CAJ | 학술논문

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(chronic 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 doraain,OD)特异性结合的嵌合泛素连接酶E3 β-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-OD-HA、突变型Ad5AF-TrCP-OD-HA及空载Ad5GFP重组腺病毒栽体,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,Western blot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,重组腺病毒载体感染效率达66.4%以上,β-TrCP-OD-HA、AF-TrCP-OD-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Western blot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%、G_0/G_1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能够在白血痛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.
목적:t(9;22)(q34;q11)돌변도치적BCR-ABL융합기인급기편마적융합단백재만성립세포백혈병(chronic myeloid leukemia,CML)발병중발휘중요작용.본연구관찰가여BCR-ABL융합단백N단과취화구역(Oligomerization doraain,OD)특이성결합적감합범소련접매E3 β-TrCP적β-TrCP-OD-HA적중조선병독재체,경범소-단백매체도경강해BCR-ABL융합단백대백혈병K562세포증식적영향.방법:HEK293세포확증야생형Ad5β-TrCP-OD-HA、돌변형Ad5AF-TrCP-OD-HA급공재Ad5GFP중조선병독재체,감염K562세포.이미감염K562세포작공백대조,류식세포술검측중조선병독재체감염효솔,Western blot검측외원성중조단백화BCR-ABL융합단백적표체,통과세포증식곡선、갑기섬유소집락형성시험、세포주기검측β-TrCP-OD-HA대K562세포증식적영향.결과:성공건립휴β-TrCP-OD-HA중조선병독재체적K562세포주,중조선병독재체감염효솔체66.4%이상,β-TrCP-OD-HA、AF-TrCP-OD-HA가재K562세포중표체.삼조중조선병독재체감염K562세포후,Ad5β-TrCP-OD-HA조Western blot검측BCR-ABL융합단백함량하강;K562세포생장화집락형성능력균수억제;세포주기현시S기세포수하강지10.88%±2.42%、G_0/G_1기승고지85.6%±5.61%,여Ad5β-TrCP-OD-HA조、Ad5GFP조급대조조상비,차이구유통계학의의(P균<0.05).결론:중조선병독개도적β-TrCP-OD-HA、△F-TrCP-OD-HA능구재백혈통K562세포중표체;β-TrCP-OD-HA가이억제K562세포적생장증식,기궤제가능여기강해BCR-ABL융합단백、조체세포주기이실현.
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 ade-noviruses 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 Ubiqui- tin-Proteasome System on the proliferation of leukemia call line K562. Methods: The recombinant adenovirus-es carrying wild-type β-TrCP gene (Ad5β-TrCP-OD-HA), mutational β-TrCP gene (Ad5 A F-TrCP-OD-HA)and green fluorescent protein gene (Ad5GFP)were amplified in 293 calls and co-infected into K562 cells respec- tively. The rates of infection were analyzed by flow cytometry (FCM). Recombinant protein and BCR-ABL ex-pression 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 con-trols. Result: The leukemia K562 cell lines with exogenous recombinant β-TrCP-OD-HA and 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 G_0/G_1 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 Ad5 △ F-TrCP-OD-HA and Ad5GFP. Conclusion: There is sustained ex-pression 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 call cycle.