CN113456675A - Application of acellular SIS in inhibition of scar tissue hyperplasia after glaucoma operation - Google Patents
Application of acellular SIS in inhibition of scar tissue hyperplasia after glaucoma operation Download PDFInfo
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Classifications
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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
- A61K35/37—Digestive system
- A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
-
- 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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- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
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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
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The invention discloses application of acellular SIS in inhibiting postoperative scar tissue hyperplasia of glaucoma, which is prepared by applying the acellular SIS to inhibiting postoperative scar formation of glaucoma filtration, and opens up a new treatment way for solving the problem of scar prevention in the field of ophthalmology and developing prevention and treatment of scar formation of eyes.
Description
Technical Field
The invention relates to application of acellular SIS in inhibiting scar tissue hyperplasia after glaucoma operation, and belongs to the technical field of biology.
Background
Glaucoma is a common disease in China, and accounts for the second place of blindness-causing eye diseases, and the incidence rate of the glaucoma in the population is about 0.21% -1.64%. Currently, the primary treatment of drug-uncontrolled glaucoma remains filtration surgery. The goal of glaucoma filtration surgery is to establish a new aqueous humor drainage pathway to lower intraocular pressure. The excised sclerotic trabecular tissue forms a fistula in the operation, the aqueous humor in the anterior chamber drains to the subconjunctival membrane through the fistula to form a filtering bleb, and then the aqueous humor is absorbed by capillary vessels or lymphatic vessels in the tissues around the filtering bleb so as to relieve pathological drainage obstacle and achieve the purpose of reducing intraocular pressure. The massive proliferation of fibroblasts following surgery, due to the disruption of the blood-aqueous barrier and the post-operative inflammatory response, can lead to fibrosis of the subconjunctival tissue and scarring of the bleb, the most common and leading cause of filtered surgical failure. Investigation has found that the failure rate of glaucoma filtration surgery within 2 years can reach 15-30%. Therefore, inhibiting excessive proliferation of fibroblasts after glaucoma surgery and reducing scar formation of filtering blebs are the key for improving the success rate of surgery and are also a great problem for glaucoma research.
In recent years, various methods have been clinically tried, such as application of drugs for conjunctival flap, transcorneal incision, beta ray irradiation, and postoperative anti-scarring during and after surgery, such as mitomycin C (MMC), 5-fluorouracil (5-FU), bleomycin, doxorubicin, tissue-type fibrinolysin activator (t-PA), cyclosporine a, interferon, and the like, and implantation of biomaterials, such as collagen rods, bio-Gel (SK-Gel), anterior lens capsule, amnion, and the like. The antimetabolic drugs MMC and 5-FU are widely applied clinically, but the drugs have high cytotoxicity, so that the surgical complications caused by the drugs, such as bleb leakage, low intraocular pressure maculopathy, endophthalmitis and the like, are gradually increased, and a new threat is generated to the glaucoma patients with damaged visual functions.
Small Intestinal Submucosa (SIS) is a collagen matrix of about 100 μm thickness obtained by mechanical removal of the mucosal and muscular layers of the porcine small intestine, consisting of highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans. SIS is widely applied to the repair and reconstruction of various tissues such as skin, abdominal wall, bladder, urethra, orbital wall, vagina, periosteum and the like [ 27-35 ] and tissue engineering scaffold materials such as nerves, blood vessels, heart valves, bones, tendons, ligaments, esophagus, skin, meniscus, dura mater, fat and the like. The SIS has wide source, convenient material acquisition, simple preparation process, low price, mass production, easy disinfection and preservation, good biocompatibility and no toxicity, thus having wide application prospect in ophthalmology. Although SIS has been used in ophthalmology, its use for inhibiting ocular scarring has not been reported.
Disclosure of Invention
The invention overcomes the defects of the prior art, provides the application of the acellular SIS in inhibiting the scar tissue hyperplasia after glaucoma operation, prepares the acellular SIS, applies the acellular SIS in inhibiting the scar formation after glaucoma filtration operation, and opens up a new treatment way for solving the scar prevention and treatment problem in the field of ophthalmology and developing the prevention and treatment of the scar formation of eyes.
Application of acellular SIS in preparing a medicine for inhibiting postoperative scar tissue hyperplasia of glaucoma.
Furthermore, in the application, the decellularized SIS can inhibit the growth and proliferation of fibroblasts.
Further, the preparation method of the decellularized SIS comprises the following steps:
taking a fresh proximal jejunum of a healthy adult pig, scraping by a mechanical method to obtain a small intestine submucosa, and carrying out freeze drying treatment after cell removal to obtain the cell-free SIS.
Further, the method for inhibiting the growth and proliferation of the fibroblast by the decellularized SIS comprises the following steps:
1) culturing fibroblast by tissue culture method;
2) and (2) adding the acellular SIS into the fibroblasts obtained in the step 1) to inhibit the growth and proliferation of the fibroblasts.
Has the advantages that:
this application is applied to the scar formation after the suppression glaucoma filtration with acellular SIS for the first time, opens up new thinking for the scar prevention and cure problem that solves the ophthalmology field. Opens up a new treatment way for the development of preventing and treating the scar formation of the eyes. The SIS is applied to inhibiting the proliferation of scar tissues after glaucoma operation, reducing the formation of scar tissues after glaucoma operation and opening up a new method for preventing and treating the scar. The research has important social significance for improving the success rate after glaucoma operation and reducing the blindness treatment rate. Is expected to convert scientific research into products for clinical application, and has considerable social and economic benefits.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.
Example 1
Firstly, experimental steps
1. Preparation of acellular SIS
Taking a fresh proximal jejunum of a healthy adult pig, scraping by a mechanical method to obtain a small intestine submucosa, and carrying out freeze drying treatment after cell removal to obtain the cell-free SIS.
2. Tissue block culture method for culturing Tenon's-free fibroblasts
Excised Tenon sac tissue was immediately placed under sterile conditions in a medium containing 500U/mL penicillin and 500mg/L streptomycin. Taking out the specimen in the superclean bench within 4 h. The specimen is placed in D-hanks liquid containing 100U/mL of penicillin and 100mg/L of streptomycin, the specimen is repeatedly washed by the D-hanks liquid under aseptic condition, and the cleaned tissue is cut into tissue blocks with the size of 3mm multiplied by 2 mm. The tissue blocks were transferred to 50mL flasks and adhered uniformly. Adding 1.5mL of DMEM medium containing 15% fetal calf serum, penicillin 100U/mL, streptomycin 100mg/L and pH7.2-7.4, and sealing the bottle mouth. Inverting the culture flask to allow the tissue mass to be placed on top, placing at 37 deg.C with 5% CO by volume2And culturing for 4 hours in an incubator with saturated humidity, and turning over the culture bottle to soak the tissue blocks in the culture medium. After 2d, 3-5mL of the aforementioned DMEM medium was supplemented, after which the medium was changed 1 time (the aforementioned DMEM medium) 3-4 d. When the fibroblast grows over the bottom of the flask, the cells are digested with 0.25% trypsin and subcultured. And (4) after repeatedly carrying out passage to remove non-fibroblast components, taking 3 rd-5 th-generation fibroblasts for detection.
3. Effect of decellularized SIS on fibroblasts
(1) And (3) observing cell morphology: well-grown cells were selected and seeded at 1 × 10 cells per well on glass slides in 6-well plates.
Two experimental groups were divided: (ii) SIS group: adding 500 μ L of SIS leach liquor, SIS leach liquor: soaking the obtained acellular SIS in the DMEM culture medium to obtain SIS leaching liquor; blank control group: add 500. mu.L of medium to each well (this medium is DMEM as described previously). And taking out the glass sheet after 24h, naturally drying, fixing, staining by hematoxylin-eosin (HE), and observing morphological change under a microscope.
(2) The MTT method is used for determining the proliferation influence of the SIS leaching liquor on the fibroblasts:
taking 3-5 generation fibroblast of logarithmic growth phase, digesting with 0.25% trypsin to obtain single cell suspension, adjusting to 0.5 × 105Each mL, inoculated in a 96-well plastic plate at a density of 0.5X 104Each well was filled with 100. mu.L of the cell suspension and 100. mu.L of a medium (which was the aforementioned DMEM medium), and the stock culture was aspirated after 24 hours of conventional culture.
Two experimental groups were divided: (ii) SIS group: adding 200 μ L of SIS leachate (prepared as described above); blank control group: add 200. mu.L of medium to each well (this medium is DMEM as described previously). Each of the two groups of the porous bodies is provided with 3 multiple pores, and the porous bodies are placed at 37 ℃ and have the volume fraction of 5 percent CO2After the continuous culture for 24h under the saturation humidity, adding 20 mu L of MTT (methyl thiazolyl tetrazolium) and 5mg/mL MTT (methyl thiazolyl tetrazolium) for shaking up, continuously culturing for 4h, then absorbing and removing the supernatant, adding 200 mu L of DMSO, oscillating for 20min, and after the crystal particles are dissolved, measuring the absorbance (A) value by using an enzyme-labeling instrument at the wavelength of 490 nm. The results were recorded and the inhibition of fibroblasts by decellularized SIS was calculated. The growth inhibition rate was (1-SIS group a/blank control group a) × 100%.
(3) Determination of Lactate Dehydrogenase (LDH): taking cells in logarithmic growth phase, and calculating cell content by 0.5 × 105The culture is inoculated in a 96-well culture plate for culture, and the experiment is divided into the following groups: (ii) SIS group: adding 200 μ L of SIS leachate (prepared as described above); blank control group: add 200. mu.L of medium to each well (this medium is DMEM as described previously). Each hole of the two groups is provided with 3 multiple holes. At 37 ℃ with a volume fraction of 5% CO2And after the saturation humidity is continuously cultured for 24 hours, measuring and analyzing by an automatic biochemical analyzer.
Second, experimental results and analysis
1. Effect of decellularized SIS on fibroblast morphology
Under the microscope, the blank control composition fiber cells are normal in shape, typical fusiform, large in kernel and rich in cytoplasm. The cells in the SIS group are disorganized, and the number of the cells is reduced.
2. Effect of decellularized SIS on fibroblast proliferation
The MTT method detection result shows that the difference of the SIS value and the blank control group has statistical significance (P values are all less than 0.05). SIS has inhibitory effect on fibroblast growth and proliferation, and cell proliferation is slowed down.
3. Effect of decellularized SIS on LDH Activity of fibroblasts
Compared with the blank control group, the SIS group has no statistical significance (P is more than 0.05) and does not show cytotoxicity.
Example 2 decellularized SIS inhibition of scar tissue proliferation after glaucoma surgery
Firstly, experimental steps
1. Preparation of acellular SIS
An acellular SIS was prepared as in the examples to obtain an acellular SIS.
2. Animal experiments: trabeculectomy model
1) Preparation of animals
36 healthy New Zealand white rabbits weigh 2.5-3.5 kg, and the male and female parts are not limited. Optionally, one eye is a test eye and the other eye is a control eye. Before the operation, 1% of the total time of the patient is subjected to the paradoxical surface anesthesia, and a Schiotz tonometer measures the basic intraocular pressure of the eyes.
2) Method of producing a composite material
After general anesthesia, trabeculectomy was performed for both eyes. Acellular SIS (SIS) with the thickness of 0.3mm and cut into 8mm multiplied by 6mm is paved under a sclera flap of an experimental eye, the peripheral part is placed on the surface of the sclera, and the SIS is fixed while the sclera flap is sutured. The control eye was left without SIS and the rest was the same as the experimental eye.
3) Observation indexes are as follows:
postoperative daily slit lamp microscopy of conjunctival, corneal, anterior chamber, iris and lens status, recording bleb status, and measuring intraocular pressure 1 time per week.
Killing rabbits at 1w, 2w, 1m, 2m, 3m and 6m in batches after operation, removing eyeballs (completely preserving filtering bubble parts), fixing, slicing, observing wound repair and SIS degradation conditions after trabeculectomy under a light mirror, measuring the thickness of scar tissue under conjunctiva by using a microstick, and observing fibroblast proliferation conditions and collagen fiber content by histochemical staining and immunohistochemical staining in parallel; and observing the ultrastructure of the filtering bubble part under an electron microscope.
Second, experimental results
And (4) performing postoperative slit lamp examination, wherein the conjunctiva of the eye in the experimental group is slightly hyperemic, the upper filtering bleb is dispersed, and the filtering bleb in the control group is limited and has local scar adhesion. The intraocular pressure of the experiment group at the same period is slightly lower than that of the control group, and the difference has statistical significance.
After 1 week of operation, the filtration channel of the experimental group is kept open, the collagen fibers are sparsely distributed, PCNA positive cells can be seen, the collagen fibers of the operation area of the control group are compact, and the PCNA positive cells are obviously more than those of the experimental group.
Claims (5)
1. Application of acellular SIS in preparing a medicine for inhibiting postoperative scar tissue hyperplasia of glaucoma.
2. The use of claim 1, wherein the decellularized SIS inhibits fibroblast growth and proliferation.
3. The decellularized SIS as claimed in claim 2 inhibits fibroblast growth and proliferation means that decellularized SIS affects the cellular morphology of fibroblasts and reduces the number of fibroblasts.
4. A method of preparing a decellularized SIS as claimed in claim 1 or 2, comprising the steps of:
taking a fresh proximal jejunum of a healthy adult pig, scraping by a mechanical method to obtain a small intestine submucosa, and carrying out freeze drying treatment after cell removal to obtain the cell-free SIS.
5. The method of inhibiting fibroblast cell growth and proliferation of decellularized SIS of claim 2, comprising the steps of:
1) culturing fibroblast by tissue culture method;
2) and (2) adding the acellular SIS into the fibroblasts obtained in the step 1) to inhibit the growth and proliferation of the fibroblasts.
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