| AML1/ETO(AE) fusion protein resulted from the t(8;21)translocation is a multifunctional protein, which can participate in celldifferentiation,proliferation, apoptosis and renew by affecting a variety ofsignal transduction pathway.Therfore, it is considered that AE fusionprotein is the one of key factor for the leukemogenesis of acutenonlymphoblastic leukemia (ANLL) patients,especially for M2 subtype,it is also a potential target molecule for target-theraping this kind ofpatients.Furthermore, target-transferring drug effectively and specificallyto inhibit the expression of AE fusion gene is the most important step fortarget-therapy of AE-positive patients.Recent studies have found thatsingle-chain antibody variable region fragment(scFv) can effectively andspecifically transfer drug to targeted cell.Thus,in this work,on the basis ofanalyzing the AE expression in ANLL-M2 patients and its clinicalsignificance, preliminary study of single-chain antibody library of scFvconstruction was performed.Chapter 1 The expression of AE fusion gene mRNA in ANLL-M2patients and its clinical significanceObjective: To investigate the expression of AE fusion gene mRNAin ANLL-M2 patients and the relationship betwee the expression of AEand clinical manifestations,the treatment efficacy and prognosis.Methods: The expression of AE fusion gene mRNA was analyzedby reverse transcription-polymerase chain reaction (RT-PCR). Based onthe result of expression of AE gene, two groups of AE-positive group (A)and AE-negative group (B) were classified.Then, the clinical manifesta-tions and the treatment efficacy of the two groups were compared.Additionally, the significance in prognosis after following up for 8months was evaluated.Results: 12 of 23 (52%) ANLL-M2 patients were found expressingAE fusion gene mRNA,Which including eleven case of M2a and a case ofM2b. Compared to AE-positive patients,the AE-positive patients were younger and easier to get fever and bleeding. After induction,75%(9/12)of AE-positive patients achieved complete remission(CR),only36%(2/11) of AE-negative patients did. Followed up for 8 months, oneAE-negative patient relapsed in the fourth month after CR and died due tocentrum nerve system leukaemia.One AE-negative patient relapsed in theseventh month.But none of the AE-positive patients relapsed.Conclusion: About 50% ANLL-M2 patients exspress the AE fusiongene. The AE-positive patients are easier to get fever and bleeding thanthat in the AE-negative patients,but their prognosis are better.Chapter 2 preliminary study of antibody library construction ofscFv from AE-positive patientsObjective: To construct single-chain antibody library of scFv fromAE-positive patients.Methods: Using RT-PCR, the genes of human immunoglobulin VHand VL were respectively amplified from human marrow lymphocytes ofpatients expressing AE fusion gene by different combination ofoligonucleotide primers.Then,the aim fragments were recovered fromagarose gel. Finally, VH-linker and VL-linker were assembled withLinker.Results: The whole of human VH gene and VL gene were amplifiedfrom marrow lymphocytes of AE-positive patients by 67 set of primercombination,Six VH gene and eleven VL gene (five Vκand six Vλ) weresuccessful amplified. Six VH-linker,five Vκ-linker and six Vλ-linkerwere assembled with Linker.Conclusion: By a mass of PCR, VH gene and VL gene weresuccessful amplified from marrow lymphocytes of patients expressed AEfusion gene. VH-linker and VL-linker were then assembled with Linkerwhich should be useful for further constructing single-chain antibodylibrary of scFv. |
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