CAJ | 학술논문

目的探讨乙型肝炎病毒X蛋白(HBX)、环氧合酶-2(COX-2)在乙型肝炎相关性肝细胞肝癌(HCC)组织微血管生成和转移中的关系及其可能的调节机制. 方法选择84例乙型肝炎相关性肝细胞肝癌组织和22例非乙型肝炎相关性肝细胞肝癌组织,免疫组织化学法检测HBX、COX-2及血管内皮细胞表面抗原(CD34)的表达,光镜下记录微血管计数(MVD).Spearman相关性分析HBX、COX-2与乙型肝炎相关性HCC组织微血管生成及转移中的关系; Western blot和RT-PCR检测稳定转染HBX肝癌细胞HepG2(HepG2-X)中COX-2的变化;酶联免疫吸附法检测HepG2-X培养上清液中前列腺素E2(PGE2)表达水平及不同浓度(10、30、50 μ mol/L和70 μmol/L)COX-2选择性抑制剂塞来昔布作用后PGE2的表达水平;锥虫蓝拒染法检测细胞生长速度.结果乙型肝炎相关性HCC组织中HBx、COX-2均高表达,HBx阳性表达组中COX-2阳性率为88.87%(55/62),明显高于HBx阴性组的31.82%(7/22,x2=27.188,P＜0.01)和非乙型肝炎相关性HCC组40.91%(9/22,x2=20.453,P＜0.01);HBx阴性表达的乙型肝炎相关性HCC组和非乙型肝炎相关性HCC组中COX-2阳性表达率差异无统计学意义(x2=0.393,P=0.531); HBx和COX-2在转移组表达高于无转移组(P＜0.01);HBx及COX-2的阳性表达组平均MVD值均明显高于HBx阴性组和非乙型肝炎相关性HCC组(P＜0.05),且转移组MVD高于无转移组(P＜0.01);门静脉侵犯组高于非侵犯组(P＜0.01);Spearman相关性分析结果提示HBx、COX-2在乙型肝炎相关性HCC组织微血管生成及转移中的表达呈正相关(Rs=0.568,P＜0.01);在HepG2-X细胞中,COX-2蛋白及mRNA表达水平与转染空载体的对照肝癌细胞HepG2 (空载体转染HepG2细胞,HepG2-PC)相比,均显著提高,并且细胞培养上清液中PGE2表达水平也显著提高;和转染空载体的对照细胞相比,塞来昔布对转染HBx基因的细胞分泌PGE2具有更强的抑制作用.结论 HBx、COX-2在乙型肝炎相关性HCC中有较高的表达水平,二者呈正相关,与HCC微血管生成和侵袭转移密切相关.COX-2可能是HBx促使肝癌组织微血管生成和转移的一个重要中间分子,其可能的调节机制是通过HBx、COX-2及PGE2作为信号转导通路在HCC的浸润与迁移,在微血管生成与转移的过程中发挥双重作用. 目的探討乙型肝炎病毒X蛋白(HBX)、環氧閤酶-2(COX-2)在乙型肝炎相關性肝細胞肝癌(HCC)組織微血管生成和轉移中的關繫及其可能的調節機製. 方法選擇84例乙型肝炎相關性肝細胞肝癌組織和22例非乙型肝炎相關性肝細胞肝癌組織,免疫組織化學法檢測HBX、COX-2及血管內皮細胞錶麵抗原(CD34)的錶達,光鏡下記錄微血管計數(MVD).Spearman相關性分析HBX、COX-2與乙型肝炎相關性HCC組織微血管生成及轉移中的關繫; Western blot和RT-PCR檢測穩定轉染HBX肝癌細胞HepG2(HepG2-X)中COX-2的變化;酶聯免疫吸附法檢測HepG2-X培養上清液中前列腺素E2(PGE2)錶達水平及不同濃度(10、30、50 μ mol/L和70 μmol/L)COX-2選擇性抑製劑塞來昔佈作用後PGE2的錶達水平;錐蟲藍拒染法檢測細胞生長速度.結果乙型肝炎相關性HCC組織中HBx、COX-2均高錶達,HBx暘性錶達組中COX-2暘性率為88.87%(55/62),明顯高于HBx陰性組的31.82%(7/22,x2=27.188,P＜0.01)和非乙型肝炎相關性HCC組40.91%(9/22,x2=20.453,P＜0.01);HBx陰性錶達的乙型肝炎相關性HCC組和非乙型肝炎相關性HCC組中COX-2暘性錶達率差異無統計學意義(x2=0.393,P=0.531); HBx和COX-2在轉移組錶達高于無轉移組(P＜0.01);HBx及COX-2的暘性錶達組平均MVD值均明顯高于HBx陰性組和非乙型肝炎相關性HCC組(P＜0.05),且轉移組MVD高于無轉移組(P＜0.01);門靜脈侵犯組高于非侵犯組(P＜0.01);Spearman相關性分析結果提示HBx、COX-2在乙型肝炎相關性HCC組織微血管生成及轉移中的錶達呈正相關(Rs=0.568,P＜0.01);在HepG2-X細胞中,COX-2蛋白及mRNA錶達水平與轉染空載體的對照肝癌細胞HepG2 (空載體轉染HepG2細胞,HepG2-PC)相比,均顯著提高,併且細胞培養上清液中PGE2錶達水平也顯著提高;和轉染空載體的對照細胞相比,塞來昔佈對轉染HBx基因的細胞分泌PGE2具有更彊的抑製作用.結論 HBx、COX-2在乙型肝炎相關性HCC中有較高的錶達水平,二者呈正相關,與HCC微血管生成和侵襲轉移密切相關.COX-2可能是HBx促使肝癌組織微血管生成和轉移的一箇重要中間分子,其可能的調節機製是通過HBx、COX-2及PGE2作為信號轉導通路在HCC的浸潤與遷移,在微血管生成與轉移的過程中髮揮雙重作用.
목적 탐토을형간염병독X단백(HBX)、배양합매-2(COX-2)재을형간염상관성간세포간암(HCC)조직미혈관생성화전이중적관계급기가능적조절궤제. 방법선택84례을형간염상관성간세포간암조직화22례비을형간염상관성간세포간암조직,면역조직화학법검측HBX、COX-2급혈관내피세포표면항원(CD34)적표체,광경하기록미혈관계수(MVD).Spearman상관성분석HBX、COX-2여을형간염상관성HCC조직미혈관생성급전이중적관계; Western blot화RT-PCR검측은정전염HBX간암세포HepG2(HepG2-X)중COX-2적변화;매련면역흡부법검측HepG2-X배양상청액중전렬선소E2(PGE2)표체수평급불동농도(10、30、50 μ mol/L화70 μmol/L)COX-2선택성억제제새래석포작용후PGE2적표체수평;추충람거염법검측세포생장속도.결과 을형간염상관성HCC조직중HBx、COX-2균고표체,HBx양성표체조중COX-2양성솔위88.87%(55/62),명현고우HBx음성조적31.82%(7/22,x2=27.188,P＜0.01)화비을형간염상관성HCC조40.91%(9/22,x2=20.453,P＜0.01);HBx음성표체적을형간염상관성HCC조화비을형간염상관성HCC조중COX-2양성표체솔차이무통계학의의(x2=0.393,P=0.531); HBx화COX-2재전이조표체고우무전이조(P＜0.01);HBx급COX-2적양성표체조평균MVD치균명현고우HBx음성조화비을형간염상관성HCC조(P＜0.05),차전이조MVD고우무전이조(P＜0.01);문정맥침범조고우비침범조(P＜0.01);Spearman상관성분석결과제시HBx、COX-2재을형간염상관성HCC조직미혈관생성급전이중적표체정정상관(Rs=0.568,P＜0.01);재HepG2-X세포중,COX-2단백급mRNA표체수평여전염공재체적대조간암세포HepG2 (공재체전염HepG2세포,HepG2-PC)상비,균현저제고,병차세포배양상청액중PGE2표체수평야현저제고;화전염공재체적대조세포상비,새래석포대전염HBx기인적세포분비PGE2구유경강적억제작용.결론 HBx、COX-2재을형간염상관성HCC중유교고적표체수평,이자정정상관,여HCC미혈관생성화침습전이밀절상관.COX-2가능시HBx촉사간암조직미혈관생성화전이적일개중요중간분자,기가능적조절궤제시통과HBx、COX-2급PGE2작위신호전도통로재HCC적침윤여천이,재미혈관생성여전이적과정중발휘쌍중작용.
Objective To investigate the expression of HBx and COX-2 in hepatitis B virus-related hepatocellular carcinoma, and Its correlation with microangiogenesis and metastasis, and possible mechanism.Methods Immunohistochemistry was used to detect the expression of hepatitis B virus X, cyclooxygenase-2 and CD34 in hepatitis B virus related hepatic carcinoma and 22 non-HBV related hepatic carcinoma tissues.The expression of hepatitis B virus x protein and cyclooxygenase-2 in hepatitis B virus-related hepatocellular carcinoma correlated with microangiogenesis and metastasis was tested by Spearman correlation analysis.The expression of COX-2 in HepG2-X was detected by Western blot and RT-PCR. PGE2 was detected by ELISA in clear supernatant liquid of HepG2-X. Trypan blue exclusion was performed to examine the inhibitory effects of COX-2 selective inhibitor (celecoxib). Results In Hepatitis B carcinoma tissue, HBx and COX-2 were expressed at high level. The positive rate of COX-2 expression was 88.87% (55/62) in HBx positive expression group, which was significantly higher than that of the positive expression 31.82% (7/22,x2 = 27.188, P ＜ 0.01) in HBx negative expression group and 40.91% (9/22, x2 = 20.453, P ＜ 0.01) in nonHBV related hepatic carcinoma tissues, but it had no statistical difference ( x2 = 0.393, P = 0.53l)between the HBx negative expression group and non-HBV related hepatic carcinoma tissue group. The expressions of HBx and COX-2 in metastasis group were higher than that in non- metastasis group (P＜0.01), MVD in HBx or COX-2 positive expression group was significantly higher than that in negative expression group and nonHBV related hepatic carcinoma tissues (P＜0.01). MVD with metastasis was higher than that without metastasis (P ＜ 0.01) and MVD with portal vein invasion was higher than that without invasion (P＜0.05).Spearman correlation analysis showed that the expression of COX-2 was significantly correlated with the expression of HBx (Rs = 0.568, P ＜ 0.01). Celecoxib suppressed the growth of both cells in a dose-dependent manner. HepG2-X was significantly susceptible to celecoxib as compared to the HepG2-PC cells. COX-2 protein and mRNA were upregulated in HepG2-X cells than in HepG2-PC. Moreover, PGE2 was upregulated in clear supernatant liquid of HepG2-X than in HepG2-PC. Conclusion The expressions of HBx and COX-2 were higer in HBV-related hepatocellular carcinoma. COX-2 was significanfiy correlated with HBx in HCC and it could be a key factor involved in HBx contributed hepatocellular carcinoma's microangiogenesis and metastasis. The possible mechanism of the dual effects might be through HBx,COX-2 and PGE2 pathways in infiltration involved metastasis and microangiogenesis involved metastasis.