes) using RiverTra Ace (TOYOBO, catalog number: TRT-101), and type I collagen using GAPDH as an internal standard using the reaction reagent TaqMan Fast Advanced mastermix (Applied Biosystems).
  • Type IV collagen and fibronectin were amplified.
  • the PCR reaction was performed by StepOne TM (Applied Biosystems) real-time PCR system using the probes shown below.
  • iFECD and iHCEC were cultured in DMEM containing 10% fetal calf serum, cultured overnight in DMEM not containing 10% fetal bovine serum, and then subjected to real-time PCR method for Snail1, ZEB1, type I collagen, type IV collagen, Expression of type VIII collagen and fibronectin was examined.
  • the PCR reaction was performed by StepOne TM (Applied Biosystems) real-time PCR system using the probes shown below.
  • a model system of Fuchs corneal endothelial dystrophy is provided, and technologies available in industries related to Fuchs corneal endothelial dystrophy treatment (cell culture industry, pharmaceuticals, etc.) are provided.

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Abstract

L'objet de la présente invention est de fournir un système modèle de la dystrophie de l'endothélium cornéen de Fuchs. L'invention concerne des cellules immortalisées de la dystrophie de l'endothélium cornéen de Fuchs. Ces cellules comprennent au moins une caractéristique choisie dans le groupe constitué par (1) un épaississement de la couche de matrice extracellulaire, (2) une surproduction d'au moins une protéine de la matrice extracellulaire choisie dans le groupe constitué par le collagène I, le collagène IV, le collagène VIII et la fibronectine, (3) une augmentation de l'expression d'au moins un marqueur associé à la transition épithélio-mésenchymateuse (TEM), choisi dans le groupe constitué par ZEB1 et Snail1, (4) une morphologie non-fibroblastique et (5) une fonction cellulaire normale.
PCT/JP2013/071096 2013-07-30 2013-07-30 Lignées cellulaires immortalisées de dystrophie de l'endothélium cornéen de fuchs et leur procédé de préparation WO2015015655A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9260678B2 (en) 2006-07-19 2016-02-16 Nazzareno De Angelis Integrated process for the production of biofuels from different types of starting materials and related products
US10813920B2 (en) 2013-11-14 2020-10-27 The Doshisha Drug for treating corneal endothelium by promoting cell proliferation or inhibiting cell damage
WO2017110093A1 (fr) * 2015-12-24 2017-06-29 学校法人同志社 Médicament permettant le traitement ou la prévention d'un trouble provoqué par des signaux tgf-â et son application
JPWO2017110093A1 (ja) * 2015-12-24 2018-10-11 学校法人同志社 ＴＧＦ−βシグナルに起因する障害を治療または予防するための医薬およびその応用
US10980787B2 (en) 2015-12-24 2021-04-20 The Doshisha Drug for treating or preventing disorder caused by TGF-B signals, and application thereof
WO2018230711A1 (fr) 2017-06-16 2018-12-20 学校法人同志社 Médicament contenant un inhibiteur de mtor destiné au traitement ou à la prévention de symptômes, de troubles ou de maladies ophtalmiques, et son application
WO2018230712A1 (fr) 2017-06-16 2018-12-20 学校法人同志社 Procédé novateur de criblage à la recherche d'un inhibiteur du tgf-bêta
WO2018230713A1 (fr) 2017-06-16 2018-12-20 学校法人同志社 Composés ayant une activité inhibitrice de caspase, agent pharmaceutique les contenant pour le traitement ou la prévention des symptômes, troubles ou maladies de l'endothélium cornéen, et application dudit agent pharmaceutique
JPWO2018230712A1 (ja) * 2017-06-16 2020-04-16 学校法人同志社 ＴＧＦ−β阻害剤の新規スクリーニング法
US11433090B2 (en) 2017-06-16 2022-09-06 The Doshisha mTOR-inhibitor-containing medicine for treating or preventing ophthalmic symptoms, disorders, or diseases, and application thereof
CN115521901A (zh) * 2022-10-12 2022-12-27 中国医学科学院医学生物学研究所 永生化树鼩视网膜微血管内皮细胞株及其构建方法与应用
CN115521901B (zh) * 2022-10-12 2023-09-05 中国医学科学院医学生物学研究所 永生化树鼩视网膜微血管内皮细胞株及其构建方法与应用

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