原發(fā)性肝細(xì)胞癌中p16、p15、p53基因缺失、突變以及HBV DNA在肝細(xì)胞癌中整合的研究.pdf

1、安徽醫(yī)科大學(xué)碩士學(xué)位論文原發(fā)性肝細(xì)胞癌中p16、p15、p53基因缺失、突變以及HBVDNA在肝細(xì)胞癌中整合的研究姓名：劉虎申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：汪淵袁凌云2000.3.1蘭塑燮蘭堡主旦塞竺蘭些堡塞!生塑些蘭皇坌三竺塑堂!些：!!塑!型生Studiesonhomozygousdeletionsandmutationsofp16，p15andp53andtheintegrationofHBVDNAinhep

2、atocelluarcarcinoma[Abstract]Pul瑚seToconstructthecDNAcloneofp16exon2，prepareitscDNAprobeandestablishitsSouthernBlotanalysismethodToinvestigatethehomozygousdeletionsofp16exonlandexon2，p15exon2，thehomozygousdeletionandpoin

3、tmutationofp53exon7inhepatocellularcarcinora(HCC)andtheintegrationofHBVprecore，SregionandXregioninthegenomeofHCCMethodseDNAofp16exon2isobtainedthroughRT—PCRandinsertedintopGEM—TvectorresultingintheconstructofpGEMp16Thepo

4、sitivecloneisscreenedthroughblue—whitecolorscreening，restrictireendonucleasemappingandsequencingTheprobeisachievedaftertheinsertiscutoff，purifiedandlabeledwithdigoxinsubsequentlyPCRproductsofp16exon2areanalyzedbySouthern

5、BlotwiththeprobewemadeMethodsofPCR，PCR—SSCPPCRSouthernBlotandPCR—RFLPareappliedtoinvestigatethehomozygousdeletionsofp16exonl，exon2，p15exon2andp53exon7andpointmutationofp53exon7PCRand3’terminalbasespecificPCRareappliedtoa

6、nalyzetheintegrationofHBVprecorewildty耐(耵)andmutanttype(MT)，SregionandXregioninthegenomeofHCCTheposiriverateofHBVprecoreintheserumisalsoexaminedResultsTherestrictiveendonucleasemappingoftherecombinantplasmidismatchedwith

7、theoriginaldesignTheinsert’s1engthis307bpwhichisidenticalwiththespanbetweentwoprimersDigoxinlabeledprobecanhybridizewithPCRproductsofp16exon2Inthe38HCCpatients。thehomozygousdeletionrateofp16exon2is342％Nohomozygousdeletio

8、nsofp16exonl，p15exon2andp53exon7aredetectedBut4sampleshavepointmutationofp53codon249whicharea11heterozygousmutationswithamutationrateof1053％ThepositiverateofHBVprecore盯is42I％10ofwhichisMrpositive(263％)ThepositiveratesofH

9、BVXregionandSregionare447％and50％respectivelyIntheserumthepositiveratesofHBVprecoreWTandMTare15896and184％respectivelyConclusionTherecombinantplasmidwegotistheconstuctofpGI瑚p16wedesignedcDNAprobeofp16ismadeandoffthatbasisp

10、16SouthernBlotanalysismethodisestablishedsuccessfullyTheresultswegotsuggestthatthehomozygousdeletionofp16exon2isanimportantwayofinactivationofp16genewhileitisnotsameforp15geneButothermethodsofp15inactivationcan’tbeexclud

11、edTheheterozygousmutationofp53exon7indicatesthatthesepatientshavegeneticsusceptibilitytoHCCThatnohomozygousmutationofp53exon7isfoundwhichsuggeststhatthepointmutationofp53exon7maYnotplayanimportantrolethecarcinogenesisofH