中文摘要：

目的：t（9；22）（q34；q11）突变导致的BCR—ABL融合基因及其编码的融合蛋白在慢性粒细胞白血病（chmnic myeloid leukemia，CML）发病中发挥重要作用。本研究观察可与BCR—ABL融合蛋白N端寡聚化区域（Oligomerization domain，OD）特异性结合的嵌合泛素连接酶E3β—TrCP的β-TrCP-OD—HA的重组腺病毒载体，经泛素-蛋白酶体途径降解BCR—ABL融合蛋白对白血病K562细胞增殖的影响。方法：HEK293细胞扩增野生型Ad5β—TrCP-0D—HA、突变型Ad5ΔF—TrCP-OD—HA及空载Ad5GFP重组腺病毒载体，感染K562细胞。以未感染K562细胞作空白对照。流式细胞术检测重组腺病毒载体感染效率，Westernblot检测外源性重组蛋白和BCR—ABL融合蛋白的表达，通过细胞增殖曲线、甲基纤维素集落形成试验、细胞周期检测β—TrCP-OD—HA对K562细胞增殖的影响。结果：成功建立携β-TrCP-OD—HA重组腺病毒载体的K562细胞株，重纽腺病毒载体感染效率达66．4％以上，β—TrCP-OD—HA、ΔF-TrCP-0D—HA可在K562细胞中表达。三组重组腺病毒载体感染K562细胞后，Ad5β-TrCP—OD—HA组Westernblot检测BCR—ABL融合蛋白含量下降；K562细胞生长和集落形成能力均受抑制；细胞周期显示S期细胞数下降至10．88％±2．42％、G0/G1期升高至85．6％±5．61％，与Ad5β-TrCP—OD-HA组、Ad5GFP组及对照组相比，差异具有统计学意义（P均〈0．05）。结论：重组腺病毒介导的β—TrCP—OD—HA、△F-TrCP—OD—HA能够在白血病K562细胞中表达；β—TrCP—OD—HA可以抑制K562细胞的生长增殖，其机制可能与其降解BCR—ABL融合蛋白、阻滞细胞周期而实现。

英文摘要：

Objective： The BCR-ABL fusion gene induced by reciprocal translocation of t （9; 22） （q34; q11） plays an important role in the pathogenesis of chronic myeloid leukemia （CML）. Using recombinant adenoviruses carrying the N-terminal oligomerizaton domain （OD） of the BCR/ABL and chimeric ubiquitin ligase β-TrCP, this study was to investigate the effect of the targeted degradation of oncoprotein BCR-ABL by Ubiquitin-Proteasome System on the proliferation of leukemia cell line K562. Methods： The recombinant adenoviruses carrying wild-type β-TrCP gene （Ad5β-TrCP-OD-HA）, mutational β-TrCP gene （Ad5 Δ F-TrCP-OD-HA）and green fluorescent protein gene （Δd5GFP）were amplified in 293 cells and co-infected into K562 cells respectively. The rates of infection were analyzed by flow cytometry （FCM）. Recombinant protein and BCR-ABL expression was detected by Western blot. Cell proliferation was determined by cell counting and methylcellu- cose clonal cell culture. Cell cycle was observed through FCM. Untreated K562 cells were used as blank controls. Result： The leukemia K562 cell lines with exogenous recombinant β-TrCP-OD-HA and Δ F-TrCP-OD-HA gene were established. The infection rates in the three groups were over 66.4% and recombinant protein sus- tained to be expressed. Ad5β-TrCP-OD-HA down-regulated the expression of BCR-ABL and inhibited prolifer-ation of K562 cells. FCM showed that the percentage of cells at S phase was decreased to 10.88%±2.42%, while that of cells at G0/G1 was increased to 85.6%±5.61%, with a significant difference （P〈0.05）. No changes were found in the cell cycle in groups of Ad5ΔF-TrCP-OD-HA and Ad5GFP. Conclusion： There is sustained expression of recombinant β-TrCP-OD-HA protein in K562 cells infected by recombinant adenovirus. β-TrCP-OD-HA could inhibit the proliferation and clonogenicity of K562 cells through targeted degradation of oncoprotein BCR-ABL and arresting the progression of cell cycle.