CAJ | 학술논문

目的：环氧合酶-2(cyclooxygenase-2,COX-2)参与食管鳞癌(esophageal squamous cell carcinoma,ESCC)的发生发展过程,但在部分 ESCC 细胞株中 COX-2呈低表达。选择低表达 COX-2基因的 ESCC 进行裸鼠移植。探讨 COX-2基因甲基化与基因表达的关系,观察联合 COX-2选择性抑制剂和去甲基化药物干预对移植瘤生长的影响。方法选择 KYSE 150细胞进行裸鼠移植,采用数字表法随机分为4组：0.9%(质量分数)氯化钠注射液对照检查组7只。分别给予①尼美舒利(nimesulide,NIM)+5-氮杂脱氧胞苷(5-Aza-deoxycytidine,5-Aza)干预②尼美舒利干预③5-Aza 干预。5周后处死动物,比较各组移植瘤的直径,计算移植瘤体积,绘制生长曲线。反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)测定组织中 COX-2 mRNA 表达；免疫组织化学方法测定 COX-2蛋白的表达；亚硫酸氢盐测序方法测定移植瘤中 COX-2启动子的甲基化状态。酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测各组移植瘤中前列腺素 E2(prostaglandin E2,PGE2)的浓度。结果各组动物体质量增长差异无统计学意义,组间具有可比性。联合干预组移植瘤明显小于其他各组,差异具有统计学意义(P<0.01)。 COX-2 mRNA 和蛋白表达结果一致,均表现为联合干预组最低,5-Aza 干预组最高。甲基化测序结果10个 CpG 位点中联合干预组和5-Aza 干预组的甲基化率低于尼美舒利组和对照组(30% vs 58.3%)。 PGE2的浓度分析显示联合干预组明显低于对照组和单独尼美舒利干预组, PGE2比值在联合干预组、尼美舒利干预组分别为0.37、0.91,联合干预组的比值与对照组比较,差异具有统计学意义(P<0.05)。结论低表达 COX-2的 ESCC 移植模型中 COX-2启动子甲基化是调节该基因表达的重要机制之一。联合选择性 COX-2抑制剂尼美舒利和去甲基化药物5-氮杂脱氧胞苷对低表达 COX-2的 ESCC 移植瘤的生长有协同抑制作用。目的：環氧閤酶-2(cyclooxygenase-2,COX-2)參與食管鱗癌(esophageal squamous cell carcinoma,ESCC)的髮生髮展過程,但在部分 ESCC 細胞株中 COX-2呈低錶達。選擇低錶達 COX-2基因的 ESCC 進行裸鼠移植。探討 COX-2基因甲基化與基因錶達的關繫,觀察聯閤 COX-2選擇性抑製劑和去甲基化藥物榦預對移植瘤生長的影響。方法選擇 KYSE 150細胞進行裸鼠移植,採用數字錶法隨機分為4組：0.9%(質量分數)氯化鈉註射液對照檢查組7隻。分彆給予①尼美舒利(nimesulide,NIM)+5-氮雜脫氧胞苷(5-Aza-deoxycytidine,5-Aza)榦預②尼美舒利榦預③5-Aza 榦預。5週後處死動物,比較各組移植瘤的直徑,計算移植瘤體積,繪製生長麯線。反轉錄-聚閤酶鏈反應(reverse transcription-polymerase chain reaction,RT-PCR)測定組織中 COX-2 mRNA 錶達；免疫組織化學方法測定 COX-2蛋白的錶達；亞硫痠氫鹽測序方法測定移植瘤中 COX-2啟動子的甲基化狀態。酶聯免疫吸附測定(enzyme-linked immunosorbent assay,ELISA)法檢測各組移植瘤中前列腺素 E2(prostaglandin E2,PGE2)的濃度。結果各組動物體質量增長差異無統計學意義,組間具有可比性。聯閤榦預組移植瘤明顯小于其他各組,差異具有統計學意義(P<0.01)。 COX-2 mRNA 和蛋白錶達結果一緻,均錶現為聯閤榦預組最低,5-Aza 榦預組最高。甲基化測序結果10箇 CpG 位點中聯閤榦預組和5-Aza 榦預組的甲基化率低于尼美舒利組和對照組(30% vs 58.3%)。 PGE2的濃度分析顯示聯閤榦預組明顯低于對照組和單獨尼美舒利榦預組, PGE2比值在聯閤榦預組、尼美舒利榦預組分彆為0.37、0.91,聯閤榦預組的比值與對照組比較,差異具有統計學意義(P<0.05)。結論低錶達 COX-2的 ESCC 移植模型中 COX-2啟動子甲基化是調節該基因錶達的重要機製之一。聯閤選擇性 COX-2抑製劑尼美舒利和去甲基化藥物5-氮雜脫氧胞苷對低錶達 COX-2的 ESCC 移植瘤的生長有協同抑製作用。
목적：배양합매-2(cyclooxygenase-2,COX-2)삼여식관린암(esophageal squamous cell carcinoma,ESCC)적발생발전과정,단재부분 ESCC 세포주중 COX-2정저표체。선택저표체 COX-2기인적 ESCC 진행라서이식。탐토 COX-2기인갑기화여기인표체적관계,관찰연합 COX-2선택성억제제화거갑기화약물간예대이식류생장적영향。방법선택 KYSE 150세포진행라서이식,채용수자표법수궤분위4조：0.9%(질량분수)록화납주사액대조검사조7지。분별급여①니미서리(nimesulide,NIM)+5-담잡탈양포감(5-Aza-deoxycytidine,5-Aza)간예②니미서리간예③5-Aza 간예。5주후처사동물,비교각조이식류적직경,계산이식류체적,회제생장곡선。반전록-취합매련반응(reverse transcription-polymerase chain reaction,RT-PCR)측정조직중 COX-2 mRNA 표체；면역조직화학방법측정 COX-2단백적표체；아류산경염측서방법측정이식류중 COX-2계동자적갑기화상태。매련면역흡부측정(enzyme-linked immunosorbent assay,ELISA)법검측각조이식류중전렬선소 E2(prostaglandin E2,PGE2)적농도。결과각조동물체질량증장차이무통계학의의,조간구유가비성。연합간예조이식류명현소우기타각조,차이구유통계학의의(P<0.01)。 COX-2 mRNA 화단백표체결과일치,균표현위연합간예조최저,5-Aza 간예조최고。갑기화측서결과10개 CpG 위점중연합간예조화5-Aza 간예조적갑기화솔저우니미서리조화대조조(30% vs 58.3%)。 PGE2적농도분석현시연합간예조명현저우대조조화단독니미서리간예조, PGE2비치재연합간예조、니미서리간예조분별위0.37、0.91,연합간예조적비치여대조조비교,차이구유통계학의의(P<0.05)。결론저표체 COX-2적 ESCC 이식모형중 COX-2계동자갑기화시조절해기인표체적중요궤제지일。연합선택성 COX-2억제제니미서리화거갑기화약물5-담잡탈양포감대저표체 COX-2적 ESCC 이식류적생장유협동억제작용。
Objective Overexpression of cyclooxygenase-2(COX-2) is known to be associated with the carcinogenesis of esophageal squamous cell carcinoma(ESCC), but in some ESCC cell lines the expression level of COX-2 may be low. In this study, methylation state of cyclooxygenase-2 gene promoter in ESCC was examined in xenografts to investigate the relationship between promoter methylation and COX-2 expression. We also studied the effect of COX-2 selective inhibitor nimesulide in conjunction with 5-aza-2 deoxycytidine on the growth of xenografts. Methods KYSE150 cell line with low COX-2 expression was used to establish ESCC xenograft in nude mice. The mice were divided into four groups, nimesulide(NIM)+5-Aza-DC(5-Aza) (group 1), NIM group(group 2), 5-Aza group(group 3), and sodium chloride control group ( group 4 ). The growth of those xenografts in nude mice was observed. Methylation state of cyclooxygenase-2 gene promoter in xenografts was monitored using bisulfate sequencing method. RT-PCR and immunohistochemistry were employed to determine the expression level of COX-2 mRNA and protein in xenograft samples, respectively. PGE2 concentration in xenografts was measured by ELISA. Results During the trial, mice grew well and no infection or death was observed. No difference of weight was found between groups(P>0. 05). Xenograft volumes were different between groups with that of the sodium chloride group smaller than other groups, and the difference was significant(P<0. 01). It was showed that methylation degree of COX-2 gene promoter was higher in NIM group and sodium choloride group than that in NIM combining 5-Aza group and 5-Aza group. For 10 CpG sites, methylation degrees were 30% and 58. 3% in group 3 and group 4, respectively. The expressions of COX-2 mRNA and protein were coincidently low in group 1 and high in group 3. PGE2 values were 0. 37, 0. 91 and 1. 22 in group 1, 2 and 3 compared with group 4, respectively. Conclusion Promoter methylation of COX-2 gene is one of the important mechanisms to regulate COX-2 expression in low COX-2 expression ESCC xenografts. Combination of selective COX-2 inhibitor and 5-Aza has synergistic effect on ESCC xenografts.