中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2010年
1期
54-57
,共4页
聂晓瑞%张阳%潘凯枫%李文庆%游伟程%张联
聶曉瑞%張暘%潘凱楓%李文慶%遊偉程%張聯
섭효서%장양%반개풍%리문경%유위정%장련
环氧合酶-2%甲基化%色谱法%高压液相%评价研究
環氧閤酶-2%甲基化%色譜法%高壓液相%評價研究
배양합매-2%갑기화%색보법%고압액상%평개연구
Cyelooxygenase 2%Methylation%Chromatography%high pressure liquid%Evaluation studies
目的 利用变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)技术,建立定量分析环氧合酶-2(cyclooxygenase-2,COX-2)基因启动子区甲基化水平的方法 ,并应用于胃黏膜组织甲基化状态的检测.方法 利用亚硫酸氢钠-DHPLC技术,在55℃部分变性条件下,乙腈洗脱浓度梯度为4.0/min时,对COX-2基因启动子重要区域甲基化水平进行检测,并对10例胃镜活检石蜡组织标本进行测定.结果 以人早幼粒细胞白血病细胞系(HL-60)和人胃癌细胞系(MGC803)分别作为完全甲基化和非甲基化对照,建立COX-2基因启动子甲基化定量分析的标准体系.甲基化含量百分率与相应色谱峰比值具有良好的线性关系,回归方程为Y=1.0608×甲基化色谱峰高(M)/甲基化和非甲基化色谱峰高之和(M+u),R~2=0.9894.在此标准体系下测定10例胃黏膜组织标本中COX-2基因启动子甲基化含量百分率,发现甲基化阳性的2例异型增生病变中COX-2启动子甲基化含量百分率均高于甲基化阳性浅表性胃炎和慢性萎缩性胃炎(24.5%、18.4%对7.6%、9.6%).结论本研究建立了COX-2启动子甲基化含量百分率定量分析体系,并首次将其应用于小样本胃黏膜组织标本的检测,为大规模筛查人群胃黏膜COX-2甲基化水平奠定了基础.
目的 利用變性高效液相色譜(denaturing high performance liquid chromatography,DHPLC)技術,建立定量分析環氧閤酶-2(cyclooxygenase-2,COX-2)基因啟動子區甲基化水平的方法 ,併應用于胃黏膜組織甲基化狀態的檢測.方法 利用亞硫痠氫鈉-DHPLC技術,在55℃部分變性條件下,乙腈洗脫濃度梯度為4.0/min時,對COX-2基因啟動子重要區域甲基化水平進行檢測,併對10例胃鏡活檢石蠟組織標本進行測定.結果 以人早幼粒細胞白血病細胞繫(HL-60)和人胃癌細胞繫(MGC803)分彆作為完全甲基化和非甲基化對照,建立COX-2基因啟動子甲基化定量分析的標準體繫.甲基化含量百分率與相應色譜峰比值具有良好的線性關繫,迴歸方程為Y=1.0608×甲基化色譜峰高(M)/甲基化和非甲基化色譜峰高之和(M+u),R~2=0.9894.在此標準體繫下測定10例胃黏膜組織標本中COX-2基因啟動子甲基化含量百分率,髮現甲基化暘性的2例異型增生病變中COX-2啟動子甲基化含量百分率均高于甲基化暘性淺錶性胃炎和慢性萎縮性胃炎(24.5%、18.4%對7.6%、9.6%).結論本研究建立瞭COX-2啟動子甲基化含量百分率定量分析體繫,併首次將其應用于小樣本胃黏膜組織標本的檢測,為大規模篩查人群胃黏膜COX-2甲基化水平奠定瞭基礎.
목적 이용변성고효액상색보(denaturing high performance liquid chromatography,DHPLC)기술,건립정량분석배양합매-2(cyclooxygenase-2,COX-2)기인계동자구갑기화수평적방법 ,병응용우위점막조직갑기화상태적검측.방법 이용아류산경납-DHPLC기술,재55℃부분변성조건하,을정세탈농도제도위4.0/min시,대COX-2기인계동자중요구역갑기화수평진행검측,병대10례위경활검석사조직표본진행측정.결과 이인조유립세포백혈병세포계(HL-60)화인위암세포계(MGC803)분별작위완전갑기화화비갑기화대조,건립COX-2기인계동자갑기화정량분석적표준체계.갑기화함량백분솔여상응색보봉비치구유량호적선성관계,회귀방정위Y=1.0608×갑기화색보봉고(M)/갑기화화비갑기화색보봉고지화(M+u),R~2=0.9894.재차표준체계하측정10례위점막조직표본중COX-2기인계동자갑기화함량백분솔,발현갑기화양성적2례이형증생병변중COX-2계동자갑기화함량백분솔균고우갑기화양성천표성위염화만성위축성위염(24.5%、18.4%대7.6%、9.6%).결론본연구건립료COX-2계동자갑기화함량백분솔정량분석체계,병수차장기응용우소양본위점막조직표본적검측,위대규모사사인군위점막COX-2갑기화수평전정료기출.
Objective To setup a quantitative assay for detection of cyclcoxygenase-2 (COX-2)methylation in human gastric mucosa samples. MethodsA standard analysis system was established by denaturing high performance liquid chromatography (DHPLC) under the condition of 55 ℃ oven temperature and a linear acetonitril gradient (4.0/min). While, a total of 10 cases of gastric biopsy samples were detected for methylation status of COX-2. ResultsThe complete methylated human promyelocytic leukemia cells (HL-60) and unmethylated gastric cancer cell line (MGC803) were used as positive and negative control. The proportion of the methylated copies of COX-2 was calculated according to the peak heights of methylated(M) and unmethylated (U) COX-2 in same PCR amplicon. The formula was Y= 1. 0608 ×M/(M +U), R~2 = 0. 9894. Among 10 biopsy samples, the proportions of methylated copies of COX-2 in 2 cases of dysplasia were higher than superficial gastritis and chronic atrophy gastritis (24. 5%, 18.4% vs 7.6% ,9. 6% ). ConclusionThe methylation of COX-2 promoter CpG islands can be detected in human gastric mucosa samples by quantitative DHPLC assay, which could be used in the population-based study of precancerous gastric lesions.
