中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2008年
9期
1034-1038
,共5页
史梦%冯文莉%黄世峰%曾建明%王小中%温健萍%张文萍%陶昆%陈新敏%曹唯希%黄宗干
史夢%馮文莉%黃世峰%曾建明%王小中%溫健萍%張文萍%陶昆%陳新敏%曹唯希%黃宗榦
사몽%풍문리%황세봉%증건명%왕소중%온건평%장문평%도곤%진신민%조유희%황종간
白血病,髓样,慢性%K562细胞%哌嗪类%嘧啶类%DNA修复
白血病,髓樣,慢性%K562細胞%哌嗪類%嘧啶類%DNA脩複
백혈병,수양,만성%K562세포%고진류%밀정류%DNA수복
Leukemia,myeloid,chronic%K562 cells%Piperasines%Pyrimidines%DNA repair
目的 建立人类慢性粒细胞白血病(CML)细胞系K562细胞在伊马替尼(Imatinib mesylate)处理前、后的DNA损伤模型,探讨伊马替尼对K562细胞的DNA损伤修复功能的影响.方法 用噻唑蓝(MTT)法确定伊马替尼预处理K562细胞的浓度;用免疫印迹法(Western blot)检测伊马替尼处理后K562细胞BCR/ABL的磷酸化状态,以反映BCR-ABL酪氨酸激酶活性受抑制情况;用彗星实验检测不同浓度过氧化氢(H2O2)诱导的K562细胞、伊马替尼预处理K562细胞的DNA损伤模型;用彗星实验对各组细胞在DNA损伤后的修复进行动态观测.结果 MTT实验结果显示,伊马替尼预处理K562细胞的最佳终浓度为1 μmol/L,作用时间24 h;Western blot实验结果显示,该浓度的伊马替尼可有效抑制BCR/ABL融合蛋白第177位酪氨酸激酶磷酸化,密度比为0.100±0.018,与对照组(0.425±0.039)相比,降低了(77.11±5.59)%,差异有统计学意义(t=4.57,P<0.05);彗星实验确定了各组细胞DNA损伤模型的建立条件,采用10 μmol/L终浓度的H2O2对K562细胞和伊马替尼预处理后K562细胞进行染毒,H2O2作用温度和时间为4℃、10 min.修复结果显示,经伊马替尼预处理的K562细胞修复时间为120 min,与未处理组修复时间60 min相比,前者DNA损伤修复时间明显延长(F=97.79,P<0.05).结论 本研究建立了伊马替尼处理前、后的白血病K562细胞的DNA损伤模型,BCR/ABL融合蛋白的酪氨酸激酶抑制剂伊马替尼能减弱K562细胞的DNA损伤后修复能力.
目的 建立人類慢性粒細胞白血病(CML)細胞繫K562細胞在伊馬替尼(Imatinib mesylate)處理前、後的DNA損傷模型,探討伊馬替尼對K562細胞的DNA損傷脩複功能的影響.方法 用噻唑藍(MTT)法確定伊馬替尼預處理K562細胞的濃度;用免疫印跡法(Western blot)檢測伊馬替尼處理後K562細胞BCR/ABL的燐痠化狀態,以反映BCR-ABL酪氨痠激酶活性受抑製情況;用彗星實驗檢測不同濃度過氧化氫(H2O2)誘導的K562細胞、伊馬替尼預處理K562細胞的DNA損傷模型;用彗星實驗對各組細胞在DNA損傷後的脩複進行動態觀測.結果 MTT實驗結果顯示,伊馬替尼預處理K562細胞的最佳終濃度為1 μmol/L,作用時間24 h;Western blot實驗結果顯示,該濃度的伊馬替尼可有效抑製BCR/ABL融閤蛋白第177位酪氨痠激酶燐痠化,密度比為0.100±0.018,與對照組(0.425±0.039)相比,降低瞭(77.11±5.59)%,差異有統計學意義(t=4.57,P<0.05);彗星實驗確定瞭各組細胞DNA損傷模型的建立條件,採用10 μmol/L終濃度的H2O2對K562細胞和伊馬替尼預處理後K562細胞進行染毒,H2O2作用溫度和時間為4℃、10 min.脩複結果顯示,經伊馬替尼預處理的K562細胞脩複時間為120 min,與未處理組脩複時間60 min相比,前者DNA損傷脩複時間明顯延長(F=97.79,P<0.05).結論 本研究建立瞭伊馬替尼處理前、後的白血病K562細胞的DNA損傷模型,BCR/ABL融閤蛋白的酪氨痠激酶抑製劑伊馬替尼能減弱K562細胞的DNA損傷後脩複能力.
목적 건립인류만성립세포백혈병(CML)세포계K562세포재이마체니(Imatinib mesylate)처리전、후적DNA손상모형,탐토이마체니대K562세포적DNA손상수복공능적영향.방법 용새서람(MTT)법학정이마체니예처리K562세포적농도;용면역인적법(Western blot)검측이마체니처리후K562세포BCR/ABL적린산화상태,이반영BCR-ABL락안산격매활성수억제정황;용혜성실험검측불동농도과양화경(H2O2)유도적K562세포、이마체니예처리K562세포적DNA손상모형;용혜성실험대각조세포재DNA손상후적수복진행동태관측.결과 MTT실험결과현시,이마체니예처리K562세포적최가종농도위1 μmol/L,작용시간24 h;Western blot실험결과현시,해농도적이마체니가유효억제BCR/ABL융합단백제177위락안산격매린산화,밀도비위0.100±0.018,여대조조(0.425±0.039)상비,강저료(77.11±5.59)%,차이유통계학의의(t=4.57,P<0.05);혜성실험학정료각조세포DNA손상모형적건립조건,채용10 μmol/L종농도적H2O2대K562세포화이마체니예처리후K562세포진행염독,H2O2작용온도화시간위4℃、10 min.수복결과현시,경이마체니예처리적K562세포수복시간위120 min,여미처리조수복시간60 min상비,전자DNA손상수복시간명현연장(F=97.79,P<0.05).결론 본연구건립료이마체니처리전、후적백혈병K562세포적DNA손상모형,BCR/ABL융합단백적락안산격매억제제이마체니능감약K562세포적DNA손상후수복능력.
Objeetive To construct the cell DNA damage models for the human CML K562 cell line before or after imarlnib mesylate treatment and observe the repairing process dynamically for investigating the iniluence of imatinib mesylate on the repair function of K562 cells after cell DNA danlage.Methods The MTT assay was used to estimate the optimal pretreatment concentration of imatinib mesylate in K562 cells and Western blot was employed to evaluate the phosphorylation status in K562 cells after imatinib mesylate treatment to estimate BCR/ABL tyrosine kinase inhibition by imatinib mesylate.The comet assay was used to detect the DNA damage induced by hydrogen peroxide at various concentrations in K562 cells with or without the pretreatment of imatinib mesylate.A dynamic observation on the repairing process after cell DNA damage was made by the comet assay.Results The pretreatment by imatinib mesylate for K562 cells was optimized to be at a final concentration of 1 μmol/L for 24 h as revealed by the MTT assay additionly imatinib mesylate treatment at this concentration could effectively inhibit the phosphorylation of the BCR-ABL fusion protein at Tyr177(Deusityrate 0.100±0.018).When compared with the control group(Deusityrate 0.425±0.039),the BCR/ABL phosphorylation at Tyr177 was significantly decreased by (77. 11±5.59) % (t=4. 57,P<0. 05). The cell DNA damage models for both imatinib mesylate-nontreated and imatinib mesylate-pretreated K562 cell groups were constructed with hydrogen peroxide treatment at a final concentration of 10 μmol/L for 10 min at 4℃ as confirmed by the comet assay. When compared with the control imatinib mesylatenontreatod K562 cell group,the time duration required for the DNA repair in imatinib mesylate-pretreated K562 cell group was significantly prolonged (F= 97.79,P<0. 05 ). Conclusions The cell DNA damage models for the leukemic K562 cell groups before or after imatinib mesylate treatment were successfully constructed and the tyrosine kinase inhibitor imatinib mesylate for BCR/ABL fusion protein was revealed to attenuate the DNA repair capacity of the K562 cells after DNA damage.