基因%急性淋巴细胞白血病%甲基化%氧化还原酶类%肿瘤抑制蛋白质类%DNA结合蛋白质类%核蛋白质类
基因%急性淋巴細胞白血病%甲基化%氧化還原酶類%腫瘤抑製蛋白質類%DNA結閤蛋白質類%覈蛋白質類
기인%급성림파세포백혈병%갑기화%양화환원매류%종류억제단백질류%DNA결합단백질류%핵단백질류
Gene%Acute lymphocytic leukemia%Methylation%Oxidoreductases%Tumor suppressor proteins%DNA-binding proteins%Nuclear proteins
目的 研究含有氧化还原酶的WW域(WW domain containing oxidoreductase,WWOX)和p73基因在急性淋巴细胞白血病(acute lymphocytic leukemia,ALL)中异常表达的临床意义及其机制.方法 采用病例对照研究,收集2010-2011年广西医科大学第一附属医院收治的ALL患者骨髓样本48份,其中包括初诊患者32例、缓解患者11例、复发患者5例；收集同期非白血病患者骨髓样本31份作为对照组.抽取骨髓3 ml,EDTA抗凝,1 ml骨髓样本采用离心柱法立即提取RNA,取纯度在1.8～2.0范围内的产物逆转录后进行逆转录聚合酶链反应(RT-PCR),检测WWOX和p73基因mRNA的表达情况；2 ml骨髓样本采用离心柱法提取DNA,取纯度在1.7 ～1.9范围内的产物进行甲基化特异性聚合酶链反应(MS-PCR),检测WWOX基因启动子区和p73基因第1外显子的甲基化情况.不同组别间甲基化状态的比较采用x2检验或Fisher确切概率法.结果 31份对照组标本中,WWOX与p73基因mRNA阳性表达率均为94.00％(29/31).48份ALL标本中,WWOX mRNA阳性表达率为48.00％(23/48),低于对照组,差异有统计学意义(x2=17.434,P =0.000)；其中初诊组为34.38％(11/32),低于缓解组的90.91％(10/11),差异有统计学意义(x2=10.471,P=0.001).p73基因mRNA阳性表达率为56.00％( 27/48),低于对照组,差异有统计学意义(x2=12.697,P=0.000)；其中初诊组为43.75％( 14/32),低于缓解组的90.91％(10/11),差异有统计学意义(P=0.012).31份对照组标本中,WWOX与p73基因均未见甲基化现象(0/31).48份ALL标本中,WWOX基因甲基化率为44.00％( 21/48),明显高于对照组,差异有统计学意义(x2=18.473,P=0.000)；其中初诊组为56.25％ (18/32),高于缓解组的9.09％ (1/11),差异有统计学意义(P=0.012);p73基因甲基化率为35.00％(17/48),明显高于对照组,差异有统计学意义(x2=13.990,P=0.000),其中初诊组为46.88％( 15/32),高于缓解组的9.09％ (1/11),差异有统计学意义(P=0.033).WWOX与p73基因mRNA阳性表达率均与各自基因甲基化状态呈负相关(r=-0.678、-0.577,P=0.000).结论 WWOX与p73基因的甲基化可能导致基因的沉默,使其mRNA减少或缺失；WWOX与p73基因甲基化的检测可能对ALL的诊断和疗效评估有一定意义.
目的 研究含有氧化還原酶的WW域(WW domain containing oxidoreductase,WWOX)和p73基因在急性淋巴細胞白血病(acute lymphocytic leukemia,ALL)中異常錶達的臨床意義及其機製.方法 採用病例對照研究,收集2010-2011年廣西醫科大學第一附屬醫院收治的ALL患者骨髓樣本48份,其中包括初診患者32例、緩解患者11例、複髮患者5例；收集同期非白血病患者骨髓樣本31份作為對照組.抽取骨髓3 ml,EDTA抗凝,1 ml骨髓樣本採用離心柱法立即提取RNA,取純度在1.8～2.0範圍內的產物逆轉錄後進行逆轉錄聚閤酶鏈反應(RT-PCR),檢測WWOX和p73基因mRNA的錶達情況；2 ml骨髓樣本採用離心柱法提取DNA,取純度在1.7 ～1.9範圍內的產物進行甲基化特異性聚閤酶鏈反應(MS-PCR),檢測WWOX基因啟動子區和p73基因第1外顯子的甲基化情況.不同組彆間甲基化狀態的比較採用x2檢驗或Fisher確切概率法.結果 31份對照組標本中,WWOX與p73基因mRNA暘性錶達率均為94.00％(29/31).48份ALL標本中,WWOX mRNA暘性錶達率為48.00％(23/48),低于對照組,差異有統計學意義(x2=17.434,P =0.000)；其中初診組為34.38％(11/32),低于緩解組的90.91％(10/11),差異有統計學意義(x2=10.471,P=0.001).p73基因mRNA暘性錶達率為56.00％( 27/48),低于對照組,差異有統計學意義(x2=12.697,P=0.000)；其中初診組為43.75％( 14/32),低于緩解組的90.91％(10/11),差異有統計學意義(P=0.012).31份對照組標本中,WWOX與p73基因均未見甲基化現象(0/31).48份ALL標本中,WWOX基因甲基化率為44.00％( 21/48),明顯高于對照組,差異有統計學意義(x2=18.473,P=0.000)；其中初診組為56.25％ (18/32),高于緩解組的9.09％ (1/11),差異有統計學意義(P=0.012);p73基因甲基化率為35.00％(17/48),明顯高于對照組,差異有統計學意義(x2=13.990,P=0.000),其中初診組為46.88％( 15/32),高于緩解組的9.09％ (1/11),差異有統計學意義(P=0.033).WWOX與p73基因mRNA暘性錶達率均與各自基因甲基化狀態呈負相關(r=-0.678、-0.577,P=0.000).結論 WWOX與p73基因的甲基化可能導緻基因的沉默,使其mRNA減少或缺失；WWOX與p73基因甲基化的檢測可能對ALL的診斷和療效評估有一定意義.
목적 연구함유양화환원매적WW역(WW domain containing oxidoreductase,WWOX)화p73기인재급성림파세포백혈병(acute lymphocytic leukemia,ALL)중이상표체적림상의의급기궤제.방법 채용병례대조연구,수집2010-2011년엄서의과대학제일부속의원수치적ALL환자골수양본48빈,기중포괄초진환자32례、완해환자11례、복발환자5례；수집동기비백혈병환자골수양본31빈작위대조조.추취골수3 ml,EDTA항응,1 ml골수양본채용리심주법립즉제취RNA,취순도재1.8～2.0범위내적산물역전록후진행역전록취합매련반응(RT-PCR),검측WWOX화p73기인mRNA적표체정황；2 ml골수양본채용리심주법제취DNA,취순도재1.7 ～1.9범위내적산물진행갑기화특이성취합매련반응(MS-PCR),검측WWOX기인계동자구화p73기인제1외현자적갑기화정황.불동조별간갑기화상태적비교채용x2검험혹Fisher학절개솔법.결과 31빈대조조표본중,WWOX여p73기인mRNA양성표체솔균위94.00％(29/31).48빈ALL표본중,WWOX mRNA양성표체솔위48.00％(23/48),저우대조조,차이유통계학의의(x2=17.434,P =0.000)；기중초진조위34.38％(11/32),저우완해조적90.91％(10/11),차이유통계학의의(x2=10.471,P=0.001).p73기인mRNA양성표체솔위56.00％( 27/48),저우대조조,차이유통계학의의(x2=12.697,P=0.000)；기중초진조위43.75％( 14/32),저우완해조적90.91％(10/11),차이유통계학의의(P=0.012).31빈대조조표본중,WWOX여p73기인균미견갑기화현상(0/31).48빈ALL표본중,WWOX기인갑기화솔위44.00％( 21/48),명현고우대조조,차이유통계학의의(x2=18.473,P=0.000)；기중초진조위56.25％ (18/32),고우완해조적9.09％ (1/11),차이유통계학의의(P=0.012);p73기인갑기화솔위35.00％(17/48),명현고우대조조,차이유통계학의의(x2=13.990,P=0.000),기중초진조위46.88％( 15/32),고우완해조적9.09％ (1/11),차이유통계학의의(P=0.033).WWOX여p73기인mRNA양성표체솔균여각자기인갑기화상태정부상관(r=-0.678、-0.577,P=0.000).결론 WWOX여p73기인적갑기화가능도치기인적침묵,사기mRNA감소혹결실；WWOX여p73기인갑기화적검측가능대ALL적진단화료효평고유일정의의.
Objective To investigate the clinical significance and mechanism of WW domain containing oxidoreductase (WWOX) gene and p73 gene abnormal expression in acute lymphocytic leukemia (ALL).Methods Case-control study was used in the research.Forty-eight cases of bone marrows from ALL patients were collected,including 32 cases newly diagnosed,11 cases with complete remission and 5 case with relapse.Thirty-one cases of bone marrows from non-leukemia patients were used as control group.All the samples were collected from First Affiliated Hospital of Guangxi Medical University from July 2010 to July 2011.The doctors punctured patients' bone marrows 3 milliliters from the left of posterior superior iliac spine.Samples were bottled up with EDTA anti-coagulation tube.1 milliliter bone marrow was used to extract genome RNA with purity from 1.8 to 2.0.And then,the level of WWOX and p73 gene transcripts were tested immediately using reverse transcriptase-polymerase chain reaction (RT-PCR).Meanwhile genome DNA was also extracted from the other 2 milliliter bone marrow with purity from 1.7 to 1.9,which was used to detect the promoter methylation of WWOX gene and the first exon methylation of p73 gene by methylation PCR (MS-PCP).x2 test and Fisher's exact test were used to compare tbe methylation status of WWOX and p73 gene.Results In 31 controls,expression of WWOX and p73 gene mRNA was 94.00％.The total expression frequency of WWOX gene mRNA in 48 ALL samples was 48.00％ (23/48),much lower than control (x2 =17.434,P =0.000 ).There was significant difference (x2 =10.471,P =0.001 ) between newly diagnosed cases 34.38％ ( 11/32),complcte remission cases (90.91％,10/11 ) and control.The total expression frequency of p73 gene mRNA in 48 ALL samples was 56.00％ (27/48),much lower than control (x2 =12.697,P =0.000).There was significant difference (P =0.012 ) between newly diagnosed cases 43.75％ (14/32) and complete remission cases 90.91％ (10/11).It was unmethylation in 31 controls.The total methylation frequency of WWOX gene promoter region in 48 ALL samples was 44.00％(21/48),much lower than control (x2 =18.473,P =0.000).There was significant difference (P =0.012) between newly diagnosed cases 56.25％ (18/32),complete remission cases 9.09％ (1/11 ) and control.The total methylation frequency of p73 gene the first exon region in 48 ALL samples was 35.00％(17/48),much lower than control (x2 =13.990,P =0.000).There was significant difference (P =0.033) between newly diagnosed cases 46.88％ (15/32),complete remission cases 9.09％ ( 1/11 ) and control.There was a negative correlation between the expression of WWOX gene mRNA and its methylation status(r =- 0.678,P =0.000),the same as p73 gene ( r =- 0.577,P =0.000).Conclusions The abnormal methylation of WWOX and p73 gene may be the major mechanism of gene silence in ALL,which leads to no expression of WWOX mRNA or p73 mRNA.And the abnormal methylation of WWOX and p73 gene may be relevant with the process of occurrence and development in ALL.It may be an effective and significant to detect methylation status of WWOX gene and p73 gene for the diagnosis and treatment of ALL patients.(Chin J Lab Med,2012,35:820-825)
