| BackgroundThe rapid development of high-tech, new concepts in the field of military weaponscontinue to be developed,based on laser technology blinding laser weapons is one of them.Meanwhile, the high-tech field of medicine has also brought to the new revolution,therehas been in the field of ophthalmology, such as: laser correction treatment of refractiveerrors, cataract laser treatment, laser treatment of retinal retinopathy, etc. These high-techappearance of great promote the development of ophthalmology, but also to bring thegospel of countless patients. However, along with these favorable factors, the use ofhigh-tech discomfort, excessive application of some unfavorable factors resultinggradually revealed. The eye is extremely sensitive to external light, it is most vulnerable todamage caused by strong light. Bouquet of the laser wavelength rang400to900nm isincidented to the cornea, after aggregation by the refractive medium,a unit area of theretina absorbed irradiation energy is nearly105-fold increase over the amount of irradiationof the cornea. Therefore, the laser radiation can cause severe damage to the retina, leadingto vision loss and blindness.Once the retina is damaged, restore visual function become amajor issue of concern.The change first appeared in the pigment epithelium after laser irradiation, pigmentgranules will convert the absorbed light energy to heat, which cause damage to the retinalpigment epithelial tight junctions between cells, and it is detached from Bruch’s membrane,resulting in the outer blood-retinal barrier breakdown. Studies have shown that there aredirectly related between the laser damage to the retina and the damage to the blood-retinalbarrier. The damage to the outer blood-retinal barrier can make the component of choroidleak into the retina,which cause the retinal edema, leading to a sharp decline in vision andeven blindness.Aquaporins(AQPs) are a class of membrane transport proteins,which are discoveredrecently that they are highly selective for water molecules. There are13subtypes(AQP0-12), which widely distributed in different tissues, they can significantly increasethe cell membrane water permeability, promote the water secretion, absorption and the intracellular water balance.They have been confirmed that they are closely related with thetissue edema which caused by a variety of pathological factors.As the most abundant water content of human organs, a number of physiologicalfunctions of the eye tissue dely on the fast, efficient water transport system, in which waterchannel protein also plays an important role,such as participate the aqueous fluidcirculation, and maintain the cornea、lens’dehydration status. Throughout the varioussubtypes of aquaporin retina, AQPl is the highest levels in the retina,mainly expressed inamacrine cells, pigment epithelial cells and photoreceptor cells, but its specific role isunclear yet.Previous studies showed that AQPs closely related to the permeability of theblood-brain barrier, it plays an important role in regulating the water balance in the brain.As part of the extension of retinal optic nerve center of the brain, the outer blood-retinalbarrier and the blood-brain barrier have much similarities: both have amazing similaritiesin the expression of polarity protein,transport function and permeability aspects. Thus,AQP1,which exists in the retinal tissue extensive,is likely to participate in the regulation ofthe outer blood retinal barrier permeability,and plays an important role in retinal edemainduced by the laser damage.This study intends to establish a retinal laser injury model in rats,on which applyimmunohistochemistry、Western-blot analysis and RT-PCR techniques to determine theexpression and distribution of AQP1in retinal tissue cells. Culture the human retinalpigment epithelial cells in vitro, observe the expression and change of AQP1in thepigment epithelium cells, establish the model of laser-induced damage in vitro, observe theeffect of laser damage on AQP1expression in the pigment epithelium cells. So as toexplore the AQP1role in the formation and development of retinal edema,which inducedby laser, and to explore the underlying mechanisms, in order to provide a new researchdirection and theoretical basis for taking advantage of the water channel proteins as drugtargets for laser retinal damage to carry out effective treatment and prevention.Purpose:1、Establish methods of the human retinal pigment epithelial cells cultured in vitro;2、Observe the AQP-1expression in human retinal pigment epithelial cells cultured invitro;3、Observe the effects of laser irradiation on AQP-1expression in human retinalpigment epithelial cells cultured in vitro; 4、Observe aquaporin-1(AQP1) changes in the rat retina damaged after laserirradiation.Method:1、Establishment of retinal model damaged by laser.Establish methods of the retinal model damaged by laser: monocular fully dilated,eyes put coverslip contact with the cornea, carry out the laser photocoagulation on dilatedeyes’retinal, each eye evenly photocoagulation50point in four quadrants outside the opticdisc of1.5-2optic disc diameter. Argon laser parameters:532nm wavelength,0.1secondsexposure time, the spot diameter is50um, laser energy is100mw, the normal eye iscomparative analysis.2、Culture the human retinal pigment epithelial cells in vitro:Select fresh healthy corneal transplant donor eyeballs, wash them with saline+gentamicin, scissors remove the extraocular connective tissue. Scissors cut circular alongthe rear edge of the cornea at about1-2mm, and in turn remove the cornea, iris, lens,vitreous and retinal neuroepithelial, expose the retinal pigment epithelium, rinse the eyecup section with D-Hank’s solution2times,add0.25%trypsin mixture and lay at37℃digest for30minutes twice, collect the RPE cell suspension,add fresh fetal bovine serum toterminate the digestion,afer the collected suspension centrifuged at1000r/min for10minutes twice,retinal pigment epithelial cells are obtained.Drop DMEM-F12culturesolution containing20%fetal bovine serum, inoculated into25ml culture flask. Cells arepurified by sticking the parietes repeatly.3、Establish the laser injuried model with the human retinal pigment epithelialcells cultured in vitro.Select the4-5th cultured human retinal pigment epithelial cells, seed them in6-wellplates by1×105cells density after recovery, incubate them in incubator containning5%CO2、95%air、90%humidity、37.5℃temperature.The next day, we observe that the cellsgrow adherence, extend pseudopodia,in irregular shape. After4-5days, cells in the poreplates fusion in the form of a cell monolayer substantially, wash the culture solution,put itin front of the retina laser separometer to carry out laser irradiation damage.After laserirradiation damage,collect the cells at5different time points,including postinjuryinstantly,12hours,24hours,72hours and normal cells, centrifugate the cells, remove thesupernatant solution and the cell pellet was collected placed in-80℃cryogenic refrigerator.Argon laser parameters:532nm wavelength,150mw power,100um diameter, 0.15s.exposure time4、 Apply immunohistochemistry method, the real-time fluorescent quantitationsemi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and WesternBlot to observe the distribution of AQP1and its mRNA and protein expression changes inrat retina at different time after laser damage.5、Apply real-time quantitative RT-PCR method to observe the AQP-1expressionchanges at different times after the laser-induced damage in human retinal pigmentepithelial cells cultured in vitro.Result:1、Morphology of human retinal pigment epithelial cells.After48-72hours primary human retinal pigment epithelial cells cultured in culturebottle can adherent growth basically,it is flat, partly extending pseudopodia, in irregularpolygonal shape.Nucleus can not be seen clear under the inverted microscope.There arerich melanin granules in the cytoplasm. About one week, the adherent cells fuse to form asingle basic cell layer.It can be seen the round nuclei,the cytoplasm rich in melaningranules. Passaged every once, cytoplasm melanin gradually fades, until around the6thgeneration, cells become more transparent.2、 The characteristics of AQP-1expression at different time after laserirradiation damage in human retinal pigment epithelial cell cultured in vitro.Real-time PCR results show that AQP1mRNA expression in normal control group is:24.63±0.15, laser injury group are: immediate:25.75±0.35,12hours:25.82±0.10,24hours:25.67±0.19,72hours:25.19±0.12.Compared with the normal group, the laserdamage instantly,12hours,24hours and72hours are all statistically significant (p<0.001). Compared to the other injuried group the laser injuried72hours group isstatistically significant (p <0.05).3、The expression characteristics of AQP-1in mouse retina after laser irradiated.Immunohistochemical staining of optic nerve tissue cell layer and inner nuclear layercells in the rat retinal all show positive brown coarse particles, revealing that AQP-1in theretina of the eye in these two parts show the main positive expression. Real-time PCRresults show that AQP1mRNA expression in normal control group is:1.15±0.01, laserdamage instantly:1.10±0.01,12hours:1.10±0.03,24hours:1.16±0.01,72hours:1.13±0.01. Compared with normal control group, the laser damage immediately (p=0.001<0.05) and laser damage12hours (p=<0.001) are statistically significant. Western Blot results show that: the expression of AQP-1immediately after the laser injury group (1.25±0.35, p=0.04),12-hour group (1.06±0.33, p=0.17),24-hour group (1.87±0.38, p=0.00),72hours group (1.15±0.36, p=0.09), compare with the expression of AQP1innormal control retina (0.73±0.17), statistical analysis found that immediately after thelaser injury group and a24-hour group difference is statistically significant (p <0.05).Conclusion:1、It reveals that in the conditions under laser damage, the expression of AQP-1mRNA in RPE show a dynamic change, that is it upregulation in the initial laser damageresponse to injury, to maximum at the12hours after injury,the latter it shows a decreasewith the reaction time increasing.2、It reveals that in the conditions under laser damage, the expression of AQP-1in theretina in vivo show a dynamic change:that it is up-regulated in the initial laser damage, tomaximum at the24hours after injury, and downregulated with the time going. |
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