中国临床康复第加卷第33朋2006—09—10出版ChineseJournalofClinicalRehabilitation，September102006Vo1．10No．33重组人内皮抑素与血管内皮生长抑制因子融合基因对角膜新生血管内皮细胞的协同作用王泳’，柳林，潘欣85·基础研究·解放军广州军区广州总医院眼科，广东省广州市510010；解放军第二军医大学，长海医院眼科，微生物教研室，上海市200433王泳★，女，1976年生，河南省临汝市人，汉族，20o3年解放军第二军医大学毕业，硕士，医师，主要从事屈光外科方面的研究。国家自然科学基金资助项目(3027l391)中图分类号：R3l8文献标识码：A文章编号：l67l一5926(2006)33—0085-04收稿日期：2006—06—26修回日期：2006-07一Il(06—50—6---4660／G·ⅢEfrectofrecombinantadenovirus-mediatedendostatin-solublevascul8rendotheliumgrowthinhibitorfusiongeneoncomealneovascularizationWangYong．LiuLin2,PanXin0DepartmentofOphthalmology，GeneralHospitalof‘GuangzhouMilitaryAreaCommandofChinesePLA，Guangzhou510010，GuangdongProvince，China；ZDeparpmentofOphthalmology，FirstAfiliatedHospital，SecondMilitaryMedicalUniversityofChinesePLA，Shanghai200433．China；DeparpmentofMicrobiology，SecondMilitaryMedicalUniversityofChinesePLA，Shanghai200433，ChinaWangYong★，Master，Physician，DepartmentofOphthalmology，GeneralHospitalofGuangzhouMilitaryAreaCommandofChinesePLA．Guangzhou5l0010，GuangdongProvince，ChinaSupportedby：theNationalNaturalScienceFoundationofChina，No．3027l39lReceived：2006—)6—26Accepted：2006—07一IlAbstractAIM：Toobservetheinhibitoryandspecialeffectsofrecombinantadenovirus．．mediatedendostatin．solublevascularendotheliumgrowthinhibitor(sVEGI)fusiongene(Ad-hENDO-sVEGI)onproliferationofvascularendotheliumcellline．METHODS：TheexperimentwascarriedoutinthelaboratoryoftheDepartmentofMicrobiologyoftheSecondMilitaryMedicalUniversityofChinesePLAbetweenJune20o2andJune20o3．Recombinantadenovirus-mediatedendostatin．solublevascularendoiheliumgrowthinhibitorfusiongenewasconstructedbyroutinemolecularbiology，andidentifiedbyenzymedigestionandpelymerasechainreaction(PCR】．Theviralparticlesweretiteredby50％tissuecultureinfectivedosemethod．Phosphatebuferedsolution(PBs】，Ad-LacZandAd-VEGIwereregardedascontrolgroupswithvirustiterofMOI=20．WhenECV304andL929wereinfectedf04hoursfnvitro．theexpressionoffusiongenemRNAandproteinweredemonstratedbyRT．PcRandWesternblotrespectively．ThebioactivitywasdeterminedbyVioletcrystalassayonday1．2，3，4，5and7andrepresentedbyabsorbance似1570／630nm；thegrowthcurveofECV304andL929wasdrawnandcomparedtoobserveitsefectsandthespecificity．REsULTS：①PCRshowedthatthefusiongenewassuccessfullyclonedandtittered2×lo8pfu／mL．②ComparisonofvioletcrystalassayresultsofECV304cellsineachgroup：ExceptthelstdayofinfectionoftheAd-VEGIgroup(P>0．05)，theAvaluesofECV3o4cellsoftheAd-VEGI，PBSandAd．LacZgroupswereallhigherthantheAd．hENDO-sVEGIgroup<0．01)．(ComparisonofvioletcrystalassayresultsofL929cellofeachgroup：NosignificantdifierencewasfoundintheAvaluesofL929cellsamongtheAd-hENDOsVEGIgroup，PBSgroupandAd-LacZgroupduringgroupcomparison(P>0．05)．C0NCLUS10N：TheconstructedAd-hENDO-sVEGIcanexpressfusegeneproductswithbioactivity，andshowsaspecificsynergisticinhibitiononproliferationofendothelialcellofvein．whichisstrongerthanthatofmonogenicproducts．Itcanbeusedinfurtherexperimentsfortreatingcornealneovascularization．WangY．LiuLPanXEfectofrecombinantadenovirus-mediatedendostatin-solublevascularendotheliumgrowthinhibitor...
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