CN116672366A - Application of choroid plexus epithelial cells in replacing cornea endothelial cells - Google Patents
Application of choroid plexus epithelial cells in replacing cornea endothelial cells Download PDFInfo
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- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2430/00—Materials or treatment for tissue regeneration
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Abstract
The application discloses application of choroid plexus epithelial cells in preparing medicines or medical instruments for relieving or treating corneal endothelial injury, corneal endothelial lesion, corneal endothelial dysfunction and corneal endothelial cell function decompensation, wherein the choroid plexus epithelial cells are used for replacing the corneal endothelial cells, can reconstruct and improve the barrier function of the corneal endothelium so as to eliminate corneal edema, reduce the thickness of the cornea and recover the visual function, and provide a new seed cell and a new treatment strategy for the clinical treatment of the corneal endothelial function decompensation.
Description
Technical Field
The application belongs to the field of medicines, and particularly relates to application of choroid plexus epithelial cells in relieving or treating corneal endothelial diseases.
Technical Field
The corneal endothelial cells are single units of the inner surface of the corneaLamellar cells play a key role in the maintenance of corneal transparency. The proliferation capacity of the adult corneal endothelial cells in vivo is limited, when the adult corneal endothelial cells are affected by factors such as trauma, operation trauma, medicines or malnutrition, irreversible damage or loss of the corneal endothelial cells can be caused, and the damaged area can only depend on expansion and migration of peripheral healthy endothelial cells for damage repair. When the endothelial cell number has fallen to a critical value (about 400-500 pieces/mm 2 ) The following will cause decompensation of corneal endothelial cell function, which is manifested as progressive corneal edema and turbidity, and seriously jeopardizes the visual quality of patients. At present, the most effective method for clinically treating corneal endothelial dysfunction is corneal transplantation surgery, but the popularization and application of the surgical formula are obviously limited due to the lack of corneal donors in the global scope.
In order to solve the problems, the construction of tissue engineering cornea endothelium to replace donor cornea becomes the mainstream of current research, and has great clinical application prospect. The selection of seed cells is a core element of the technology, and the main substitute seed cells at present comprise cultured primary cornea endothelial cells, adult stem cells, embryonic stem cells, cornea endothelial-like cells derived from induced pluripotent stem cells and the like, but the cells still have certain limitations in application. Human primary corneal endothelial cells cannot be separated from the dependence on high-quality donor cornea, and the cells are easy to change fibrosis in vitro culture and cannot be greatly amplified. The cornea endothelial-like substitute cells are widely available, but a standard directional differentiation method is not available, and the identification and purification of the cells are difficult, so that the safety and effectiveness of the treatment still need to be further studied. Therefore, the research of ideal cornea endothelial to replace seed cells to realize long-term transparency of cornea and wide clinical application are still the research bottleneck of tissue engineering cornea endothelium.
Disclosure of Invention
In one aspect of the application, there is provided the use of choroidal plexus epithelial cells for corneal endothelial replacement cells; in another aspect, the application provides the use of choroid plexus epithelial cells in the manufacture of a medicament or medical device for alleviating or treating corneal endothelial injury, corneal endothelial lesions, corneal endothelial dysfunction, corneal endothelial cell dysfunction; further provided is the use of choroidal epithelial cells in the preparation of a medicament or medical device for alleviating or treating corneal thickness abnormalities, reduced corneal transparency, corneal edema, reduced or lost vision, dry eyes, pain.
In the present application, choroid plexus epithelial cells are used as the replacement seed cells for corneal endothelial cells.
Alternatively, the choroid plexus epithelial cells are selected from the group consisting of choroid plexus epithelial cell lines, primary vein plexus epithelial cells, or choroid plexus epithelial cells from which stem cells differentiate.
In the present application, choroid plexus epithelial cells are administered to the eye of a subject suffering from dysfunction of corneal endothelial cells in an effective amount to alleviate or treat symptoms of corneal thickness abnormalities, reduced corneal transparency, corneal edema, reduced or lost vision, dry eyes, pain.
Optionally, the drug is a cell suspension or an injection; the medical instrument is a cell sheet, a tissue engineering plant sheet or a kit. The application can be proportioned by a conventional method by a technician before application, and finally mixed into a required pharmaceutical dosage form. Can be dripping pill, water suspension, water solution, eye drop, injection, colloid solution, nanometer preparation, cell sheet, tissue engineering implant or other forms for human or animal.
Alternatively, the choroid plexus epithelial cells are applied to the patient's eye in the form of tissue engineering implants having a thickness of 50-200 μm and a suprachoroid plexus epithelial cell density of 500/mm 2 -2000/mm 2 。
Alternatively, the choroid plexus epithelial cells are administered to the patient's eye in the form of a cell suspension injection in which the number of choroid plexus epithelial cells is 2×10 4 - 8× 10 5 And each. Optionally, the cell suspension further comprises DMEM low sugar medium and Y-27632.
As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.
In the present application, the term "individual" or "patient" is used interchangeably herein and refers to a vertebrate, preferably a mammal. The mammal may be a human, non-human primate, mouse, rat, rabbit, dog, cat, horse, or cow, but is not limited to these examples. Preferably, the patient is a human. Such "individuals" or "patients" typically suffer from or are susceptible to a condition that can be prevented or treated by administration of the above-described drugs, medical devices of the present application, including, but not limited to, corneal endothelial injury, corneal endothelial lesions, corneal endothelial dysfunction, corneal endothelial cell dysfunction symptoms: abnormal corneal thickness, reduced corneal transparency, corneal edema, reduced or lost vision, dry eyes, pain, etc.
In the present application, the terms "effective amount", "effective dose" refer to that amount which imparts a therapeutic or inhibitory effect (e.g., controls, relieves, improves, mitigates or slows progression) to "an individual" or "patient"; or the aforementioned drugs, medical devices that prevent (e.g., delay onset or reduce risk of developing) a disease, disorder or condition or symptoms thereof. The amount effective for this use will depend on, for example, the route of delivery, the particular cell number or cell activity employed, the severity of the corneal endothelial injury, the individual's weight and general health, and the experience's advice from one situation to another. The dose may be administered once a week, or for two days or once a day, or even several times a day. Dosage units may be administered for a short period (e.g., weeks to months) or longer period (months to years). Can be made into any preparation formulation.
Advantageous effects
The application firstly brings human choroid plexus epithelial cells into the field of cornea endothelial treatment, and the provided cell suspension and the preparation method thereof can ensure the activity of transplanted cells, and can effectively replace cornea endothelial cells to restore cornea thickness and maintain cornea transparency.
Drawings
FIG. 1, (A) morphological observations of human choroid plexus epithelial cells (HCPEPI); (B) Immunofluorescent staining of human choroid plexus epithelial cells (hcp epi).
Fig. 2, OCT images of anterior ocular segment of human choroid plexus epithelial cells (hcp epi) taken 1 day, 14 days, 30 days after implantation.
FIG. 3, photographs of anterior ocular segment of a slit lamp at 1 day and 14 days after mechanical scraping of rabbit corneal endothelial cells.
FIG. 4, (A) morphology of rabbit corneal endothelial cells; (B) Slit lamp eye anterior segment photographic images 1 day and 14 days after rabbit corneal endothelial cell transplantation.
Detailed Description
The following examples, which are given by way of illustration of the application, are given by way of illustration only and should not be construed to limit the scope of the application. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods, conditions, materials, or materials similar or equivalent to those disclosed herein can be used in the practice of the present application, the preferred methods, conditions, materials, or materials are described herein. In the present application, the term "comprising" is synonymous with "including". The terms "comprising," "including," "having," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not specific to the manufacturer and are all conventional products available through conventional channels.
The material sources are as follows:
human choroid plexus epithelial cells: purchased from Shanghai Bayer Biotechnology Co.Ltd
New Zealand white rabbits: purchased from the Jinan Kaolin horn breeding center.
EXAMPLE 1 treatment of human choroid plexus epithelial cells corneal endothelial cell function decompensation
1. In vitro culture of human choroid plexus epithelial cells
Human chorionic epithelial cells (HCPEPI) were obtained from Shanghai Bayer Biotechnology Co., ltd. By adding 10% fetal bovine serum and penicillin-streptomycin to conventional DMEM medium, and placing at 37deg.C and 5% CO 2 Amplification culture is performed in the environment.
Animal experiment
The corneal endothelial cells within the diameter range of 7.0mm in the center phi of the right eye cornea of the experimental rabbit are gently scraped by a 20G soft silica gel needle, an elastic layer after injury is avoided, and a rabbit corneal endothelial cell function decompensation model is established. Will be 3X 10 5 Human choroid plexus epithelial cells were resuspended in 250 μl DMEM low sugar medium, 10 μ M Y-27632 mixed solution and injected with anterior chamber cells using a 2mL syringe, and the experimental rabbits were kept in right lateral position for 3h, ensuring that the transplanted cells were attached to the surface of the posterior elastic layer of the cornea. And (5) observing the recovery condition of corneal edema of the experimental rabbit after operation.
Results
As shown in fig. 1, the human choroid plexus epithelial cells (hcp epi) were uniformly sized in morphology and arranged in a paving stone-like manner under an inverted phase contrast microscope; cell immunofluorescent staining showed that human choroid plexus epithelial cells (hcpeppi) expressed the functional markers ZO1, ATP1A1 and the epithelial marker E-cadherein.
As shown in fig. 2, human choroid plexus epithelial cells (hcp epi) were left as corneal edema and turbidity and iris display 1 day after anterior chamber transplantation; at 14 days post-surgery, the central cornea of the animal model without transplanted cells was continuously turbid (comparative example 1), the cornea receiving the transplantation of human choroid plexus epithelial cells had been completely restored to be transparent, and the cornea thickness had been restored to the normal cornea thickness, with an effect similar to that of rabbit autologous cornea endothelial cells (comparative example 2); the normal transparency and thickness of rabbit cornea, which received human choroid plexus epithelial cell (hcp epi) transplantation, was maintained continuously 30 days after transplantation.
The results demonstrate that human choroid plexus epithelial cells can be used as corneal endothelial replacement cells to reconstruct the corneal endothelial barrier and pump water functions, eliminate corneal edema, and maintain corneal transparency and normal corneal thickness.
Comparative example 1 rabbit corneal endothelial cells without transplanted cells
The corneal endothelial cells within the range of phi 7.0mm of the right eye cornea of the New Zealand white rabbits are gently scraped by a 20G soft silica gel needle, so that an elastic layer after injury is avoided, a corneal endothelial cell function decompensation model is built, and the cornea repair condition is observed after operation.
As shown in fig. 3, 1 day after mechanical scraping of rabbit corneal endothelial cells, corneal edema, corneal haze and iris and pupil were generated in the center of rabbit cornea; without any treatment, the central region of the cornea remained white edematous, turbid and the central pupil was not clear 14 days after surgery. It was suggested that, without any treatment, central corneal endothelial cells were scraped off, and central cornea continued to be cloudy with edema within 14 days post-surgery, affecting normal visual function.
Comparative example 2 autologous corneal endothelial cell transplantation
1. In vitro culture and amplification of rabbit primary corneal endothelial cells
Descemet's membrane and corneal endothelial cells were isolated under a microscope and cultured overnight in DMEM supplemented with 10. Mu. M Y-27632. Corneal endothelial cells were digested with collagenase I at 0.6U/mL for 1 hour, and collected by centrifugation. Inoculated into complete medium (DMEM medium, 10% fetal bovine serum, 1% penicillin-streptomycin, 2ng/mL human basic fibroblast growth factor, 1% insulin-transferrin, 1. Mu.M SB431542 and 10. Mu. M Y-27632) at 37℃with 5% CO 2 And (5) culturing.
Animal experiment
The endothelial cells of the inner cornea within the range of phi 7.0mm of the right eye cornea of the New Zealand white rabbit are gently scraped by a 20G soft silica gel needle, so that an elastic layer after injury is avoided, and a rabbit cornea endothelial cell function decompensation model is established. Rabbit corneal endothelial cells (3×10) 5 Cell/cell, resuspended in DMEM low sugar medium, 10 μ M Y-27632 mix) was injected with anterior chamber cells using a 2mL syringe, and the experimental rabbits were kept in the right lateral recumbent position for 3 hours, ensuring that the cells were attached to the surface of the posterior elastic layer of the cornea. And (5) observing the recovery condition of corneal edema of the experimental rabbit after operation.
Results
As shown in fig. 4A, rabbit corneal endothelial cells were uniform in morphology and size, regular polygonal, and tightly connected to each other under an inverted phase contrast microscope.
As shown in fig. 4B, when rabbit corneal endothelial cells were injected via the anterior chamber for 1 day, the central cornea remained edematous and cloudy, with an unclear iris; 14 days after implantation, the cornea had completely recovered transparency and the cornea thickness had recovered to normal cornea thickness and normal visual function. Consistent with literature reports, transplanted rabbit corneal endothelial cells can reconstruct corneal endothelial function, eliminate corneal edema and restore normal corneal transparency and corneal thickness.
The foregoing has outlined rather broadly the more detailed description of embodiments of the application in order that the detailed description of the principles and embodiments of the application may be implemented in conjunction with the detailed description of embodiments of the application that follows. Meanwhile, based on the idea of the present application, those skilled in the art can make changes or modifications on the specific embodiments and application scope of the present application, which belong to the protection scope of the present application. In view of the foregoing, this description should not be construed as limiting the application.
Claims (10)
1. Use of choroid plexus epithelial cells for corneal endothelial replacement cells.
2. Use of choroid plexus epithelial cells for the preparation of a medicament or medical device for alleviating or treating corneal endothelial injury, corneal endothelial lesions, corneal endothelial dysfunction, corneal endothelial cell dysfunction, wherein the choroid plexus epithelial cells are used in place of corneal endothelial cells.
3. The use according to claim 1 or 2, wherein the choroid plexus epithelial cells are selected from the group consisting of choroid plexus epithelial cell lines, primary vein plexus epithelial cells, and choroid plexus epithelial cells from which stem cells differentiate.
4. The use of claim 1 or 2, wherein the choroidal epithelial cells are administered to the eye of a subject with decompensation of corneal endothelial cell function in an effective amount to alleviate or treat symptoms of corneal thickness abnormalities, reduced corneal transparency, corneal oedema, reduced or lost vision, dry eyes, pain.
5. The use according to claim 2, wherein the choroid plexus epithelial cells are administered to the eye of the patient in the form of a tissue engineering implant having a thickness of 50-200 μm and a suprachoroid plexus epithelial cell density of 500/mm 2 -2000/mm 2 。
6. The use according to claim 2, wherein the choroid plexus epithelial cells are administered to the eye of the patient in the form of a cell suspension injection, the number of choroid plexus epithelial cells in the cell suspension being 2 x 10 4 - 8× 10 5 And each.
7. The use of claim 6, wherein the cell suspension further comprises DMEM low sugar medium and Y-27632.
8. The use according to claim 1 or 2, wherein the medicament is a cell suspension or an injection; the medical instrument is a cell sheet, a tissue engineering plant sheet or a kit.
9. The use according to claim 1 or 2, wherein the subject is a mammal.
10. The use of claim 9, wherein the subject is a human, rabbit, mouse, cat, or dog.
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