1、遼寧師范大學碩士學位論文三種重組七鰓鰻RGD毒素蛋白對Hela、HepG2細胞的活性影響姓名：張丕橋申請學位級別：碩士專業(yè)：動物學指導教師：李慶偉20090501Abstract巧一RGD1、LjRGD2、Lj—RGD3wasatoxinproteinfromthesalivaglandofLampetrajaponicaTostudytheantitumorfunctionofrLjRGD1、rLj—RGD2、rLjRGD3andco

2、nfirmitsbiologicalstatusandsignificance，weextractedtotalRNAfromthesalivaglandandamplifiedtheeDNAofLjRGDl、Lj—RGD2、Lj—RGD3byRTPCRTheeDNAofLj—RGDl、LjRGD2、LjRGD3Was417、210、357bplong，whichincluding1、2and3RGD(Arg—Gly—Asp)motif

3、sWeclonedtheeDNAintotheplasmidpET23b，andexpressedtherecombinantproteinrLj—RGDl、rLj—RGD2、rLj—RGD3inEscherichiacoliBL21FusionrURGDl、rLj—RGD2、rLjRGD3witlltheC—terminalhistagwasa17kD、96kD、15kDsolubleproteinUsingtheHisBindaff

4、initychromatographywepurifiedrLj—RGDl、rLjRGD2、rLj—RGD3Furthermore，wedeterminedthebiologicalactivitiesofrLjRGDl、rLjRGD2、rLjRGD3ToexaminetheabilityofrLjRGDl、rLj—RGD2、rLjRGD3inhibitingHela、HepG2cellsproliferationweusedMTTas

5、sayTheresultsshowedthat，rLj—RGD1、rLjRGD2、rLjRGD3inhibitedbFGFinducedproliferationofHela、HepG2cellsinadosedependentmanlierHoechststainingassayrevealedthat，thenucleiofthecellstreated、ⅣitllrLjRGDl、rLjRGD2、rLjRGD3werestained

6、muchbrighterthanthatofuntreatedcellsduetochromatincondensationFurthermore，theDNAladderpatternsfromthecellstreatedwithrU—RGD1、rLj—RGD2、r功RGD3werealsoobservedTheseresultsdemonstratedthatrLjRGDl、rLj—RGD2、rLj—RGD3couldinduce

7、apoptosisofHela、HepG2cellsCelladhesion，migrationandinvasionarecriticalprocessesintunlormetastasisrLj—RGD1、rLj—RGD2、rLj—RGD3significantlyinhibitedadhesionofHela、HepG2cellstovironectininadose—dependentmannerInordertodeterm

8、inetheeffectofrLj—RGDl、rLj—RGD2、rLjRGD3onHela、HepG2cellsmigrationtowardbFGFweusedTranswellcontaininginsertfilterrLj—RGD1、rLjRGD2、rLjRGD3showedasignificantinhibitiononHela、HepG2cellsmigrationIntheinvasionassaytheMatrigela

9、ndTranswellwereusedtoimitateenvironmentinvivoTheresultsofinvasionassayrevealedthatrLj—RGD1、rLjRGD2、rLjRGD3significantlyinhibitedbFGFinducedinvasionofHelacellsTakentogethertheseresultsrevealedthatrLj—RGD1、rLjRGD2、rLj—RGD3