CN105031668B - A kind of device and its construction method for realizing live body rabbit corneal expansion model - Google Patents
A kind of device and its construction method for realizing live body rabbit corneal expansion model Download PDFInfo
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- CN105031668B CN105031668B CN201510570341.7A CN201510570341A CN105031668B CN 105031668 B CN105031668 B CN 105031668B CN 201510570341 A CN201510570341 A CN 201510570341A CN 105031668 B CN105031668 B CN 105031668B
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Abstract
The invention discloses a kind of device and its construction method for realizing live body rabbit corneal expansion model, it is related to medical faunae modelling technique field.The device of rabbit corneal expansion model is built in the present invention to be included removing epithelium instrument, cornea immersion instrument and anesthesia tools etc..Using the device, it is operated, such as goes the clostridiopetidase A corneal of corneal epithelium, various concentrations to carry out immersion treatment, so as to realize the successful structure of live body rabbit corneal.The operation technique construction method that instrument described in the present apparatus and material source are sufficient, cost is low, are related to is simple；The model constructed is consistent with the pathological state of clinical keratoconus, the mechanism Journal of Sex Research available for keratoconus.
Description
Technical field
The present invention relates to medical faunae modelling technique field, more particularly to a kind of dress for realizing live body rabbit corneal expansion model
Put and its construction method.
Background technology
Keratoconus is a kind of non-inflammatory kerectasis disease, and it can cause Central corneal or other central progressive thinning
Bulging, high astigmatism or corneal scar, have a strong impact on patient's vision.Keratoconus is more to be fallen ill in puberty, it was reported that the disease is sent out
Sick rate is about 1/2000.Clinically keratoconus is relatively conventional to distribute independent case, but keratoconus can be with many disease phases
Close, including some congenital hereditaries sick such as Down syndromes, Marfan syndromes and some knot hoof tissue abnormalities diseases are such as
Ehlers-Danlos syndromes, mitral valve prolapse etc..
The keratoconus cause of disease is unclear at present, it is generally recognized that keratoconus is made jointly by environmental factor and inherent cause
With caused.It can find that the expression of many cell factors and enzyme changes, and has scholar to think proteolysis in keratoconus
Enzyme and the protease inhibitors lowered can cause collagen to be degenerated, so as to cause corneal stroma thinning.The day and clostridiopetidase A can degrade
Right collagen；Therefore, topical application clostridiopetidase A establishes kerectasis model and certain feasibility be present.Keratoconus animal model can mould
Intend the pathological state of clinically keratoconus, and the model of live body relative in vitro model closer to true internal feelings
Condition, there is incomparable advantage for instructing keratoconus to treat and detecting and observe more afterwards, also advantageously justify in exploration
The pathogenesis of cone angle film, the live body keratoconus model for thus establishing this near ideal seem and are highly desirable.
The content of the invention
The technical problems to be solved by the invention are to establish a kind of reliable kerectasis model, to explore keratoconus
Pathogenesis provides great convenience.
To realize the purpose of the present invention, one aspect of the present invention provides a kind of device for realizing rabbit corneal expansion model, described
Device is sealed in a control box or other containers that can place following instruments, for building rabbit corneal extension module
Type, described device specifically include：
Epithelium instrument is removed, for removing the epithelium of rabbit corneal；
Cornea soaks instrument, for carrying out immersion treatment to the rabbit corneal for removing epithelium, obtains the rabbit angle after immersion treatment
Film；
Cream instrument is applied, for anti-infectious agent to be applied to the rabbit corneal after immersion treatment.
Particularly, described device also includes anesthesia tools, and before the epithelium for removing rabbit corneal, fiber crops are carried out to rabbit corneal
Liquor-saturated processing.
Wherein, it is described to go epithelium instrument to include：Small dise knife, corneal scraper.
Wherein, described cornea immersion instrument includes：
Act on cornea, for soaking II Collagenase Type solution of cornea；
For the pipettor being applied to the II Collagenase Type solution on cornea；
For rinsing the PBS solution of cornea and syringe for PBS solution corneal to be rinsed to processing.
Particularly, the concentration of II Collagenase Type is 3-18mg/ml in the II Collagenase Type solution.
Especially, the dextran that mass percent is 10-24% is included in the II Collagenase Type solution.
Especially, the II Collagenase Type solution also includes PBS solution, solution ph 7.4.
Wherein, the painting cream instrument includes：
For the anti-infection inorganic agent on the rabbit corneal after preventing pathogen from invading immersion treatment；
For putting the aseptic cotton carrier for infecting inorganic agent and being applied on rabbit corneal by described.
Particularly, the anti-infection inorganic agent is Ofloxacin salve, erythromycin ophthalmic ointment or Tobrex eye ointment.
Particularly, it is described to be used for the aseptic cotton carrier put infection inorganic agent and be applied on rabbit corneal, can also be by eye
Ointment is directly painted into rabbit conjunctival sac with hand.
Wherein, the anesthesia tools include：
For anaesthetizing rabbit corneal, it is set to lose the anesthetic of perception；
For anesthetic to be acted on to the syringe of rabbit corneal.
Particularly, the anesthetic is Oxybuprocaine hydrochloride eye drops, oxybuprocaine gel or oxybuprocaine eye drops.
To realize the purpose of the present invention, another aspect of the present invention provides a kind of construction method of rabbit corneal expansion model, bag
Include：
Using epithelium instrument is removed, the epithelial structure of rabbit corneal is removed, the rabbit corneal for the epithelium that is removed；
Instrument is soaked using cornea, immersion treatment is carried out to the rabbit corneal for removing epithelium, drops the collagen on cornea
Solution, obtain the rabbit corneal of collagen content reduction；
Using cream instrument is applied, anti-infectious agent is applied on the rabbit corneal of collagen degradation, obtains that there is resistance pathogen to invade
The rabbit corneal entered；
The rabbit corneal with resistance pathogen intrusion is raised 5-15 days, obtains rabbit corneal expansion model.
Particularly, it is described also to include anesthesia tools, before the epithelium for removing rabbit corneal, rabbit corneal is carried out at anesthesia
Reason.
It is wherein, described that to remove epithelium instrument be small dise knife, corneal scraper.
Wherein, described cornea immersion instrument includes：
Act on cornea, for soaking II Collagenase Type solution of cornea；
For the pipettor being applied to the II Collagenase Type solution on cornea；
For rinsing the PBS solution of cornea and syringe for PBS solution corneal to be rinsed to processing.
Particularly, the concentration of II Collagenase Type is 3-18mg/ml in the II Collagenase Type solution.
Especially, the dextran that mass percent is 10-24% is included in the II Collagenase Type solution.
Especially, the II Collagenase Type solution also includes PBS solution.
Wherein, instrument is soaked using cornea, including for immersion treatment is carried out to the rabbit corneal for removing epithelium：
II Collagenase Type solution is added dropwise in anterior corneal surface using pipettor, immersion treatment is carried out, makes the collagen in cornea
Degrade；
After the immersion treatment terminates, II Collagenase Type solution on cornea is blotted using cotton swab；
Finally PBS solution is carried out in the anterior corneal surface corneal blotted after II Collagenase Type solution using syringe
Rinse, obtain removing the cornea of II Collagenase Type solution.Wherein, the immersion treatment time is 20-40min.
Preferably, the immersion treatment time is 30min.
Wherein, the painting cream instrument includes：
For the anti-infection inorganic agent on the rabbit corneal after preventing pathogen from invading immersion treatment；
For putting the aseptic cotton carrier for infecting inorganic agent and being applied on rabbit corneal by described.
Particularly, the anti-infection inorganic agent is Ofloxacin salve, erythromycin ophthalmic ointment or Tobrex eye ointment.
Particularly, it is described to be used for the aseptic cotton carrier put infection inorganic agent and be applied on rabbit corneal, can also be by eye
Ointment is directly painted into rabbit conjunctival sac with hand.
Wherein, the anesthesia tools include：
For anaesthetizing rabbit corneal, it is set to lose the anesthetic of perception；
For anesthetic to be acted on to the syringe of rabbit corneal.
Particularly, the anesthetic is Oxybuprocaine hydrochloride eye drops, oxybuprocaine gel or oxybuprocaine eye drops.
The advantage of the invention is that：
1st, the device provided by the invention for realizing rabbit corneal expansion model only includes anesthesia tools, skinner, cornea leaching
Bubble instrument and painting cream instrument can establish rabbit corneal expansion model, and the instrument is all commercially available prod, and cost is low, easily obtains
.
2nd, the tools build rabbit corneal expansion method that model is expanded using rabbit corneal provided by the invention is simple, it is only necessary to rabbit
Cornea is anaesthetized, goes epithelium, immersion cornea, smearing eye ointment to complete building process, it is not necessary to staff training expense is put into,
Therefore this method cost is low, and operating personnel do not specially require.
3rd, the rabbit corneal built using device provided by the invention expands the curvature increase of model and central thickness reduce with
It is consistent that the curvature that the pathological state of clinical keratoconus is presented, which increases, central thickness is reduced, available for keratoconus song
Rate change mechanism Journal of Sex Research.
4th, the kerectasis model that the people's cornea used relative to prior art is established, rabbit corneal expansion provided by the invention
The cornea of model derives from rabbit, therefore cost is low, is readily available.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.
Experiment is through Peking University First Hospital's Medical Ethics Committee approval, it then follows statements of the ARVO on zoopery.
Material：
NZw：Peking University First Hospital's Experimental Animal Center provides, and is in a good state of health, female adult body weight
2.0-3.0kg, examination of eyes no abnormality seen, 30.
II Collagenase Type (Worthington companies of the U.S.)
Dextran (Adamas companies of the U.S.)
Handheld electronic keratometer (Tianjin Suowei Electronic Technology Co., Ltd.)
Hand-held cornea audigage (Accutome companies of the U.S.)
Back springing type tonometer (iCare companies of Finland)
Embodiment 1
1st, the preparation of II Collagenase Types solution
The preparation of 1.1PBS solution
By 0.27g potassium dihydrogen phosphate (KH2PO4), 1.42g disodium hydrogen phosphate (Na2HPO4), 8g sodium chloride
(NaCl), 0.2g potassium chloride (KCl) is sufficiently stirred dissolving with 800mL deionized water, and constant volume is adjusted to 1L, and using concentrated hydrochloric acid
PH to 7.4, obtains PBS solution.
The preparation of 1.2II Collagenase Type solution
15mg dextrans powder and 0.3g II Collagenase Type powder are dissolved in 100ml PBS, gently
Vortex oscillation, it is set fully to dissolve, then the membrane filtration with low protein bound 0.22um is degerming, is distributed into small deal, and -20
DEG C preserve lucifuge freeze.
2nd, the foundation of live body keratoconus model
It is preoperative that 10 experimental rabbits are carried out with the inspection such as conventional slit-lamp, direct eyeground line, corneal curvature, central corneal thickness
Look into, exclude anterior ocular segment lesion, it for experimental eye and left eye is to compare eye that 10 NZws, which use right eye,.Experimental rabbit is fixed on
In test operation platform, 5% pentobarbital sodium injection is slowly injected rabbit anesthesia with 0.5ml/Kg row experimental rabbit auricular veins,
Hydrochloric acid oka makes rabbit corneal topical anaesthsia because eye drip drop 3 drips；Surveyed using hand-held keratometer and hand-held cornea ultrasound
Thick instrument observation cornea of right eye curvature Ki (i=1,2 ... 10), cornea of left eye curvature K ' i (i=1,2 ... 10) and corneal central thickness
(CCT) Hi (i=1,2 ... 10)；Corneal center diameter 8mm corneal epithelium is then removed using small circular knife and corneal scraper),
The collagenase solution for the 3mg/ml for being configured step 1 using pipettor instills right eye, infusion volume 200ul, soaks cornea of right eye
30min, while cornea of left eye uses the PBS solution immersion 30min containing 15% dextran.Then blotted using cotton swab molten
Liquid, PBS solution rinse repeatedly clean residual solution, Ofloxacin salve prevention infection are smeared in operation eye, after processing 15 days
Observe the corneal curvature Kj (j=1,2 ... 10) and corneal central thickness (CCT) Hj (j=1,2 ... 10) of cornea of both eyes.
3rd, interpretation of result
Using SPSS17.0 statistics softwares to before processing with processing 15 days after cornea of both eyes average curvature
(Km=1/10 (K1+K2+ ... K10), K ' m=1/10 (K ' 1+K ' 2+ ...+K ' 10)), corneal central thickness (CCT)
(Hm=1/10 (H1+H2+ ... H10), H ' m=1/10 (H ' 1+H ' 2+ ... H ' 10)) is analyzed, and all data results are with mean
± standard deviation represents that experimental data is shown in Tables 1 and 2.The cornea average curvature and Central corneal of the right and left eyes of every rabbit
Thickness change value carries out paired t-test, P respectively<0.05 is that difference is statistically significant.
Embodiment 2
1st, the preparation of II Collagenase Types solution
The preparation of 1.1PBS solution
By 0.27g potassium dihydrogen phosphate (KH2PO4), 1.42g disodium hydrogen phosphate (Na2HPO4), 8g sodium chloride
(NaCl), 0.2g potassium chloride (KCl) is sufficiently stirred dissolving with 800mL deionized water, and constant volume is adjusted to 1L, and using concentrated hydrochloric acid
PH to 7.4, obtains PBS solution.
The preparation of 1.2II Collagenase Type solution
24mg dextrans powder and 1.8g II Collagenase Type powder are dissolved in 100ml PBS, gently
Vortex oscillation, it is set fully to dissolve, then the membrane filtration with low protein bound 0.22um is degerming, is distributed into small deal, and -20
DEG C preserve lucifuge freeze.
2nd, the foundation of live body keratoconus model
It is preoperative that 10 experimental rabbits are carried out with the inspection such as conventional slit-lamp, direct eyeground line, corneal curvature, central corneal thickness
Look into, exclude anterior ocular segment lesion, it for experimental eye and left eye is to compare eye that 10 NZws, which use right eye,.Experimental rabbit is fixed on
In test operation platform, 5% pentobarbital sodium injection is slowly injected rabbit anesthesia with 0.5ml/Kg row experimental rabbit auricular veins,
Hydrochloric acid oka makes anterior corneal surface infiltration anesthesia because eye drip drop 3 drips；Using hand-held keratometer and hand-held cornea ultrasonic thickness measurement
Instrument observation cornea of right eye curvature Ki (i=1,2 ... 10), cornea of left eye curvature K ' i (i=1,2 ... 10) and corneal central thickness
(CCT) Hi (i=1,2 ... 10).Go to strike off corneal center diameter 8mm corneal epithelium using small circular knife and corneal scraper, adopt
The collagenase solution for the 18mg/ml for being configured step 1 with pipettor instills right eye, infusion volume 200ul, soaks cornea of right eye
20min, while cornea of left eye uses the PBS solution immersion 20min containing 24% dextran.Then blotted using cotton swab molten
Liquid, PBS solution, which is rinsed, repeatedly cleans residual solution, smears Ofloxacin salve prevention infection in operation eye, is seen after handling 5 days
Examine the corneal curvature Kj (j=1,2 ... 10) and corneal central thickness (CCT) Hj (j=1,2 ... 10) of cornea of both eyes.
3rd, interpretation of result
Using SPSS17.0 statistics softwares to before processing with processing 5 days after cornea of both eyes average curvature
(Km=1/10 (K1+K2+ ... K10), K ' m=1/10 (K ' 1+K ' 2+ ...+K ' 10)), corneal central thickness (CCT)
(Hm=1/10 (H1+H2+ ... H10), H ' m=1/10 (H ' 1+H ' 2+ ... H ' 10)) is analyzed, and all data results are with mean
± standard deviation represents that experimental data is shown in Tables 1 and 2.The cornea average curvature and Central corneal of the right and left eyes of every rabbit
Thickness change value carries out paired t-test, P respectively<0.05 is that difference is statistically significant.
Embodiment 3
1st, the preparation of II Collagenase Types solution
The preparation of 1.1PBS solution
By 0.27g potassium dihydrogen phosphate (KH2PO4), 1.42g disodium hydrogen phosphate (Na2HPO4), 8g sodium chloride
(NaCl), 0.2g potassium chloride (KCl) is sufficiently stirred dissolving with 800mL deionized water, and constant volume is adjusted to 1L, and using concentrated hydrochloric acid
PH to 7.4, obtains PBS solution.
The preparation of 1.2II Collagenase Type solution
10mg dextrans powder and 1.0g II Collagenase Type powder are dissolved in 100ml PBS, gently
Vortex oscillation, it is set fully to dissolve, then the membrane filtration with low protein bound 0.22um is degerming, is distributed into small deal, and -20
DEG C preserve lucifuge freeze.
2nd, the foundation of live body keratoconus model
It is preoperative that 10 experimental rabbits are carried out with the inspection such as conventional slit-lamp, direct eyeground line, corneal curvature, central corneal thickness
Look into, exclude anterior ocular segment lesion, it for experimental eye and left eye is to compare eye that 10 NZws, which use right eye,.Experimental rabbit is fixed on
In test operation platform, 5% pentobarbital sodium injection is slowly injected rabbit anesthesia with 0.5ml/Kg row experimental rabbit auricular veins,
Hydrochloric acid oka makes anterior corneal surface infiltration anesthesia because eye drip drop 3 drips；Using hand-held keratometer and hand-held cornea ultrasonic thickness measurement
Instrument observation cornea of right eye curvature Ki (i=1,2 ... 10), cornea of left eye curvature K ' i (i=1,2 ... 10) and corneal central thickness
(CCT) Hi (i=1,2 ... 10).Go to strike off corneal center diameter 8mm corneal epithelium using small circular knife and corneal scraper, adopt
The collagenase solution for the 10mg/ml for being configured step 1 with pipettor instills right eye, infusion volume 200ul, soaks cornea of right eye
40min, while cornea of left eye uses the PBS solution immersion 40min containing 10% dextran.Then blotted using cotton swab molten
Liquid, PBS solution rinse repeatedly clean residual solution, Ofloxacin salve prevention infection are smeared in operation eye, after processing 10 days
Observe the corneal curvature Kj (j=1,2 ... 10) and corneal central thickness (CCT) Hj (j=1,2 ... 10) of cornea of both eyes.
3rd, interpretation of result
Using SPSS17.0 statistics softwares to before processing with processing 10 days after cornea of both eyes average curvature
(Km=1/10 (K1+K2+ ... K10), K ' m=1/10 (K ' 1+K ' 2+ ...+K ' 10)), corneal central thickness (CCT)
(Hm=1/10 (H1+H2+ ... H10), H ' m=1/10 (H ' 1+H ' 2+ ... H ' 10)) is analyzed, and all data results are with mean
± standard deviation represents that experimental data is shown in Tables 1 and 2.The cornea average curvature and Central corneal of the right and left eyes of every rabbit
Thickness change value carries out paired t-test, P respectively<0.05 is that difference is statistically significant.
Table 1 tests front and rear cornea average curvature data statistics and analysis result
According to table 1, the front and rear cornea average curvature of right eye experiment is all apparently higher than before processing in different embodiments above
Cornea average rate, and P values are respectively less than 0.01, and left eye control group tests front and rear cornea average curvature difference unobvious, meter
Obtained P values are not statistically significant, it was demonstrated that have by the front and rear cornea average curvature of each embodiment processing scheme experiment
There are significant difference, left eye control group cornea average curvature indifference before and after experiment.
Table 2 tests front and rear cornea average central thickness data statistics and analysis result
According to table 2, the front and rear corneal central thickness of right eye experiment is all significantly lower than before processing in different embodiments above
Cornea average rate, and P values are respectively less than 0.01, and left eye control group tests front and rear corneal central thickness difference unobvious, point
The P values being calculated are analysed much larger than 0.05, it is not statistically significant, it was demonstrated that by the front and rear angle of each embodiment processing scheme experiment
Film central thickness is respectively provided with significant difference, left eye control group corneal central thickness indifference before and after experiment.
Handled by the II Collagenase Types of various concentrations in different embodiments above and detect angle under the different disposal time
Film curvature data and corneal central thickness difference data (CCT), further analysis result, it was demonstrated that the rabbit angle of live body before and after experiment
The average curvature of film dramatically increases, and the central thickness of cornea also substantially reduces, this pathological state with clinical keratoconus
Curvature increase, the central thickness reduction presented is consistent.Thus, it can show that the model can be used as effectively reliable live body angle
Film expands model, can be used to study pathogenesis of keratoconus and explores safely and effectively treatment method.In addition, institute in the present apparatus
The operation technique construction method that the instrument and material source stated are sufficient, cost is low, are related to is simple, advantageously in clinically
Wide popularization and application.
Claims (7)
- A kind of 1. device for realizing rabbit corneal expansion living sample, it is characterised in that including：Epithelium instrument is removed, for removing the epithelium of rabbit corneal；Cornea soaks instrument, for carrying out immersion treatment to the rabbit corneal for removing epithelium, expands rabbit corneal；Cream instrument is applied, for anti-infectious agent to be applied to the rabbit corneal after immersion treatment；Wherein, described cornea immersion instrument includes：Act on cornea, for soaking II Collagenase Type solution of cornea；For the pipettor being applied to the II Collagenase Type solution on cornea；Due to removing the cotton swab of the collagenase solution remained；For rinsing the PBS solution of cornea；For PBS solution corneal to be rinsed to the syringe of processing.
- 2. device as claimed in claim 1, it is characterised in that also including anesthesia tools, for remove rabbit corneal epithelium it Before, anaesthetic treatment is carried out to rabbit corneal.
- 3. device as claimed in claim 1, it is characterised in that the concentration of II Collagenase Type is 3-18mg/ml.
- 4. device as claimed in claim 1, it is characterised in that the painting cream instrument includes：The anti-infection inorganic agent on the rabbit corneal after immersion treatment is invaded for anti-pathogen；For the aseptic cotton carrier being applied to the anti-infection inorganic agent on rabbit corneal.
- 5. device as claimed in claim 4, it is characterised in that the anti-infection inorganic agent is selected from Ofloxacin salve, erythromycin One kind in eye ointment or Tobrex eye ointment.
- 6. device as claimed in claim 2, it is characterised in that the anesthesia tools include：For anaesthetizing rabbit corneal, it is set to lose the anesthetic of perception；For fix to be entered to the syringe of rabbit corneal.
- 7. device as claimed in claim 6, it is characterised in that the anesthetic is selected from Oxybuprocaine hydrochloride eye drops, Ao Bu One kind in cacaine gel or oxybuprocaine eye drops.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1398644A (en) * | 2001-07-27 | 2003-02-26 | 北京科宇联合干细胞生物技术有限公司 | Stem cell regenerating surface cornea and its application in corneal transplantation |
| WO2008013557A1 (en) * | 2006-07-24 | 2008-01-31 | International Stem Cell Corporation | Synthetic cornea from retinal stem cells |
| CN101745152A (en) * | 2008-12-09 | 2010-06-23 | 上海交通大学医学院附属第九人民医院 | Corneal posterior lamella and use thereof |
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2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1398644A (en) * | 2001-07-27 | 2003-02-26 | 北京科宇联合干细胞生物技术有限公司 | Stem cell regenerating surface cornea and its application in corneal transplantation |
| WO2008013557A1 (en) * | 2006-07-24 | 2008-01-31 | International Stem Cell Corporation | Synthetic cornea from retinal stem cells |
| CN101745152A (en) * | 2008-12-09 | 2010-06-23 | 上海交通大学医学院附属第九人民医院 | Corneal posterior lamella and use thereof |
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| Collagenase-Mediated Tissue Modeling of Corneal Ectasia and Collagen Cross-Linking Treatments;Cheng W. Hong et al;《Investigative Ophthalmology & Visual Science》;20120429;第53卷(第4期);第2321页最后一段 * |
| 兔眼LASIK术后角膜扩张模型的建立;姚达强 等;《广东医学》;20100131;第31卷(第2期);摘要,"1.1实验动物" 和"1.2方法"小节，"讨论"小节的第2段 * |
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