Application of choroid plexus epithelial cells in replacing corneal endothelial cells
By using choroidal plexus epithelial cells as alternative seeds for corneal endothelial cells, the problem of insufficient donor and cell stability in the decompensated treatment of corneal endothelial cells was solved, and the recovery of corneal transparency and thickness was achieved, significantly improving the patient's vision .
Patent Information
- Application Number
- CN202310527518.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-11
- Publication Date
- 2025-01-03
- Estimated Expiration
- 2043-05-11
AI Technical Summary
The prior art has problems such as insufficient donor, cell fibrosis, difficulty in directed differentiation and failure to meet the safety and effectiveness of the treatment in treating corneal endothelial cells, resulting in decreased corneal transparency and visual impairment.
Choroid plexus epithelial cells are used as alternative seed cells for corneal endothelial cells and are applied to the patient's eyes through cell suspension or tissue engineering transplantation. They are used to alleviate or treat corneal endothelial injury and dysfunction and restore corneal transparency and thickness.
Effectively replace corneal endothelial cells, restore corneal transparency and thickness, significantly alleviate corneal edema and vision loss, and improve the healthy state of the cornea.
Smart Images
Figure CN116672366B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medicine, and in particular relates to the application of choroid plexus epithelial cells in alleviating or treating corneal endothelial diseases. Background Art
[0002] Corneal endothelial cells are a single layer of cells on the inner surface of the cornea, and they play a key role in maintaining corneal transparency. Adult corneal endothelial cells have limited proliferation capacity in vivo. When affected by trauma, surgical trauma, drugs or malnutrition, they can cause irreversible damage or loss of corneal endothelial cells. The damaged area can only rely on the expansion and migration of surrounding healthy endothelial cells for damage repair. When the number of endothelial cells drops to a critical value (about 400-500 / mm 2 ) The following will cause corneal endothelial cell decompensation, manifested as progressive corneal edema and opacity, seriously endangering the patient's visual quality. At present, the most effective method for clinical treatment of corneal endothelial decompensation is corneal transplantation, but due to the global shortage of corneal donors, the promotion and application of this procedure is significantly limited.
[0003] In order to solve the above problems, the construction of tissue-engineered corneal endothelium to replace the donor cornea has become the mainstream of current research and has great prospects for clinical application. The selection of seed cells is the core element of this technology. The main replacement seed cells currently include cultured human primary corneal endothelial cells, adult stem cells, embryonic stem cells, and corneal endothelial-like cells derived from induced pluripotent stem cells, but these cells still have certain limitations in application. Human primary corneal endothelial cells cannot be separated from the dependence on high-quality donor corneas, and cells are prone to fibrosis changes in vitro culture and cannot be expanded in large quantities. Corneal endothelial-like replacement cells are widely available, but there is still a lack of standard directed differentiation methods, and cell identification and purification are difficult. The safety and effectiveness of treatment still need further research. Therefore, the study of ideal corneal endothelial replacement seed cells to achieve long-term corneal transparency and widespread clinical application is still a bottleneck in the research of tissue-engineered corneal endothelium. Summary of the invention
[0004] On the one hand, the present invention provides the use of choroid plexus epithelial cells for corneal endothelial replacement cells; on the other hand, the present invention provides the use of choroid plexus epithelial cells for preparing drugs or medical devices for relieving or treating corneal endothelial damage, corneal endothelial lesions, corneal endothelial dysfunction, and corneal endothelial cell decompensation; further provides the use of choroid plexus epithelial cells for preparing drugs or medical devices for relieving or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or loss of vision, dry eyes, and pain.
[0005] In the present invention, choroid plexus epithelial cells are used as substitute seed cells for corneal endothelial cells.
[0006] Optionally, the choroid plexus epithelial cells are selected from choroid plexus epithelial cell lines, primary choroid plexus epithelial cells or choroid plexus epithelial cells derived from stem cell differentiation.
[0007] In the present invention, choroid plexus epithelial cells are administered to the eyes of individuals with corneal endothelial cell decompensation at an effective dose to alleviate or treat symptoms of abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or lost vision, dry eyes, and pain.
[0008] Optionally, the drug is a cell suspension or an injection; the medical device is a cell sheet, a tissue engineering implant or a kit. The present invention can be mixed by a technician using conventional methods before administration, and finally mixed into the required drug dosage form. It can be a pill, an aqueous suspension, an aqueous solution, an eye drop, an injection, a colloid, a colloidal solution, a nano preparation, a cell sheet, a tissue engineering implant or other dosage forms for human or animal use.
[0009] Optionally, the choroid plexus epithelial cells are applied to the patient's eye in the form of a tissue engineering graft, wherein the graft thickness is 50-200 μm and the density of the choroid plexus epithelial cells on the graft is 500 cells / mm 2 -2000pcs / mm 2 .
[0010] Optionally, the choroid plexus epithelial cells are administered to the patient's eye in the form of a cell suspension injection, wherein the number of choroid plexus epithelial cells in the cell suspension is 2×10 4 - 8× 10 5 Optionally, the cell suspension further comprises DMEM low-glucose medium and Y-27632.
[0011] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
[0012] In the present invention, 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 cattle, but is not limited to these examples. Preferably, the patient is a human. Such an "individual" or "patient" typically suffers from or is susceptible to a condition that can be prevented or treated by administering the above-mentioned drugs and medical devices of the present invention, including but not limited to the following corneal endothelial damage, corneal endothelial lesions, corneal endothelial dysfunction, corneal endothelial cell decompensation symptoms: abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or lost vision, dry eyes, pain, etc.
[0013] In the present invention, the term "effective amount" or "effective dose" refers to the above-mentioned drugs and medical devices that impart therapeutic or inhibitory effects (e.g., control, alleviate, improve, mitigate or slow down the progress) to "individuals" or "patients"; or prevent (e.g., delay the onset or reduce the risk of development) diseases, disorders or conditions or their symptoms. The amount effective for this purpose will depend on, for example, the route of delivery, the specific number of cells or cell activity adopted, the severity of corneal endothelial damage, the individual's body weight and overall health, and the advice of experienced people according to different situations. The dosage can be given once a week, or once every two days or every day, or even several times a day. The dosage unit can be given in a short period of time (e.g., weeks to months) or longer periods of time (months to years). Any usable formulation can be made.
[0014] Beneficial Effects
[0015] This application introduces human choroid plexus epithelial cells into the field of corneal endothelial treatment for the first time. The provided cell suspension and preparation method can effectively replace corneal endothelial cells to restore corneal thickness and maintain corneal transparency while ensuring the vitality of transplanted cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 , (A) Morphological observation of human choroid plexus epithelial cells (HCPEpi); (B) Immunofluorescence staining of human choroid plexus epithelial cells (HCPEpi).
[0017] Figure 2 , slit lamp photography and anterior segment OCT images 1 day, 14 days, and 30 days after human choroid plexus epithelial cell (HCPEpi) transplantation.
[0018] Figure 3 The anterior segment of the rabbit cornea was photographed under slit lamp 1 day and 14 days after mechanical scraping of corneal endothelial cells.
[0019] Figure 4 , (A) Morphology of rabbit corneal endothelial cells; (B) Slit lamp anterior segment photography images 1 day and 14 days after rabbit corneal endothelial cell transplantation. DETAILED DESCRIPTION
[0020] The following examples further illustrate the present invention, which are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Unless otherwise specified, the technical and scientific terms used herein are the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any method, condition, substance or material similar to or equivalent to that disclosed herein can be used in the implementation of the present invention, preferred methods, conditions, substances or materials are described herein. In the present invention, the term "including" is synonymous with "comprising". The terms "including", "comprising", "having", "containing" or any other variations thereof used herein are intended to cover non-exclusive inclusions. For example, a composition, step, method, product or device comprising the listed elements need not be limited to those elements, but may include other elements not explicitly listed or elements inherent to such a composition, step, method, product or device.
[0021] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through conventional channels.
[0022] Sources:
[0023] Human choroid plexus epithelial cells: purchased from Shanghai Baili Biotechnology Co., Ltd.
[0024] New Zealand White Rabbit: purchased from Xilingjiao Breeding and Propagation Center in Jinan.
[0025] Example 1 Treatment of corneal endothelial cell dysfunction with human choroid plexus epithelial cells
[0026] 1. In vitro culture of human choroid plexus epithelial cells
[0027] Human choroid plexus epithelial cells (HCPEpi) were purchased from Shanghai Baili Biotechnology Co., Ltd. and cultured in a conventional DMEM medium supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37°C and 5% CO2.
[0028] Animal experiments
[0029] A 20G soft silicone needle was used to gently scrape the corneal endothelial cells within a diameter of Φ7.0 mm in the center of the right cornea of the experimental rabbit to avoid damaging the posterior elastic layer, and a rabbit corneal endothelial cell decompensation model was established. 5 Human choroid plexus epithelial cells were resuspended in 250 μL of DMEM low-glucose medium and 10 μM Y-27632 mixture and injected into the anterior chamber using a 2 mL syringe. The rabbits were kept in the right lateral position for 3 h to ensure that the transplanted cells adhered to the surface of the corneal Descemet's membrane. The recovery of corneal edema in the rabbits was observed after surgery.
[0030] result
[0031] like Figure 1 As shown, under an inverted phase contrast microscope, human choroid plexus epithelial cells (HCPEpi) were uniform in size and arranged in a cobblestone-like pattern; cell immunofluorescence staining showed that human choroid plexus epithelial cells (HCPEpi) expressed functional markers ZO1, ATP1A1 and epithelial marker E-cadherin.
[0032] like Figure 2 As shown, one day after human choroid plexus epithelial cells (HCPEpi) were transplanted into the anterior chamber, the cornea was edematous and turbid, and the iris was unclear; 14 days after the operation, the central cornea of the animal model without transplanted cells continued to be turbid (Comparative Example 1), and the cornea transplanted with human choroid plexus epithelial cells had completely recovered its transparency, and the corneal thickness had returned to normal corneal thickness, and the effect was similar to that of rabbit autologous corneal endothelial cells (Comparative Example 2); 30 days after transplantation, the rabbit cornea transplanted with human choroid plexus epithelial cells (HCPEpi) continued to maintain normal transparency and thickness.
[0033] The above results prove that human choroid plexus epithelial cells can be used as corneal endothelial replacement cells to rebuild the corneal endothelial barrier and water pumping function, eliminate corneal edema, and maintain corneal transparency and normal corneal thickness.
[0034] Comparative Example 1 Rabbit corneal endothelial cells without transplanted cells
[0035] A 20G soft silicone needle was used to gently scrape the corneal endothelial cells within the Φ7.0 mm range of the central cornea of the right eye of the experimental New Zealand white rabbit to avoid damaging the posterior Descemet's membrane. A corneal endothelial cell decompensation model was established, and the corneal repair was observed after surgery.
[0036] like Figure 3 As shown in the figure, one day after mechanical scraping of the rabbit corneal endothelial cells, corneal edema and corneal opacity occurred in the central area of the rabbit cornea, and the iris and pupil were not visible; without any treatment, the central area of the cornea still showed white edema and opacity 14 days after surgery, and the central pupil was not clear. This suggests that without any treatment, scraping the central corneal endothelial cells will cause continuous edema and opacity in the central cornea within 14 days after surgery, affecting normal visual function.
[0037] Comparative Example 2 Autologous corneal endothelial cell transplantation
[0038] 1. In vitro culture and expansion of primary rabbit corneal endothelial cells
[0039] Descemet's membrane and corneal endothelial cells were separated under a microscope and cultured overnight in DMEM supplemented with 10 μM Y-27632. Corneal endothelial cells were digested with 0.6U / mL collagenase I for 1 hour and collected by centrifugation. They were inoculated into complete culture medium (DMEM medium, 10% fetal bovine serum, 1% penicillin-streptomycin, 2ng / mL human basic fibroblast growth factor, 1% insulin-transferrin, 1μM SB431542 and 10μM Y-27632) and cultured at 37°C and 5% CO2.
[0040] Animal experiments
[0041] A 20G soft silicone needle was used to gently scrape the corneal endothelial cells within the Φ7.0 mm range of the central cornea of the right eye of the New Zealand white rabbit to avoid damaging the Descemet's membrane, and a rabbit corneal endothelial cell decompensation model was established. 5 The cells were resuspended in a mixture of DMEM low-glucose medium and 10 μM Y-27632) and injected into the anterior chamber using a 2 mL syringe. The rabbits were kept in the right lateral position for 3 hours to ensure that the cells adhered to the surface of the corneal Descemet's membrane. The recovery of corneal edema in the rabbits was observed after surgery.
[0042] result
[0043] like Figure 4 As shown in A, under an inverted phase contrast microscope, the rabbit corneal endothelial cells were uniform in size and shape, regular polygonal, and the cells were closely connected.
[0044] like Figure 4 As shown in Figure B, one day after the rabbit corneal endothelial cells were injected into the anterior chamber, the central cornea was still edematous and turbid, and the iris was unclear; 14 days after transplantation, the cornea had completely recovered its transparency, and the corneal thickness had returned to normal corneal thickness and normal visual function. Consistent with literature reports, transplantation of rabbit corneal endothelial cells can reconstruct corneal endothelial function, eliminate corneal edema, and restore normal corneal transparency and corneal thickness.
[0045] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.
Claims
1. Use of human choroid plexus epithelial cells in the preparation of drugs or medical devices for alleviating or treating corneal endothelial cell decompensation, characterized in that: The human choroid plexus epithelial cells are used to replace corneal endothelial cells.
2. The use according to claim 1, characterized in that The human choroid plexus epithelial cells are selected from choroid plexus epithelial cell lines, primary choroid plexus epithelial cells or choroid plexus epithelial cells derived from stem cell differentiation.
3. The use according to claim 1, characterized in that The human choroid plexus epithelial cells are administered to the eye of an individual with corneal endothelial cell decompensation at an effective dose to alleviate or treat symptoms of abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or lost vision, or dry or painful eyes.
4. The use according to claim 1, characterized in that The human choroid plexus epithelial cells are applied to the individual eye in the form of a tissue engineering implant, the implant thickness is 50-200 μm, and the density of the choroid plexus epithelial cells on the implant is 500 cells / mm 2 -2000pcs / mm 2 .
5. The use according to claim 1, characterized in that The human choroid plexus epithelial cells are administered to the individual eye in the form of a cell suspension injection, wherein the number of choroid plexus epithelial cells in the cell suspension is 2×10 4 -8×10 5 indivual.
6. The use according to claim 5, characterized in that The cell suspension also includes DMEM low-glucose medium and Y-27632.
7. The use according to claim 1, characterized in that The medicine is a cell suspension or an injection; the medical device is a tissue engineering implant or a kit.
8. The use according to claim 4, characterized in that The individual is a mammal.