中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2011年
8期
722-726
,共5页
孙利%何晓东%孙余婕%许维东%李道静%张白银%张永娟%刘锐%沈佐君
孫利%何曉東%孫餘婕%許維東%李道靜%張白銀%張永娟%劉銳%瀋佐君
손리%하효동%손여첩%허유동%리도정%장백은%장영연%류예%침좌군
细胞系,肿瘤%白血病%甲氨蝶呤%肽合酶类%抗药性,肿瘤%聚合酶链反应%立体异构现象
細胞繫,腫瘤%白血病%甲氨蝶呤%肽閤酶類%抗藥性,腫瘤%聚閤酶鏈反應%立體異構現象
세포계,종류%백혈병%갑안접령%태합매류%항약성,종류%취합매련반응%입체이구현상
Cell line,tumor%Leukemia%Methotrexate%Peptide synthases%Drug resistance,neoplasm%Polymerase chain reaction%Stereoisomerism
目的 建立实时荧光定量PCR检测肿瘤细胞中FPGS mRNA表达的方法,研究MTX对映体[L-(+)-MTX和D-(-)-MTX]耐药细胞株中FPGS的基因表达差异,并用于观察白血病患者MTX治疗耐药前后FPGS mRNA表达水平的变化.方法 用SYBR Green Ⅰ为荧光染料,以β-actin作参照,建立检测FPGS mRNA的实时荧光定量PCR方法.根据Ct值、标准曲线相关系数、斜率、重复性曲线、熔解曲线、扩增效率曲线等进行方法学评价.并用该方法检测MTX对映体耐药细胞株及应用MTX耐药的14例白血病患者骨髓细胞中FPGS mRNA的表达.结果 建立的标准曲线Ct值与模板浓度有良好的线性关系,FPGS和β-actin标准曲线相关系数分别为0.996 8和0.998 7,斜率分别为-3.595和-3.740,批内CV为1.27%～2.95%,批间CV为3.82%;熔解曲线均呈单个特异峰,扩增效率相似(斜率为0.021 7);L-(+)-MTX耐药细胞和D-(-)-MTX耐药细胞中FPGS mRNA相对含量分别为(3.51±0.66)和(0.16±0.01),A549亲本细胞(S)中FPGS mRNA相对含量为(1.00±0.31),差异有统计学意义(F=64.45,P＜0.01);L-(+)-MTX耐药细胞和D-(-)-MTX耐药细胞间FPGS mRNA相对含量差异有统计学意义(q=9.29,P＜0.01).白血病患者应用MTX治疗耐药后,FPGS mRNA表达水平为(0.35±0.04),用药前为(1.00±0.44),差异有统计学意义(t=8.83,P＜0.01).结论 建立的实时荧光定量PCR检测FPGS mRNA的方法重复性好、特异度高,可用于FPGSmRNA含量分析.MTX诱导耐药后细胞株及白血病患者骨髓细胞中FPGS mRNA表达发生了变化,MTX 2种对映体形式在细胞耐药机制中可能发挥了不同的作用.
目的 建立實時熒光定量PCR檢測腫瘤細胞中FPGS mRNA錶達的方法,研究MTX對映體[L-(+)-MTX和D-(-)-MTX]耐藥細胞株中FPGS的基因錶達差異,併用于觀察白血病患者MTX治療耐藥前後FPGS mRNA錶達水平的變化.方法 用SYBR Green Ⅰ為熒光染料,以β-actin作參照,建立檢測FPGS mRNA的實時熒光定量PCR方法.根據Ct值、標準麯線相關繫數、斜率、重複性麯線、鎔解麯線、擴增效率麯線等進行方法學評價.併用該方法檢測MTX對映體耐藥細胞株及應用MTX耐藥的14例白血病患者骨髓細胞中FPGS mRNA的錶達.結果 建立的標準麯線Ct值與模闆濃度有良好的線性關繫,FPGS和β-actin標準麯線相關繫數分彆為0.996 8和0.998 7,斜率分彆為-3.595和-3.740,批內CV為1.27%～2.95%,批間CV為3.82%;鎔解麯線均呈單箇特異峰,擴增效率相似(斜率為0.021 7);L-(+)-MTX耐藥細胞和D-(-)-MTX耐藥細胞中FPGS mRNA相對含量分彆為(3.51±0.66)和(0.16±0.01),A549親本細胞(S)中FPGS mRNA相對含量為(1.00±0.31),差異有統計學意義(F=64.45,P＜0.01);L-(+)-MTX耐藥細胞和D-(-)-MTX耐藥細胞間FPGS mRNA相對含量差異有統計學意義(q=9.29,P＜0.01).白血病患者應用MTX治療耐藥後,FPGS mRNA錶達水平為(0.35±0.04),用藥前為(1.00±0.44),差異有統計學意義(t=8.83,P＜0.01).結論 建立的實時熒光定量PCR檢測FPGS mRNA的方法重複性好、特異度高,可用于FPGSmRNA含量分析.MTX誘導耐藥後細胞株及白血病患者骨髓細胞中FPGS mRNA錶達髮生瞭變化,MTX 2種對映體形式在細胞耐藥機製中可能髮揮瞭不同的作用.
목적 건립실시형광정량PCR검측종류세포중FPGS mRNA표체적방법,연구MTX대영체[L-(+)-MTX화D-(-)-MTX]내약세포주중FPGS적기인표체차이,병용우관찰백혈병환자MTX치료내약전후FPGS mRNA표체수평적변화.방법 용SYBR Green Ⅰ위형광염료,이β-actin작삼조,건립검측FPGS mRNA적실시형광정량PCR방법.근거Ct치、표준곡선상관계수、사솔、중복성곡선、용해곡선、확증효솔곡선등진행방법학평개.병용해방법검측MTX대영체내약세포주급응용MTX내약적14례백혈병환자골수세포중FPGS mRNA적표체.결과 건립적표준곡선Ct치여모판농도유량호적선성관계,FPGS화β-actin표준곡선상관계수분별위0.996 8화0.998 7,사솔분별위-3.595화-3.740,비내CV위1.27%～2.95%,비간CV위3.82%;용해곡선균정단개특이봉,확증효솔상사(사솔위0.021 7);L-(+)-MTX내약세포화D-(-)-MTX내약세포중FPGS mRNA상대함량분별위(3.51±0.66)화(0.16±0.01),A549친본세포(S)중FPGS mRNA상대함량위(1.00±0.31),차이유통계학의의(F=64.45,P＜0.01);L-(+)-MTX내약세포화D-(-)-MTX내약세포간FPGS mRNA상대함량차이유통계학의의(q=9.29,P＜0.01).백혈병환자응용MTX치료내약후,FPGS mRNA표체수평위(0.35±0.04),용약전위(1.00±0.44),차이유통계학의의(t=8.83,P＜0.01).결론 건립적실시형광정량PCR검측FPGS mRNA적방법중복성호、특이도고,가용우FPGSmRNA함량분석.MTX유도내약후세포주급백혈병환자골수세포중FPGS mRNA표체발생료변화,MTX 2충대영체형식재세포내약궤제중가능발휘료불동적작용.
Objective To establish a real-time fluorescence quantitative PCR method for detection of the different expression level of FPGS in methotrexate enantiomer-resistant A549 cell lines,and observe FPGS mRNA expression in patients with leukemia.Methods A real-time fluorescence quantitative PCR method for detection FPGS mRNA was established using SYBR Green Ⅰ as fluorescence and β-actin as reference.The method was evaluated by Ct,correlation coefficient,slope,repeatability curve,melting curve and amplification efficiency curve.The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method.Results The standard curves had a high linear relationship between cycle threshold and template concentration.The correlation coefficients of FPGS and β-actin were 0.996 8 and 0.998 7,and the slopes were -3.595 and -3.740,respectively.The inter-coefficient of variation was from 1.27% to 2.95%.The intra-coefficient of variation was 3.82%.The method was characterized with specific melting curve and similar amplification efficiency(slope was 0.021 7).The relative contents of FPGS mRNA were(3.51 ±0.66),(0.16 ±0.01) and(1.00 ±0.31) in L-(+)-MTX/A549 cells(L),D-(-)-MTX/A549 cells(D)and A549 parent cells,and there was statistically difference among the three groups(F = 64.45 ,P＜ 0.01)Statistical difference was observed between L and D(q =9.29,P＜0.01).After treated with MTX,the expression level of FPGS mRNA was(0.35 ± 0.04) in methotrexate resistant leukemia patients,compared with(1.00 ± 0.44) before treatment.Statistical difference was observed(t = 8.83 ,P＜ 0.01).Conclusions The real-time fluorescence quantitative PCR is suitable for the quantification of FPGS.The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow and drug resistant cells are different.Two enantiomer forms of methotrexate may play different roles in drug resistance mechanisms.
