CN107796674B - Method for evaluating eye irritation injury and repair by long-term culture of animal cornea - Google Patents
Method for evaluating eye irritation injury and repair by long-term culture of animal cornea Download PDFInfo
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- CN107796674B CN107796674B CN201710538216.7A CN201710538216A CN107796674B CN 107796674 B CN107796674 B CN 107796674B CN 201710538216 A CN201710538216 A CN 201710538216A CN 107796674 B CN107796674 B CN 107796674B
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Abstract
A method for evaluating the irritation injury and repair effect of eyes by using an animal isolated cornea long-term culture model comprises the following steps: 1. preparing an in vitro cornea; 2. long-term culture of cornea; 3. a cornea chemical damage model and evaluation; 4. cornea physical damage model and evaluation; 5. evaluation of the repair effect after corneal injury; 6. and (5) performing statistical analysis and result judgment. The invention utilizes the reversible degeneration of corneal epithelial cells and matrix damage caused by exogenous stimuli such as chemical substances and ultraviolet rays, and evaluates the degree of eye irritation through a prediction model; or a method for evaluating the cornea self-repair after the injury by using reversible stimulation, slight abrasion or loss injury of corneal epithelial cells and stroma caused by a chemical method, a physical method or a mechanical method or adding medicaments to evaluate the effect of promoting the cornea repair after the injury occurs. The method is simple to operate, has good correlation with in vivo experiments and high sensitivity, and can meet the requirements of detection and research on the corneal irritant damage and repair thereof.
Description
Technical Field
The invention relates to a method for evaluating eye irritation injury and repairing effect evaluation by using an isolated cornea long-term culture model of animals such as pigs or cattle and the like, which is applied to toxicological and pharmacological experimental evaluation of eye irritation and recovery of chemicals, cosmetics, pesticides and medicines.
Background
Many exogenous chemicals, such as cosmetics, drugs, pesticides, and atmospheric pollutants, may contact the eyes and cause health hazards to the human eyes. Therefore, detecting and evaluating the potential eye irritation of drugs, daily consumables (such as personal care products, household chemicals, cleaning and disinfecting products, and the like), and environmental pollutants (such as pesticides, smoke, particulate matters, pollen, and the like) become important contents for guaranteeing personal safety. The eye irritation test is a mandatory test item for safety evaluation before eye contact products come into the market, and is also a mandatory requirement for chemical identification and classification, and the purpose is to adopt a scientific method to detect and judge the safety of the products so as to reduce the human health risk. Therefore, rapid classification and hazard identification of the analyte are the primary tasks of risk decision. For toxicological evaluation of eye irritants, the traditional method continues to use the rabbit eye irritation in vivo experiment proposed by Draize in 1944, and the disadvantages of the rabbit eye experiment include great suffering of animals, long test period, high inspection cost, strong subjectivity of result judgment, difference of animal and human body reactions and the like.
On the other hand, the treatment of the clinical common corneal trauma or mechanical injury, or after operation (such as after corneal foreign body removal operation, after excimer operation or after cataract operation) usually needs to use some drugs to promote the corneal healing, and the observation of whether these drugs have irritation and their effect of promoting corneal repair is also the basic requirement of pharmacological research. Conventional evaluation methods generally use living animals to create corneal defects or irritative damage models, and then evaluate the effects of drugs by examining corneal morphology, structure and function after application of the drugs. The animal experiment generally has the defects of great pain of animals, long experiment period, poor operation stability, high research cost, difference in reaction with human bodies and the like.
With the internationalization of the "3R principle", i.e. reduction (reduction), replacement (replacement) and optimization (refinement) animal experimental principles in life science research and test testing, and in particular the introduction of the "21 st century toxicity test vision" and the development of new technologies, test systems no longer based on animal models have become a hotspot in scientific research, and some methods of detection and evaluation have been accepted by regulations in many countries. For example, European Union cosmetic Regulation 1223/2009/EC prohibits cosmetic animal experiments, and European Union chemical REACH Regulation 1907/2006/EC (registration, evaluation, approval, and restriction of chemicals) encourages the development and application of alternative methods for animal testing. Eye irritation evaluation using in vitro experimental systems and methods has been highly successful, and for testing acute irritation and corrosivity, the organization for development of economic synergy (OECD) has identified and recognized several methods, including methods for ex vivo bovine cornea, methods for short-term exposure of corneal cells, methods for fluorescein leakage of epithelial cells, and methods for reconstructing corneal models, among others. How to reasonably and scientifically use the methods and further innovate a new testing system and an evaluation method on the basis of the methods, solve the existing methods and the defects, and still remain the main research content of the ophthalmic toxicology and pharmacology evaluation.
According to the mechanism of in vivo eye injury, currently developed eye stimulation substitution methods can be divided into four types, namely a cell system, a chick embryo, an artificial cornea and an isolated organ, and the methods have the advantages, the disadvantages and the application range. The cell system has the highest standardization degree, low cost and quickness, but is only suitable for testing dissoluble substances. The chick embryo method has low standardization degree, is suitable for simulating conjunctival injury, and has poor prediction on corneal injury. The artificial cornea reconstructed in vitro lacks corneal endothelial cell layer, and has high construction cost and great operation difficulty. Moreover, none of the above methods can be used for the study and testing of the recovery effect after corneal damage, since some slightly irritating substances cause a temporary reversible damage to the cornea, which can only be detected by test systems with long in vitro culture times. In addition, for mechanical damage of cornea caused by various reasons, such as corneal epithelial abrasion (e.g. scratch of foreign body like nail, branch, contact lens, etc.), corneal foreign body injury (e.g. iron filings, chaff, sand, dust, etc.), corneal surgery (e.g. laser treatment of myopia, minimally invasive resection), there is also a need to develop drugs for promoting corneal repair and study the action mechanism thereof. In vitro methods such as monolayer cell culture, chick embryo culture or artificial cornea cannot simulate the complete structure and mechanical damage of the cornea.
The in vitro cornea used in the invention is derived from animal waste eyeballs in animal husbandry, and the active cornea tissue is cut for culture and experiment, so that the cornea tissue can be used for replacing an in vivo animal experiment to detect the irritation of the cornea, and can also be used for the inspection and evaluation of the recovery capability of the cornea after being damaged. In addition, the cornea derived from large animals is more suitable for testing eye irritation than the cornea of rabbits, mice or chickens, particularly, the cornea tissue structure of pigs is closer to that of human beings, and the cornea tissue structure is an ideal model for researching the cornea repairing effect. At present, no relevant patent and literature reports exist on an in vitro method established based on porcine and bovine isolated cornea models.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for evaluating the irritation and repair effect of eyes by using an in-vitro pig cornea or ox horn membrane model, which is universal, quick, simple, convenient, low in cost and free of special requirements on a detection instrument.
The technical scheme adopted by the invention is as follows.
The above object of the present invention is achieved by the following technical solutions: a method for evaluating the irritation injury and repair effect of eyes by using an isolated cornea long-term culture system comprises the following steps:
a method for evaluating eye irritation injury and repairing effect evaluation by using an animal isolated cornea long-term culture model is characterized by comprising the following steps:
step 1, preparing an in vitro cornea;
step 2, cornea long-term culture:
taking a cleaned cornea, suspending the cornea with the epithelial surface facing downwards in a 6-hole plate or a culture dish with the diameter of 35mm, which is added with HBSS liquid, and completely immersing the cornea in the culture liquid;
slowly dropwise adding 0.5-5% agar-gelatin-M199 mixture in a molten state into the inner surface of the corneal fossa 2-3 drops at a time until the inner fossa is completely filled, and completely solidifying the agar-gelatin at room temperature;
inverting the cornea filled with the solidified agar-gelatin, and transferring to a plurality of 35mm culture dishes;
aspirating 40-60ml of M199 medium into each dish until the limbus is covered and the bare corneal epithelial surface is exposed to air;
placing the culture dish on a small-sized oscillator, and placing the culture dish at the temperature of 32 +/-2 ℃ and 5% CO2Culturing for 8-24h in an incubator with 90% relative humidity, and shaking the culture dish to 45 degrees from a horizontal position every 2-3h by using a small oscillator so that the cornea can be soaked in the culture medium for infiltrating epithelial tissues for a short time;
step 3, a cornea chemical damage model and evaluation;
step 4, cornea physical damage model and evaluation;
step 5, evaluation of the repairing effect after corneal injury:
(1) after the compound damages the cornea, the cornea is transferred to a new culture dish, 40ml of new M199 culture medium is added into each culture dish, and then the culture dish is incubated for 2 hours; replacing M199 culture medium containing active substance epidermal growth factor, basic fibroblast growth factor or vitamins, placing at 32 + -2 deg.C and 5% CO2An incubator with 90% relative humidity, and continuously culturing the cornea to the 14 th day according to the cornea long-term culture method in the step 2; 20-30 parallel samples of the experimental group; an M199 culture medium containing active substances is an active repairing group, an M199 culture medium without the active substances is a natural repairing group, and a blank control is set at the same time; setting reference contrast when necessary;
(2) during the culture period, the M199 culture solution containing the active substances is replaced every 2 days; respectively performing cytokine test on culture solution at 2h, 14h, 26h, 50h, 74h, 98h, 170h and 14d +/-2 h after chemical injury, performing turbidity test, fluorescein retention score and histological observation on 2-3 corneas in the experimental group, and continuously culturing the rest corneas;
(3) corneal fluid cytokine assay: before replacing with new medium, the medium was removed from the culture dish and the levels of inflammatory and repair factors in the medium were measured: including IL-1a, IL-6, IL-8;
(4) turbidity change: before turbidity measurements, the cornea was removed from the petri dish and fixed in a holder, the rear and front chambers of which were filled in turn with sterile phenol red-free MEM medium, respectively, and the turbidity values of each cornea were measured and recorded with a keratometer, and the turbidimetry values, OP, were recorded separately for corneas from different groupsActivity h，OPRepair h，OPBlank space(ii) a Calculating the turbidity difference between the experimental group and the blank group;
(5) resistance measurement and fluorescein observation: after measuring the turbidity, measuring the resistance value (omega) of the cornea by using a resistance meter; then the cornea is taken down from the holder, and PBS containing 2% fluorescein sodium (NaFL) is dripped on the upper surface of the cornea; excess NaFL was immediately washed with PBS and observed under UV light for NaFl retention, the extent of retention indicating the severity of corneal injury;
calculating the percentage value of the retention area S in the whole corneal area C, and scoring the damage/recovery degree according to the S/C value; 0 minute: <2%, 1 point: 2-25%, 2 min: 26-50%, 3 min: 51-75%, 4 min: 76% -100%;
(6) histological evaluation: after the fluorescein scoring is finished, adding the cornea into 10% neutralized formalin buffer solution for fixation for 24 hours; embedding paraffin, slicing, and taking part of the slices for HE staining; observing and evaluating histological changes under a microscope;
taking a part of the section for immunohistochemical staining: staining of corneal epithelial cell tight junction protein Occludin or ZO-1 or staining of cadherin, which is used for observing the improvement of epithelial barrier function in the corneal repair process;
step 6, statistical analysis and result judgment
Taking the average value of each group of experimental data obtained in the step as +/-standard deviation
Representing that SPSS22.0 statistical software is adopted for analysis, single-factor variance analysis is used for comparing differences among multiple groups, an SNK (single-noise keying) test method is adopted for pairwise comparison among the groups, the significance level alpha is 0.05, and P < 0.05 represents that the differences have statistical significance;
repairing after chemical injury and comprehensively analyzing resistance measurement values, fluorescein staining, turbidity measurement values, inflammatory factor detection and histological observation results; the mechanical damage repair is not carried out with resistance measurement and turbidity test; repairing the reference chemical damage after the ultraviolet damage;
according to the significance of different detection parameters, comprehensive judgment of the repairing effect is made through statistical result analysis:
(1) resistance measurement fluorescein staining: along with the prolonging of the culture time, the resistance values of the natural restoration group and the active restoration group gradually increase; the ratio of S/C of fluorescein staining gradually decreased, and the score decreased; compared with a blank control, the ratio of S/C of the natural repair group and the active repair group has statistical difference p less than 0.05; compared with the natural restoration group, the increase of the resistance value of the active restoration group has statistical significance, the grading values of retention of fluorescein are different from grade 1 and grade 1, and the active substances are considered to have promotion effect on restoration;
(2) turbidity test: the turbidity (OP) values of the natural repair group and the active repair group gradually decrease with the increase of the culture time; the turbidity difference of the repair group compared with the blank control is gradually reduced; compared with the natural restoration group, the activity restoration group has at least 2 and more than 2 time points, the turbidity difference has statistical difference p less than 0.05, and the active substance is considered to have promotion effect on restoration;
(3) inflammatory factor detection: the values of inflammatory factors of a natural repair group and an active repair group are in a descending or ascending trend along with the prolonging of the culture time, and the statistical difference p is less than 0.05; at the same time, compared with the natural repair group, the level of the characteristic inflammatory factor or repair factor of the active repair group is statistically different, which indicates that the active substance has a promoting effect on repair;
(4) and (3) histological observation: histological changes of the cornea were observed by HE staining and immunohistochemical staining, and detailed recordings were made:
after the chemical irritation injury of the cornea occurs, the histology usually shows that the epithelium is obviously damaged, the epithelial cells are deleted, the permeability is increased, and the structures of zon-1 and Occludin disappear; over time, the epithelial cells proliferate and migrate to the defect, the permeability of the epithelial cells gradually decreases, and the tight junction protein structure gradually forms; at 24h, epithelial cells cover the corneal epithelial defect, but the permeability of the epithelial cells is still higher than that of the normal group, and the tight connection between the cells is loose and the arrangement is disordered; after 48 hours, the corneal permeability is recovered to be normal, the corneal epithelial cells are tightly connected, and a tight connection structure is formed among the cells; if the repair-promoting group is compared with the natural repair group, the histological repair time is shortened by more than 12 hours or the repair degree is improved, namely the observation time of similar histological structures and characteristics is advanced by more than 12 hours, which indicates that the active substances have promotion effect on repair.
Preferably, the step 1 of ex vivo corneal preparation comprises the following substeps:
the eyeballs are picked from the eye sockets within 2-3 hours after slaughtering of slaughterhouse animals, and the cornea is prevented from being damaged in the separation process;
completely immersing the isolated eyeball in ice-cold HBSS balanced salt solution containing antibiotics and conveying the isolated eyeball to a laboratory;
the excision of the cornea of the eye is carried out in a sterile area in a laboratory, the integrity of the cornea is checked by naked eyes before the cornea is excised, whether scratches, pigmentation, turbidity or mechanical scratches exist or not is checked, and the eyeball with the defects is discarded;
soaking the pig eyeball in 1% povidone iodine solution for 2min, washing with sterile PBS, soaking in PBS containing 0.1% gentamicin for 15min for sterilization, and performing corneal resection;
bovine cornea was isolated directly from the intact eye, leaving a 2-4mm intact circular sclera at the corneal limbus when the cornea was isolated, and excised cornea was rinsed 3-5 times with 5-7ml sterile HBSS in succession.
Preferably, the step 3 compound stimulation injury cornea model and evaluation comprises the following sub-steps:
(1) after the cornea is cultured for about 4-24h, beginning a formal experiment, and sucking out the culture solution from the culture dish; placing a polytetrafluoroethylene resin O-shaped ring or a Teflon O-shaped ring with the diameter of 1.0-1.5 cm on the corneal epithelium surface;
the test substance is chemical liquid with appropriate concentration expected to produce stimulation, such as 3% SLS, 100% ethanol or 3% H2O2, and the liquid stimulant is added into the ring at 40-100ul and exposed for 10-20 min; if ultraviolet rays are selected as a damage model, the wavelength is UVB, and the irradiation dose is 40-70mJ/cm 2;
after the exposure is finished, gently rinsing the cornea with 2-3ml of PBS for 2-3 times until the residues of the test substances are completely removed; immersing it in a culture dish containing 40-60ml of M199 culture medium at 32 + -2 deg.C and 5% CO2The incubator with 90% relative humidity was incubated for 2 hours; 6-10 corneas were used for each injury model;
(2) corneal haze change: after the incubation is finished, taking 2 corneas, removing the polytetrafluoroethylene resin or the Teflon O-shaped ring, and fixing in a cornea holder; then adding fresh MEM culture solution in the sequence of filling the first chamber and then filling the second chamber and then filling the front chamber; turbidity of each cornea was measured by nephelometerRecording the haze value (OP) as OPStimulating for 0hOr OPRepair for 0h；
Meanwhile, a blank group of corneas are taken for turbidity test, and the turbidity is recorded as OPBlank space；
(3) Resistance measurement and fluorescein observation: the same as step 5 (5);
(4) histological evaluation: the same as step 5 (6);
(5) evaluation of chemically damaged cornea:
if the resistance measurement value is more than 1 and less than or equal to 5, the corneal barrier function is considered to be damaged, if the resistance value is less than or equal to 1, the damage is considered to be too serious and can not be used for a repair test, and the resistance value of a blank group is more than 5; if the turbidity value changes by more than or equal to 3 and less than 10, the mild-moderate injury is considered to be caused; if the turbidity value is more than or equal to 10 and less than 25, the medium-serious damage is considered to be caused; if the turbidity value is more than or equal to 25, the damage degree is considered to be too serious, and the turbidity value cannot be used for the repairability test;
the fluorescein staining score should be 3 points, namely the damage area should be more than 50%, less than 75%, otherwise the damage degree is too serious, and the damage cannot be used for damage repair evaluation;
histological examination: after corneal injury, epithelial defects of different degrees usually appear, and the epithelial cells are separated, so that the structures of the tight junction proteins ZO-1 and Occludin are reduced and disappear; edema of different degrees of the stroma layer and disorganized arrangement of collagen bundles; some chemical substances, such as oxidant hydrogen peroxide and the like, damage the stroma layer, but the epithelial barrier function is not obviously damaged; histological lesions should correspond to turbidity tests and fluorescein staining;
(6) the remaining cornea was immersed in a culture dish containing 40-60ml of M199 medium, and placed at 32 ℃. + -. 2 ℃ with 5% CO2And (3) continuously culturing the cornea in the incubator with the relative humidity of 90% according to the cornea long-term culture method in the step (2) to carry out subsequent natural repair or repair promotion research;
(7) and simultaneously setting a blank control and a negative control, wherein the negative control is deionized water, and the same operation steps are adopted.
Preferably, the step 4 of damaging the corneal model and evaluating by physical stimulation comprises the following sub-steps:
(1) after the cornea is cultured for about 24 hours, the culture solution is sucked out of the culture dish; placing a polytetrafluoroethylene resin O-ring or Teflon ring with a diameter of 1.0cm on the cornea; scraping the central epithelium, the deep epithelium and the corneal epithelial cell basal layer with the diameter of 8mm from the center of the cornea of the eye by using a sterile ophthalmic surgical blade to prepare a mechanical corneal injury model; 6-10 corneas were used for each injury model; 2 of these corneas were subjected to the following irritation level evaluation test:
(2) and (3) fluorescein observation: the same as step 5 (5); NaFL staining revealed damaged cornea, the size of the yellow areas indicating the severity of the damage;
(3) histological evaluation of physical stimulus model introduction: the same as step 5 (6);
(4) evaluation of physically damaged cornea:
the fluorescein staining score should be 3 points, i.e. the damage area should be more than 50% and less than 75%, otherwise the damage degree is too severe to be used for damage repair evaluation
Histological examination: after corneal injury, obvious epithelial defect appears, which is manifested by epithelial cell loss, and the structures of zon-1 and Occludin disappear; (ii) a The upper layer of the matrix layer is broken or lost, and histological damage caused by physical factors corresponds to fluorescein dyeing;
(5) the remaining cornea was immersed in a culture dish containing 40-60ml of M199 medium, and placed at 32 ℃. + -. 2 ℃ with 5% CO2And (3) continuously culturing the cornea in the incubator with the relative humidity of 90% according to the cornea long-term culture method in the step (2) to carry out subsequent natural repair or repair promotion research;
(6) and simultaneously setting a negative control, wherein the negative control is deionized water, and the same operation steps are adopted.
Preferably, any of said steps 1-4, involve isolation of the cornea and long-term culture:
the in-vitro cornea is obtained by separating the discarded pig or cattle eyeballs in a slaughterhouse;
the agar-pigskin gelatin-M199 mixture is 1% of low freezing point agar, 1% of pigskin gelatin or M199 culture medium, and is maintained at 37 ℃;
the HBSS buffer solution refers to a commercial Hank's balanced salt solution;
the M199 culture medium is an M199 culture medium, 10% of calf serum, gentamicin, penicillin/streptomycin are added, and amphotericin B is also added in the culture of the pig cornea;
the povidone iodine is a preservative, and the concentration is 1%;
the O-shaped ring is a white ring or a Teflon ring prepared from polytetrafluoroethylene resin inert materials, has no toxicity or irritation to the cornea, does not react with and adhere to a sample, and can be sterilized under high pressure for repeated use;
if the ring is 9-11mm in outer diameter and 8-10mm in inner diameter, the sample loading amount is 40 mu L; if the ring is 13-15mm in outer diameter and 11-13mm in inner diameter, the sample loading amount is 100 mu L; for fixing the test object and defining the observation area.
Preferably, any one of said steps 3-4, relates to a method for corneal injury wherein:
the chemical injury refers to chemical stimulation of cornea caused by chemical substances or mixture entering eyeballs, common stimulators are 1.5% -3% of SLS, 0.2-0.4 mol of NaOH, 3% -6% of tricho-acetic acid (TCA) and 1.5% -3% of H2O2, the exposure time of the chemical substances is 10-20 min, and the chemical injury is selected according to the types and degrees of the chemical stimulators: selecting short duration of action for compounds with moderate to severe eye irritation, longer duration of action for compounds with weak to moderate eye irritation, or determining exposure time using pre-experiments;
the ultraviolet injury belongs to physical factors, and refers to corneal injury caused by UVB or UVA, and the UVB irradiation dose is 40mJ/cm2-70mJ/cm2The UVA irradiation dose is 10J/cm2-50J/cm2；
According to the purpose of research, the mechanical injury is realized by selecting a perforator with the diameter of 2-3mm to damage corneal epithelium and stromal surface layer, but not to damage corneal stroma deep layer; a blade, a needle or a blunt instrument is selected to slightly cut, scratch, wipe, grind or peel off the surface of the cornea without damaging the deep layer of the cornea; other physical means should be chosen to ensure that the procedure is well-controlled and that the individual differences between corneas are as small as possible.
Preferably, any one of said steps 5-6, relating to the detection and assessment of corneal damage repair:
the number of parallel corneas for detecting the repairing effect of the active substances depends on the parameters of the detection time points, if 5 detection time points including histological observation, fluorescein determination and turbidity test are considered, 2 to 5 corneas are taken at each time for testing, and the number of the corneas is at least 10 to 25 are required in the experimental group, wherein 5 detection time points including 2h, 14h, 26h, 36h, 50h, 74h, 86h, 90h, 170h and 14d +/-2 h are considered; the repair-promoting group, placebo and baseline controls should also employ a corresponding number of corneas;
the repairing effect of the active substance after mechanical damage is detected, and the turbidity test is not considered;
the corneal resistance is characteristic of the function of a reaction barrier and can be measured by a low-voltage alternating-current Wheatstone bridge;
the turbidity value is mainly reflected to the integrity of the corneal stroma layer and can be measured by a corneal turbidimeter; the inflammatory factor or the repairing factor refers to a specific substance secreted by corneal cells in the corneal repairing process, and comprises the following components: IL-1 α, IL-1 β, IL-6, IL-7, IL-10, IL-12, IL-12, IL-15, MIP-1 α, MIP-1 β, MIP-2, TNF- α or PGE2, a proliferation factor, an enzyme: LDH;
the commercial kit comprises flow cytometry, ELISA and immunohistochemical methods, performs content measurement on collected cells and corresponding culture media, detects the generation condition of target secretion compared with a test control group, and statistically analyzes whether the increase or decrease of certain secretion in the repair process has significant change;
the immunohistochemistry refers to staining a histological section by using the principle of antigen-antibody combination reaction, wherein the markers for indicating the corneal barrier function comprise zon-1, Occludin and cadherin, polyclonal rabbit antibody bovine or porcine antibody can be selected as primary antibody, and the dilution is 1: 100-1: 200; the secondary antibody may be selected from a murine anti-rabbit or goat anti-rabbit antibody.
The experimental control refers to a blank control which does not contain any active substances;
the evaluation of the repair promotion effect of the active substance takes natural repair as a contrast; if necessary, a reference substance control is added, and the reference substance is a substance known to have a definite repair promoting effect.
Preferably, the active substance for promoting corneal repair is prepared by adding basic fibroblast growth factor, deproteinized calf blood extract, recombinant epidermal growth factor (rhEGF) or vitamin A palmitate into the culture medium, or directly using eye drop containing medicine;
the statistical method refers to that all groups of experimental data are obtained according to the mean value plus or minus standard deviation
Representing that SPSS22.0 statistical software is adopted for analysis, single-factor variance analysis is used for comparing differences among multiple groups, an SNK (single-noise keying) test method is adopted for pairwise comparison among the groups, the significance level alpha is 0.05, and P < 0.05 represents that the differences have statistical significance;
the chemical injury repair should be comprehensively analyzed for fluorescein staining, turbidity test, inflammatory factor detection and histological observation; the weights are as follows from big to small: fluorescein staining, resistance value measurement, turbidity test, histological observation and inflammatory factor detection;
the physical injury repair should be comprehensively analyzed for fluorescein, inflammatory factor detection and histological observation; the weights are as follows from big to small: fluorescein staining > histological observation > inflammatory factor detection.
The method for evaluating the eye irritation and the repair process thereof by using the pig cornea or ox horn membrane model provided by the invention utilizes the reversible corneal epithelial cell degeneration and stroma injury caused by external stimuli such as chemical substances and ultraviolet rays, and evaluates the degree of the eye irritation by a prediction model; or a method for evaluating the cornea self-repair after the injury by using reversible stimulation, slight abrasion or loss injury of corneal epithelial cells and stroma caused by a chemical method, a physical method or a mechanical method or adding medicaments to evaluate the effect of promoting the cornea repair after the injury occurs.
The method is simple to operate, has good correlation with in vivo experiments and high sensitivity, and can meet the requirements of detection and research related to corneal irritant damage, repair and the like.
The extent of ocular damage by exogenous compounds is not limited to the epithelium, but may also involve the upper lamellar stroma, with mild damage being reversible and recoverable, and most in vitro evaluation methods fail to detect recovery after damage. Therefore, the long-term culture of the isolated cornea can be used as an in vitro model for evaluating the potential irritation of a chemical and the recovery condition after the chemical exposure, and can also be used as an in vitro model for evaluating the self-repair or drug-promoted repair condition of the cornea after mechanical damage. The cornea tissue structure of the isolated large animals (pig cornea and cow cornea) is similar to that of the human cornea, and the generation mechanism and the repair process of the irritant reaction are similar to those of the human cornea. The indexes for quantitatively indicating the corneal irritant damage and repairing the corneal irritant damage comprise: degree of corneal clouding, corneal tissue morphology, changes in corneal inflammation, changes in corneal barrier function, and the like. Corneal turbidity was quantitatively measured by a corneal turbidimeter, corneal microstructure changes were observed by making histological sections and scored, corneal inflammation changes were measured by quantitatively detecting component reactions in the culture solution, and corneal barrier function changes were measured by the amount of fluorescein sodium retention.
The prediction model established based on the two indexes of the corneal injury and the recovery effect can be used for judging whether the object to be detected has no irritation or irritation degree, can be used for distinguishing the eye irritation injury and the recovery after the injury, and can be used for detecting the difference of self-repairing or auxiliary repairing effects. Can completely provide complete information including product safety, cornea injury degree, repair process and the like.
The invention relates to an in vitro cornea culture technology used in experiments, which is mature, easy to operate, good in repeatability and low in price.
Has the advantages that: the invention can be used for replacing living animals to detect the recovery effect of the corneal irritation caused by exogenous chemical injury or mechanical injury. Compared with the prior art, the invention has the following advantages:
(1) the invention establishes a long-term culture model of the isolated animal cornea, can keep active culture for more than 14 days in vitro, and solves the defect that the in vitro method can not be used for repairing function evaluation after injury;
(2) the in vitro animal cornea used by the invention is a three-dimensional tissue material which has stable and reliable source, has a structure and activity similar to those of a human cornea, can be directly contacted with an irritant for injury, and is suitable for testing any chemical injury and physical injury effects and recovery effects;
(3) the invention detects the multi-dimensional parameter changes such as corneal turbidity, fluorescein retention, inflammatory medium level, histological observation and the like, completely evaluates the corneal turbidity degree, the corneal tissue morphological structure, the corneal inflammation change and the dynamic change of the corneal barrier function in the injury action and the repair process, has comprehensive detection parameters, sensitive method, high specificity and accurate prediction model;
(4) compared with the two-dimensional cell which can only detect the stimulus damage of soluble chemical substances, the three-dimensional system can detect the eye cornea damage caused by single compound, mixed pollutant, ultraviolet exposure and mechanicalness by using a long-term culture model of the natural cornea;
(5) the method established by the invention can replace the experiment of living animals, provide the detection and research of the natural and accelerated repair functions of the damaged cornea quantitatively and qualitatively, and can be directly used for the prediction of the cornea stimulation and recovery functions of products and samples such as cosmetics, medicines, air pollutants and the like;
drawings
FIG. 1 shows the long-term culture of isolated cornea after the addition of agar-pigskin gelatin-M199 mixture;
FIG. 2 porcine corneal histology (blank control HE staining pattern for a certain experiment);
FIG. 3 bovine corneal fluorescein sodium staining after chemical injury (injury degree score 2);
FIG. 4 bovine corneal fluorescein sodium staining after 7 days of natural recovery (injury score of 0);
figure 5 bovine corneal histology after chemical injury (corresponding to a haze value of 18);
figure 6 bovine corneal histology (HE staining) after 4 days of recovery was promoted;
FIG. 7 Tight junction immunohistology (ZO-1 immunostaining) after mild injury to bovine corneal membranes;
FIG. 8 tight junction immunohistology (Occludin immunostaining) after 4 days of repair of bovine corneal lesions;
Detailed Description
Example 1 evaluation of the effects of SLS on the injury and Natural repair of porcine cornea
1. In vitro porcine cornea preparation
1.1 preparation of porcine cornea: the eyeballs are picked from the eye sockets within 2 hours after the pig is slaughtered, and the cornea is prevented from being damaged in the separation process. Ex vivo porcine eyeball was transported to the laboratory completely immersed in ice-cold HBSS balanced salt solution containing amphotericin B. The excision of the pig eye cornea is carried out in a sterile area in a laboratory, the integrity of the cornea is checked by naked eyes before the cornea is cut, and whether scratches, pigmentation, turbidity and grape sores exist or not is checked; eyes with the above defects should be discarded. Soaking the eyeball in 1% povidone iodine solution for 2min, washing with sterile PBS, soaking in 0.1% gentamicin-containing PBS for 15min, sterilizing, and performing corneal resection. The cornea was excised from the intact eyeball and the limbus was left with a 2-4mm intact circular sclera. The excised cornea was washed 3-5 times successively with 5-7ml of sterile HBSS.
1.2 gel filling, namely taking a clean pig cornea, suspending the cornea from the epithelial surface of the cornea downwards in a 12-hole plate, adding HBSS into culture wells to support the suspended state of the cornea, and slowly and dropwise adding (2-3 drops each time) agar-gelatin-M199 mixture in a molten state to the inner skin surface of a corneal pit so as to enable the gel to be in direct contact with the endothelium. The filling procedure was repeated until the endothelial cell was completely filled and the agar-gelatin completely solidified at room temperature. The cornea filled with the solidified gel was then inverted and transferred to a 12-well plate.
1.32 isolated porcine cornea culture
Approximately 40ml of M199 medium was aspirated into each dish until the limbus was covered and the bare corneal epithelial surface was exposed to air. Will cultivatePlacing the culture dish on a small oscillator, and placing the culture dish at 37 ℃ and 5% CO2And culturing in an incubator with 90% relative humidity for about 24 +/-2 h. The mini-shaker programmed the dish to shake from a horizontal position to an angle of about 45 degrees every 2 hours so that the cornea could be briefly soaked in the medium to infiltrate the epithelial tissue.
2. Compound injury model
2.1 sample adding: after the cornea was cultured for about 6 hours, the culture solution was aspirated from the dish. A polytetrafluoroethylene resin O-ring having a diameter of 1.0cm was placed on the cornea. 40 μ L of 3% SLS and negative control (DI water) were added to the loop. 20 corneas were used for each test.
2.2 Exposure: the exposure time was 10min, and after exposure was complete, the cornea was gently rinsed with approximately 2ml PBS until the test substance residue was completely removed. The cornea was then transferred to a new petri dish containing 40ml of M199 medium at 32 ℃. + -. 2 ℃ with 5% CO2And the incubator with 90% relative humidity is continued to incubate;
3 Damage detection and self-repair
3.1 assessment of degree of injury: 2 hours, taking 2 corneas of the damaged group and the blank control group for turbidity, fluorescein sodium staining and histological flaking, and evaluating the stimulation degree; the remaining cornea was immersed in a culture dish containing 40ml of M199 medium and placed at 32. + -. 2 ℃ with 5% CO2An incubator at 90% relative humidity;
3.2 repair assessment: culturing for 14h, 26h, 50h and 98h, increasing 173h if necessary, taking 2 corneas of the damaged group and the blank control group to perform turbidity measurement, resistance measurement, fluorescein sodium staining, inflammatory factor detection and histological slide, and performing self-repair degree evaluation;
and (3) turbidity detection: before detection, the Teflon O-ring is removed and fixed in a special cornea holder. Fresh MEM medium is then added in the order of filling the first chamber and then refilling the first chamber. The opacity of each cornea was measured using a BASF3.0 turbidimeter and the opacity value (OP) was recorded as OPStimulating for 0hOr OPRepair for 0hMeanwhile, a blank group of corneas are taken for turbidity test, and the turbidity is recorded as OPBlank space。
Testing of corneal fluid inflammatory factor: before replacing new culture solution, removing the culture solution from the culture dish, and detecting the levels of inflammatory factors and repair factors including IL-1a, IL-6, IL-8 and the like in the culture solution by using the kit;
and (3) resistance measurement: after turbidity is measured, a Millipore resistance tester is used for detecting the corneal resistance value according to the instrument instruction;
and (3) fluorescein staining: the cornea was removed from the holder and PBS containing 2% sodium fluorescein (NaFL) was applied dropwise to the upper surface. Excess NaFL was immediately flushed with PBS and NaFl retention was observed under UV light, the extent of retention indicating the severity of corneal injury. Calculating the percentage value of the retention area (S) in the whole cornea area (C), and scoring the damage/recovery degree according to the S/C value. 0 minute: <2%, 1 point: 2-25%, 2 min: 26-50%, 3 min: 51-75%, 4 min: 76 to 100 percent.
Histological evaluation: after completion of fluorescein scoring, the cornea was fixed for 24h by adding to 10% neutralized formalin buffer. Paraffin embedding, slicing, taking partial section and performing HE staining. Observing and evaluating histological changes under a microscope; and taking a part of the section to perform immunohistochemical staining, such as staining of corneal epithelial cell tight junction protein Occludin and ZO-1, and observing the improvement condition of the epithelial barrier function in the corneal repair process.
4. Observation of repair Effect results
4.1 turbidity assay
Turbidity value OP2h12, the change in turbidity was greater than or equal to 3 and less than 15 compared to the blank OP0h, which is considered to be mild-to-moderate damage;
4.2 resistance measurement and fluorescein staining
Treating cornea with 3% SLS for 10min, and detecting cornea resistance value of 1.12 Ω 2h after exposure; more than 50% of cornea area can be seen to be stained by fluorescein sodium staining, the damaged area is 75-85%, and the score is 3.5; approximately 30% of the corneal area stained after 26h, scored 2 points; about 5% in 50h, and the score is 1; almost no staining of the corneal area was visible at 98h, suggesting a restoration of the corneal epithelium.
4.3 cytokine detection
Treatment of the cornea with 3% SLS resulted in the release of a large amount of inflammatory factors, the levels of which correlated with the extent of injury, and decreased as the injury was repaired. The detection results at different time points are shown in table 1;
4.4 histological observations
After treatment of the cornea with 3% SLS, complete damage or loss of epithelial cells was shown, and loss of viable corneal cells also occurred in the upper endothelium; 26h after exposure, the damaged areas were shown to migrate from epithelial cells to basement membrane; after exposure for 50h, only eosinophilic hyperkeratocyte of corneal epithelial cells is increased, which indicates that the epithelium is recovered obviously; after 98h exposure, corneal tissue structure was essentially normal, suggesting complete restoration of the corneal epithelium.
TABLE 1 corneal recovery following SLS chemical stimulation
Example 2 assessment of mechanical Damage and Natural repair action of bovine horn Membrane
1. Ex vivo corneal preparation and culture
1.2 preparation of ox horn membrane: the eyeballs are picked from the eye sockets within 5 hours after the cattle are slaughtered, and the cornea is prevented from being damaged in the separation process. Ex vivo bovine eyeballs were shipped to the laboratory completely immersed in HBSS containing penicillin/streptomycin at 4 ℃. The bovine eye was examined for status before cutting the cornea and discarded if any significant vascular changes, pigmentation, turbidity, grape sores or mechanical scratches were observed. The cornea was excised from the intact eyeball and the limbus was left with a 2-4mm intact circular sclera. The excised cornea was washed 3-5 times successively with 5-7ml of sterile HBSS.
1.2 gel filling: taking a clean ox horn membrane, suspending the corneal epithelium side downwards in a 6-well plate, adding HBSS into culture wells to support the suspended state of the cornea, and slowly dropwise adding (2-3 drops each time) a mixture of agar-gelatin-M199 in a molten state to the corneal pit endothelium side to make the gel directly contact with the endothelium. The filling procedure was repeated until the endothelial cell was completely filled and the agar-gelatin completely solidified at room temperature. The cornea filled with the coagulated gel was then inverted and transferred to a 35mm petri dish.
1.3 corneal culturing: approximately 50ml of M199 medium was aspirated into each dish until the limbus was covered and the bare corneal epithelial surface was exposed to air. Placing the culture dish on a small-sized oscillator, and placing the culture dish at 37 ℃ and 5% CO2And culturing in an incubator with 90% relative humidity for about 24 +/-2 h. The mini-shaker programmed the dish to shake from a horizontal position to an angle of about 45 degrees every 2.75 hours so that the cornea could be briefly soaked in the medium to infiltrate the epithelial tissue.
2. Mechanical injury model
After the cornea was cultured for about 24 hours, the culture solution was aspirated from the dish. A1.3 cm diameter Teflon O-ring was placed over the cornea. A mechanical corneal injury model was prepared by scraping corneal epithelium, corneal epithelial cell basal layer, and depth of 8mm in diameter from the center of the cornea of the eye with a sterile ophthalmic surgical blade. 6-10 corneas were used for each injury model.
3. Assessment of repair action
3.1 mechanical injury 2 hours, taking 2 corneas in the injured group and the blank control group for fluorescein sodium staining and histological slide making, and evaluating the stimulation degree;
the remaining cornea was immersed in a culture dish containing 40ml of M199 medium and placed at 32. + -. 2 ℃ with 5% CO2An incubator at 90% relative humidity; taking 2 corneas of the damaged group and the blank control group for carrying out fluorescein sodium staining, inflammatory factor detection and histological slide observation in 2h, 26h, 50h and 170h respectively, and carrying out self-repair action evaluation;
testing of corneal fluid inflammatory factor: before replacing new culture solution, removing the culture solution from the culture dish, and detecting the levels of inflammatory factors and repair factors including IL-1a, IL-6, IL-8 and the like in the culture solution by using the kit;
and (3) fluorescein staining: after turbidity was measured, the cornea was removed from the holder and PBS containing 2% sodium fluorescein (NaFL) was added dropwise to the upper surface. Excess NaFL was immediately flushed with PBS and NaFl retention was observed under UV light, the extent of retention indicating the severity of corneal injury. Calculating the percentage value of the retention area (S) in the whole cornea area (C), and scoring the damage/recovery degree according to the S/C value. 0 minute: <2%, 1 point: 2-25%, 2 min: 26-50%, 3 min: 51-75%, 4 min: 76 to 100 percent.
Histological evaluation: after completion of fluorescein scoring, the cornea was fixed for 24h by adding to 10% neutralized formalin buffer. Paraffin embedding, slicing, taking partial section and performing HE staining. Observing and evaluating histological changes under a microscope; and taking a part of the section to perform immunohistochemical staining, such as staining of corneal epithelial cell tight junction protein Occludin and ZO-1, and observing the improvement condition of the epithelial barrier function in the corneal repair process.
4. Observation of repair Effect results
4.1 fluorescein staining
After 2 hours, the mechanically damaged cornea is used for detecting the retention of the fluorescein sodium, more than 50 percent of corneal area is stained, the damaged area is more than 50 percent, and the score is 3; approximately 30% of the corneal area stained after 26h, scored 2 points; about 5% in 50h, and the score is 1; almost no staining of the corneal area was visible at 98h, suggesting a restoration of the corneal epithelium.
4.2 cytokine detection
The release of intracellular enzymes and inflammatory factors caused by mechanical damage is shown as the increase of LDH, IL-1a, IL-1b and the like in the culture solution, and the change of different time points is shown in a table 2;
4.3 histological observations
After mechanical injury of the cornea, complete defect of epithelial cells and shedding of stromal cells are shown; 26h after exposure, epithelial cells were shown to migrate from the undamaged area to the defect; after exposure for 50h, the defect is filled with new epithelial cells, indicating that the epithelium is recovered obviously; after 98h exposure, corneal tissue structure was essentially normal, suggesting complete restoration of the corneal epithelium. The epithelial cells are exfoliated, and the structures of zon-1 and Occludin of the tight junction protein disappear;
TABLE 2 corneal recovery and scoring after mechanical injury
Example 3 repair of porcine cornea after injury by recombinant human fibroblast growth factor
1. In vitro porcine cornea preparation and culture
1.1 preparation of porcine cornea: the eyeballs are picked from the eye sockets within 24 hours after the pig is slaughtered, and the cornea is prevented from being damaged in the separation process. Ex vivo porcine eyeball was transported to the laboratory completely immersed in ice-cold HBSS balanced salt solution containing amphotericin B. The excision of the porcine eye cornea should be performed in a sterile field in a laboratory, and the cornea is excised for visual inspection as intact and flawless. Soaking the eyeball in 1% povidone iodine solution for 2min, washing with sterile PBS, soaking in 0.1% gentamicin-containing PBS for 15min, sterilizing, and performing corneal resection. The cornea was excised and separated from the intact eyeball, leaving a 2-4mm intact annular sclera at the corneal limbus. The excised cornea was washed 3-5 times successively with 5-7ml of sterile HBSS.
1.2 gel filling: the cleaned porcine cornea is taken, the corneal epithelial surface is suspended downwards in a 12-hole plate, HBSS is added into culture wells to support the suspended state of the cornea, and then 2% agar-gelatin-M199 mixture in a molten state is slowly and dropwise added (2-3 drops at a time) to the corneal pit endothelial surface, so that the gel is directly contacted with the endothelium. The filling procedure was repeated until the endothelial cell was completely filled and the agar-gelatin completely solidified at room temperature. The gel-filled cornea was then inverted and transferred to a 12-well plate.
1.3 corneal culturing: approximately 50ml of M199 medium was aspirated into each dish until the limbus was covered and the bare corneal epithelial surface was exposed to air. Placing the culture dish on a small-sized oscillator, and placing the culture dish at 37 ℃ and 5% CO2And culturing in an incubator with 90% relative humidity for about 24 +/-2 h. The mini-shaker programmed the dish to shake from a horizontal position to an angle of about 45 degrees every 2.75 hours so that the cornea could be briefly soaked in the medium to infiltrate the epithelial tissue.
2. Compound exposure
2.1 sample adding: after the cornea was cultured for about 24 hours, the culture solution was aspirated from the dish. Placing polytetrafluoroethylene resin O-ring with diameter of 1.0cm at the cornerAnd (3) a membrane. Respectively taking 3% of H2O2Solution and negative control (deionized water) 40. mu.l were added to the loop. 6 corneas were used per subject.
2.2 Exposure: the hydrogen peroxide exposure time was 5min, and after exposure was complete, the cornea was gently rinsed with approximately 2ml PBS to completely remove H2O2And (4) remaining. The cornea was then transferred to a new dish and 40ml of fresh M199 medium containing 90 μ M/ml recombinant human epidermal growth factor was added to each dish. The culture was continued to day 21 as in step 1.3 above, and the repair of the damaged cornea was observed by changing the culture medium of M199 containing growth factor every day. M199 culture solution without growth factor is used as a natural repair control.
3. Assessment of Damage repair
3.1 detection of damage degree: 2 hours, taking 2 corneas of the damaged group and the blank control group for turbidity, fluorescein sodium staining and histological flaking, and evaluating the stimulation degree; the remaining cornea was immersed in a culture dish containing 40ml of M199 medium and placed at 32. + -. 2 ℃ with 5% CO2An incubator at 90% relative humidity;
3.2 repair assessment: continuously culturing for 2h, 14h, 26h, 50h and 98h, taking 2 corneas in the damaged group and the blank control group for turbidity determination, resistance value determination, fluorescein sodium staining, inflammatory factor detection and histological slide preparation, and evaluating the self-repairing degree;
and (3) turbidity detection: before detection, the Teflon O-ring is removed and fixed in a special cornea holder. Fresh MEM medium is then added in the order of filling the first chamber and then refilling the first chamber. The opacity of each cornea was measured using an turbidimeter and the opacity value (OP) was recorded as OPStimulating for 0hOr OPRepair for 0hMeanwhile, a blank group of corneas are taken for turbidity test, and the turbidity is recorded as OPBlank space。
Testing of corneal fluid inflammatory factor: before replacing new culture solution, removing the culture solution from the culture dish, and detecting the levels of inflammatory factors and repair factors including IL-1a, IL-6, IL-8 and the like in the culture solution by using the kit; and (3) fluorescein staining: after turbidity was measured, the cornea was removed from the holder and PBS containing 2% sodium fluorescein (NaFL) was added dropwise to the upper surface. Excess NaFL was immediately flushed with PBS and NaFl retention was observed under UV light, the extent of retention indicating the severity of corneal injury. Calculating the percentage value of the retention area (S) in the whole cornea area (C), and scoring the damage/recovery degree according to the S/C value. 0 minute: <2%, 1 point: 2-25%, 2 min: 26-50%, 3 min: 51-75%, 4 min: 76 to 100 percent.
Histological evaluation: after completion of fluorescein scoring, the cornea was fixed for 24h by adding to 10% neutralized formalin buffer. Paraffin embedding, slicing, taking partial section and performing HE staining. Observing and evaluating histological changes under a microscope; taking partial sections to perform immunohistochemical staining, such as staining of corneal epithelial cell tight junction protein Occludin and ZO-1, and detecting a cell proliferation marker Ki 67.
4. Results of repair action
4.1 turbidity assay
Turbidity value OP2h23, the turbidity change was greater than or equal to 15 and less than 25 compared to the blank OP0h, and moderate damage was considered to have occurred; the turbidity changes at different time points are shown in table 3.
4.2 resistance measurement and fluorescein staining
Treating cornea with 3% H2O2SLS for 10min, and detecting cornea resistance value of 3.34 Ω 2H after exposure; over 70% of corneal area staining was seen by fluorescein sodium staining, the area of injury was > 50 and < 75%, scoring 3 points; the scoring results for the groups with 12h, 26h, 50h and 98h at different time points for the groups with enhanced and natural repair are shown in table 3.
4.3 cytokine detection
The level of the inflammatory factor IL-1b in the culture broth was measured and is shown in Table 3. The inflammatory factors in the repair promoting group begin to decline after reaching the peak value in 14 hours, the peak value is smaller than that in the natural repair group, and the decline amplitude is larger than that in the natural repair group.
4.4 histological observations
Histological examination: after the cornea is treated by 3% H2O2 for 2 hours, the loss of epithelial cells is not obvious, a small amount of cells fall off, and the structures of zon-1 and Occludin are basically complete; a large amount of vacuoles of the matrix layer caused by oxidative damage appear, and are closely related to the rise of turbidity value; after 14 hours of exposure, the vacuoles of the matrix layer begin to decrease, the accelerated repair group only sees a small amount of vacuoles of the partial matrix layer after 26 hours of exposure, and after 50 hours of exposure, the epithelium and the matrix layer are basically intact; after 98h exposure, corneal tissue structure was essentially normal, suggesting complete restoration of the corneal epithelium.
Table 3 corneal recovery and scoring after H2O2 chemical stimulation
5. Conclusion
The culture solution containing the epidermal growth factor has a promoting effect on the repair of the cornea after chemical injury caused by H2O 2.
Example 4 evaluation of epidermal growth factor on repair of bovine corneal UV injury
1. Preparation and culture of isolated bovine and porcine cornea
1. Ex vivo corneal preparation and culture
1.2 preparation of ox horn membrane: the eyeballs are picked from the eye sockets within 5 hours after the cattle are slaughtered, and the cornea is prevented from being damaged in the separation process. Ex vivo bovine eyeballs were shipped to the laboratory completely immersed in HBSS containing penicillin/streptomycin at 4 ℃. The bovine eye was examined for status before cutting the cornea and discarded if any significant vascular changes, pigmentation, turbidity, grape sores or mechanical scratches were observed. The cornea was excised from the intact eyeball and the limbus was left with a 2-4mm intact circular sclera. The excised cornea was washed 3-5 times successively with 5-7ml of sterile HBSS.
1.2 gel filling: taking a clean ox horn membrane, suspending the corneal epithelium side downwards in a 12-well plate, adding HBSS into culture wells to support the suspended state of the cornea, and slowly dropwise adding (2-3 drops each time) a mixture of agar-gelatin-M199 in a molten state to the corneal pit endothelium side to make the gel directly contact with the endothelium. The filling procedure was repeated until the endothelial cell was completely filled and the agar-gelatin completely solidified at room temperature. The cornea filled with the solidified gel was then inverted and transferred to a 12-well plate.
1.3 corneal culturing: approximately 60ml of M199 medium was aspirated into each dish until the limbus was covered and the bare corneal epithelial surface was exposed to air. Placing the culture dish on a small-sized oscillator, and placing the culture dish at 37 ℃ and 5% CO2And culturing in an incubator with 90% relative humidity for about 24 +/-2 h. The mini-shaker programmed the dish to shake from a horizontal position to an angle of about 45 degrees every 2.75 hours so that the cornea could be briefly soaked in the medium to infiltrate the epithelial tissue.
2. Ultraviolet exposure
After the cornea was cultured for about 24 hours, the culture solution was aspirated from the dish. A Teflon resin O-ring with a diameter of 1.3cm was placed on the cornea. Irradiating the cornea with UVB at a dose of 60mJ/cm2Time 10 minutes, after exposure was complete, the cornea was transferred to a new dish and 40ml of fresh M199 medium containing 50 μ M/ml recombinant human epidermal growth factor was added to each dish. The culture was continued to day 21 as in step 1.3 above, and the repair of the damaged cornea was observed by changing the culture medium of M199 containing growth factor every day. M199 culture solution without growth factor is used as a natural repair control.
3. Evaluation of Effect of promoting repair of Damage
3.1 detection of damage degree: 2 hours, taking 2 corneas of the damaged group and the blank control group for turbidity, fluorescein sodium staining and histological flaking, and evaluating the stimulation degree; the remaining cornea was immersed in a culture dish containing 40ml of M199 medium and placed at 32. + -. 2 ℃ with 5% CO2An incubator at 90% relative humidity;
3.2 repair assessment: culturing for 2h, 14h, 26h, 50h and 98h, taking 2 corneas of the damaged group and the blank control group for turbidity determination, resistance value determination, fluorescein sodium staining, inflammatory factor detection and histological slide making, and evaluating the self-repairing degree;
and (3) turbidity detection: before detection, the Teflon O-ring is removed and fixed in a special cornea holder. Fresh MEM medium is then added in the order of filling the first chamber and then refilling the first chamber. The opacity of each cornea was measured using an turbidimeter and the opacity value (OP) was recorded as OPStimulating for 0hOr OPRepair for 0hMeanwhile, a blank group of corneas are taken for turbidity test, and the turbidity is recorded as OPBlank space。
Testing of corneal fluid inflammatory factor: before replacing new culture solution, removing the culture solution from the culture dish, and detecting the levels of inflammatory factors and repair factors including IL-1a, IL-6, IL-8 and the like in the culture solution by using the kit;
and (3) fluorescein staining: after turbidity was measured, the cornea was removed from the holder and PBS containing 2% sodium fluorescein (NaFL) was added dropwise to the upper surface. Excess NaFL was immediately flushed with PBS and NaFl retention was observed under UV light, the extent of retention indicating the severity of corneal injury. Calculating the percentage value of the retention area (S) in the whole cornea area (C), and scoring the damage/recovery degree according to the S/C value. 0 minute: <2%, 1 point: 2-25%, 2 min: 26-50%, 3 min: 51-75%, 4 min: 76 to 100 percent.
Histological evaluation: after completion of fluorescein scoring, the cornea was fixed for 24h by adding to 10% neutralized formalin buffer. Paraffin embedding, slicing, taking partial section and performing HE staining. Observing and evaluating histological changes under a microscope; and taking a part of the section to perform immunohistochemical staining, such as staining of corneal epithelial cell tight junction protein Occludin and ZO-1, and observing the improvement condition of the epithelial barrier function in the corneal repair process.
4. Observation of repair Effect results
4.1 turbidity assay
Turbidity value OP2h14, the turbidity change was greater than or equal to 5 < 15 compared to the blank OP0h, and mild injury was considered to have occurred; 4.2 resistance measurement and fluorescein staining
Treating cornea with UVB irradiation for 10min, and detecting cornea resistance value of 0.87 omega 2h after exposure; more than 55% of corneal area staining can be seen by fluorescein sodium staining, the damaged area is more than 50 and less than 75%, and the score is 3; staining of approximately 45% of corneal area after 26h, scoring 2 points; about 20% in 50h, and the score is 1; almost no staining of the corneal area was visible at 98h, suggesting a restoration of the corneal epithelium.
4.3 cytokine detection
Changes in macrophage inflammatory protein (MIP-1) in the culture broth were measured as shown in Table 4.
4.4 histological observations
Histological examination: after the cornea is treated by UVB for 2 hours, the loss of epithelial cells is not obvious, a small amount of cells drop off, and the structures of zon-1 and Occludin are tightly connected; but a large amount of vacuoles of the matrix layer caused by oxidative damage appear, and are closely related to the rise of turbidity value; 26h after exposure, the accelerated repair group showed that the damaged area migrated from epithelial cells to basement membrane; after exposure for 50h, only eosinophilic hyperkeratocyte of corneal epithelial cells is increased, which indicates that the epithelium is recovered obviously; after 98h exposure, corneal tissue structure was essentially normal, suggesting complete restoration of the corneal epithelium.
Table 4 corneal recovery and scoring after UVB irradiation
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention. For example, the present invention only exemplifies a mode of corneal damage such as partial chemical, ultraviolet ray, and mechanical damage, and an example of promoting corneal repair such as epidermal growth factor and basic fibroblast growth factor. However, other substances mentioned in the present invention, such as chemical irritants (chlorhexidine, 5% benzalkonium chloride, dichlorobenzoyl chloride, trichloroacetic acid, etc.), consumer products with known eye irritation, ultraviolet rays with different dosages and wavelengths, mechanically-induced corneal epithelial abrasions (such as nail, branch, contact lens, etc. foreign body scratches), corneal foreign body injuries (such as scrap iron, chaff, sand, dust, etc.), corneal surgeries (such as laser treatment of myopia, minimally invasive resection), etc. can also cause eye irritation injuries by using the method of the present invention, and vitamins, amino acids, small molecule peptides, etc. can also be detected by using the method of the present invention to promote repair. This is not further enumerated here.
Claims (7)
1. A method for evaluating eye irritation injury and repair by long-term culture of animal cornea is characterized by comprising the following steps:
step 1, preparing an in vitro cornea;
step 2, cornea long-term culture:
taking a cleaned cornea, and suspending the cornea with the epithelial surface facing downwards in a 6-hole plate, a 12-hole plate or a culture dish with the diameter of 35mm, which is added with HBSS liquid, so that the cornea is completely immersed in the culture liquid;
slowly dropwise adding 0.5-5% agar-gelatin-M199 mixture in a molten state into the inner surface of the corneal fossa 2-3 drops at a time until the inner fossa is completely filled, and completely solidifying the agar-gelatin-M199 mixture at room temperature;
inverting the cornea filled with the coagulated agar-gelatin-M199 mixture and transferring to several 35mm petri dishes;
aspirating 40-60ml of M199 medium into each dish until the limbus is covered and the bare corneal epithelial surface is exposed to air;
place the Petri dish on a Mini shaker at 32oC±2oC、5%CO2Culturing for 8-24h in an incubator with 90% relative humidity, and shaking the culture dish to an angle of 45 degrees from a horizontal position within every 2-3h by using a small oscillator so that the cornea can be soaked in the culture medium for infiltrating epithelial tissues for a short time;
step 3, a cornea chemical damage model and evaluation;
step 4, cornea physical damage model and evaluation;
step 5, evaluation of the repairing effect after corneal injury:
(1) after the compound damages the cornea, the cornea is transferred to a new culture dish, 40ml of new M199 culture medium is added into each culture dish, and then the culture dish is incubated for 2 hours; replacing M199 culture medium containing active substance epidermal growth factor, basic fibroblast growth factor or vitamins, and placing at 32oC ±2oC、5% CO2An incubator with 90% relative humidity, and continuously culturing the cornea to the 14 th day according to the cornea long-term culture method in the step 2; 20-30 parallel samples of the experimental group; m containing active substances199 culture medium is active repairing group, M199 culture medium without active substance is natural repairing group, and blank control or reference control is set at the same time;
(2) during the culture period, the M199 culture solution containing the active substances is replaced every 2 days; respectively taking culture solution to perform cytokine test at the time points of 2h, 14h, 26h, 38h, 50h, 62h, 74h, 86h, 98h, 110, 170h and 14d +/-2 h after chemical injury, taking 2-3 corneas in the experimental group to perform turbidity test, resistance test, fluorescein score and histological observation, and continuously culturing the rest corneas;
(3) corneal fluid cytokine assay: before replacing with new medium, the medium was removed from the culture dish and the levels of inflammatory and repair factors in the medium were measured: including IL-1a, IL-6, IL-8;
(4) turbidity change: before turbidity measurements, the cornea was removed from the petri dish and fixed in a holder, the rear and front chambers of which were filled in turn with sterile phenol red-free MEM medium, respectively, and the turbidity values of each cornea were measured and recorded with a keratometer, and the turbidimetry values, OP, were recorded separately for corneas from different groupsActivity h，OPRepair h，OPBlank space(ii) a Calculating the turbidity difference between the experimental group and the blank group;
(5) resistance measurement and fluorescein observation: after the turbidity is measured, measuring the resistance value of the cornea by using a resistance meter; then taking the cornea from the holder, and dripping PBS containing 2% fluorescein sodium NaFL on the upper surface; excess NaFL immediately washed with PBS, under ultraviolet lamp observation of NaFL retention, retention degree indicates the severity of corneal injury;
calculating the percentage value of the retention area S in the whole corneal area C, and scoring the damage/recovery degree according to the S/C value; 0 minute: <2%, 1 point: 2-25%, 2 min: 26-50%, 3 min: 51-75%, 4 min: 76% -100%;
(6) histological evaluation: after the fluorescein scoring is finished, adding the cornea into 10% neutral formalin buffer solution for fixation for 24 hours; embedding paraffin, slicing, and taking part of the slices for HE staining; observing and evaluating histological changes under a microscope;
taking a part of the section for immunohistochemical staining: staining of corneal epithelial cell tight junction protein Occludin or ZO-1 or staining of cadherin, which is used for observing the improvement of epithelial barrier function in the corneal repair process;
step 6, statistical analysis and result judgment
Expressing each group of experimental data obtained in the steps by mean +/-standard deviation, analyzing by SPSS22.0 statistical software, comparing differences among groups by single-factor variance analysis, comparing every two groups by SNK test method, and obtaining significance levelα=0.05，p＜0.05Indicating that the difference is statistically significant;
after chemical injury, repairing and comprehensively analyzing cornea resistance values, fluorescein staining, turbidity testing, inflammatory factor detection and histological observation results; the mechanical damage repair is not carried out with resistance test and turbidity test; repairing the reference chemical damage after the ultraviolet damage;
according to the significance of different detection parameters, comprehensive judgment of the repairing effect is made through statistical result analysis:
(1) resistance measurement and fluorescein staining: along with the prolonging of the culture time, the resistance values of the natural restoration group and the active restoration group gradually increase; the ratio of S/C of fluorescein staining gradually decreased, and the score decreased; compared with a blank control, the ratio of S/C of the natural repair group to the active repair group is statistically differentpLess than 0.05; compared with the natural restoration group, the increase of the resistance value of the active restoration group has statistical significance, the grading value of retention of fluorescein is different from 1 grade or more, and the active substance is considered to have promotion effect on restoration;
(2) turbidity test: the values of turbidity OP of the natural restoration group and the active restoration group are gradually reduced along with the prolonging of the culture time; the turbidity difference of the repair group compared with the blank control is gradually reduced; compared with the natural restoration group, the activity restoration group has more than 2 time points, the turbidity difference has statistical difference p less than 0.05, and the active substances are considered to have promotion effect on restoration;
(3) inflammatory factor detection: the values of inflammatory factors of a natural repair group and an active repair group are in a descending or ascending trend along with the prolonging of the culture time, and the statistical difference p is less than 0.05; at the same time, compared with the natural repair group, the level of the characteristic inflammatory factor or repair factor of the active repair group is statistically different, which indicates that the active substance has a promoting effect on repair;
(4) and (3) histological observation: histological changes of the cornea were observed by HE staining and immunohistochemical staining, and detailed recordings were made:
after the chemical irritation injury of the cornea occurs, the histology shows that the epithelium is obviously damaged, the epithelial cells are deleted, the permeability is increased, and the structures of zon-1 and Occludin of tight junction protein disappear; over time, the epithelial cells proliferate and migrate to the defect, the permeability of the epithelial cells gradually decreases, and the tight junction protein structure gradually forms; after 24h, epithelial cells cover the corneal epithelial defect, but the permeability of the epithelial cells is still higher than that of a normal group, and the tight connection between the cells is loose and the arrangement is disordered; after 48 hours, the corneal permeability is recovered to be normal, the corneal epithelial cells are tightly connected, and a tight connection structure is formed among the cells; if the repair promoting group is compared with the natural repair group, the histological repair time is shortened by more than 12 hours or the repair degree is improved, namely the observation time of similar histological structures and characteristics is advanced by more than 12 hours, which indicates that the active substances have promotion effect on repair;
step 4, the physical stimulation injury cornea model and evaluation comprise the following sub-steps:
(1) after the cornea is cultured for 24 hours, the culture solution is sucked out of the culture dish; placing a 1.0cm diameter Teflon O-ring on the cornea; scraping the central epithelium, the deep epithelium and the corneal epithelial cell basal layer with the diameter of 8mm from the center of the cornea of the eye by using a sterile ophthalmic surgical blade to prepare a mechanical corneal injury model; 6-10 corneas were used for each injury model; 2 corneas from each model were subjected to the following irritation level evaluation test:
(2) and (3) fluorescein observation: the same as step 5 (5); NaFL staining revealed damaged cornea, the size of the yellow areas indicating the severity of the damage;
(3) histological evaluation of physical stimulus model introduction: the same as step 5 (6);
(4) evaluation of physically damaged cornea:
if the fluorescein staining score does not meet the condition that the damage area is more than 50 percent and less than 75 percent, the damage degree is considered to be too serious and the fluorescein staining score cannot be used for damage repair evaluation;
histological examination: after corneal injury, obvious epithelial defect appears, which is represented by epithelial cell deletion, and the structures of zon-1 and Occludin disappear; the upper layer of the matrix layer is broken or lost, and histological damage caused by physical factors corresponds to fluorescein dyeing;
(5) the remaining cornea was immersed in a petri dish containing 40-60ml of M199 medium and placed at 32 deg.CoC±2oC、5%CO2And (3) continuously culturing the cornea in the incubator with the relative humidity of 90% according to the cornea long-term culture method in the step (2) to carry out subsequent natural repair or repair promotion research;
(6) and simultaneously setting a negative control, wherein the negative control is deionized water, and the same operation steps are adopted.
2. The method for evaluating the irritating injury and repair of eye by long-term culture of animal cornea as claimed in claim 1, characterized by: step 1 ex vivo corneal preparation comprising the following substeps:
the eyeballs are picked from the eye sockets within 2-3 hours after slaughtering of slaughterhouse animals, and the cornea is prevented from being damaged in the separation process;
completely immersing the isolated eyeball in ice-cold HBSS balanced salt solution containing antibiotics and conveying the isolated eyeball to a laboratory;
the excision of the cornea is carried out in a sterile area in a laboratory, the integrity of the cornea is checked by naked eyes before the cornea is cut, whether scratches, pigmentation and turbidity exist or not is checked, and the eyeballs with the defects are discarded;
soaking the pig eyeball in 1% povidone iodine solution for 2min, washing with sterile PBS, soaking in PBS containing 0.1% gentamicin for 15min for sterilization, and performing corneal resection;
bovine cornea was isolated directly from the intact eye, leaving a 2-4mm intact circular sclera at the corneal limbus when the cornea was isolated, and excised cornea was rinsed 3-5 times with 5-7ml sterile HBSS in succession.
3. The method for evaluating the irritating injury and repair of eye by long-term culture of animal cornea as claimed in claim 1, characterized by: step 3 compound stimulation injury corneal model and evaluation includes the following substeps:
(1) after the cornea is cultured for about 4-24h, beginning a formal experiment, and sucking out the culture solution from the culture dish; placing a polytetrafluoroethylene resin O-shaped ring with the diameter of 1.0-1.5 cm on the corneal epithelium surface;
the test substance is chemical liquid with proper concentration expected to generate stimulation, 40-100ul of liquid stimulation is taken and added into the ring, and exposure is carried out for 10-20 min; if ultraviolet rays are selected as a damage model, the wavelength is UVB, and the irradiation dose is 40-70mJ/cm 2;
after the exposure is finished, gently rinsing the cornea with 2-3ml of PBS for 2-3 times until the residues of the test substances are completely removed; it was immersed in a petri dish containing 40-60ml of M199 medium, 32%oC±2oC、5% CO2The incubator with 90% relative humidity was incubated for 2 hours; 6-10 corneas were used for each injury model;
(2) corneal haze change: after the incubation is finished, taking 2 corneas, removing the polytetrafluoroethylene resin O-shaped ring, and fixing in a cornea holder; then adding fresh MEM culture solution in the sequence of filling the first chamber and then filling the second chamber and then filling the front chamber; the opacity of each cornea was measured with an turbidimeter, and the opacity value OP was recorded as OPStimulating for 0hOr OPRepair for 0h；
Meanwhile, a blank group of corneas are taken for turbidity test, and the turbidity is recorded as OPBlank space；
(3) Measurement of resistance value and fluorescein observation: the same as step 5 (5);
(4) histological evaluation: the same as step 5 (6);
(5) evaluation of chemically damaged cornea:
if the turbidity value changes by more than or equal to 3 and less than 10, the mild-moderate injury is considered to be caused;
if the turbidity value is more than or equal to 10 and less than 25, the medium-serious damage is considered to be caused;
if the turbidity value is more than or equal to 25, the damage degree is considered to be too serious, and the turbidity value cannot be used for the repairability test;
if the resistance value is more than 1 and less than or equal to 5, the corneal barrier function is considered to be damaged, if the resistance value is less than or equal to 1, the damage is considered to be too serious and can not be used for a repair test, and the resistance value of a blank group is more than 5;
if the fluorescein staining score does not meet the condition that the damage area is more than 50 percent and less than 75 percent, the damage degree is considered to be too serious and the fluorescein staining score cannot be used for damage repair evaluation;
histological examination: after corneal injury, epithelial defects of different degrees appear, and the epithelial cells are exfoliated, and the structures of zon-1 and Occludin are reduced and disappear; edema of different degrees of the stroma layer and disorganized arrangement of collagen bundles; chemical substances damage the stromal layer, while epithelial barrier function is not significantly impaired; histological lesions should correspond to turbidity tests and fluorescein staining;
(6) the remaining cornea was immersed in a petri dish containing 40-60ml of M199 medium and placed at 32 deg.CoC±2oC、5%CO2And (3) continuously culturing the cornea in the incubator with the relative humidity of 90% according to the cornea long-term culture method in the step (2) to carry out subsequent natural repair or repair promotion research;
(7) and simultaneously setting a blank control and a negative control, wherein the negative control is deionized water, and the same operation steps are adopted.
4. The method for evaluating the irritating injury and repair of eye by long-term culture of animal cornea as claimed in claim 1, characterized by: any of steps 1-4, which relate to the isolation and long-term culture of the cornea:
the in vitro cornea is obtained by separating the discarded pig or cattle eyeballs in a slaughterhouse;
agar-pigskin gelatin-M199 mixture containing 1% low freezing point agar, 1% pigskin gel and M199 medium, maintained at 37 deg.C;
HBSS buffer, which refers to commercial Hank's balanced salt solution; the M199 culture medium is an M199 culture medium, 10% of calf serum, gentamicin, penicillin/streptomycin are added, and amphotericin B is also added in the culture of the pig cornea; povidone iodine is used as a preservative, and the concentration is 1%;
the O-shaped ring is a white ring prepared from polytetrafluoroethylene resin inert materials;
if the ring is 9-11mm in outer diameter and 8-10mm in inner diameter, the sample loading amount is 40 mu L; if the ring is 13-15mm in outer diameter and 11-13mm in inner diameter, the sample loading amount is 100 mu L; for fixing the test object and defining the observation area.
5. The method for evaluating the irritating injury and repair of eye by long-term culture of animal cornea as claimed in claim 1, characterized by: any one of steps 3-4, which relates to a corneal injury method, wherein:
chemical injury refers to chemical stimulation of cornea caused by chemical substances entering eyeballs, common stimulators are 1.5% -3% of SLS, 0.2-0.4 mol of NaOH, 3% -6% of trichoro-acetic acid TCA and 1.5% -3% of H2O2, the exposure time of the chemical substances is 10-20 min, and the chemical injury is selected according to the type and degree of the chemical stimulators: selecting short duration of action for compounds with moderate to severe eye irritation, longer duration of action for compounds with weak to moderate eye irritation, or determining exposure time using pre-experiments;
UV damage, which is a physical factor, refers to corneal damage caused by UVB or UVA, and the dose of UVB irradiation is 40mJ/cm2-70 mJ/cm2The UVA irradiation dose is 10J/cm2-50J/cm2；
Mechanical damage, according to the research purpose, a puncher with the diameter of 2-3mm is selected to damage the corneal epithelium and the stroma surface layer but not the corneal stroma deep layer; a blade, a needle or a blunt instrument is selected to slightly cut, scratch, wipe, grind or peel off the surface of the cornea without damaging the deep layer of the cornea; other physical means should be chosen to ensure that the procedure is well-controlled and that the individual differences between corneas are as small as possible.
6. The method for evaluating the irritating injury and repair of eye by long-term culture of animal cornea as claimed in claim 1, characterized by: any one of steps 5-6, relating to the detection and assessment of corneal damage repair:
the number of parallel corneas for the restoration of the active substance is detected, and depending on the parameters of the time points of detection, if 5 detection time points including 2h, 14h, 26h, 38h, 50h, 62h, 74h, 86h, 90h, 110h, 170h and 14d +/-2 h are considered, and 2-5 corneas are taken for testing at each time, the number of the corneas of the experimental group is at least 10-25; the repair-promoting group, placebo and baseline controls should also employ a corresponding number of corneas;
corneal resistance is a characteristic of the response barrier function, measured with a low voltage alternating-current wheatstone bridge;
haze values are measured using a corneal turbidimeter, which reflects primarily the integrity of the corneal stroma layer;
detecting the repairing effect of the active substance after mechanical damage, and not considering the turbidity test;
inflammatory or repair factors, refers to specific substances secreted by corneal cells during corneal repair, including inflammatory cytokines: IL-1 α, IL-1 β, IL-6, IL-7, IL-10, IL-12, IL-15, MIP-1 α, MIP-1 β, MIP-2, TNF- α or PGE2, a proliferative factor, an enzyme: LDH;
immunohistochemistry refers to staining a histological section by using the principle of antigen-antibody binding reaction with characteristic markers, wherein the markers indicating the corneal barrier function comprise zonulin ZO-1, Ocgludin and cadherin, polyclonal rabbit antibody bovine or porcine antibody is selected as a primary antibody, and the primary antibody is diluted by 1: 100-1: 200; selecting a second antibody as a mouse anti-rabbit or goat anti-rabbit antibody;
measuring the content of the collected cells and corresponding culture media by using a commercial kit comprising flow cytometry, ELISA and immunohistochemical methods, comparing with a test control group, detecting the generation condition of target secretion, and statistically analyzing whether the increase or decrease of certain secretion in the repair process has significant change;
experimental control means that no blank containing active substance was performed;
the repair-promoting action of the active substance was evaluated, and natural repair or a reference substance, which is known to have a definite repair-promoting action, was used as a control.
7. The method for evaluating the irritating damage and repair of the eye by long-term culture of the animal cornea as claimed in any one of claims 1 to 6, characterized in that:
the active substance for promoting cornea repair is prepared by adding alkaline fibroblast growth factor, deproteinized calf blood extract, recombinant epidermal growth rhEGF or vitamin A palmitate into culture medium, or directly using medicinal eye drop;
the statistical method is characterized in that each group of obtained experimental data is expressed by mean +/-standard deviation, SPSS22.0 statistical software is adopted for analysis, the difference among multiple groups is analyzed by single-factor variance, the two groups are compared by an SNK (selective non-subsampling) test method, and the significance level is improvedα=0.05，p＜0.05Indicating that the difference is statistically significant;
the chemical injury repair should comprehensively analyze fluorescein staining, turbidity test, inflammatory factor detection and histological observation; the weights are as follows from big to small: fluorescein staining > turbidity test > histological observation > inflammatory factor detection;
the physical damage repair should comprehensively analyze fluorescein, inflammatory factor detection and histological observation; the weights are as follows from big to small: fluorescein staining > histological observation > inflammatory factor detection.
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| CN110568173B (en) * | 2019-07-26 | 2022-09-27 | 广州市华代生物科技有限公司 | Method for predicting eye irritation by using combination of various corneal structural cells |
| CN112574884A (en) * | 2020-11-19 | 2021-03-30 | 深圳先进技术研究院 | Multifunctional organ chip based on microfluidic technology, preparation method and application thereof |
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| CN113974888A (en) * | 2021-11-11 | 2022-01-28 | 苏州药明康德新药开发有限公司 | Preparation method of rat disease model of neurotrophic keratitis |
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