中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2009年
11期
1289-1293
,共5页
罗文娟%许文林%吕旭晶%邱志远%陈巧云%王法春
囉文娟%許文林%呂旭晶%邱誌遠%陳巧雲%王法春
라문연%허문림%려욱정%구지원%진교운%왕법춘
多药耐药蛋白质类%丝裂原活化蛋白激酶%细胞外信号调节MAP激酶类
多藥耐藥蛋白質類%絲裂原活化蛋白激酶%細胞外信號調節MAP激酶類
다약내약단백질류%사렬원활화단백격매%세포외신호조절MAP격매류
Multidrug resistance-associated protein%Mitogen-activated protein kinase%Extracellular signal-regulated MAP kinases
目的 观察多柔比星(doxorubicin,DOX)诱导K562细胞mdr1基因表达过程中丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶(ERK)信号通路的作用,探讨mdr1基因的转录调控机制.方法 多柔比星初始浓度为0.01 μg/ml,诱导K562细胞24 h后撤药继续培养至细胞状态恢复,加入多柔比星继续诱导24 h,浓度增加为0.02μg/ml.依上述方法 多柔比星浓度逐渐增加,直至0.05μg/ml.收集DOX浓度为0.01、0.03和0.05μg/ml时的细胞.RT-PCR检测mdr1基因表达,流式细胞仪检测mdr1基因编码的P糖蛋白(P-gP)的表达,Western blot 检测ERK活化情况.MAPK的抑制剂PD98059预处理K562细胞1 h后与多柔比星共同作用,逆转录(RT)-PCR和流式细胞仪分别检测mdr1基因和P-gP的表达情况.结果 经多柔比星作用后K562细胞中ERK磷酸化增强,同时mdr1基因转录上调,及其蛋白产物P-gP的表达也增加,当多柔比星浓度为0.05μg/ml时两者的表达均增加了5倍之多.而用PD98059预处理细胞后能明显抑制多柔比星诱导mdr1基因转录和蛋白表达,多柔比星浓度为0.03μg/ml时PD98059对mdr1基因表达的抑制率为(74.1±0.11)%,多柔比星浓度为0.05 μg/ml时抑制率为(70.2±0.14)%.结论 多柔比星能够诱导K562细胞mdr1基因表达,同时激活MAPK/ERK信号通路,阻断ERK的活化能抑制mdr1基因的诱导性表达.
目的 觀察多柔比星(doxorubicin,DOX)誘導K562細胞mdr1基因錶達過程中絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/細胞外信號調節激酶(ERK)信號通路的作用,探討mdr1基因的轉錄調控機製.方法 多柔比星初始濃度為0.01 μg/ml,誘導K562細胞24 h後撤藥繼續培養至細胞狀態恢複,加入多柔比星繼續誘導24 h,濃度增加為0.02μg/ml.依上述方法 多柔比星濃度逐漸增加,直至0.05μg/ml.收集DOX濃度為0.01、0.03和0.05μg/ml時的細胞.RT-PCR檢測mdr1基因錶達,流式細胞儀檢測mdr1基因編碼的P糖蛋白(P-gP)的錶達,Western blot 檢測ERK活化情況.MAPK的抑製劑PD98059預處理K562細胞1 h後與多柔比星共同作用,逆轉錄(RT)-PCR和流式細胞儀分彆檢測mdr1基因和P-gP的錶達情況.結果 經多柔比星作用後K562細胞中ERK燐痠化增彊,同時mdr1基因轉錄上調,及其蛋白產物P-gP的錶達也增加,噹多柔比星濃度為0.05μg/ml時兩者的錶達均增加瞭5倍之多.而用PD98059預處理細胞後能明顯抑製多柔比星誘導mdr1基因轉錄和蛋白錶達,多柔比星濃度為0.03μg/ml時PD98059對mdr1基因錶達的抑製率為(74.1±0.11)%,多柔比星濃度為0.05 μg/ml時抑製率為(70.2±0.14)%.結論 多柔比星能夠誘導K562細胞mdr1基因錶達,同時激活MAPK/ERK信號通路,阻斷ERK的活化能抑製mdr1基因的誘導性錶達.
목적 관찰다유비성(doxorubicin,DOX)유도K562세포mdr1기인표체과정중사렬원활화단백격매(mitogen-activated protein kinase,MAPK)/세포외신호조절격매(ERK)신호통로적작용,탐토mdr1기인적전록조공궤제.방법 다유비성초시농도위0.01 μg/ml,유도K562세포24 h후철약계속배양지세포상태회복,가입다유비성계속유도24 h,농도증가위0.02μg/ml.의상술방법 다유비성농도축점증가,직지0.05μg/ml.수집DOX농도위0.01、0.03화0.05μg/ml시적세포.RT-PCR검측mdr1기인표체,류식세포의검측mdr1기인편마적P당단백(P-gP)적표체,Western blot 검측ERK활화정황.MAPK적억제제PD98059예처리K562세포1 h후여다유비성공동작용,역전록(RT)-PCR화류식세포의분별검측mdr1기인화P-gP적표체정황.결과 경다유비성작용후K562세포중ERK린산화증강,동시mdr1기인전록상조,급기단백산물P-gP적표체야증가,당다유비성농도위0.05μg/ml시량자적표체균증가료5배지다.이용PD98059예처리세포후능명현억제다유비성유도mdr1기인전록화단백표체,다유비성농도위0.03μg/ml시PD98059대mdr1기인표체적억제솔위(74.1±0.11)%,다유비성농도위0.05 μg/ml시억제솔위(70.2±0.14)%.결론 다유비성능구유도K562세포mdr1기인표체,동시격활MAPK/ERK신호통로,조단ERK적활화능억제mdr1기인적유도성표체.
Objective To investigate the effect of mitogen-activated protein kinase(MAPK)pathway on the transcriptional expression of mdr1 gene induced by doxorubicin ( DOX)and study the transcription regulation of mdr1 gene.Methods K562 cells were treated with DOX(0.01 μg/ml)with the initial concentration of 0.01 μg/ml for 24 hours,then change the culture media without DOX.K562 cells were cultured until the its status wag recovered.Subsequently the cells were treated with DOX(0.02μg/ml)for 24 hours again.The concentration of DOX was increaged until 0.05 μg/ml by following the protocol above.K562 cells were collected at the concentration of 0.01 μg/ml,0.03μg/ml and 0.05μS/ml DOX.Expression of mdr1 gene were examined by reverse transcription-polymerase chain reaction(RT-PCR).Pglycoprotein(P-gP)wag detected by flow cytometry.Western blot wag performed to detect ERK and P-ERk.K562 cells were pretreated with MAPK inhibitor PD98059 for 1 hour.and then DOX was added.RT-PCR and FCM were used to detect the expression of mdr1 mRNA and P-gp.Results When K562 cells were exposured to DOX.the phosphorylation of ERK wag increaged.the mdr1 gene wag highly expressed as well as its corresponding protein P-gp.When the concentration of DOX was 0.05μg/ml,the expression of mdr1 gene and P-gp were increased over 5 fold.When K562 cells were pretreated with MAPK inhibitor PD98059,the expression of mdr1 gene induced by DOX(the concentration was 0.03 μg/ml and 0.05 μg/m1)was effectively inhibited by(74.1±0.11)%and(70.2±0.14)%respectively.Conclusions DOX could induce the expression of mdr1 gene in K562 cells accompanied by the activation of MAPK/ERK pathway.The block of activation of ERK could inhibit the induced expression of mdr1 gene.
