国际输血及血液学杂志
國際輸血及血液學雜誌
국제수혈급혈액학잡지
INTERNATIONAL JOURNAL OF BLOOD TRANSFUSION AND HEMATOLOGY
2014年
2期
106-111
,共6页
孟凡静%李莉%曹江%吕超%周俊%程海%陈伟%陈翀%徐开林
孟凡靜%李莉%曹江%呂超%週俊%程海%陳偉%陳翀%徐開林
맹범정%리리%조강%려초%주준%정해%진위%진충%서개림
基因,NANOG%白血病,淋巴样%基因,p53%慢病毒感染
基因,NANOG%白血病,淋巴樣%基因,p53%慢病毒感染
기인,NANOG%백혈병,림파양%기인,p53%만병독감염
Gene,NANOG%Leukemia,limphoid%Gene,p53%Lentiviral vector infections
目的 探讨NANOG基因在人类急性淋巴细胞白血病(ALL)细胞株中的表达,并研究下调该基因表达对ALL细胞增殖和细胞周期的影响以及可能机制.方法 选取ALL的MOLT-4、CCRF-HSB2、Jurkat 3种细胞系为研究对象.利用逆转录-聚合酶链反应(RT-PCR)及Western blot技术检测MOLT-4、CCRF-HSB2、J urkat细胞系中NANOG的表达情况.通过构建携带NANOG基因的特异性shRNA的慢病毒载体包装病毒颗粒(pLB-shNANOG-1和pLB-shNANOG-2),该病毒感染MOLT-4细胞后,经分选获得稳定表达株作为实验组:shNANOG-1组和shNANOG-2组;以空质粒(pLB-sh control)感染的细胞和正常MOLT-4细胞分别作为阴性对照组和空白对照组.在基因及蛋白水平下,检测各组NANOG的干扰效率.采用CCK-8法及流式细胞仪检测实验组和对照组细胞增殖能力及细胞周期的变化,并利用实时定量PCR技术检测p53通路相关基因的表达变化.结果 包装病毒颗粒超速离心后病毒滴度达(1.83~3.12)×108 IU/mL.2种shRNA可有效下调病毒感染MOLT-4细胞的NANOG基因及蛋白的表达.CCK-8法检测显示shNANOG-1及shNANOG-2组细胞在不同时间点其光密度值(OD)较空白对照组及阴性对照组下降,在培养72 h后最为明显,实验组与对照组比较,差异均有统计学意义(P<0.001).细胞周期检测显示,shNANOG-1及shNANOG-2组细胞在G0/G1期细胞比例较空白以对照组及阴性对照组高(P<0.001),但两组细胞在S期细胞比例较空白对照组及阴性对照组低(P<0.01),而在G2/M期细胞比例在各组间差异无统计学意义(P>0.05).实时定量PCR法证实shNANOG-1及shNANOG-2组细胞中TP53及CDKN1A基因表达上调,而MDM2及CCND1基因表达下调,实验组与对照组相比差异均有统计学意义(P<0.05).结论 NANOG在部分人类ALL细胞系中有表达,下调NANOG后可通过MDM2-TP53-CDKN1A通路抑制白血病细胞的增殖,并使细胞阻滞于G0/G1期.
目的 探討NANOG基因在人類急性淋巴細胞白血病(ALL)細胞株中的錶達,併研究下調該基因錶達對ALL細胞增殖和細胞週期的影響以及可能機製.方法 選取ALL的MOLT-4、CCRF-HSB2、Jurkat 3種細胞繫為研究對象.利用逆轉錄-聚閤酶鏈反應(RT-PCR)及Western blot技術檢測MOLT-4、CCRF-HSB2、J urkat細胞繫中NANOG的錶達情況.通過構建攜帶NANOG基因的特異性shRNA的慢病毒載體包裝病毒顆粒(pLB-shNANOG-1和pLB-shNANOG-2),該病毒感染MOLT-4細胞後,經分選穫得穩定錶達株作為實驗組:shNANOG-1組和shNANOG-2組;以空質粒(pLB-sh control)感染的細胞和正常MOLT-4細胞分彆作為陰性對照組和空白對照組.在基因及蛋白水平下,檢測各組NANOG的榦擾效率.採用CCK-8法及流式細胞儀檢測實驗組和對照組細胞增殖能力及細胞週期的變化,併利用實時定量PCR技術檢測p53通路相關基因的錶達變化.結果 包裝病毒顆粒超速離心後病毒滴度達(1.83~3.12)×108 IU/mL.2種shRNA可有效下調病毒感染MOLT-4細胞的NANOG基因及蛋白的錶達.CCK-8法檢測顯示shNANOG-1及shNANOG-2組細胞在不同時間點其光密度值(OD)較空白對照組及陰性對照組下降,在培養72 h後最為明顯,實驗組與對照組比較,差異均有統計學意義(P<0.001).細胞週期檢測顯示,shNANOG-1及shNANOG-2組細胞在G0/G1期細胞比例較空白以對照組及陰性對照組高(P<0.001),但兩組細胞在S期細胞比例較空白對照組及陰性對照組低(P<0.01),而在G2/M期細胞比例在各組間差異無統計學意義(P>0.05).實時定量PCR法證實shNANOG-1及shNANOG-2組細胞中TP53及CDKN1A基因錶達上調,而MDM2及CCND1基因錶達下調,實驗組與對照組相比差異均有統計學意義(P<0.05).結論 NANOG在部分人類ALL細胞繫中有錶達,下調NANOG後可通過MDM2-TP53-CDKN1A通路抑製白血病細胞的增殖,併使細胞阻滯于G0/G1期.
목적 탐토NANOG기인재인류급성림파세포백혈병(ALL)세포주중적표체,병연구하조해기인표체대ALL세포증식화세포주기적영향이급가능궤제.방법 선취ALL적MOLT-4、CCRF-HSB2、Jurkat 3충세포계위연구대상.이용역전록-취합매련반응(RT-PCR)급Western blot기술검측MOLT-4、CCRF-HSB2、J urkat세포계중NANOG적표체정황.통과구건휴대NANOG기인적특이성shRNA적만병독재체포장병독과립(pLB-shNANOG-1화pLB-shNANOG-2),해병독감염MOLT-4세포후,경분선획득은정표체주작위실험조:shNANOG-1조화shNANOG-2조;이공질립(pLB-sh control)감염적세포화정상MOLT-4세포분별작위음성대조조화공백대조조.재기인급단백수평하,검측각조NANOG적간우효솔.채용CCK-8법급류식세포의검측실험조화대조조세포증식능력급세포주기적변화,병이용실시정량PCR기술검측p53통로상관기인적표체변화.결과 포장병독과립초속리심후병독적도체(1.83~3.12)×108 IU/mL.2충shRNA가유효하조병독감염MOLT-4세포적NANOG기인급단백적표체.CCK-8법검측현시shNANOG-1급shNANOG-2조세포재불동시간점기광밀도치(OD)교공백대조조급음성대조조하강,재배양72 h후최위명현,실험조여대조조비교,차이균유통계학의의(P<0.001).세포주기검측현시,shNANOG-1급shNANOG-2조세포재G0/G1기세포비례교공백이대조조급음성대조조고(P<0.001),단량조세포재S기세포비례교공백대조조급음성대조조저(P<0.01),이재G2/M기세포비례재각조간차이무통계학의의(P>0.05).실시정량PCR법증실shNANOG-1급shNANOG-2조세포중TP53급CDKN1A기인표체상조,이MDM2급CCND1기인표체하조,실험조여대조조상비차이균유통계학의의(P<0.05).결론 NANOG재부분인류ALL세포계중유표체,하조NANOG후가통과MDM2-TP53-CDKN1A통로억제백혈병세포적증식,병사세포조체우G0/G1기.
Objective To explore the expression of NANOG gene in acute lymphoblastic leukemia (ALL) cell lines and the effect of down-regulation of NANOG on the proliferation and cell cycle of ALL cells.Methods Three kinds of ALL cell line MOLT-4,CCRF-HSB2 and Jurkat were chosen as study object.The expression of NANOG was detected by reverse transcription-polymerase chain reaction (RTPCR) and Western blot in MOLT-4,CCRF-HSB2 and Jurkat cells.The lentiviral vector (pLB-shNANOG-1 and pLB-shNANOG-2) carrying NANOG specific shRNA were constructed.After infection of MOLT-4 cells with the lentivirus constructs,green fluorescent protein positive cells were harvested by flow cytometry as interference group.With empty plasmid (pLB-sh control) infected MOLT-4 cells and normal cells were used as wild-type group and control group.The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot.Cell proliferation was evaluated using CCK-8 cell counting kit.Cell cycle was analyzed by flow cytometry.The expression of p53-related gene was detected by real-time quantitative PCR.Results The virus titers were (1.83-3.12)×108 IU/mL.Each interfering sequence cloud stably down-regulate the expression of NANOG.Analysis of cell proliferation indicated that the MOLT-4 cells expressing NANOG shRNA grew significantly slower than wild-type cells and control cells,and this difference became more obvious after 72 hours (P<0.001).Cell cycle analysis indicated that the percentage of S-phase cells in wild-type or control group cells was obviously increased when compared to MOLT-4 cells expressing NANOG shRNAs (P<0.01).The percentage of MOLT-4 cells expressing NANOG shRNAs in the G0/G1-phase was higher than in wild-type cells and control cells (P<0.001),while the percentage of cells in the G2/M-phase was not statistical different among these cells (P>0.05).The expression of gene TP53 and CDKN1A were increased in groups of shNANOG-1 and shNANOG-2 than that of groups of control and wild-type (P<0.05).But the expression of gene MDM2 and CCND1 was decreased (P<0.05).Conclusions NANOG is expressed in some human ALL cell lines.Down-regulation of NANOG could inhibit cell proliferation and arrest in the cell cycle of G0/G1 phase through a MDM2-TP53 CDKN1A pathway in leukemic cells.