中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2010年
5期
309-314
,共6页
王巍%孟凡义%黄走方%黄明%刘理想
王巍%孟凡義%黃走方%黃明%劉理想
왕외%맹범의%황주방%황명%류이상
白血病,非淋巴细胞,急性%淀粉样前体蛋白基因%RNA干扰
白血病,非淋巴細胞,急性%澱粉樣前體蛋白基因%RNA榦擾
백혈병,비림파세포,급성%정분양전체단백기인%RNA간우
Leukemia,non-lymphoblastic,acute%Amyloid precursor protein%RNA interference
目的 探讨淀粉样前体蛋白(APP)基因在急性髓系白血病(AML)细胞的表达及其生物学行为.方法 应用实时定量PCR方法(2-ΔCt)检测85例初诊AML和20例非恶性血液病患者(对照)骨髓细胞APP mRNA的表达,并研究其表达水平对AML患者的临床特征和治疗反应的影响;应用实时定量PCR和Western blot方法检测APP基因和蛋白在AML细胞株的表达,并与AML原代细胞亚型的结果相比较;化学合成针对APP基因的小干扰RNA(siRNA),利用脂质体转染HL-60细胞,下调APP基因表达.RNA干扰24、48、72 h后,采用MTT法及锥虫蓝拒染细胞计数检测细胞增殖;瑞特-姬姆萨染色观察细胞形态;PI染色流式细胞术分析细胞周期;Annexin V/PI双染色流式细胞术及Hoechst染色检测细胞凋亡;RNA干扰48 h,Western blot法检测凋亡相关蛋白NF-κB、bcl-2和caspase-3的表达;MTT法检测细胞对阿霉素的敏感性.结果 APP mRNA表达在AML亚型间差异有统计学意义(P=0.019),伴t(8;21)的M2b表达最高(中位值0.1080),其次是AML-未定型(0.0467)、M3(0.0266)、M2a(0.0221)、M4a(0.0167)、M5b(0.0151)、M4b(0.0025),两两比较分析显示,M2b患者APPmRNA表达显著高于M5b患者(P=0.000).APP mRNA表达水平对AML亚型以外患者临床特征及治疗反应无显著影响(P＞0.05).Kasumi-1细胞APP基因表达显著高于U937细胞(P＜0.05),与AML各亚型原代细胞中的结果相一致.APP-siRNA转染HL-60细胞后,其APP mRNA表达下凋64%,在培养24、48及72 h后检测HL-60细胞增殖、分化、凋亡、细胞周期及对阿霉素敏感性均无明显变化(P＞0.05).结论 伴t(8;21)异位的M2型白血病高表达APP mRNA,单核细胞白血病低表达APPmRNA.APP基因表达沉默对HL-60细胞的生物学作用无显著影响.
目的 探討澱粉樣前體蛋白(APP)基因在急性髓繫白血病(AML)細胞的錶達及其生物學行為.方法 應用實時定量PCR方法(2-ΔCt)檢測85例初診AML和20例非噁性血液病患者(對照)骨髓細胞APP mRNA的錶達,併研究其錶達水平對AML患者的臨床特徵和治療反應的影響;應用實時定量PCR和Western blot方法檢測APP基因和蛋白在AML細胞株的錶達,併與AML原代細胞亞型的結果相比較;化學閤成針對APP基因的小榦擾RNA(siRNA),利用脂質體轉染HL-60細胞,下調APP基因錶達.RNA榦擾24、48、72 h後,採用MTT法及錐蟲藍拒染細胞計數檢測細胞增殖;瑞特-姬姆薩染色觀察細胞形態;PI染色流式細胞術分析細胞週期;Annexin V/PI雙染色流式細胞術及Hoechst染色檢測細胞凋亡;RNA榦擾48 h,Western blot法檢測凋亡相關蛋白NF-κB、bcl-2和caspase-3的錶達;MTT法檢測細胞對阿黴素的敏感性.結果 APP mRNA錶達在AML亞型間差異有統計學意義(P=0.019),伴t(8;21)的M2b錶達最高(中位值0.1080),其次是AML-未定型(0.0467)、M3(0.0266)、M2a(0.0221)、M4a(0.0167)、M5b(0.0151)、M4b(0.0025),兩兩比較分析顯示,M2b患者APPmRNA錶達顯著高于M5b患者(P=0.000).APP mRNA錶達水平對AML亞型以外患者臨床特徵及治療反應無顯著影響(P＞0.05).Kasumi-1細胞APP基因錶達顯著高于U937細胞(P＜0.05),與AML各亞型原代細胞中的結果相一緻.APP-siRNA轉染HL-60細胞後,其APP mRNA錶達下凋64%,在培養24、48及72 h後檢測HL-60細胞增殖、分化、凋亡、細胞週期及對阿黴素敏感性均無明顯變化(P＞0.05).結論 伴t(8;21)異位的M2型白血病高錶達APP mRNA,單覈細胞白血病低錶達APPmRNA.APP基因錶達沉默對HL-60細胞的生物學作用無顯著影響.
목적 탐토정분양전체단백(APP)기인재급성수계백혈병(AML)세포적표체급기생물학행위.방법 응용실시정량PCR방법(2-ΔCt)검측85례초진AML화20례비악성혈액병환자(대조)골수세포APP mRNA적표체,병연구기표체수평대AML환자적림상특정화치료반응적영향;응용실시정량PCR화Western blot방법검측APP기인화단백재AML세포주적표체,병여AML원대세포아형적결과상비교;화학합성침대APP기인적소간우RNA(siRNA),이용지질체전염HL-60세포,하조APP기인표체.RNA간우24、48、72 h후,채용MTT법급추충람거염세포계수검측세포증식;서특-희모살염색관찰세포형태;PI염색류식세포술분석세포주기;Annexin V/PI쌍염색류식세포술급Hoechst염색검측세포조망;RNA간우48 h,Western blot법검측조망상관단백NF-κB、bcl-2화caspase-3적표체;MTT법검측세포대아매소적민감성.결과 APP mRNA표체재AML아형간차이유통계학의의(P=0.019),반t(8;21)적M2b표체최고(중위치0.1080),기차시AML-미정형(0.0467)、M3(0.0266)、M2a(0.0221)、M4a(0.0167)、M5b(0.0151)、M4b(0.0025),량량비교분석현시,M2b환자APPmRNA표체현저고우M5b환자(P=0.000).APP mRNA표체수평대AML아형이외환자림상특정급치료반응무현저영향(P＞0.05).Kasumi-1세포APP기인표체현저고우U937세포(P＜0.05),여AML각아형원대세포중적결과상일치.APP-siRNA전염HL-60세포후,기APP mRNA표체하조64%,재배양24、48급72 h후검측HL-60세포증식、분화、조망、세포주기급대아매소민감성균무명현변화(P＞0.05).결론 반t(8;21)이위적M2형백혈병고표체APP mRNA,단핵세포백혈병저표체APPmRNA.APP기인표체침묵대HL-60세포적생물학작용무현저영향.
Objective To investigate the expression of amyloid precursor protein (APP) gene in acute myeloid leukemia(AML) and its biological behaviour in AML cells. Methods The expressions of APP mRNA in 85 AML and 20 nonmalignant hematological diseases patients (as control) were measured by realtime PCR. The expression of APP in AML cell lines was also examined by real-time PCR and Western blot and the results were compared with those in their original subtypes. Small interfering RNAs(siRNAs) targeting APP gene were synthesized and transfected into HL-60 cell by lipofectamine 2000 for 24 h, 48h and 72 h. Cell growth was measured by trypan blue dye exclusion and MTT, differentiation by Wright-Giemsa staining, cell cycle by PL/RNase staining, apoptosis by Annexin V/PI and Hoechst33342 staining. Apoptosisrelated protein NF-κB, bcl-2 and Caspase-3 were detected by Western blot after siRNAs transfection for 48 h.Sensitivity to adriamycin was measured by MTT. Results The expression of APP mRNA among AML subtypes differed significantly ( P = 0. 019 ), the highest expression subtype was M2 with t ( 8;21 ) ( median 0. 1080), followed in order by AML-undefined ( 0. 0467 ), M3 ( 0. 0266 ), M2a (0.0221 ), M4a ( 0. 0167 ),M5b (0.0151), and M4b (0.0025). APP expression had no significant effect on AML clinical characteristics excepting for subtypes. The expression of APP in Kasumi-1 cells was significantly higher than that of U937cells ( P ＜0.05 ), which was in agreement with APP expression in their original AML subtypes. After siRNAs transfection for 24 h, 48 h, and 72 h, no significant difference in proliferation, differentiation, apoptosis,cell cycle and sensitivity to adriamycin was detected between interfering group and control groups( P ＞ 0.05 ).Conclusions The APP mRNA expression was highest in M2 with t(8;21 ) and lowest in M5b. Down-regulation of APP expression has no significant effects on biological behaviour of HL-60 cells.
