【作者】 吴光杨；

【导师】 李建生； 许戈良； 黄强； 胡何节； 马金良； 英卫东；

【作者基本信息】 安徽医科大学 ， 外科学， 2008， 硕士

【摘要】 肝细胞性肝癌（hepatocellular carcinoma, HCC）是我国常见的恶性肿瘤之一,近二十年来,我国HCC的死亡率增加了41.7%,约占全世界HCC死亡人数的42%。目前,手术切除仍是治疗HCC的最佳手段,但本病起病隐匿,病情发展迅速,大多数患者就诊时已发展到中晚期,失去了手术治疗的机会,患者5年生存率低于10%。环氧合酶（cylooxygenases,COX）和脂氧合酶（lipoxygenase,LOX）是体内催化花生四烯酸（arachidonic acid, AA）代谢的两个关键酶。细胞膜上的磷脂在磷脂酶A₂作用下释放出AA,AA经COX和LOX两条通路代谢产生一系列的生物活性物质,如前列腺素（PG）、血栓烷素A2 （TXA2）、羟基廿碳四烯酸（HETES）和白细胞三烯（LTS）等。通过这些活性物质,COX及LOX影响细胞信号转导及代谢,进而对包括肿瘤,炎症在内的多种疾病产生作用。NS-398是一种选择性COX-2抑制剂,与传统的非甾体类抗炎药（nonsteroidal anti-inflammatory drugs,NSAIDS）不同的是其抑制COX-1的作用非常弱,从而减轻了胃肠道的不良反应。目前其已被发现能够抑制肝癌细胞增殖和诱导细胞周期阻滞。LOX抑制剂NDGA对于人肝癌细胞株HepG2细胞具有明显生长抑制作用,同时还可以诱导HepG₂细胞凋亡,引起细胞周期被阻滞于G1/S期。本研究选用人肝癌细胞株HepG₂作为研究对象,探讨NS-398和NDGA联合应用对人肝癌细胞的增殖抑制和诱导凋亡作用及对bcl-2mRNA表达的影响,为将NS-398和NDGA联合应用于HCC的治疗提供初步的实验依据。目的:探讨单独和联合应用选择性环氧合酶-2抑制剂（cyclooxygenase-2 selective inhibitor） NS-398和脂氧合酶抑制剂（lipoxygenase inhibitor）NDGA对于人肝癌细胞株HepG2的增殖、凋亡和对bcl-2mRNA表达的影响。方法:（1）采用四甲基偶氮唑蓝（MTT）比色法检测单独及联合应用NS-398和NDGA对于人肝癌细胞株HepG2的增殖抑制作用并应用金正均法判断两药的相互作用。（2）采用DNA末端原位标记染色法（TUNEL法）观察药物作用后人肝癌细胞株HepG2的形态学改变并计算凋亡指数（apoptosis index, AI）。（3）采用流式细胞术检测药物作用后人肝癌细胞株HepG2细胞凋亡率。（4）采用RT-PCR检测药物作用后人肝癌细胞株HepG2中bcl-2 mRNA的表达。结果:（1） MTT结果显示NS-398 （20¹60μmol/L）或NDGA （25²00μmol/L）单独应用对人肝癌细胞株HepG2均有增殖抑制作用,药物浓度越高,抑制作用越强,呈现明显的剂量效应关系。系列浓度NS-398或NDGA作用于细胞24h、48h、72h后观察细胞的生长情况,结果显示NS-398或NDGA作用时间越长,对上述细胞的抑制作用越强,呈明显的时间效应关系。依据金正均法NS-398在20¹60μmol·L^-1浓度范围内与NDGA 25¹00μmol·L^-1浓度范围内及NS-398 20μmol·L^-1与NDGA 200μmol·L^-1联合应用显示出协同作用（q >1.15）,余浓度联合应用显示相加作用（0.85<q<1.15）。联合用药组与同等剂量NDGA各单独用药组相比差异具有显著性（*P<0.05,**P<0.01）。（2） TUNEL染色结果显示经NS-398（160μmol/L）和NDGA（200μmol/L）单独和联合应用48h后均可诱导人肝癌细胞株HepG2发生凋亡,凋亡细胞胞核成棕色,核固缩,大小不一;非凋亡细胞细胞核淡蓝紫色,核形态大小较一致。联合用药组较各单独用药组及对照组凋亡指数增高。（3）流式细胞术结果显示经NS-398（160μmol/L）和NDGA（200μmol/L）单独和联合应用48h后均可诱导人肝癌细胞株HepG2发生凋亡,在DNA直方图上G0/G1期前可见亚G1峰即凋亡峰,NS-398与NDGA联合应用48h后凋亡率明显高于各单独用药组,提示两药合用对诱导人肝癌细胞株HepG2凋亡有协同作用。（4） RT-PCR检测bcl-2 mRNA的结果显示NS-398（160μmol/L）或NDGA（200μmol/L）单独和联合用药48h后HepG2细胞bcl-2 mRNA的表达均降低,两药联合用药组人肝癌HepG2细胞bcl-2 mRNA表达明显低于NS-398或NDGA单独用药组,差异具有显著性（**P<0.01）。结论:（1）单独应用NS-398或NDGA对于人肝癌细胞株HepG2均有增殖抑制作用,且呈剂量和时间效应关系。NS-398和NDGA联合用药对人肝癌细胞株HepG2的增殖抑制具有协同作用。（2）单独应用NS-398或NDGA对于人肝癌细胞株HepG2均有诱导凋亡作用; NS-398和NDGA联合用药对于人肝癌细胞株HepG2有协同诱导凋亡作用,并可能与抑制bcl-2 mRNA表达的作用增强有关。更多还原

【Abstract】 Hepatocellular carcinoma （HCC） is one of the most common malignancies in China, which tends to present at an advanced stage before it is detected clinically. It is not amenable to curative therapies and the prognosis of such patients is extremely poor, with a 5-year survival of less than 10%. Cylooxygenases （COX） and lipoxygenase （LOX） are two key enzymes in the metabolism of arachidonic acid （AA）. Phospholipid （PHL） in cellular membrane released AA under the effect of phosphatidolipase. AA are catalyzed to a series of biotic activators such as PG, TXA₂, HETES, LTS etc. through COX and LOX. COX and LOX effect on cell signal transduction and metabolism through these biotic activators and play an important role in many kinds of disease including tumor and inflammation. NS-398 is a selective COX-2 inhibitor, different from the traditional nonsteroidal anti-inflammatory drugs （NSAIDS）, its inhibition effect on COX-1 is poor, so palliate adverse reaction of gastrointestinal tract. NS-398 can inhibit the proliferation and induce cell life cycle arrest in human hepatoma carcinoma cell. NDGA is a LOX inhibitor, also can inhibit the proliferation, induce apoptosis and cell cycle arrest at the G₁/S phase in human hepatoma carcinoma cell HepG2. The present study was designed to explore the effect of NS-398 in combination with NDGA on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2, in order to develop an effective combination therapy for HCC.OBJECTIVETo explore the effect of NS-398 in combination with NDGA or alone on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2.METHODS（1） The inhibitory effect of NS-398 in combination with NDGA or alone on the proliferation of HepG2 in vitro was measured by MTT assay. The Jin’s formula was used to analyze the synergic inhibitory effect.（2） Morphological evidence of apoptosis induced by NS-398 in combination with NDGA or alone of HepG2 cells was detected by TUNEL. The apoptosis index （AI） was also calculated.（3） Apoptosis of HepG2 cells caused by NS-398 in combination with NDGA or alone was detected by flow cytometry （FCM）.（4） The expression of bcl-2 mRNA of HepG2 treated with NS-398 in combination with NDGA or alone was detected by RT-PCR.RESULTS（1） The MTT results suggested that NS-398 or NDGA had inhibitory effect on the proliferation of HepG2. The higher the concentration of NS-398 or NDGA was, the stronger the cytotoxic effect reached, which suggested obvious dose-dependent manner of NS-398 or NDGA. And the inhibitory effects were augmented with the prolongation of culture time （24h, 48h to 72h）, which had time-dependence. NS-398（20、40、80、 160μmol/L） in combination with NDGA（25、50、100μmol/L） showed synergic effect on the proliferation of human cell line HepG2 （q >1.15）, and others were addition（0.85< q <1.15 ）effect. There was significant difference between the combined group and the NDGA group alone（*P<0.05,**P<0.01）.（2） Results of TUNEL suggested that treatment with NS-398 （160μmol/L） or NDGA （200μmol/L） or a combination of the two agents for 48h. The AI of combination group was higher than drugs used alone, which demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.（3） FCM assay showed that treatment with NS-398 （160μmol/L） or NDGA （200μmol/L） or a combination of the two agents for 48h, the sub-G1 peak appeared before G1 phase, which represents apoptotic cell population, was observed clearly in the HepG2 cells, and the apoptotic rate of combination group was higher than drugs used alone. The results demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.（4） The results of RT-PCR showed that the expression of bcl-2 mRNA of HepG2 cells in combined group or alone all decreased. There was significant difference between combined group than alone（**P<0.01）.CONCLUSIONS（1） NS-398 or NDGA used alone has antiproliferative effect on the human hepatoma cell line HepG2, with an obvious dose- and time-dependent manner. NS-398 in combination with NDGA had synergistic growth-inhibitory effect on the HepG2 cells.（2） NS-398 or NDGA used alone could induce apoptosis on the human hepatoma cell line HepG2. NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells and correlated with potentialize reducing the expression of bcl-2 mRNA probably.更多还原