[1]郭芳,魏瑞华,孙笑笑,等.Cochlin及其编码基因凝血因子C同源物（COCH）在形觉剥夺性近视豚鼠眼球后极部组织的表达[J].眼科新进展,2019,39(3):201-206.[doi:10.13389/j.cnki.rao.2019.0046]
　GUO Fang,WEI Rui-Hua,SUN Xiao-Xiao,et al.Expression of Cochlin and its coding gene coagulation factor C homology (COCH) in the ocular posterior polar tissues of guinea pigs with form-deprived myopia[J].Recent Advances in Ophthalmology,2019,39(3):201-206.[doi:10.13389/j.cnki.rao.2019.0046]
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Cochlin及其编码基因凝血因子C同源物（COCH）在形觉剥夺性近视豚鼠眼球后极部组织的表达/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
39卷
期数:
2019年3期
页码:
201-206
栏目:
实验研究
出版日期:
2019-03-05

文章信息/Info

Title:
Expression of Cochlin and its coding gene coagulation factor C homology (COCH) in the ocular posterior polar tissues of guinea pigs with form-deprived myopia
作者:
郭芳; 魏瑞华; 孙笑笑; 陈思童; 吴绵绵; 李亚红; 张琰
300384　天津市，天津医科大学眼科医院，天津医科大学眼科研究所，天津医科大学眼视光学院
Author(s):
GUO Fang; WEI Rui-Hua; SUN Xiao-Xiao; CHEN Si-Tong; WU Mian-Mian; LI Ya-Hong; ZHANG Yan
Tianjin Medical University Eye Hospital，Tianjin Medical University Eye Institute，College of Optometry and Ophthalmology of Tianjin Medical University，Tianjin 300384，China
关键词:
形觉剥夺性近视; 蛋白质组学; 凝血因子C同源物; 基因表达
Keywords:
form-deprived myopia; proteomics; coagulation factor C homology; gene expression
分类号:
R778
DOI:
10.13389/j.cnki.rao.2019.0046
文献标志码:
A
摘要:
目的　探讨Cochlin及其编码基因凝血因子C同源物（coagulation factor C homology，COCH）在形觉剥夺性近视（form-deprived myopia，FDM）豚鼠眼球后极部组织的表达。方法　选取3周龄的雄性健康三色豚鼠46只，随机分为2组，每组23只，饲养6周。正常对照组的双眼不予任何处理，FDM组的右眼为实验眼，左眼为自身对照，遮盖FDM组豚鼠的右眼，但不压迫右眼角膜和眼睑，保证右眼能自由瞬目。在遮盖后0周、2周、4周及6周时，分别测量两组豚鼠的屈光度、眼轴长度和角膜曲率半径；HE染色观察两组豚鼠后极部巩膜的厚度及形态变化；进行高通量蛋白质组学分析；实时荧光定量PCR检测COCH mRNA的表达量；Western blot检测Cochlin的蛋白表达水平。结果　遮盖前，两组豚鼠眼球的屈光度和眼轴长度双眼间差值（右眼-左眼）差异均无统计学意义（均为P>0.05）。遮盖后2周，相比对侧的左眼，FDM组右眼诱导出近视，眼轴相对延长；与正常对照组相比，FDM组右眼的屈光度下降，眼轴长度增加，差异均有统计学意义（均为P<0.05）。遮盖后4周和6周，FDM组的右眼相对左眼近视进一步加深，眼轴相对更加延长，近视度数和眼轴长度双眼间差值较正常对照组差异进一步加大，差异均有统计学意义（均为P<0.05）。遮盖前，遮盖后2周、4周及6周，两组豚鼠眼球的角膜曲率半径双眼间差值差异均无统计学意义（均为P>0.05）。遮盖后6周，HE染色结果显示，正常对照组右眼后极部巩膜的厚度正常，胶原纤维排列致密规则，未见断裂现象。然而FDM组右眼的后极部巩膜变薄，胶原纤维变细，变稀疏，间隙变大，且部分纤维出现断裂现象。蛋白质组学分析结果显示，正常对照组和FDM组间表达差异在1.3倍以上的蛋白共221种，其中100种上调，121种下调。其中Cochlin在FDM组豚鼠眼球后极部组织的表达量是正常对照组的3.77倍，升高趋势最明显。实时荧光定量PCR检测结果显示，遮盖后6周，在正常对照组和FDM组右眼后极部组织中，COCH mRNA的相对表达量分别为0.38±0.15和1.86±0.35。FDM组的相对表达量明显高于正常对照组，差异有统计学意义（P<0.05）。Western blot检测结果显示，在正常对照组和FDM组右眼后极部组织中，Cochlin与GAPDH的灰度比值分别为0.37±0.14和0.73±0.15。FDM组Cochlin的表达水平显著高于正常对照组，差异有统计学意义（P<0.05）。结论　FDM豚鼠眼球后极部组织中可检测到COCH mRNA和蛋白Cochlin的表达上调，提示COCH可能在FDM的发生发展中起到重要作用。
Abstract:
ObjectiveToinvestigatetheexpressionofCochlinanditscodinggenecoagulationfactorChomology(COCH)intheocularposteriorpolartissuesofguineapigswithform-deprivedmyopia(FDM).Methods　Totally46threeweek-oldmalehealthytricolorguineapigswereselectedandrandomlyassignedintotwogroups：normalcontrolgroupandFDMgroup,eachofwhichhad23animalsandbothgroupswerefedfor6weeks.Botheyesofthenormalcontrolgroupwerefreeofanytreatment,whiletherighteyesoftheFDMgroupwerethetreated-eyes,leavingthelefteyesasself-controls.TherighteyesofguineapigsintheFDMgroupwerecovered,butthecorneaandeyelidoftherighteyeswerenotcompressedtoensurethattherighteyescouldblinkfreely.At0,2,4and6weekaftertheocclusion,refraction,axiallength,andradiusofcornealcurvatureofguineapigsinthetwogroupsweremeasured.HEstainingwasusedtoobservetheposteriorscleralthicknessandmorphologicalchangesofguineapigsinthetwogroups,andhigh-throughputproteomicsanalysiswasalsoperformed.TheexpressionlevelofCOCHmRNAwasdetectedbyreal-timefluorescencequantitativePCR.WesternblotwasusedtodetecttheproteinexpressionlevelofCochlin.Results　Beforetheocclusion,therewerenosignificantdifferencesinthebinoculardifferences(thevariationbetweentherighteyeandthelefteye)oftherefractionandtheaxiallengthbetweenguineapigseyeballsofthetwogroups(bothP>0.05).At2weekaftertheocclusion,comparedwiththeoppositelefteyes,myopiawasinducedintherighteyesoftheFDMgroupandtheaxiallengthwasrelativelyelongated.Ascomparedwiththenormalcontrolgroup,therighteyesoftheFDMgroupshowedadecreaseintherefractionandanelongationintheaxiallength,andthedifferenceswerestatisticallysignificant(bothP<0.05).At4and6weekaftertheocclusion,therighteyesoftheFDMgroupexhibitedfurtherdeepermyopicdegreeandrelativelylongeraxiallengththanthatofthelefteyes,andcomparedwiththenormalcontrolgroup,thedifferencesinthemyopicdegreeandbinoculardifferencesoftheaxiallengthwerefurtherenlarged,withstatisticalsignificance(bothP<0.05).Therewerenosignificantdifferencesinthebinoculardifferenceoftheradiusofcornealcurvaturebetweenguineapigseyeballsofthetwogroupsbeforetheocclusionandat2,4,and6weekaftertheocclusion(allP>0.05).At6weekaftertheocclusion,theHEstainingresultsshowedthatintherighteyesofthenormalcontrolgroup,theposteriorscleralthicknesswasnormalandthecollagenfiberswerearrangedinadenseandregularmanner,withoutfractures.However,intherighteyesoftheFDMgroup,theposteriorscleralthicknesswasthinner,andthecollagenfibersbecamethinnerandsparser,withthelargergap.Inaddition,thereexistedfracturesinsomefibers.Theproteomicsanalysisresultsrevealedthattherewereatotalof221proteinswithmorethan1.3-foldoftheexpressiondifferencebetweenthenormalcontrolgroupandtheFDMgroup,ofwhich100wereup-regulatedand121weredown-regulated.TheexpressionlevelofCochlinintheocularposteriorpolartissuesoftheFDMgroupwas3.77-foldthatofthenormalcontrolgroupandthetrendofincreasewasthemostobvious.Theresultofreal-timefluorescencequantitativePCRshowedthatat6weekaftertheocclusion,therelativeexpressionlevelofCOCHmRNAwas0.38±0.15and1.86±0.35respectively,intherightocularposteriorpolartissuesofthenormalcontrolgroupandtheFDMgroup.TherelativeexpressionlevelintheFDMgroupwassignificantlyhigherthanthatinthenormalcontrolgroup,andthedifferencewasstatisticallysignificant(P<0.05).WesternblotresultshowedthatintherightocularposteriorpolartissuesofthenormalcontrolgroupandFDMgroup,thegrayscaleratiosofCochlintoGAPDHwere0.37±0.14and0.73±0.15respectively.TheexpressionlevelofCochlinintheFDMgroupwassignificantlyhigherthanthatinthenormalcontrolgroup,andthedifferencewasstatisticallysignificant(P<0.05).Conclusion　TheupregulationofCOCHmRNAandproteinCochlincanbedetectedintheocularposteriorpolartissuesofFDMguineapigs,indicatingthatCOCHmayplayanimportantroleintheformationanddevelopmentofFDM.

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备注/Memo

备注/Memo:
国家级和市级大学生创新创业训练计划项目（编号：201810062036）；天津市自然科学基金重点项目（编号：17JCZDJC35600）；2017天津市高校“中青年骨干创新人才培养计划”；高水平创新人才计划——杰出学者/中青年领军人才（编号：YDYYRCXM-B2018-02）
更新日期/Last Update: 2019-03-15