CN107796674A - A kind of method assessed using the damage of animal isolated cornea long-term cultivation model evaluation eye irritation and repair - Google Patents
A kind of method assessed using the damage of animal isolated cornea long-term cultivation model evaluation eye irritation and repair Download PDFInfo
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- CN107796674A CN107796674A CN201710538216.7A CN201710538216A CN107796674A CN 107796674 A CN107796674 A CN 107796674A CN 201710538216 A CN201710538216 A CN 201710538216A CN 107796674 A CN107796674 A CN 107796674A
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Abstract
A kind of method assessed using the damage of animal isolated cornea long-term cultivation model evaluation eye irritation and repair, is comprised the following steps：1. prepared by isolated cornea；2. cornea long-term cultivation；3. corneal chemical damage model and evaluation；4. cornea physical damnification model and evaluation；5. repair is assessed after corneal injury；6. statistical analysis and result judgement.The present invention utilize exogenous irritant, as chemical substance, ultraviolet cause the denaturation of reversible corneal epithelial cell and matrix damage, by forecast model, assesses the degree of eye irritation；The reversible sexual stimulus, mild wear or missing for either causing corneal epithelial cell and matrix using chemical method, physical method or mechanical means damage, itself reparation of cornea after damaging is assessed, or the method that drug evaluation medicine promotion corneal restoration acts on is added after damage generation.This method is simple to operate, good with experiment in vivo correlation, and sensitivity is high, can meet the needs of corneal irritancy damage and its detection and research repaired.
Description
Technical field
The present invention relates to a kind of toxicology applied to chemicals, cosmetics, agricultural chemicals and medicine eye irritation and recovery and
Pharmacological experiment evaluation utilizes the damage of the isolated cornea long-term cultivation model evaluation eye irritations such as animal such as pig or ox and reparation
Act on the method assessed.
Background technology
Many exogenous chemical substances, such as cosmetics, medicine, agricultural chemicals and atmosphere pollution, it is possible to eyes can be touched
Cause the health hazard of human eye.So detection and assessment medicine, daily necessities (such as personal care articles, household chemistry
Product, cleaning-sterilizing product etc.), environmental contaminants (such as agricultural chemicals, flue dust, particulate matter, pollen) potential eye irritation turn into ensure
The important content of personal safety.Eye irritant test is the forced examination project of safety evaluation before the listing of eye contact product, and
Chemicals identifies and the compulsive requirement of classification, and the purpose is to use scientific method to detect and judge Product Safety to reduce people
Class health risk.Therefore the Fast Classification to determinand and harm identification are the top priorities of decision in the face of risk.For Eye irritation thing
Toxicological evaluation, conventional method continue to use nineteen forty-four Draize proposition lagophthalmos stimulate experiment in vivo, lagophthalmos test the shortcomings that wrap
Include animal by considerable distress, the test period is long, inspection cost is high, result judgement subjectivity is strong, animals and human beingses precursor reactant is present
Difference etc..
On the other hand, the treatment of clinical common ocular surface burns or mechanical injuries, or Post operation (such as foreign body on cornea
Reject that postoperative, quasi-molecule is postoperative or postcataract) usually require to promote corneal healing using some medicines, to these medicines
Whether there is excitant, and its promote the observation of corneal restoration effect and the basic demand of pharmacological research.Traditional evaluation side
Method causes corneal defect or excitant damage model, then by checking cornea shape after applying medicine usually using living animal
State, 26S Proteasome Structure and Function, assess the effect of medicine.Zoopery generally existing animal extremely pain, test period length, operation is steady
The deficiencies of qualitative poor, research cost is high and human response has differences.
With life science and examine in detection " 3R principles ", that is, reduce (reduction), replace
(replacement) and optimization (refinement) zoopery principle internationalization, particularly " 21 century toxotest is willing to
The it is proposed of scape " and the development of new technology, the test system no longer based on zootype have turned into the focus of scientific research,
Some detections and the method assessed are received by many national regulations.Such as European Union's cosmetics regulation《1223/2009/EC》
For forbidding for cosmetics zoopery, EU chemicals REACH regulations《1907/2006/EC》(registration of chemicals, comment
Estimate, permit and limit) encourage animal experiment alternative development and application.Eye is carried out using extracorporeal experiment system and method
Excitant evaluation has obtained howling success, for acute irritation and corrosive test, the development institution (OECD) of economic cooperation
Certain methods are had confirmed that and be recognized, include the method for in vitro ox horn film, the short-term process for exposing of keratocyte, epithelial cell
Fluorescein breakthrough method and rebuild the method etc. of cornea model.How these methods are scientifically rationally used, and on this basis
New test system and appraisal procedure are further innovated, existing method and deficiency is solved, is still ophthalmology toxicology and pharmacology
The main research of evaluation.
The mechanism occurred according to internal eye injury, the Eye irritation alternative in developing at present can be divided into cell system, chicken
Embryo, artificial cornea and the class of isolated organ four, this few class method respectively have its advantage and disadvantage and the scope of application.Cell system standardizes journey
It is low, quick to spend highest, cost, but is only applicable to the test of alkaline soluble materials.The standardization level of chicken embryo method is not high, is adapted to
The damage of conjunctiva is simulated, and corneal damage forecast is bad.The artificial cornea of reconstruction in vitro lacks endothelial cell layer, structure
This height is built up, operation difficulty is big.Moreover, these above-mentioned methods all cannot be used for the research and survey of restitution after corneal injury
Examination, because the material of some minimal irritations can cause the of short duration reversible lesion of cornea, only Time in Vitro is longer
Pilot system could detect this effect.In addition, wiped for cornea mechanical injuries caused by a variety of causes, such as corneal epithelium
Wound (such as nail, branch, contact lenses foreign matter scratch), High-risk (such as iron filings, husk, dust storm, dust), cornea
Perform the operation (such as laser treatment of myopia eye, minimally invasive excision), it is also desirable to which exploitation promotes the medicine of corneal restoration and studies its effect machine
System.And use the in-vitro method such as monolayer cell culture, chick embryo culture or artificial cornea be unable to analog cornea complete structure and
Mechanical injuries.
The isolated cornea that the present invention uses, the animal eyeball discarded from animal husbandry, cuts active cornea tissue and is trained
Support and test, in addition to it can be used for substituting interior animal experiment detection corneal irritancy, after can be also used for corneal damage
The inspection and assessment of recovery capability.In addition, the cornea from larger animal is more properly used for eye compared with the cornea of rabbit, mouse or chicken
The cornea tissue structure of irritating test, particularly pig is the preferable mould for studying the effect of angle corneal restoration closer to the mankind
Type.The in-vitro method for being currently based on pig and the foundation of ox isolated cornea model yet there are no Patents and document report.
The content of the invention
The technical problems to be solved by the invention, just it is to provide one kind using in vitro porcine cornea or ox horn membrane modle evaluation eye
The method that excitant and repair are assessed, this method is general, quick, easy, cost is low, to detecting instrument without particular/special requirement.
Solves above-mentioned technical problem, the technical solution adopted by the present invention is as follows.
The above-mentioned purpose of the present invention is achieved by the following technical solution：One kind utilizes isolated cornea long-term cultivation system
The method that system evaluation eye irritation damage and repair are assessed, it is comprised the steps of：
A kind of method assessed using the damage of animal isolated cornea long-term cultivation model evaluation eye irritation and repair,
It is characterized in that comprise the following steps：
It is prepared by step 1. isolated cornea；
Step 2. cornea long-term cultivation：
The cornea cleaned up is taken, epithelium is suspended in the culture of 6 orifice plates or diameter 35mm added with HBSS liquid down
Ware, cornea is set to be totally submerged in nutrient solution；
The 0.5-5% of state agar-gelatin-M199 mixtures will be melted, slowly each 2-3 is added dropwise to corneal pocket dropwise
Interior surface, until endothelium nest is fully filled with, agar-gelatin solidifies completely at room temperature；
The cornea that will be filled with the agar-gelatin of solidification is inverted, and is transferred in some 35mm culture dishes；
The M199 culture mediums for drawing 40-60ml are added in each culture dish, until covering corneal limbus, exposed corneal epithelium
Face is exposed in air；
Culture dish is placed on mini-vibrator, is placed in 32 DEG C ± 2 DEG C, 5%CO2, 90% relative humidity incubator in
8-24h is cultivated, mini-vibrator should be such that culture dish is shaken in every 2-3h from horizontal level to 45° angle so that cornea can be with
Of short duration immersion infiltrates epithelial tissue in the medium；
Step 3. corneal chemical damage model and evaluation；
Step 4. cornea physical damnification model and evaluation；
Repair is assessed after step 5. corneal injury：
(1) after compound corneal damage, cornea is transferred in new culture dish, the M199 culture mediums for taking 40ml new add
Enter in each culture dish, it is rear to be incubated 2 hours；Change containing active material EGF, basic fibroblast growth because
The M199 culture mediums of son or vitamin, are placed in 32 DEG C ± 2 DEG C, 5%CO2, 90% relative humidity incubator, by step 2 cornea
Long-period culture method continued culture to the 14th day；Experimental group Duplicate Samples 20-30；M199 culture mediums containing active material are work
Property reparation group, the M199 culture mediums without active material are nature reparation group, while set blank control；Benchmark is set if necessary
Control；
(2) during cultivating, the M199 nutrient solutions containing active material are changed within every 2 days；2h after chemical damage,
14h 26h, 50h, 74h, 98h, 170h and 14d ± 2h, take nutrient solution to carry out cytokine test, and take experimental group wherein 2-3
Cornea carries out turbidity test, fluorescein stays scoring and histological observation, remaining cornea to continue to cultivate；
(3) liquor coneae cytokine test：Before the nutrient solution more renewed, nutrient solution, detection training are removed from culture dish
Inflammatory factor and reparative factor in nutrient solution is horizontal：Including IL-1a, IL-6, IL-8；
(4) turbidity changes：Before Turbidity measurement, cornea is taken out from culture dish, is fixed in clamper, clamper
Rear chamber and cup be sequentially filled respectively it is sterile be free of phenol red MEM culture mediums, measured with cornea transmissometer and record each angle
The turbidity value of film, turbidity value, OP are recorded respectively from different groups of corneasActive h, OPRepair h, OPBlank；Experiment with computing group and blank group
Turbidity difference；
(5) resistance measurement and fluorescein observation：After having surveyed turbidity, the resistance value (Ω) of cornea is determined with resistance instrument；Then
Cornea is removed from clamper, upper surface instills the PBS containing 2% fluorescein sodium (NaFL)；Excessive NaFL is rushed with PBS immediately
Wash, NaFl retention is observed under uviol lamp, the degree of retention shows the seriousness of corneal injury；
The percent value that retention area S accounts for whole cornea area C is calculated, damage/recovery extent is carried out according to S/C values
Scoring；0 point：<2%, 1 point：2~25%, 2 points：26~50%, 3 points：51~75%, 4 points：76%~100%；
(6) Histological evaluation：After the completion of fluorescein scoring, cornea is taken to add in 10% and consolidate in formalin buffer
Determine 24h；FFPE, cut into slices, take partially sliced progress HE dyeing；Micro- Microscopic observation simultaneously assesses histological change；
Take partially sliced carry out immunohistochemical staining：Corneal epithelial cell Tight junction protein occludin or ZO-1's
Dyeing or Cadherins dyeing, for observing in cornea repair process, the improvement situation of epithelial barrier function；
Step 6. statistical analysis and result judgement
The each group experimental data that above-mentioned steps are obtained, with mean ± standard deviationRepresent, using SPSS22.0
Statistical software is analyzed, it is multigroup between difference comparison one-way analysis of variance, compare two-by-two between group and examined using SNK
Method, level of significance α=0.05, P ＜ 0.05 represent that difference has statistical significance；
Comprehensive analysis resistance measurements, fluorescent staining, turbidity measurements, inflammatory factor detection are repaired after chemical damage
And histological findings；Mechanical damage reparation is tested without resistance measurement and turbidity；Referenceization is repaired after ultraviolet injury
Learn damage；
According to the meaning of different detection parameters, from analysis of statistical results, the comprehensive descision of repair is made：
(1) resistance measurement fluorescent staining：With the extension of incubation time, natural reparation group and active reparation group resistance
Value gradually rises；The S/C of fluorescent staining ratio is gradually reduced, and scoring reduces；Natural reparation group and active reparation group and sky
White control is compared, and S/C ratio has significant difference p ＜ 0.05；Active reparation group is compared with natural reparation group, resistance value
Increase has statistical significance, and the score value that fluorescein is detained differs 1 grade and more than 1 grade, it is believed that active material has rush to reparation
Enter effect；
(2) turbidity is tested：With the extension of incubation time, the value of natural reparation group and active reparation group turbidity (OP) by
Gradually decline；Turbidity difference of the reparation group compared with blank control is gradually reduced；Active reparation group is compared with natural reparation group, extremely
Less in 2 and 2 more than time point, turbidity difference, which is compared, has significant difference p ＜ 0.05, it is believed that active material is to repairing
There is facilitation；
(3) inflammatory factor detects：With the extension of incubation time, natural reparation group and active reparation group inflammatory factor
Numerical value should be in decline or raise trend, and have significant difference p ＜ 0.05；Under the same time, active reparation group with repairing naturally
Multiple group is compared, and the level of characteristic inflammatory factor or reparative factor has significant difference, prompts active material to have rush to reparation
Enter effect；
(4) histological observation：By HE dyeing and immunohistochemical staining, the histological change of cornea is observed, and remember in detail
Record：
After the damage of corneal chemical excitant occurs, histology is usually expressed as the obvious defect of epithelium, and epithelial cell lacks
Lose, permeability increase, tight junction protein ZO-1, Occludin structures disappear；Over time, epithelial cell proliferation and to
Migrated at defect, its permeability gradually reduces, and tight junction protein structure gradually forms；During 24h, epithelial cell covering cornea
At epithelial defect, but its permeability is still high compared with normal group, and its intercellular tight junction is loose, arrangement disorder；After 48h, cornea leads to
Permeability recovers normal, and its corneal epithelial cell is completely embedded, and iuntercellular forms tight connecting device；If promote reparation group with
Natural reparation group is compared, and histology repair time, which shortens more than 12h or repairs degree, improves, i.e. similar histologic structure and spy
Levy the time advance of observation more than 12 hours, prompt active material to have facilitation to reparation.
Preferably, prepared by described step 1 isolated cornea includes following sub-step：
Eyeball is won from eye socket out of 2-3 hours after the animal slaughtering of slaughterhouse, cornea is avoided damage in separation process；
In-vitro eyeball is totally submerged is transported to laboratory in the ice-cold HBSS balanced salt solutions containing antibiotic；
Aseptic area of the excision of cornea in laboratory is carried out, and is cut visual inspection cornea integrality before cornea, is checked
There are no marking, pigmentation, muddiness or mechanical scratch, the eyeball for having drawbacks described above then abandons；
Pig eyeball is immersed containing 1% Betagen Solution 2min, is rinsed with sterile PBS, is immersed big mould containing 0.1% celebrating
After the PBS 15min sterilizations of element, corneal ablation operation is carried out；
Ox horn film is directly separated from intact eye, and during isolated cornea, it is completely annular to retain 2-4mm in edge of cornea
Sclera, the isolated cornea after excision are continuously rinsed 3-5 times with HBSS sterile 5-7ml.
Preferably, described step 3 compound stimulates corneal damage model and evaluation to include following sub-step：
(1) start formal experiment after cornea culture about 4-24h, nutrient solution is suctioned out from culture dish；Diameter 1.0~
1.5cm polyflon O-ring or Teflon O-ring is placed in corneal epithelium face；
Tested material for the expected debita spissitudo for producing stimulation chemical liquor, such as 3% SLS, 100% ethanol or
3% H2O2, liquid stimulant 40-100ul is taken to add in ring, 10~20min of exposure；If it is damage model to select ultraviolet,
Wavelength is UVB, exposure dose 40-70mJ/cm2；
After exposure terminates, cornea is gently rinsed 2-3 times with 2-3ml PBS, until removing tested material residual completely；By its
It is immersed in the culture dish containing 40-60ml M199 culture mediums, 32 DEG C ± 2 DEG C, 5%CO2, 90% relative humidity incubator
Continue to be incubated 2 hours；Each damage model uses 6-10 cornea；
(2) cornea turbidity changes：After incubation terminates, wherein 2 corneas are taken, remove polyflon or Teflon
O-ring, it is fixed in cornea clamper；Then fresh MEM cultures are added by the order that cup is first refilled full of rear chamber
Liquid；The turbidity of each cornea is detected with transmissometer, it is OP to record muddy angle value (OP)Stimulate 0hOr OPRepair 0h；
Meanwhile taking blank group cornea to carry out turbidity test, record turbidity is OPBlank；
(3) resistance measurement and fluorescein observation：With step 5 its (5)；
(4) Histological evaluation：With step 5 its (6)；
(5) chemical damage cornea is evaluated：
If resistance measurements ＞ 1, and≤5, it is believed that cornea barrier function damage, if resistance value≤1, it is believed that
Damage is excessively serious, is not useable for recovery test, blank group resistance value answers ＞ 5；If turbidity value changes >=3 and ＜ 10, it is believed that
Mild-moderate damage has resulted in；If turbidity value >=10 and ＜ 25, it is believed that moderate-major injury has resulted in；If turbidity value
>=25, it is believed that degree of injury is excessively serious, is not useable for prosthetic test；
Fluorescent staining scoring should be 3 points, i.e. damaged area answers ＞ 50%, and less than 75%, otherwise degree of injury is excessively tight
Weight, it is impossible to be used in injury repair is assessed；
Histological examination：After corneal injury, the different degrees of defect of epithelium is generally occurred within, epithelial cell is shown as and takes off
Fall, tight junction protein ZO-1, Occludin structures, which are reduced, to disappear；The different degrees of oedema of hypothallus, collagen beam arrangement disorder；
Some chemical substances, such as oxidants hydrogen peroxide, hypothallus is damaged, and epithelial barrier function is damaged unobvious；Histology is damaged
Wound response is tested with turbidity and fluorescent staining is corresponding；
(6) remaining cornea is immersed in the culture dish containing 40-60ml M199 culture mediums, be placed in 32 DEG C ± 2 DEG C,
5%CO2, 90% relative humidity incubator, continue to cultivate by step 2 cornea long-period culture method, carry out follow-up reparation naturally
Or promote repair research；
(7) while blank control and negative control are set, negative control is deionized water, operates and walks using identical
Suddenly.
Preferably, described step 4 physical stimulation corneal damage model and evaluation include following sub-step：
(1) after cornea culture about 24h, nutrient solution is suctioned out from culture dish；Diameter 1.0cm polyflon
O-ring or Teflon ring are placed in cornea；With sterile ophthalmologic operation blade a diameter of 8mm cornea is struck off in cornea center
Central epithelium, depth and corneal epithelial cell basalis, processing machinery corneal injury model；Each damage model uses 6-10
Individual cornea；Take 2 corneas therein to carry out following stimulation scale evaluation to test：
(2) fluorescein is observed：With step 5 its (5)；The cornea of NaFL dyeing display damages, the size of yellow area represent
The order of severity of damage；
(3) Histological evaluation that physical stimulation model is introduced：With step 5 its (6)；
(4) physical damnification cornea is evaluated：
Fluorescent staining scoring should be 3 points, i.e. damaged area answers ＞ 50%, and less than 75%, otherwise degree of injury is excessively tight
Weight, it is impossible to be used in injury repair is assessed
Histological examination：After corneal injury, the obvious defect of epithelium should occur, show as epithelial cell missing, closely connect
Connect the disappearance of albumen ZO-1, Occludin structure；；Fault rupture or missing on hypothallus, Histological injury caused by physical factor should
It is corresponding with fluorescent staining；
(5) remaining cornea is immersed in the culture dish containing 40-60ml M199 culture mediums, be placed in 32 DEG C ± 2 DEG C,
5%CO2, 90% relative humidity incubator, continue to cultivate by step 2 cornea long-period culture method, carry out follow-up reparation naturally
Or promote repair research；
(6) while negative control is set, negative control is deionized water, using identical operating procedure.
Preferably, described step 1-4 any one, is related in the separation and long-term cultivation of cornea：
Described isolated cornea is separating obtained in the eyeball of the discarded pig in slaughterhouse or ox；
Described agar-pigskin gelatin-M199 mixtures, refer to containing 1% low-freezing agar, 1% pigskin gel or
M199 culture mediums, maintain 37 DEG C；
The HBSS buffer solutions, refer to the Hank's balanced salt solutions of commercialization；
The M199 culture mediums, it is M199 culture mediums, adds 10% calf serum, gentamicin, penicillin/streptomycin,
The culture of porcine cornea is additionally added by amphotericin B；
The PVP-I is preservative, concentration 1%；
Described " O-ring " is white annulus or Teflon ring annulus prepared by polyflon inert material,
Corneal is non-toxic, it is nonirritant, do not react and adhere to sample, can autoclaving reuse；
If external diameter is 9-11mm, internal diameter 8-10mm ring, sample-adding amount is 40 μ L；If external diameter is 13-15mm, internal diameter
For 11-13mm ring, sample-adding amount is 100 μ L；For fixing tester and limiting viewing area.
Preferably, described step 3-4 any one, is related in corneal injury method：
Described chemical damage, refer to that chemical substance or mixture enter corneal chemical sexual stimulus caused by eyeball, commonly use
Stimulant is 1.5%-3% SLS, 0.2mol-0.4mol NaOH, 3%-6% trichloro-acetic acid
(TCA), (1.5-3) % H2O2, chemical exposing time is 10~20min, according to the type and extent of chemical irritant
Selected：Moderate selects short action time, the weak compound to moderate Eye irritation to the compound of serious Eye irritation
Selection longer action time, or open-assembly time is determined using preliminary experiment；
Described ultraviolet injury, belongs to physical factor, refers to corneal damage caused by UVB or UVA, UVB exposure doses
For 40mJ/cm2-70mJ/cm2, UVA exposure doses are 10J/cm2-50J/cm2；
Described mechanical damage, according to research purpose, selection diameter 2-3mm card punch corneal damage epithelium and matrix
Top layer, but corneal stroma deep layer is not injured；Blade, syringe needle or blunt corneal surface is selected to carry out slightly cutting, cut, wiping
Wipe, grind or peel off, but do not injure cornea deep layer；Other physics modes are selected to should ensure that skilled operation is stable between cornea
Individual difference is as small as possible.
Preferably, described step 5-6 any one, is related in the detection and assessment of corneal injury reparation：
The parallel cornea quantity of described detection active material repair, depending on the parameter at the time point that inspection is surveyed, such as
Fruit consider histological observation, fluorescein measure and turbidity test, while consider 2h, 14h, 26h, 36h, 50h, 74h, 86h,
5 detection time points including 90h, 170h and 14d ± 2h, each time take 2-5 cornea to be tested, and experimental group is at least
Need 10-25 cornea quantity；Reparation group, blank control and primary standard substance control is promoted to should also be as the angle using respective numbers
Film；
Repair after the mechanical damage of described detection active material, does not consider the test of turbidity；
Described cornea resistance is the feature of reactive barrier function, low-voltage AC favour Dent electric bridge can be used to determine；
Described turbidity value is the integrality of key reaction corneal stroma, cornea transmissometer can be used to determine；Described inflammation
Sex factor or reparative factor, refer to the specificity substance that keratocyte is secreted during corneal restoration, including inflammatory cell because
Son：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, proliferation factor, enzyme：LDH；
The commercialized kit of described utilization, including flow cytometry, ELISA and ImmunohistochemistryMethods Methods, to collection
Cell and corresponding culture medium carry out assay, compared with test control group, detect the production of target secretion, and
Whether have significant change by increasing or decreasing for some secretion in statistical analysis repair process；
Described SABC, refer to the principle using Ag-Ab association reaction, feature is carried out to Histological section
Property mark dyeing, show cornea barrier function mark include tight junction protein ZO-1, Occludin, calcium adhesion
Albumen, it is primary antibody that multi-clone rabbit antibody ox or pig antibody, which can be selected, 1:100~1:200 dilutions；It is mouse anti-rabbit that secondary antibody, which may be selected,
Or goat anti-rabbit antibodies.
Described experiment contrast refers to without any blank control containing active material；
Described evaluation active material promotes repair, and control is used as to repair naturally；Increase primary standard substance if necessary
Control, primary standard substance refer to the known promotion clear and definite material of repair.
Preferably, the active material promoted used in corneal restoration, refers to add basic fibroblast in the medium thin
The intracellular growth factor, calf blood protein-removed extraction, restructuring epidermal growth (rhEGF) or Retinol Palmitate, or directly make
With the ophthalmically acceptable liquid medicine of drug containing；
Described statistical method, refer to each group experimental data of acquisition, with mean ± standard deviationRepresent,
Analyzed using SPSS22.0 statistical softwares, it is multigroup between difference comparison one-way analysis of variance, compare two-by-two between group
Using SNK methods of inspection, level of significance α=0.05, P ＜ 0.05 represents that difference has statistical significance；
The chemical damage reparation answers comprehensive analysis fluorescent staining, turbidity test, inflammatory factor detection and histology to see
Examine；Weight therein is followed successively by from big to small：Fluorescent staining ＞ resistance values measure ＞ turbidity test ＞ histological observations ＞ is scorching
Sex factor detects；
Comprehensive analysis fluorescein, inflammatory factor detection and histological observation are answered in the physical damnification reparation；Weight therein
It is followed successively by from big to small：Fluorescent staining ＞ histological observation ＞ inflammatory factors detect.
The above-mentioned side that eye irritation and its repair process are evaluated using porcine cornea or ox horn membrane modle provided by the present invention
Method, it is to utilize exogenous irritant, as chemical substance, ultraviolet cause reversible corneal epithelial cell to be denatured and matrix damage,
By forecast model, the degree of eye irritation is assessed；Either angle is caused using chemical method, physical method or mechanical means
The film epithelial cell and reversible sexual stimulus of matrix, mild wear or missing damage, itself reparation of cornea after damage is assessed, or
Person adds the method that drug evaluation medicine promotes corneal restoration effect after damaging generation.
This method is simple to operate, good with experiment in vivo correlation, and sensitivity is high, can meet corneal irritancy damage and
The needs of the related detection and research such as its reparation.
Xenobiontics are not only limited to epithelium to the degree of eye injury, it is also possible to are related to upper strata interstitial, slight damage
It is reversible and can recover, and most in-vitro evaluation methods can not detect the recovery after damage.Therefore, isolated cornea is grown
Phase culture be able to can also be made as the external model of recovery situation after assessment chemicals potential stimulus and chemicals exposure
To assess the external model of itself reparation or medicine promotion reparation situation after cornea mechanical injuries.In vitro big animal (porcine cornea
With ox horn film) corneal tissue structure it is close with people's cornea, the mechanism and repair process of irritative response and people's cornea
It is similar.The present invention is used to quantitatively indicate that corneal irritancy damage and its index repaired include：Opacity of the cornea degree, cornea group
Knit morphosis, Corneal inflammation change, the change of cornea barrier function etc..Cornea opacity is quantitative determined with cornea transmissometer, angle
The change of film microstructure is by making Histological section's observation and scoring, and Corneal inflammation change is by quantitatively detecting in nutrient solution
Composition reacts, the measure that the change of cornea barrier function passes through fluorescein sodium hold-up.
Forecast model of the invention based on two Index Establishments of corneal injury and restitution can be used for determinand whether there is
The judgement of excitant and stimulation degree size, it can be used for distinguishing the recovery after Eye irritation damage and damage, available for inspection certainly
Body reparation or the difference for aiding in repair.Can completely it provide including Product Safety, corneal injury degree and repair process etc.
Sufficiently complete information.
The present invention relates to isolated cornea culture technique used in experiment, and the culture technique is ripe, and operation is easy, repeats
Property is good and cheap.
Beneficial effect：The present invention can be used for angle caused by substituting living animal detection external source chemical damage or mechanical damage
The irritating restitution of film.Compared with prior art, the invention has the advantages that：
(1) present invention establishes the long-term cultivation model of in vitro animal corneal, active culture can be kept in vitro 14 days with
On, solve the deficiency that in-vitro method is not used to repair assessment after damage；
(2) the in vitro animal corneal that uses of the present invention, is that steady sources are reliable, have structure similar to human cornea and
Three-dimensional tissue's material of activity, it can directly damage contact stimulus thing, be acted on suitable for any chemical damage and physical damnification and extensive
The test acted on again；
(3) present invention have detected the various dimensions such as cornea turbidity, fluorescein retention, inflammatory mediator level and histological observation
Parameters variation, intactly have rated opacity of the cornea degree in damaging action and repair process, cornea tissue morphosis, angle
Film inflammation change, the dynamic change of cornea barrier function, detection parameters are comprehensive, and method is sensitive, and specificity is high, and forecast model is accurate
Really；
(4) present invention uses the long-term cultivation model of natural cornea, and dissolvable chemical substance can only be detected with two-dimentional cell
Excitant damage is compared, and three dimension system can detect single compound, mixed pollutants, UV light exposure and mechanicalness and cause
Canthus membrane damage；
(5) method that the present invention establishes can replace living animal to test, there is provided after corneal damage qualitatively and quantitatively certainly
Detection and research that is right and accelerating repair, it is directly used in the angle of the products such as cosmetics, medicine, air pollutants and sample
Film stimulates and the prediction of restitution；
Brief description of the drawings
After Fig. 1 isolated corneas addition agar-pigskin gelatin-M199 mixtures during long-term cultivation；
Fig. 2 porcine corneas histological structure (the blank control group HE colored graphs of certain experiment)；
Ox horn film fluorescein sodium dyeing after the damage of Fig. 3 chemicals (degree of injury is chosen as 2 points)；
Ox horn film fluorescein sodium dyeing of Fig. 4 clear-cutting forestlands after 7 days (injury severity score is 0 point)；
Ox horn membrane tissue structure after the damage of Fig. 5 chemicals (corresponding turbidity value is 18)；
Fig. 6 promotes the ox horn membrane tissue structure (HE dyeing) after recovering 4 days；
Close connection immunohistology (ZO-1 immunostainings) after Fig. 7 ox horn film minor injuries；
Fig. 8 ox horns membrane damage repair 4 days after close connection immunohistology (Occludin immunostainings)；
Embodiment
Damages and natural repair of the embodiment 1SLS to porcine cornea are assessed
1. prepared by vitro porcine cornea
It is prepared by 1.1 porcine corneas：Pig is butchered in rear 2h and wins eyeball from eye socket, and cornea is avoided damage in separation process.In vitro
Pig eyeball is totally submerged is transported to laboratory in the ice-cold HBSS balanced salt solutions containing amphotericin B.Pig cornea
Excision should be carried out in the aseptic area in laboratory, answered visual inspection cornea integrality before cutting cornea, checked for cut, color
Plain calm, muddy and scratches；Eyeball if any drawbacks described above should abandon.Pig eyeball is immersed containing 1% Betagen Solution
2min, rinsed with sterile PBS, immerse the PBS 15min containing 0.1% gentamicin, after sterilization, carry out corneal ablation operation.
Isolated cornea is cut off from intact eye, edge of cornea retains 2-4mm completely annular scleras.Isolated cornea is continuously used into 5-
Rinsed 3-5 times in HBSS sterile 7ml.
It is 1.2 gel-filled:The porcine cornea cleaned up is taken, corneal epithelium is suspended in 12 orifice plates, added in culture hole down
Add HBSS to support the suspended state of cornea, it is then that the 2% agar-gelatin-M199 mixtures for melting state is slow dropwise
Surface in (each 2-3 drops) corneal pocket is added, gel is directly contacted with endothelium.The operating procedure of filling is repeated, until endothelium
Nest is fully filled with, and agar-gelatin solidifies completely at room temperature.Then the cornea that will be filled with solidifying gel is inverted, and is transferred to 12 holes
In culture plate.
1.32 in vitro porcine cornea cultures
The M199 culture mediums for drawing about 40ml are added in each culture dish, until covering corneal limbus, exposed corneal epithelium face
In air.Culture dish is placed on mini-vibrator, is placed in 37 DEG C, 5%CO2, 90% relative humidity incubator in
Cultivate about 24 ± 2h.Mini-vibrator establishment vibration program, make culture dish in every 2h, can from horizontal level shake to
About 45° angle so that cornea can infiltrate epithelial tissue in the medium with of short duration immersion.
2. compound damage model
2.1 sample-adding：After cornea culture about 6h, nutrient solution is suctioned out from culture dish.Diameter 1.0cm polytetrafluoroethylene (PTFE) tree
Fat O-ring shape is placed in cornea.The 3%SLS and μ L of negative control (deionized water) 40 is taken to add in ring respectively.Each measured object is with 20
Individual cornea.
2.2 exposure：Open-assembly time is 10min, after exposure terminates, cornea is gently rinsed with about 2ml PBS, until complete
Remove tested material residual.Then cornea is transferred in the new culture dish containing 40ml M199 culture mediums, 32 DEG C ± 2 DEG C,
5%CO2, 90% relative humidity incubator continue to be incubated；
3 degree of injury are detected and itself is repaired
3.1 degree of injury are assessed：Wherein each 2 corneas of impaired group and blank control group are taken within 2 hours to carry out a turbidity, glimmering
Light element sodium dyes and histology film-making, carries out stimulation scale evaluation；Remaining cornea is immersed in containing 40ml M199 culture mediums
In culture dish, 32 DEG C ± 2 DEG C, 5%CO are placed in2, 90% relative humidity incubator；
3.2 repairs are assessed：Continue culture to 14h, 26h, 50h, 98h, increase 173h if necessary, take impaired group and
Wherein each 2 corneas of blank control group carry out turbidimetric analysis turbidimetry, resistance measurement, fluorescein sodium dyeing, inflammatory factor detection and group
Length of schooling piece is knitted, itself is carried out and repairs scale evaluation；
Turbidity measurement：Before detection, polyflon O-ring is removed, is fixed in special cornea clamper.Then
Order by cup is first refilled full of rear chamber adds fresh MEM nutrient solutions.Each cornea is detected with BASF3.0 transmissometers
Turbidity, it is OP to record muddy angle value (OP)Stimulate 0hOr OPRepair 0hMeanwhile take blank group cornea to carry out turbidity test, record muddy
Turbidity is OPBlank。
Liquor coneae inflammatory factor is tested：Before the nutrient solution more renewed, nutrient solution is removed from culture dish, uses kit
The inflammatory factor and reparative factor level in nutrient solution are detected, including IL-1a, IL-6, IL-8 etc.；
Resistance measurement：After having surveyed turbidity, with Millipore resistance determinators, cornea resistance is detected according to instrument specification
Value；
Fluorescent staining：Cornea is removed from clamper, upper surface instills the PBS containing 2% fluorescein sodium (NaFL).Cross
The NaFL of amount is rinsed with PBS immediately, and NaFl retention can be observed under uviol lamp, and the degree of retention shows corneal injury
Seriousness.The percent value that retention area (S) accounts for whole cornea area (C) is calculated, damage/recovery extent is entered according to S/C values
Row scoring.0 point：<2%, 1 point：2~25%, 2 points：26~50%, 3 points：51~75%, 4 points：76%~100%.
Histological evaluation：After the completion of fluorescein scoring, cornea is taken to add in 10% and fixed in formalin buffer
24h.FFPE, cut into slices, take partially sliced progress HE dyeing.Micro- Microscopic observation simultaneously assesses histological change；Take part
Section carries out immunohistochemical staining, such as corneal epithelial cell Tight junction protein occludin and ZO-1 dyeing, for observing
During corneal restoration, the improvement situation of epithelial barrier function.
4. repair result is observed
4.1 Turbidity measurement
Turbidity value OP2hFor 12, compared with blank group OP0h, turbidity change change >=3, ＜ 15, it is believed that slight-moderate
Damage has resulted in；
4.2 resistance measurements and fluorescent staining
Cornea 10min is handled with 3%SLS, 2h detections cornea resistance value is 1.12 Ω after exposure；Dyed through fluorescein sodium
It can be seen that being dyed more than 50% pupil region, damaged area scores as 3.5 points between 75~85%；About 30% cornea after 26h
Region is dyed, and is scored 2 points；50h about 5%, score 1 point；The almost invisible pupil region dyeing of 98h, prompts corneal epithelium extensive
It is multiple.
4.3 cytokines measurement
3%SLS processing corneas can cause inflammatory factor largely to discharge, and inflammatory factor is horizontal related to degree of injury, with
The reparation of damage, inflammatory factor is horizontal to be declined.The testing result of different time points is shown in Table 1；
4.4 histological observation
After handling cornea with 3%SLS, active cornea also occurs for the damage or loss, upper strata endothelium completely of display epithelial cell
Cell loss；26h after exposure, display affected area is from migration of epithelial cells to basilar memebrane；After exposure 50h, rarely seen corneal epithelium
Cell eosinocyte skin increases, and prompts epithelium to recover obvious；After exposure 98h, cornea tissue structure is normal, prompts angle
Film epithelium recovers completely.
Cornea restitution after the SLS chemical stimulations of table 1
The ox horn film mechanical damage of embodiment 2 and natural repair are assessed
1. isolated cornea prepares and culture
1.2 ox horn film preparations：Ox wins eyeball in 5 hours after butchering from eye socket, and cornea is avoided damage in separation process.
In vitro buphthalmos ball is totally submerged is transported to laboratory in 4 DEG C of HBSS containing penicillin/streptomycin.Checked before cutting cornea
Buphthalmos state, it was observed that thering is any obvious Vascular change, pigmentation, muddiness, scratches or mechanical scratch then to be lost
Abandon.Isolated cornea is cut off from intact eye, edge of cornea retains 2-4mm completely annular scleras.Isolated cornea is continuously used
Rinsed 3-5 times in HBSS sterile 5-7ml.
It is 1.2 gel-filled：The ox horn film cleaned up is taken, corneal epithelium is suspended in 6 orifice plates, added in culture hole down
Add HBSS to support the suspended state of cornea, the agar-gelatin-M199 mixtures that then will melt the % of state are slow dropwise
Surface in (each 2-3 drops) corneal pocket is added, gel is directly contacted with endothelium.The operating procedure of filling is repeated, until endothelium
Nest is fully filled with, and agar-gelatin solidifies completely at room temperature.Then the cornea that will be filled with solidifying gel is inverted, and is transferred to 35mm
In culture dish.
1.3 cornea cultures：The M199 culture mediums for drawing about 50ml are added in each culture dish, naked until covering corneal limbus
Reveal corneal epithelium face to be exposed in air.Culture dish is placed on mini-vibrator, is placed in 37 DEG C, 5%CO2, it is 90% relatively wet
About 24 ± 2h is cultivated in the incubator of degree.Mini-vibrator establishment vibration program, makes culture dish in every 2.75h, Ke Yicong
Horizontal level is shaken to about 45° angle so that cornea can infiltrate epithelial tissue in the medium with of short duration immersion.
2. mechanical injury model
After cornea culture about 24h, nutrient solution is suctioned out from culture dish.Diameter 1.3cm polyflon O- shapes
Ring is placed in cornea.A diameter of 8mm corneal epithelium is struck off in cornea center with Sterile ophthalmic knife blade, in depth and cornea
Chrotoplast basalis, processing machinery corneal injury model.Each damage model uses 6-10 cornea.
3. repair is assessed
3.1 mechanical damages 2 hours take wherein each 2 corneas of impaired group and blank control group to carry out fluorescein sodium dyeing
With histology film-making, stimulation scale evaluation is carried out；
Remaining cornea is immersed in the culture dish containing 40ml M199 culture mediums, is placed in 32 DEG C ± 2 DEG C, 5%CO2, 90%
The incubator of relative humidity；Respectively in 2h, 26h, 50h and 170h, impaired group and wherein each 2 corneas of blank control group are taken
Fluorescein sodium dyeing, inflammatory factor detection and histology film-making observation are carried out, carries out itself repair assessment；
Liquor coneae inflammatory factor is tested：Before the nutrient solution more renewed, nutrient solution is removed from culture dish, uses kit
The inflammatory factor and reparative factor level in nutrient solution are detected, including IL-1a, IL-6, IL-8 etc.；
Fluorescent staining：After having surveyed turbidity, cornea is removed from clamper, upper surface, which instills, contains 2% fluorescein sodium
(NaFL) PBS.Excessive NaFL is rinsed with PBS immediately, and NaFl retention, the degree of retention can be observed under uviol lamp
Show the seriousness of corneal injury.The percent value that retention area (S) accounts for whole cornea area (C) is calculated, according to S/C values pair
Damage/recovery extent is scored.0 point：<2%, 1 point：2~25%, 2 points：26~50%, 3 points：51~75%, 4 points：
76%~100%.
Histological evaluation：After the completion of fluorescein scoring, cornea is taken to add in 10% and fixed in formalin buffer
24h.FFPE, cut into slices, take partially sliced progress HE dyeing.Micro- Microscopic observation simultaneously assesses histological change；Take part
Section carries out immunohistochemical staining, such as corneal epithelial cell Tight junction protein occludin and ZO-1 dyeing, for observing
During corneal restoration, the improvement situation of epithelial barrier function.
4. repair result is observed
4.1 fluorescent staining
With the cornea after mechanical damage, fluorescein sodium retention is detected after 2h, it is seen that dyed more than 50% pupil region, damage
Hinder area ＞ 50%, score as 3 points；About 30% pupil region is dyed after 26h, is scored 2 points；50h about 5%, score 1 point；98h
Almost invisible pupil region dyeing, prompts corneal epithelium to recover.
4.2 cytokines measurement
Mechanical damage can cause desmoenzyme and inflammatory factor to discharge, and show as LDH, IL-1a, IL-1b etc. in nutrient solution
Increase, the change of different time points are shown in Table 2；
4.3 histological observation
After mechanical damage cornea, epithelial cell complete collyriculum is shown, stromal cells come off；26h after exposure, in display
Chrotoplast never affected area migrates at defect；After exposure 50h, filled by the epithelial cell of new life at defect, in prompting
Skin recovers obvious；After exposure 98h, cornea tissue structure is normal, prompts corneal epithelium to recover completely.It is thin to show as epithelium
Born of the same parents come off, and tight junction protein ZO-1, Occludin structures disappear；
Cornea after the mechanical damage of table 2 recovers and scoring
The recombinant human fibroblast growth factor of embodiment 3 is to the repair after porcine cornea damage
1. in vitro porcine cornea prepares and culture
It is prepared by 1.1 porcine corneas：Pig is butchered in rear 24h and wins eyeball from eye socket, and cornea is avoided damage in separation process.From
Body pig eyeball is totally submerged is transported to laboratory in the ice-cold HBSS balanced salt solutions containing amphotericin B.Pig cornea
Excision should laboratory aseptic area carry out, cut the perfect sunken cornea of visual inspection.Pig eyeball is immersed and contained
1% Betagen Solution 2min, rinsed with sterile PBS, immerse the PBS 15min containing 0.1% gentamicin, after sterilization,
Carry out corneal ablation operation.Isolated cornea is cut off in intact eye, edge of cornea retains 2-4mm completely annular scleras.Will be from
Body cornea is continuously rinsed 3-5 times with HBSS sterile 5-7ml.
It is 1.2 gel-filled：The porcine cornea cleaned up is taken, corneal epithelium is suspended in 12 orifice plates, added in culture hole down
Add HBSS to support the suspended state of cornea, it is then that the 2% agar-gelatin-M199 mixtures for melting state is slow dropwise
Surface in (each 2-3 drops) corneal pocket is added, gel is directly contacted with endothelium.The operating procedure of filling is repeated, until endothelium
Nest is fully filled with, and agar-gelatin solidifies completely at room temperature.Then the cornea that will be filled with gel is inverted, and is transferred to the culture of 12 holes
In plate.
1.3 cornea cultures：The M199 culture mediums for drawing about 50ml are added in each culture dish, naked until covering corneal limbus
Reveal corneal epithelium face to be exposed in air.Culture dish is placed on mini-vibrator, is placed in 37 DEG C, 5%CO2, it is 90% relatively wet
About 24 ± 2h is cultivated in the incubator of degree.Mini-vibrator establishment vibration program, makes culture dish in every 2.75h, Ke Yicong
Horizontal level is shaken to about 45° angle so that cornea can infiltrate epithelial tissue in the medium with of short duration immersion.
2. compound exposes
2.1 sample-adding：After cornea culture about 24h, nutrient solution is suctioned out from culture dish.Diameter 1.0cm polytetrafluoroethylene (PTFE)
Resin O-ring shape is placed in cornea.3%H is taken respectively2O2Solution and the μ l of negative control (deionized water) 40 are added in ring.It is each tested
6 corneas of thing.
2.2 exposure：Hydrogen peroxide open-assembly time is 5min, and after exposure terminates, cornea is gently rinsed with about 2ml PBS, complete
It is complete to remove H2O2Residual.Then cornea is transferred in new culture dish, takes the new M199 culture mediums of 40ml to add each culture
In ware, culture medium contains 90 μm/ml recombined human epidermis growth factor.Continue culture to the 21st day by above-mentioned steps 1.3, daily
The M199 nutrient solutions containing growth factor are changed, observe the reparation of corneal damage.Using be not added with the M199 nutrient solutions of growth factor as
Naturally control is repaired.
3. injury repair effect is assessed
3.1 degree of injury detect：Wherein each 2 corneas of impaired group and blank control group are taken within 2 hours to carry out a turbidity, glimmering
Light element sodium dyes and histology film-making, carries out stimulation scale evaluation；Remaining cornea is immersed in containing 40ml M199 culture mediums
In culture dish, 32 DEG C ± 2 DEG C, 5%CO are placed in2, 90% relative humidity incubator；
3.2 repairs are assessed：Continue culture to 2h, 14h, 26h, 50h and 98h, take impaired group and blank control group its
In each 2 corneas carry out turbidimetric analysis turbidimetry, resistance value measure, fluorescein sodium dyeing, inflammatory factor detection and histology film-making,
Carry out itself and repair scale evaluation；
Turbidity measurement：Before detection, polyflon O-ring is removed, is fixed in special cornea clamper.Then
Order by cup is first refilled full of rear chamber adds fresh MEM nutrient solutions.The muddiness of each cornea is detected with transmissometer
Degree, it is OP to record muddy angle value (OP)Stimulate 0hOr OPRepair 0hMeanwhile taking blank group cornea to carry out turbidity test, record turbidity is
OPBlank。
Liquor coneae inflammatory factor is tested：Before the nutrient solution more renewed, nutrient solution is removed from culture dish, uses kit
The inflammatory factor and reparative factor level in nutrient solution are detected, including IL-1a, IL-6, IL-8 etc.；Fluorescent staining：Survey turbid
After degree, cornea is removed from clamper, upper surface instills the PBS containing 2% fluorescein sodium (NaFL).Excessive NaFL is used immediately
PBS is rinsed, and NaFl retention can be observed under uviol lamp, the degree of retention shows the seriousness of corneal injury.Calculate retention
Area (S) accounts for the percent value of whole cornea area (C), and damage/recovery extent is scored according to S/C values.0 point：<
2%, 1 point：2~25%, 2 points：26~50%, 3 points：51~75%, 4 points：76%~100%.
Histological evaluation：After the completion of fluorescein scoring, cornea is taken to add in 10% and fixed in formalin buffer
24h.FFPE, cut into slices, take partially sliced progress HE dyeing.Micro- Microscopic observation simultaneously assesses histological change；Take part
Section carries out immunohistochemical staining, and such as the dyeing of corneal epithelial cell Tight junction protein occludin, ZO-1, cell grows increasing
Grow mark Ki67 detection.
4. repair result
4.1 Turbidity measurement
Turbidity value OP2hFor 23, compared with blank group OP0h, turbidity change change >=15, ＜ 25, it is believed that moderate lesion
Have resulted in；The turbidity change of different time points is shown in Table 3.
4.2 resistance measurements and fluorescent staining
Cornea 10min is handled with 3%H2O2SLS, 2h detections cornea resistance value is 3.34 Ω after exposure；Through fluorescein sodium
Dye it is visible dyed more than 70% pupil region, damaged area ＞ 50, and ＜ 75% scores as 3 points；12h, 26h, 50h and
98h different time points, promote and the appraisal result of natural reparation group is shown in Table 3.
4.3 cytokines measurement
The level of inflammatory factor IL-1b in nutrient solution is detected, is shown in Table 3.The inflammatory factor of reparation group is promoted to reach for 14 hours
Begun to decline after peak value, peak value is less than nature reparation group, and fall is more than nature reparation group.
4.4 histological observation
Histological examination：With 2 hours after 3%H2O2 processing corneas, display epithelial cell lacked unobvious, and cell is a small amount of
Come off, tight junction protein ZO-1, Occludin structures are substantially complete；Hypothallus vacuole largely occurs caused by oxidative damage,
Raised with turbidity value in close relations；After exposure 14h, hypothallus vacuole starts to reduce, and exposure 26h, accelerates the rarely seen part of reparation group
The a small amount of vacuole of hypothallus, after exposing 50h, epithelium and hypothallus are substantially intact；After exposure 98h, cornea tissue structure is substantially just
Often, prompting corneal epithelium recovers completely.
Cornea after the H2O2 chemical stimulations of table 3 recovers and scoring
5. conclusion
Nutrient solution containing EGF has facilitation to the reparation after corneal chemical damage caused by H2O2.
Assessment of the EGF of embodiment 4 to the repair after ox horn film ultraviolet injury
1. in vitro ox porcine cornea prepares and culture
1. isolated cornea prepares and culture
1.2 ox horn film preparations：Ox wins eyeball in 5 hours after butchering from eye socket, and cornea is avoided damage in separation process.
In vitro buphthalmos ball is totally submerged is transported to laboratory in 4 DEG C of HBSS containing penicillin/streptomycin.Checked before cutting cornea
Buphthalmos state, it was observed that thering is any obvious Vascular change, pigmentation, muddiness, scratches or mechanical scratch then to be lost
Abandon.Isolated cornea is cut off from intact eye, edge of cornea retains 2-4mm completely annular scleras.Isolated cornea is continuously used
Rinsed 3-5 times in HBSS sterile 5-7ml.
It is 1.2 gel-filled：The ox horn film cleaned up is taken, corneal epithelium is suspended in 12 orifice plates, added in culture hole down
Add HBSS to support the suspended state of cornea, the agar-gelatin-M199 mixtures that then will melt the % of state are slow dropwise
Surface in (each 2-3 drops) corneal pocket is added, gel is directly contacted with endothelium.The operating procedure of filling is repeated, until endothelium
Nest is fully filled with, and agar-gelatin solidifies completely at room temperature.Then the cornea that will be filled with solidifying gel is inverted, and is transferred to 12 holes
In culture plate.
1.3 cornea cultures：The M199 culture mediums for drawing about 60ml are added in each culture dish, naked until covering corneal limbus
Reveal corneal epithelium face to be exposed in air.Culture dish is placed on mini-vibrator, is placed in 37 DEG C, 5%CO2, it is 90% relatively wet
About 24 ± 2h is cultivated in the incubator of degree.Mini-vibrator establishment vibration program, makes culture dish in every 2.75h, Ke Yicong
Horizontal level is shaken to about 45° angle so that cornea can infiltrate epithelial tissue in the medium with of short duration immersion.
2. UV light exposure
After cornea culture about 24h, nutrient solution is suctioned out from culture dish.Diameter 1.3cm polyflon O- rings
Shape is placed in cornea.Cornea, dosage 60mJ/cm are irradiated with UVB2, the time is 10 minutes, and after exposure terminates, cornea is transferred to
In new culture dish, the new M199 culture mediums of 40ml are taken to add in each culture dish, culture medium contains 50 μm/ml restructuring fell
Table growth factor.Continue culture to the 21st day by above-mentioned steps 1.3, change the M199 nutrient solutions containing growth factor, observation daily
The reparation of corneal damage.Compareed using the M199 nutrient solutions for being not added with growth factor as nature reparation.
3. injury repair effect is promoted to assess
3.1 degree of injury detect：Wherein each 2 corneas of impaired group and blank control group are taken within 2 hours to carry out a turbidity, glimmering
Light element sodium dyes and histology film-making, carries out stimulation scale evaluation；Remaining cornea is immersed in containing 40ml M199 culture mediums
In culture dish, 32 DEG C ± 2 DEG C, 5%CO are placed in2, 90% relative humidity incubator；
3.2 repairs are assessed：Culture takes impaired group and blank control group therein to 2h, 14h, 26h, 50h and 98h
Each 2 corneas carry out turbidimetric analysis turbidimetry, resistance value measure, fluorescein sodium dyeing, inflammatory factor detection and histology film-making, carry out
Itself repairs scale evaluation；
Turbidity measurement：Before detection, polyflon O-ring is removed, is fixed in special cornea clamper.Then
Order by cup is first refilled full of rear chamber adds fresh MEM nutrient solutions.The muddiness of each cornea is detected with transmissometer
Degree, it is OP to record muddy angle value (OP)Stimulate 0hOr OPRepair 0hMeanwhile taking blank group cornea to carry out turbidity test, record turbidity is
OPBlank。
Liquor coneae inflammatory factor is tested：Before the nutrient solution more renewed, nutrient solution is removed from culture dish, uses kit
The inflammatory factor and reparative factor level in nutrient solution are detected, including IL-1a, IL-6, IL-8 etc.；
Fluorescent staining：After having surveyed turbidity, cornea is removed from clamper, upper surface, which instills, contains 2% fluorescein sodium
(NaFL) PBS.Excessive NaFL is rinsed with PBS immediately, and NaFl retention, the degree of retention can be observed under uviol lamp
Show the seriousness of corneal injury.The percent value that retention area (S) accounts for whole cornea area (C) is calculated, according to S/C values pair
Damage/recovery extent is scored.0 point：<2%, 1 point：2~25%, 2 points：26~50%, 3 points：51~75%, 4 points：
76%~100%.
Histological evaluation：After the completion of fluorescein scoring, cornea is taken to add in 10% and fixed in formalin buffer
24h.FFPE, cut into slices, take partially sliced progress HE dyeing.Micro- Microscopic observation simultaneously assesses histological change；Take part
Section carries out immunohistochemical staining, such as corneal epithelial cell Tight junction protein occludin and ZO-1 dyeing, for observing
During corneal restoration, the improvement situation of epithelial barrier function.
4. repair result is observed
4.1 Turbidity measurement
Turbidity value OP2hFor 14, compared with blank group OP0h, turbidity change change >=5, ＜ 15, it is believed that minor injury is
Cause；4.2 resistance measurements and fluorescent staining
With UVB treatment with irradiation cornea 10min, 2h detections cornea resistance value is 0.87 Ω after exposure；Dyed through fluorescein sodium
It can be seen that about dyed more than 55% pupil region, damaged area ＞ 50, and ＜ 75%, score as 3 points；About 45% cornea area after 26h
Domain is dyed, and is scored 2 points；50h about 20%, score 1 point；The almost invisible pupil region dyeing of 98h, prompts corneal epithelium to recover.
4.3 cytokines measurement
The change of macrophage inflammatory protein (MIP-1) in detection nutrient solution is shown in Table 4.
4.4 histological observation
Histological examination：With 2 hours after UVB processing corneas, display epithelial cell missing unobvious, cell came off on a small quantity,
Tight junction protein ZO-1, Occludin structures；But hypothallus vacuole largely occurs caused by oxidative damage, with turbidity value liter
It is high in close relations；26h after exposure, the display of reparation group is accelerated to show affected area from migration of epithelial cells to basilar memebrane；Exposure
After 50h, rarely seen corneal epithelial cell eosinocyte skin increases, and prompts epithelium to recover obvious；After exposure 98h, cornea tissue knot
Structure is normal, prompts corneal epithelium to recover completely.
Cornea after the UVB of table 4 irradiations recovers and scoring
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included in protection scope of the present invention.Such as although the present invention only lists part chemistry
The corneal injury mode such as product, ultraviolet, mechanical damage, and EGF, basic fibroblast growth factor etc. promote
Enter the example of corneal restoration.But other materials mentioned in the present invention such as chemical irritant (chlorhexidine, 5% BZK, two
Chlorobenzoyl chloride, trichloroacetic acid etc.), the consumer goods of known Eye irritation effect, the ultraviolet of various dose and wavelength, Yi Jiji
Abrasio corneae caused by tool (such as nail, branch, contact lenses foreign matter scratch), High-risk (such as iron filings, husk,
Dust storm, dust etc.), operation on cornea (such as laser treatment of myopia eye, minimally invasive excision) can also cause with the method for the invention
Eye irritation is damaged, and vitamin, amino acid, small-molecular peptides etc. can also be carried out promoting repair inspection with the method for the invention
Survey.It will not enumerate herein.
Claims (8)
1. a kind of method assessed using the damage of animal isolated cornea long-term cultivation model evaluation eye irritation and repair, its
It is characterized in comprising the following steps：
It is prepared by step 1. isolated cornea；
Step 2. cornea long-term cultivation：
The cornea cleaned up is taken, epithelium is suspended in 6 orifice plates added with HBSS liquid, 12 orifice plates or diameter 35mm training down
Ware is supported, cornea is totally submerged in nutrient solution；
The 0.5-5% of state agar-gelatin-M199 mixtures will be melted, slowly each 2-3 is added dropwise to corneal pocket endothelium dropwise
Face, until endothelium nest is fully filled with, agar-gelatin solidifies completely at room temperature；
The cornea that will be filled with the agar-gelatin of solidification is inverted, and is transferred in some 35mm culture dishes；
The M199 culture mediums for drawing 40-60ml are added in each culture dish, until covering corneal limbus, exposed corneal epithelium face exposure
In air；
Culture dish is placed on mini-vibrator, is placed in 32 DEG C ± 2 DEG C, 5%CO2, 90% relative humidity incubator in cultivate 8-
24h, mini-vibrator should be such that culture dish is shaken in every 2h-3h from horizontal level to 45° angle so that cornea can be with of short duration immersion
Epithelial tissue is infiltrated in the medium；
Step 3. corneal chemical damage model and evaluation；
Step 4. cornea physical damnification model and evaluation；
Repair is assessed after step 5. corneal injury：
(1) after compound corneal damage, cornea is transferred in new culture dish, takes the new M199 culture mediums of 40ml to add each
It is rear to be incubated 2 hours in culture dish；Replacing contains active material EGF, basic fibroblast growth factor or dimension
The M199 culture mediums of raw element, are placed in 32 DEG C ± 2 DEG C, 5%CO2, 90% relative humidity incubator, trained for a long time by step 2 cornea
Foster method continued culture to the 14th day；Experimental group Duplicate Samples 20-30；M199 culture mediums containing active material are repaired for activity
Group, the M199 culture mediums without active material are nature reparation group, while set blank control；Baseline control is set if necessary；
(2) during cultivating, the M199 nutrient solutions containing active material are changed within every 2 days；2h, 14h after chemical damage,
26h, 38h, 50h, 62h, 74h, 86h, 98h, 110,170h and 14d ± 2h equi-time points, nutrient solution is taken to carry out cell factor survey
Examination, and take experimental group wherein 2-3 the test of cornea progress turbidity, resistance test, fluorescein to stay scoring and histological observation, remaining
Cornea continues to cultivate；
(3) liquor coneae cytokine test：Before the nutrient solution more renewed, nutrient solution is removed from culture dish, detects nutrient solution
In inflammatory factor and reparative factor it is horizontal：Including IL-1a, IL-6, IL-8；
(4) turbidity changes：Before Turbidity measurement, cornea is taken out from culture dish, is fixed in clamper, the rear chamber of clamper
Be sequentially filled respectively with cup it is sterile be free of phenol red MEM culture mediums, measured with cornea transmissometer and record the turbid of each cornea
Angle value, turbidity value, OP are recorded respectively from different groups of corneasActive h, OPRepair h, OPBlank；The turbidity of experiment with computing group and blank group
Difference；
(5) resistance measurement and fluorescein observation：After having surveyed turbidity, the resistance value of cornea is determined with resistance instrument；Then from clamper
In remove cornea, upper surface instills the PBS containing 2% fluorescein sodium (NaFL)；Excessive NaFL is rinsed with PBS immediately, ultraviolet
NaFl retention is observed under lamp, the degree of retention shows the seriousness of corneal injury；
The percent value that retention area S accounts for whole cornea area C is calculated, damage/recovery extent is scored according to S/C values；0
Point：<2%, 1 point：2~25%, 2 points：26~50%, 3 points：51~75%, 4 points：76%~100%；
(6) Histological evaluation：After the completion of fluorescein scoring, take cornea to add in 10% and fix 24h in formalin buffer；
FFPE, cut into slices, take partially sliced progress HE dyeing；Micro- Microscopic observation simultaneously assesses histological change；
Take partially sliced carry out immunohistochemical staining：Corneal epithelial cell Tight junction protein occludin or ZO-1 dyeing or
Cadherins dye, for observing in cornea repair process, the improvement situation of epithelial barrier function；
Step 6. statistical analysis and result judgement
The each group experimental data that above-mentioned steps are obtained, with mean ± standard deviationRepresent, count soft using SPSS22.0
Part is analyzed, it is multigroup between difference comparison one-way analysis of variance, compare two-by-two between group using SNK methods of inspection, significantly
Property horizontal α=0.05, P ＜ 0.05 represent difference there is statistical significance；
Comprehensive analysis cornea resistance value, fluorescent staining, turbidity test, inflammatory factor detection and histology are repaired after chemical damage
Observe result；Mechanical damage reparation is tested without resistance test and turbidity；Repaired after ultraviolet injury and refer to chemical damage；
According to the meaning of different detection parameters, from analysis of statistical results, the comprehensive descision of repair is made：
(1) resistance measurement and fluorescent staining：With the extension of incubation time, natural reparation group and active reparation group resistance value by
Edge up height；The S/C of fluorescent staining ratio is gradually reduced, and scoring reduces；Natural reparation group and active reparation group and blank pair
Photograph ratio, S/C ratio have significant difference p ＜ 0.05；Compared with natural reparation group, resistance value increases active reparation group
With statistical significance, the score value that fluorescein is detained differs 1 grade and more than 1 grade, it is believed that active material has promotion to make reparation
With；
(2) turbidity is tested：With the extension of incubation time, under the value of natural reparation group and active reparation group turbidity (OP) is gradual
Drop；Turbidity difference of the reparation group compared with blank control is gradually reduced；Active reparation group is compared with natural reparation group, at least 2
Individual and 2 more than time point, turbidity difference is compared to having significant difference p ＜ 0.05, it is believed that active material has promotion to repairing
Effect；
(3) inflammatory factor detects：With the extension of incubation time, the numerical value of natural reparation group and active reparation group inflammatory factor should
In decline or rise trend, and there is significant difference p ＜ 0.05；Under the same time, active reparation group and natural reparation group phase
Than the level of characteristic inflammatory factor or reparative factor has significant difference, prompts active material to have facilitation to reparation；
(4) histological observation：By HE dyeing and immunohistochemical staining, the histological change of cornea is observed, and record in detail：
After the damage of corneal chemical excitant occurs, histological appearance is the obvious defect of epithelium, and epithelial cell missing, permeability increase
Add, tight junction protein ZO-1, Occludin structures disappear；Over time, epithelial cell proliferation and to migrating at defect, its
Permeability gradually reduces, and tight junction protein structure gradually forms；During 24h, at epithelial cell covering corneal epithelial defect, but its
Permeability is still high compared with normal group, and its intercellular tight junction is loose, arrangement disorder；After 48h, cornea permeability recovers normal, its
Corneal epithelial cell is completely embedded, and iuntercellular forms tight connecting device；If promote reparation group compared with natural reparation group, group
Knit to learn repair time shortening more than 12h or repair degree and improve, be i.e. the time advance 12 of similar histologic structure and features observation
More than hour, active material is prompted to have facilitation to reparation.
2. it is according to claim 1 using cornea long-term cultivation model evaluation eye irritation damage and repair assess
Method, it is characterized in that：Prepared by described step 1 isolated cornea include following sub-step：
Eyeball is won from eye socket out of 2-3 hours after the animal slaughtering of slaughterhouse, cornea is avoided damage in separation process；
In-vitro eyeball is totally submerged is transported to laboratory in the ice-cold HBSS balanced salt solutions containing antibiotic；
Aseptic area of the excision of cornea in laboratory is carried out, and is cut visual inspection cornea integrality before cornea, is checked for
Cut, pigmentation, muddiness or mechanical scratch, the eyeball for having drawbacks described above then abandon；
Pig eyeball is immersed containing 1% Betagen Solution 2min, is rinsed, is immersed containing 0.1% gentamicin with sterile PBS
After PBS 15min sterilizations, corneal ablation operation is carried out；
Ox horn film is directly separated from intact eye, during isolated cornea, retains 2-4mm completely annular scleras in edge of cornea,
Isolated cornea after excision is continuously rinsed 3-5 times with HBSS sterile 5-7ml.
3. it is according to claim 1 using cornea long-term cultivation model evaluation eye irritation damage and repair assess
Method, it is characterized in that：Described step 3 compound stimulates corneal damage model and evaluation to include following sub-step：
(1) start formal experiment after cornea culture about 4-24h, nutrient solution is suctioned out from culture dish；1.0~1.5cm's of diameter
Polyflon O-ring or Teflon O-ring are placed in corneal epithelium face；
Tested material is the chemical liquor of the expected debita spissitudo for producing stimulation, takes liquid stimulant 40-100ul to add ring
It is interior, 10~20min of exposure；If selection ultraviolet is damage model, wavelength UVB, exposure dose 40-70mJ/cm2；
After exposure terminates, cornea is gently rinsed 2-3 times with 2-3ml PBS, until removing tested material residual completely；It is immersed in
In culture dish containing 40-60ml M199 culture mediums, 32 DEG C ± 2 DEG C, 5%CO2, 90% relative humidity incubator continue to incubate
Educate 2 hours；Each damage model uses 6-10 cornea；
(2) cornea turbidity changes：After incubation terminates, wherein 2 corneas are taken, remove polyflon or Teflon O- shapes
Ring, it is fixed in cornea clamper；Then fresh MEM nutrient solutions are added by the order that cup is first refilled full of rear chamber；With
Transmissometer detects the turbidity of each cornea, and it is OP to record muddy angle value (OP)Stimulate 0hOr OPRepair 0h；
Meanwhile taking blank group cornea to carry out turbidity test, record turbidity is OPBlank；
(3) resistance value measure and fluorescein observation：With step 5 its (5)；
(4) Histological evaluation：With step 5 its (6)；
(5) chemical damage cornea is evaluated：
If turbidity value changes >=3 and ＜ 10, it is believed that mild-moderate damage has resulted in；
If turbidity value >=10 and ＜ 25, it is believed that moderate-major injury has resulted in；
If turbidity value >=25, it is believed that degree of injury is excessively serious, is not useable for prosthetic test；
If resistance measurements ＞ 1, and≤5, it is believed that cornea barrier function damage, if resistance value≤1, it is believed that damage is excessively tight
Weight, is not useable for recovery test, blank group resistance value answers ＞ 5；
If fluorescent staining scoring is unsatisfactory for damaged area ＞ 50%, less than 75%, then it is assumed that degree of injury is excessively serious, it is impossible to
Assessed for injury repair；
Histological examination：After corneal injury, there is the different degrees of defect of epithelium, show as epithelial cell shedding, closely connect
Connect albumen ZO-1, Occludin structure and reduce and disappear；The different degrees of oedema of hypothallus, collagen beam arrangement disorder；Chemical substance is damaged
Hinder hypothallus, and epithelial barrier function is damaged unobvious；Histological injury should be tested with turbidity and fluorescent staining is corresponding；
(6) remaining cornea is immersed in the culture dish containing 40-60ml M199 culture mediums, is placed in 32 DEG C ± 2 DEG C, 5%CO2、
The incubator of 90% relative humidity, continue to cultivate by step 2 cornea long-period culture method, carry out follow-up reparation naturally or promote to repair
Multiple Effect study；
(7) while blank control and negative control are set, negative control is deionized water, using identical operating procedure.
4. it is according to claim 1 using cornea long-term cultivation model evaluation eye irritation damage and repair assess
Method, it is characterized in that：Described step 4 physical stimulation corneal damage model and evaluation includes following sub-step：
(1) after cornea culture about 24h, nutrient solution is suctioned out from culture dish；Diameter 1.0cm polyflon O-ring
Or Teflon ring is placed in cornea；With sterile ophthalmologic operation blade on the Central corneal that a diameter of 8mm is struck off in cornea center
Skin, depth and corneal epithelial cell basalis, processing machinery corneal injury model；Each damage model uses 6-10 cornea；
Take 2 corneas therein to carry out following stimulation scale evaluation to test：
(2) fluorescein is observed：With step 5 its (5)；The cornea of NaFL dyeing display damages, the size of yellow area represent damage
The order of severity；
(3) Histological evaluation that physical stimulation model is introduced：With step 5 its (6)；
(4) physical damnification cornea is evaluated：
If fluorescent staining scoring is unsatisfactory for damaged area ＞ 50%, less than 75%, then it is assumed that degree of injury is excessively serious, it is impossible to
Assessed for injury repair；
Histological examination：After corneal injury, there is the obvious defect of epithelium, show as epithelial cell missing, tight junction protein
ZO-1, Occludin structure disappear；Fault rupture or missing on hypothallus, Histological injury caused by physical factor should be with fluoresceins
Dyeing is corresponding；
(5) remaining cornea is immersed in the culture dish containing 40-60ml M199 culture mediums, is placed in 32 DEG C ± 2 DEG C, 5%CO2、
The incubator of 90% relative humidity, continue to cultivate by step 2 cornea long-period culture method, carry out follow-up reparation naturally or promote to repair
Multiple Effect study；
(6) while negative control is set, negative control is deionized water, using identical operating procedure.
5. it is according to claim 1 using cornea long-term cultivation model evaluation eye irritation damage and repair assess
Method, it is characterized in that：Described step 1-4 any one, is related in the separation and long-term cultivation of cornea：
Described isolated cornea is separating obtained in the eyeball of the discarded pig in slaughterhouse or ox；
Described agar-pigskin gelatin-M199 mixtures, refer to containing 1% low-freezing agar, 1% pigskin gel or M199 trainings
Base is supported, maintains 37 DEG C；
The HBSS buffer solutions, refer to the Hank's balanced salt solutions of commercialization；The M199 culture mediums, it is M199 culture mediums,
10% calf serum, gentamicin, penicillin/streptomycin are added, the culture of porcine cornea is additionally added by amphotericin B；It is described poly-
Dimension ketone iodine is preservative, concentration 1%；
Described " O-ring " is white annulus or Teflon ring annulus prepared by polyflon inert material；
If external diameter is 9-11mm, internal diameter 8-10mm ring, sample-adding amount is 40 μ L；If external diameter is 13-15mm, internal diameter 11-
13mm ring, sample-adding amount are 100 μ L；For fixing tester and limiting viewing area.
6. it is according to claim 1 using cornea long-term cultivation model evaluation eye irritation damage and repair assess
Method, it is characterized in that：Described step 3-4 any one, is related in corneal injury method：
Described chemical damage, refer to that chemical substance or mixture enter corneal chemical sexual stimulus caused by eyeball, commonly using stimulates
Agent be 1.5%-3% SLS, 0.2mol-0.4mol NaOH, 3%-6% trichloro-acetic acid (TCA),
(1.5-3) % H2O2, chemical exposing time are 10~20min, are selected according to the type and extent of chemical irritant
Select：Moderate selects short action time to the compound of serious Eye irritation, the weak compound to moderate Eye irritation select compared with
Long action time, or open-assembly time is determined using preliminary experiment；
Described ultraviolet injury, belongs to physical factor, refers to corneal damage caused by UVB or UVA, and UVB exposure doses are
40mJ/cm2-70mJ/cm2, UVA exposure doses are 10J/cm2-50J/cm2；
Described mechanical damage, according to research purpose, selection diameter 2-3mm card punch corneal damage epithelium and matrix top layer,
But corneal stroma deep layer is not injured；Blade, syringe needle or blunt corneal surface is selected to carry out slightly cutting, cut, wiping, grinding
Or peel off, but do not injure cornea deep layer；Other physics modes are selected to should ensure that the stable individual difference between cornea of skilled operation
It is as small as possible.
7. it is according to claim 1 using cornea long-term cultivation model evaluation eye irritation damage and repair assess
Method, it is characterized in that：Described step 5-6 any one, is related in the detection and assessment of corneal injury reparation：
The parallel cornea quantity of described detection active material repair, depending on the parameter at the time point that inspection is surveyed, if considering
Histological observation, fluorescein measure and turbidity test, while consider 2h, 14h, 26h, 38h, 50h, 62h, 74h, 86h, 90h,
5 detection time points including 110h, 170h and 14d ± 2h, each time take 2-5 cornea to be tested, then experimental group is extremely
10-25 cornea quantity is needed less；Reparation group, blank control and primary standard substance control is promoted to should also be as the angle using respective numbers
Film；
Described cornea resistance is the feature of reactive barrier function, is determined with low-voltage AC favour Dent electric bridge；
Described turbidity value is the integrality of key reaction corneal stroma, is determined with cornea transmissometer；
Repair after the mechanical damage of described detection active material, does not consider the test of turbidity；
Described inflammatory factor or reparative factor, refer to the specificity substance of keratocyte secretion during corneal restoration, including
Inflammatory cytokine：IL-1α,IL-1β,IL-6,IL-7,IL-10,IL-12,IL-12,IL-15,MIP-1α,MIP-1β,MIP-
2, TNF-α or PGE2, proliferation factor, enzyme：LDH；
Described SABC, refer to the principle using Ag-Ab association reaction, characteristic mark is carried out to Histological section
The dyeing of thing, showing the mark of cornea barrier function includes tight junction protein ZO-1, Occludin, Cadherins, choosing
It is primary antibody with multi-clone rabbit antibody ox or pig antibody, 1:100~1:200 dilutions；Selection secondary antibody resists for mouse anti-rabbit or goat antirabbit
Body；
The commercialized kit of described utilization, including flow cytometry, ELISA and ImmunohistochemistryMethods Methods, to the cell of collection
Assay is carried out with corresponding culture medium, compared with test control group, detects the production of target secretion, and pass through system
Whether increasing or decreasing for some secretion has significant change in meter analysis repair process；
Described experiment contrast refers to without any blank control containing active material；
Described evaluation active material promotes repair, and control is used as to repair naturally；Increase primary standard substance control if necessary,
Primary standard substance refers to the known promotion clear and definite material of repair.
8. utilize the damage of cornea long-term cultivation model evaluation eye irritation and reparation according to claim 1-7 any one
The method assessed is acted on, it is characterized in that：
Active material used in the promotion corneal restoration, refer to add in the medium basic fibroblast growth factor,
Calf blood protein-removed extraction, restructuring epidermal growth (rhEGF) or Retinol Palmitate, or directly use the eye of drug containing
Use liquid medicine；
Described statistical method, refer to each group experimental data of acquisition, with mean ± standard deviationRepresent, use
SPSS22.0 statistical softwares are analyzed, it is multigroup between difference comparison one-way analysis of variance, compare use two-by-two between group
SNK methods of inspection, level of significance α=0.05, P ＜ 0.05 represent that difference has statistical significance；
Comprehensive analysis fluorescent staining, turbidity test, inflammatory factor detection and histological observation are answered in the chemical damage reparation；Its
In weight be followed successively by from big to small：The test ＞ histological observation ＞ inflammatory factor detections of fluorescent staining ＞ turbidity；
Comprehensive analysis fluorescein, inflammatory factor detection and histological observation are answered in the physical damnification reparation；Weight therein is from big
It is followed successively by small：Fluorescent staining ＞ histological observation ＞ inflammatory factors detect.
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| CN110568173A (en) * | 2019-07-26 | 2019-12-13 | 广州市华代生物科技有限公司 | Method for predicting eye irritation by using combination of multiple corneal structural cells |
| 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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