中华血液学杂志
中華血液學雜誌
중화혈액학잡지
Chinese Journal of Hematology
2013年
6期
527-531
,共5页
潘飞%许莲蓉%王宏伟%朱孟霞%刘艳%覃艳红%陈秀花%任方刚
潘飛%許蓮蓉%王宏偉%硃孟霞%劉豔%覃豔紅%陳秀花%任方剛
반비%허련용%왕굉위%주맹하%류염%담염홍%진수화%임방강
K562细胞%NF-E2相关因子2%硫氧还蛋白还原酶%RNA干扰%细胞凋亡
K562細胞%NF-E2相關因子2%硫氧還蛋白還原酶%RNA榦擾%細胞凋亡
K562세포%NF-E2상관인자2%류양환단백환원매%RNA간우%세포조망
K562 cells%NF-E2 related factor 2%Thioredoxin reductase%RNA interference%Apoptosis
目的 研究核因子NF-E2相关因子2(Nrf2)及硫氧还蛋白还原酶(TrxR)基因表达对慢性髓性白血病(CML)细胞增殖的影响并初步探讨其作用机制.方法 根据siRNA序列设计原则,设计并合成四条针对Nrf2的小分子干扰RNA (siRNA)及一条阴性对照siRNA,并构建慢病毒载体,转染CML细胞系K562细胞,以未转染的细胞作为空白对照；应用激光共聚焦显微镜观察转染效果,流式细胞术检测转染效率；实时荧光定量PCR检测siRNA的抑制效应；CCK-8法检测细胞增殖抑制率；膜联蛋白A5(Annexin V-PE)/碘化丙锭(PI)双染法流式细胞术检测细胞凋亡率,激光共聚焦显微镜观察细胞凋亡状态.结果 慢病毒转染效率达65％,实时荧光定量PCR检测显示明显抑制Nrf2表达的细胞克隆为K562-C3,其Nrf2相对表达水平(1.003±0.093)高于对照组(0.344±0.032)；TrxR相对表达水平(1.090±0.549)高于对照组(0.395±0.029),差异均有统计学意义(P值均＜0.001);CCK-8法检测显示转染Nrf2特异siRNA 24、48、72 h细胞的K562-C3细胞增殖抑制率分别为(4.74±0.39)％、(6.13±1.78)％和(25.36±3.77)％；转染72 h的K562-C3细胞凋亡率(29.9％)与对照组(7.9％)比较明显提高；激光共聚焦显微镜显示Annexin V-PE标记阳性细胞出现核固缩、核碎裂及凋亡小体形成等凋亡特征.结论 在细胞水平上,Nrf2特异的siRNA转染K562细胞可抑制Nrf2表达并引起下游调控的抗氧化酶如TrxR表达下调,抑制K562细胞增殖,促进细胞凋亡.
目的 研究覈因子NF-E2相關因子2(Nrf2)及硫氧還蛋白還原酶(TrxR)基因錶達對慢性髓性白血病(CML)細胞增殖的影響併初步探討其作用機製.方法 根據siRNA序列設計原則,設計併閤成四條針對Nrf2的小分子榦擾RNA (siRNA)及一條陰性對照siRNA,併構建慢病毒載體,轉染CML細胞繫K562細胞,以未轉染的細胞作為空白對照；應用激光共聚焦顯微鏡觀察轉染效果,流式細胞術檢測轉染效率；實時熒光定量PCR檢測siRNA的抑製效應；CCK-8法檢測細胞增殖抑製率；膜聯蛋白A5(Annexin V-PE)/碘化丙錠(PI)雙染法流式細胞術檢測細胞凋亡率,激光共聚焦顯微鏡觀察細胞凋亡狀態.結果 慢病毒轉染效率達65％,實時熒光定量PCR檢測顯示明顯抑製Nrf2錶達的細胞剋隆為K562-C3,其Nrf2相對錶達水平(1.003±0.093)高于對照組(0.344±0.032)；TrxR相對錶達水平(1.090±0.549)高于對照組(0.395±0.029),差異均有統計學意義(P值均＜0.001);CCK-8法檢測顯示轉染Nrf2特異siRNA 24、48、72 h細胞的K562-C3細胞增殖抑製率分彆為(4.74±0.39)％、(6.13±1.78)％和(25.36±3.77)％；轉染72 h的K562-C3細胞凋亡率(29.9％)與對照組(7.9％)比較明顯提高；激光共聚焦顯微鏡顯示Annexin V-PE標記暘性細胞齣現覈固縮、覈碎裂及凋亡小體形成等凋亡特徵.結論 在細胞水平上,Nrf2特異的siRNA轉染K562細胞可抑製Nrf2錶達併引起下遊調控的抗氧化酶如TrxR錶達下調,抑製K562細胞增殖,促進細胞凋亡.
목적 연구핵인자NF-E2상관인자2(Nrf2)급류양환단백환원매(TrxR)기인표체대만성수성백혈병(CML)세포증식적영향병초보탐토기작용궤제.방법 근거siRNA서렬설계원칙,설계병합성사조침대Nrf2적소분자간우RNA (siRNA)급일조음성대조siRNA,병구건만병독재체,전염CML세포계K562세포,이미전염적세포작위공백대조；응용격광공취초현미경관찰전염효과,류식세포술검측전염효솔；실시형광정량PCR검측siRNA적억제효응；CCK-8법검측세포증식억제솔；막련단백A5(Annexin V-PE)/전화병정(PI)쌍염법류식세포술검측세포조망솔,격광공취초현미경관찰세포조망상태.결과 만병독전염효솔체65％,실시형광정량PCR검측현시명현억제Nrf2표체적세포극륭위K562-C3,기Nrf2상대표체수평(1.003±0.093)고우대조조(0.344±0.032)；TrxR상대표체수평(1.090±0.549)고우대조조(0.395±0.029),차이균유통계학의의(P치균＜0.001);CCK-8법검측현시전염Nrf2특이siRNA 24、48、72 h세포적K562-C3세포증식억제솔분별위(4.74±0.39)％、(6.13±1.78)％화(25.36±3.77)％；전염72 h적K562-C3세포조망솔(29.9％)여대조조(7.9％)비교명현제고；격광공취초현미경현시Annexin V-PE표기양성세포출현핵고축、핵쇄렬급조망소체형성등조망특정.결론 재세포수평상,Nrf2특이적siRNA전염K562세포가억제Nrf2표체병인기하유조공적항양화매여TrxR표체하조,억제K562세포증식,촉진세포조망.
Objective To explore the effect of nuclear factor erythroid-2 related factor 2 (Nrf2) and thioredoxin reductase (TrxR) gene on proliferation of chronic myeloid leukemia (CML) line cells and its mechanism.Methods Four interfering sequences of Nrf2 and one negative control sequence were designed and synthesised based on the principle of target sequence of siRNA,then constructed lentivirus vectors,which were transfected into K562 cell lines.The transfection effect was observed by laser scanning confocal microscope (LSCM) and flow cytometer (FCM) ; The depressing effect of siRNA was analyzed by real-time PCR.The cell proliferation inhibiting rate was measured with CCK-8 assay,the apoptotic rate by Annexin V-PE/PI with FCM and the apoptotic morphology of cells by LSCM.Results The transfection efficiency of lentivirus was 65％.One cell line K562-C3 which significantly inhibited Nrf2 mRNA was obtained by real-time PCR,Nrf2 relative quantitation (RQ) expressions were 1.003 ±0.093 and 0.344-±0.032 in the control group and K562-C3 respectively; TrxR expression also decreased with RQ as 1.090 ±0.549 and 0.395 ±0.029 respectively.The cellular proliferation inhibition rates of K562-C3 were (4.74 ± 0.39)％,(6.13 ± 1.78)％ and (25.36 ± 3.77)％,respectively at 24,48 and 72 h.The apoptotic rate induced by K562-C3 (29.9％) at 72 hours was obviously higher than in the control group (7.9％).The Annexin V-PE positive K562-C3 cells presented the following apoptotic characteristics,such as karyopyknosis,nuclear fragmentation and apoptotic bodies observed by LSCM.Conclusion Nrf2 specific siRNA could repress its expression at the cellular level and down-regulate the expression of its downstream antioxidant enzyme,such as TrxR,which lead to increased apoptotic rate and decreased cell proliferation.
