CN116999466A - Application of neural crest cells in preparation of medicines or medical instruments for relieving or treating trabecular meshwork abnormality diseases - Google Patents
Application of neural crest cells in preparation of medicines or medical instruments for relieving or treating trabecular meshwork abnormality diseases Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
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Abstract
The invention provides an application of neural crest cells in preparing a medicament for relieving or treating trabecular meshwork abnormality, and belongs to the technical field of biological medicines. In the invention, the neural crest cells are used for replacing trabecular meshwork cells, so that trabecular meshwork structure, quantity and function abnormality of glaucoma patients can be relieved or treated, normal intraocular pressure is maintained, eye pain, dryness, vision decline or loss, cornea transparency decline and other symptoms are relieved, visual functions are recovered, and a new seed cell and a new treatment strategy are provided for clinical treatment of trabecular meshwork abnormality.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of neural crest cells in preparation of medicines or medical instruments for relieving or treating trabecular meshwork abnormality diseases.
Background
Trabecular meshwork cells are the main components of the traditional aqueous humor outflow pathway, and have very important significance in maintaining the normal drainage process of aqueous humor. The pathological elevated intraocular pressure caused by the decrease in trabecular meshwork cell number or dysfunction is closely related to the occurrence and development of glaucoma disease. Ocular hypertension can cause mechanical strain, stress, and vascular damage to the optic nerve, ultimately leading to irreversible vision loss. At present, the main methods for reducing intraocular pressure and preventing or slowing down further vision loss are drug, laser and operation treatment, but none of the above treatment modes can fundamentally solve the problems of trabecular meshwork cell function loss and degeneration.
In order to solve the problems, stem cells replace trabecular meshwork cells, which are the main stream of current researches, have great clinical application prospect. The selection of seed cells is a core element of the technology, and the primary replacement seed cells at the present stage comprise cultured human primary small Liang Wanggan cells, adult stem cells, embryonic stem cells, trabecular meshwork-like cells derived from induced pluripotent stem cells and the like, but the cells still have certain limitations in application. Most primary open angle glaucoma patients have had their own small Liang Wanggan cells dysfunctional or depleted, and it is difficult to meet their own therapeutic needs. Trabecular meshwork-like surrogate cells are widely available, but lack standard methods of directed differentiation, and have difficulty in cell identification and purification, and further research is still needed for therapeutic safety and efficacy. Therefore, developing an ideal trabecular meshwork to replace seed cells to achieve long-term maintenance of ocular pressure stability and promote wide clinical application remains a research bottleneck in the field of stem cell therapy for glaucoma.
Disclosure of Invention
The invention aims to provide an application of neural crest cells in preparing medicines for relieving or treating trabecular meshwork abnormal diseases, wherein the neural crest cells can be used as trabecular meshwork to replace seed cells for relieving or treating the trabecular meshwork abnormal diseases such as glaucoma.
The invention provides application of neural crest cells in preparing medicines or medical instruments for relieving or treating trabecular meshwork abnormality diseases.
Preferably, the trabecular meshwork disorder includes glaucoma.
Preferably, said alleviating or treating a trabecular meshwork abnormality comprises one or more of the following aspects 1) to 5):
1) Relieving or treating elevated intraocular pressure in individuals with trabecular meshwork abnormality;
2) Relieving or treating eye pain in individuals with trabecular meshwork disorder;
3) Relieving or treating vision decline or loss of individuals with trabecular meshwork abnormality;
4) Relieving or treating the dry eyes of individuals with abnormal trabecular meshwork diseases;
5) Alleviating or treating the decrease in corneal transparency in individuals with trabecular meshwork disorder.
Preferably, the individual comprises a mammal; the mammal preferably comprises a human, rabbit, mouse, cat or dog.
Preferably, the trabecular meshwork abnormality includes: trabecular meshwork cell structural lesions, a reduction in trabecular meshwork cell numbers, and impairment of trabecular meshwork function.
Preferably, the medicament or medical device is a unit dose formulation; the unit dose formulation comprises 1X 10 4 ～1×10 7 And (3) neural crest cells.
Preferably, the dosage form of the medicament comprises cell suspension, dripping pill, aqueous suspension, aqueous solution, eye drop, injection, colloid solution or nano preparation; the medical apparatus is a cell sheet or a kit.
Preferably, the cell suspension further comprises DMEM low-sugar medium and Y-27632.
The invention also provides application of the neural crest cells in preparing alternative seed cells of trabecular meshwork cells.
Preferably, the neural crest cells comprise neural crest cell lines, human primary neural crest cells or neural crest cells derived from stem cells induced differentiation.
The invention provides application of neural crest cells in preparing medicines or medical instruments for relieving or treating trabecular meshwork abnormality diseases. In the invention, the neural crest cells are used for replacing trabecular meshwork cells, so that trabecular meshwork structure, quantity and function abnormality of glaucoma patients can be relieved or treated, normal intraocular pressure is maintained, symptoms such as dry eyes, pain, vision decline or loss, cornea transparency decline and the like are relieved, visual functions are recovered, and a new seed cell and a new treatment strategy are provided for clinical treatment of trabecular meshwork abnormality.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a human embryonic stem cell-derived neural crest cell; wherein, A is the morphological expression of neural crest cells; b is immunofluorescence staining of neural crest cells;
FIG. 2 shows cell differentiation and maturation following neural crest cell transplantation; wherein A is immunofluorescence staining of 7-day rabbit trabecular meshwork tissue after cell transplantation, and B is immunofluorescence staining of 14-day rabbit trabecular meshwork tissue after cell transplantation;
fig. 3 is an evaluation of effects 1 day, 7 days, and 14 days after neural crest cell transplantation, wherein a is intraocular pressure, B is corneal thickness, C is slit lamp anterior ocular segment photograph, and D is corneal OCT image.
Detailed Description
The invention provides application of neural crest cells in preparing medicines or medical instruments for relieving or treating trabecular meshwork abnormality diseases.
In the present invention, the neural crest cells are used to replace trabecular meshwork cells; the neural crest cells can relieve or reconstruct trabecular meshwork dysfunction of glaucoma patients, further maintain normal intraocular pressure, relieve eye pain, dryness, vision decline or loss, cornea transparency decline and other symptoms, recover visual functions, and provide new seed cells and new treatment strategies for clinical treatment of the trabecular meshwork dysfunction.
In the invention, the neural crest cells are applied to the eyes of individuals with abnormal trabecular meshwork in an effective dose to relieve or treat symptoms such as elevated intraocular pressure, eye pain, vision loss or loss, dry eyes, reduced cornea transparency and the like.
In the present invention, the trabecular meshwork abnormality preferably includes primary or secondary glaucoma and/or ocular hypertension.
In the present invention, the relief or treatment of abnormal trabecular meshwork disease preferably includes one or more of the following aspects 1) to 5):
1) Relieving or treating elevated intraocular pressure in individuals with trabecular meshwork abnormality;
2) Relieving or treating eye pain in individuals with trabecular meshwork disorder;
3) Relieving or treating vision decline or loss of individuals with trabecular meshwork abnormality;
4) Relieving or treating the dry eyes of individuals with abnormal trabecular meshwork diseases;
5) Alleviating or treating the decrease in corneal transparency in individuals with trabecular meshwork disorder.
In the present invention, the individual preferably includes a mammal; the mammal preferably comprises a human, rabbit, mouse, cat or dog.
In the present invention, the trabecular meshwork abnormality preferably includes: one or more of trabecular meshwork cell structural lesions, reduced trabecular meshwork cell numbers, and trabecular meshwork functional lesions; the trabecular meshwork dysfunction is equivalent to trabecular meshwork dysfunction or trabecular meshwork dysfunction.
In the present invention, the drug or medical device is preferably a unit dose formulation; the unit dose formulation preferably comprises 1X 10 4 ～1×10 7 And (3) neural crest cells.
In the present invention, the dosage form of the drug preferably includes a cell suspension, a dripping pill, an aqueous suspension, an aqueous solution, an eye drop, an injection, a colloid, a colloidal solution or a nano-preparation; the medical device is preferably a cell sheet or a kit. The medicaments can be proportioned by a conventional method by a technician before administration, and finally mixed into a required medicament form.
In the present invention, the neural crest cells are administered to the eyes of a human or animal body in the form of cell suspension injection.
In the present invention, the cell suspension preferably further comprises DMEM low sugar medium and Y-27632. In the present invention, the cell suspension is based on DMEM low sugar medium, comprising neural crest cells and Y-27632; the concentration of Y-27632 in the cell suspension is preferably 1-20 mu M; y-27632 is a ROCK inhibitor for reducing apoptosis in the process of cell transplantation; the concentration of neural crest cells in the cell suspension is preferably 1.0X10 4 -1.0×10 7 2-200 mu L.
The invention also provides application of the neural crest cells in preparing alternative seed cells of trabecular meshwork cells.
In the present invention, the neural crest cells preferably include neural crest cell lines, human primary neural crest cells, or neural crest cells derived from stem cells induced differentiation.
In the present invention, the stem cells preferably include pluripotent stem cells, more preferably include human pluripotent stem cells; the human pluripotent stem cells are preferably suitably sized human embryonic stem cells; the human embryonic stem cells with the proper size are that each human embryonic stem cell clone contains about 60-100 cells under an inverted phase-contrast microscope, and the clone size is uniform and the density is moderate.
In the present invention, the neural crest cells derived from the stem cell induced differentiation are preferably prepared by the following method:
culturing human pluripotent stem cells to the proper size, and then culturing the cells by using a neural crest induction differentiation medium containing all-trans retinoic acid (all trans retinoic acid, RA) for induction culture; the time of the induction culture is preferably 5 days to obtain neural crest cells. Neural crest cell digestion and counting are ready for use.
In the invention, the neural crest induction differentiation medium containing RA is preferably based on a knockout DMEM/F12 medium, and further comprises the following components with the following concentration: beta-mercaptoethanol 0.1mM, glutamine 0.1mM, bFGF 4-8ng/ml, NEAA 0.1mM, KSR at a volume concentration of 10-20% and RA 0.5-2. Mu.M.
After induction culture, the human pluripotent stem cells are cloned and expanded under the microscope and connected, the cell volume is increased, the cell morphology is changed from round or oval shape into polygonal shape, the arrangement is compact, and the nuclear-cytoplasmic ratio is reduced.
In the present invention, the cell suspension is preferably prepared by the following method:
digesting the neural crest cells derived from the human pluripotent stem cells obtained by the scheme to obtain a mixed solution containing cells;
mixing the mixed solution containing the cells with a low-sugar culture medium, performing cell counting and centrifugation, discarding the supernatant, and collecting cell sediment;
will be 1.0X10 4 ～1.0×1 7 The cell sediment with the cell density is suspended in 2-200 mu L of DMEM low-sugar culture medium, and Y-27632 with the final concentration of 1-20 mu M is added at the same time;
the preferred digestive enzyme for the digestion is Actutase digestive enzyme; the consumption of the Ackutase digestive enzyme is preferably 1-3 ml in a culture dish with the thickness of 60 mm; the temperature of the digestion is preferably 37 ℃; the digestion time is preferably 5 to 15 minutes, more preferably 10 minutes, based on the cells in a dispersed state.
In the present invention, the neural crest cells or the cell suspension containing the neural crest cells are used for anterior chamber cell injection transplantation.
The invention applies neural crest cells to the field of glaucoma/trabecular meshwork abnormality treatment for the first time, and the provided cell suspension and the preparation method thereof can effectively maintain the normal structure of trabecular meshwork, stabilize normal intraocular pressure and balance of aqueous humor outflow while guaranteeing the activity of transplanted cells.
As used in this specification, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
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 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 invention, including, but not limited to, lesions of trabecular meshwork cell structure, reduced trabecular meshwork cell numbers, and symptoms of trabecular meshwork dysfunction: elevated ocular pressure, ocular pain, reduced or lost vision, dry eyes, reduced corneal transparency, and the like.
In the present invention, 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.
For further explanation of the present invention, the application of neural crest cells provided by the present invention in preparing a medicine or a medical device for alleviating or treating abnormal trabecular meshwork diseases is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
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 embryonic stem cell line H1: the subject group benefit is given by the first affiliated hospital of army medical university's ophthalmic yin-force professor.
New Zealand white rabbits: purchased from the Jinan Kaolin horn breeding center.
EXAMPLE 1 application of human embryonic Stem cell-derived neural crest cells to trabecular meshwork tissue remodeling
1. In vitro neural crest cell culture expansion
Human embryonic stem cell line H1 was from the task group of eye-professor in the first affiliated hospital of army university of medical science, mTESR1 was used TM After culturing the stem cell culture medium for about 4 days to about 30% fusion, the human embryonic stem cell culture medium mTESR1 is used for culturing the stem cells TM Changing to neural crest induction differentiation medium (knockout DMEM/F12 as basic medium, and also composed of the following concentration components of beta-mercaptoethanol 0.1mM, glutamine 0.1mM, bFGF 4ng/ml, NEAA 0.1mM, KSR with volume concentration of 20% and 1 μM RA), changing liquid every day, adding 1 μM retinoic acid for inducing neural crest cells when changing liquid, continuously inducing for 5 days, and inducing cells to differentiate into neural crest cells after 5 days.
2. Animal experiment
Healthy, male, 6 month old New Zealand white rabbits were selected as subjects. The skeletal muscle relaxant ketamine hydrochloride (40 mg/kg) was intramuscular injected into New Zealand white rabbits, and then the eye surface of the rabbits was added dropwise with the eye surface preparation, promecaine hydrochloride eye drops, 1×10 6 The neural crest cells derived from the individual embryonic stem cells are resuspended in 100. Mu.l of DMEM low-sugar medium and 10. Mu.MY-27632 mixed solution, and the anterior chamber cells are injected by using a 2mL syringe, and no liquid exudation after injection indicates that the transplantation is successful. After the anterior chamber injection is finished, the experimental rabbits are put back into a rearing cage, and the action of the skeletal muscle relaxant is gradually recovered after the drug effect of the skeletal muscle relaxant is finished. Post-operative detection of transplanted cell localization and observation of experimental rabbits for ocular pressure, corneal transparency, and central corneal thickness.
3. Results
As shown in FIG. 1, the morphology of neural crest cells derived from human embryonic stem cells is polygonal and the cells are closely arranged under an inverted phase contrast microscope. Cell immunofluorescent staining showed that differentiated neural crest cells expressed well the typical neural crest cell markers PITX2, AP-2. Beta. And HNK-1.
As shown in fig. 2, human embryonic stem cell-derived neural crest cells that positively expressed human specific MTCO2 were located in the trabecular meshwork region of rabbits 7 days and 14 days after the transplantation of neural crest cells through the anterior chamber, while the trabecular meshwork cell markers AQP1, CHI3L1 and TIMP3 were positively expressed.
As shown in fig. 3, in early stage of neural crest cell transplantation, there was slight fluctuation in intraocular pressure in the experimental group, but there was no significant difference (P > 0.05) in intraocular pressure value at each time point compared with the control group, and the intraocular pressure was substantially stabilized between 10 and 15 mmHg. Slight edema of rabbit cornea can be seen 1 day after cell transplantation, a small amount of transplanted cells are adhered to the pupil edge, then the rabbit cornea is gradually transparent, and the cornea edema is obviously relieved; cell adhesion at the pupil margin of the experimental group rabbits was substantially lost 14 days after cell transplantation. OCT detection results meet the change trend, and the central cornea thickness of the rabbits in the experimental group is slightly increased 1 day after cell transplantation compared with that of the rabbits in the normal control group, but the central cornea thickness of the rabbits in the experimental group is normal 7 days and 14 days after cell transplantation.
The results prove that the neural crest cells derived from the human embryonic stem cells transplanted by the anterior chamber injection mode can be positioned to the trabecular meshwork area and further differentiated into the trabecular meshwork cells, the transplanted cells are well integrated with the original trabecular meshwork tissue, and the normal intraocular pressure and the aqueous humor outflow balance of the transplanted rabbits are jointly maintained; meanwhile, the transplanted neural crest cells do not have adverse effects on other structures and functions of the anterior chamber, and the cornea transparency and the central cornea thickness are basically normal. Therefore, the neural crest cells can be used as the sources of the trabecular meshwork cells to replace seed cells for treating diseases such as glaucoma caused by trabecular meshwork abnormality.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The application of neural crest cells in preparing medicaments or medical instruments for relieving or treating trabecular meshwork abnormality diseases.
2. The use according to claim 1, wherein the trabecular meshwork disorder comprises glaucoma.
3. The use according to claim 1, wherein said alleviation or treatment of trabecular meshwork abnormality comprises one or more of the following aspects 1) to 5):
1) Relieving or treating elevated intraocular pressure in individuals with trabecular meshwork abnormality;
2) Relieving or treating eye pain in individuals with trabecular meshwork disorder;
3) Relieving or treating vision decline or loss of individuals with trabecular meshwork abnormality;
4) Relieving or treating the dry eyes of individuals with abnormal trabecular meshwork diseases;
5) Alleviating or treating the decrease in corneal transparency in individuals with trabecular meshwork disorder.
4. The use of claim 3, wherein the individual comprises a mammal; the mammal preferably comprises a human, rabbit, mouse, cat or dog.
5. The use of claim 1, wherein the trabecular meshwork abnormality comprises: trabecular meshwork cell structural lesions, a reduction in trabecular meshwork cell numbers, and impairment of trabecular meshwork function.
6. The use according to claim 1, wherein the medicament or medical device is a unit dose formulation; the unit dose formulation comprises 1X 10 4 ～1×10 7 And (3) neural crest cells.
7. The use according to claim 1, wherein the pharmaceutical dosage form comprises a cell suspension, a drop pill, an aqueous suspension, an aqueous solution, an eye drop, an injection, a colloid, a colloidal solution or a nano-preparation; the medical apparatus is a cell sheet or a kit.
8. The use according to claim 7, wherein the cell suspension further comprises DMEM low sugar medium and Y-27632.
9. Use of neural crest cells in the preparation of replacement seed cells for trabecular meshwork cells.
10. The use according to any one of claims 1 to 9, wherein said neural crest cells comprise neural crest cell lines, human primary neural crest cells or neural crest cells derived from stem cells induced differentiation.
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