【作者】 姚怀齐；

【导师】 郭光华；

【作者基本信息】 汕头大学 ， 消化内科学， 2010， 硕士

【摘要】 背景及目的：近年来脂氧化酶(LOX)及其代谢产物羟基二十碳四烯酸(HETE)与肿瘤的发生发展的关系越来越受到重视。12-LOX作为脂氧合酶家族的一员,是催化AA转化为12-羟廿碳四烯酸(12-HETE)的关键酶,而12-HETE与12-LOX一起,通过各种机制共同参与肿瘤的发生发展过程。两者与恶性肿瘤的关系已成为研究重点。黄芩素(baicalein,BAI)是从唇形科植物黄芩中提取出来的具有抗菌、抗病毒、抗炎、抗变态反应、抗氧化、清除氧自由基、抗癌、抗凝、抗血栓形成和保护肝脏、心脑血管、神经元等多种药理活性等的黄酮类化合物。有关黄芩素的一些制剂已在临床应用于上呼吸道感染、急性扁桃体炎、咽炎、慢性阻塞性肺病、传染性肝炎、急慢性胃肠炎、细菌性痢疾、肾盂肾炎等多种疾病的治疗,并显示出较好的疗效。近年来,随着中药抗癌研究的深入,黄芩素抗肿瘤的机制得到大量研究,结果表明,作为12-脂氧化酶(12-LOX)抑制剂,黄芩素可抑制多种肿瘤细胞增殖,包括胰腺癌、肝癌、前列腺癌、卵巢癌等,而对食管癌细胞是否具有生长抑制作用及其机制尚未见报道。本实验旨在探讨12-脂氧化酶(12-LOX)抑制剂黄芩素对人食管癌EC-109细胞增殖及细胞周期的影响及可能的机制,为黄芩素的进一步开发利用提供理论依据。材料和方法：体外培养的人食管癌EC-109细胞,用含不同浓度黄芩素(0、5、10、20、40、80μmol/L)的培养基处理,倒置相差显微镜观察EC-109细胞的形态学变化；噻唑蓝比色法(MTT法)检测黄芩素对食管癌细胞增殖的影响；经碘化丙啶(PI)染色后,用流式细胞分析仪(FCM)检测黄芩素对EC-109细胞周期的影响。结果:1.倒置相差显微镜下观察未经黄芩素干预的细胞包膜完整,细胞贴壁生长,状态良好。以不同浓度黄芩素作用后,细胞形态发生明显变化。当低浓度且作用时间较短时,细胞体积稍增大、变得扁平。当作用时间达48小时后,细胞开始出现皱缩,伪足形成,细胞折光性差,模糊,与周围细胞分离,形态不规则,且随浓度增加可见部分细胞呈“发芽”状态改变,类似凋亡细胞形态改变,最终漂浮。给予大剂量黄芩素与细胞一起培养,细胞在短时间内缩小、脱落、漂浮,产生许多细胞碎片,可能形成细胞坏死。2.应用MTT法研究黄芩素对EC-109细胞的作用,结果发现,黄芩素对EC-109细胞有细胞毒性作用,.能抑制细胞增殖,且随着黄芩素终浓度(0、5、10、20、40、80μmol/L)增加和作用时间(24、48、72h)的延长,细胞存活率明显下降,24h后分别为98.4%、87.7%、87.6%、81.9%、79.0%；48h后分别为71.6%、67.6%、64.1%、60.0%、46.7%；72h后分别为43.8%、43.1%、43.1%、40.0%、23.0%。与对照组相比,这种抑制作用呈一定的时间、浓度依赖性。3.流式细胞术PI单染法检测黄芩素对EC-109细胞周期的影响,结果显示,0、5、10、20、40、80μmol/L的黄芩素作用于EC-109细胞24h后,随着黄芩素浓度的增高,G1期细胞的比例升高,分别为37.7%、43.6%、48.2%、56.7%、68.5%,G2期细胞的比例降低,分别为24.5%、18.5%、12.4%、9.5%、0.0%。结论：1.黄芩素在一定的浓度范围内能够抑制食管癌EC-109细胞的生长,并且随着黄芩素浓度的增加及作用时间的延长,该抑制作用更为明显。2.黄芩素可改变EC-109细胞的周期分布,并呈一定的时间、浓度依赖性。更多还原

【Abstract】 Background and ObjectivesIn recent years, researchers found that PGs, HETE and LTs, which are produced by arachidonic acids (AA) through cyclosygease and lipoxygenase pathy ways, is closely related to tumors.12-LOX is one menber of the lipoxygenase family as well as the key enzyme to catalyze AA into 12-HETE.12-LOX and 12-HETE participate in the occurrence of tumors through certain pathways.Baicalein (BAI), extracting from scutellaria baicalensis, is one of the flavonoids compounds which has effects of antioxidation, anti-inflammatory, anti-tumor and anti-allergic. So far, many preparations made from BAI are used for the treatment of upper respiratory tract infection, acute tonsillitis, pharyngitis, chronic obstructive pulmonary disease, infective hepatitis, gastro-enteritis, bacillary dysentery, pyelonephritis and show fine effects. According to many researches, as an inhibitor of 12-LOX, BAI could inhibit many tumor cells proliferation by different mechanisms, such as pancreatic cancer, hepatoma, prostatic cancer, oophoroma and so on. The main mechanisms include affecting on the AA pathway, inhibition of cell proliferation, inducing apoptosis and antiangiogenesis. So far as concern, there are still rare reports about whether baicalein has effects on esophageal carcinoma. The aim of our study was to investigate the the possible mechanisms of BAI on the proliferation and cell cycle of human esophageal carcinoma cell line EC-109.MethodsEC-109 cells were treated with different concerntrations (0、5、10、20、40、80μmol/L) of BAI and observed with inverted phase contrast microscope. MTT assay was used to evaluate the effect of BAI on cell proliferation. PI staining and flow cytometry were uesed to detect the effect of BAI on the cell cycle of EC-109 cells.Results1. Tumor cells which were not treated with BAI showed complete cell membrane and grew well. The growth of cells turned slowly and growth inhibition became obvious as the rise of BAI concerntration. Brim shrinkage and cell cast-off could be observed with the microscope. Cell fragments could be observed soon after the treatment of BAI.2. At the concentration from 0 to 80μmol/L, BAI inhibited the proliferation of EC-109 cells in a concentration and time-dependent manner from 24 to 72 hours. The survival rate of the cells was 98.4%、87.7%、87.6%、81.9%、79.0% after 24h,71.6%、67.6%、64.1%、60.0%、46.7% after 48h, and 43.8%、43.1%、43.1 %、40.0%、23.0% after 72h respectively.3. Cell cycle analysis revealed a marked increase from 43.6% to 68.5% in the G1 phase and a decrease from 24.5% to 0.0% in the G2 phase following 24h exposure to BAI from 0 to 80μmol/L.Conlusion1.BAI could inhibit the proliferation of EC-109 cell in a concentration and time-dependent manner.2. BAI could inhibit the proliferation of EC-109 cell in a concentration and time-dependent manner.更多还原