毛叶香茶菜素F对人食管癌EC9706细胞生长的抑制作用及其机制初步研究

发布时间：2018-01-03 18:21

本文关键词：毛叶香茶菜素F对人食管癌EC9706细胞生长的抑制作用及其机制初步研究　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的食管癌是最常见的消化道疾病之一,其死亡率几乎与胃癌相当,治疗效果较差,5年存活率还不足10%,严重威胁着人类健康。食管癌分为食管鳞癌和食管腺癌,我国以食管鳞癌居多,食管腺癌较少,尤其是河南安阳、林州等地是食管癌的高发地区。目前食管癌还没有特效治疗药物,而且其治疗方案的选择也与发病时期有关,处于发病早期和部分中期的患者可以采用手术治疗,而晚期患者只能采用放化疗方案,并且效果都不是特别理想。毛叶香茶菜是唇形科植物,主要含有二萜、三萜、木质素和黄酮类等化合物,已有体内外研究表明,毛叶香茶菜素A、G和E在体外对艾氏腹水癌(ECA)的增殖具有明显的抑制作用,在体内同样表现出一定的抗肿瘤效应,毛叶香茶菜素B对L1210白血病小鼠模型的生命延长表现出可观的效果,毛叶香茶菜素C和D则没有抗肿瘤作用。目前对毛叶香茶菜素F的研究较少,关于其对食管癌的作用文献中尚未见报道,本研的目的在于初步探讨毛叶香茶菜素F对人食管癌EC9706细胞增殖的抑制作用及机制。材料和方法1.MTT法粗测样品活性以食管癌EC9706细胞为模型,试验组分别加100、80、50、40、30μg/ml的毛叶香茶菜素F样品,阴性对照组加相同体积的完全培养基,在37℃5%的CO2培养箱中分别培养24h、48h、72h后每孔加20μl MTT溶液,继续培养4h后吸掉上清并每孔加入160μl DMSO,用酶标仪检测吸光度。2.观察样品对细胞生长状态的影响试验组分别加入80、50、40、30μg/ml的毛叶香茶菜素F样品,阴性对照组加相同体积的完全培养基,在37℃5%的CO2培养箱中培养72h后在显微镜下观察细胞生长状态并拍照。3.细胞划痕试验阴性对照组不加药,试验组分别加入50、40、30μg/ml的毛叶香茶菜素F样品,阳性对照组加入50μg/ml顺铂,作用24h后,在显微镜下观察、取样、拍照并测量各划痕区域的面积。4.Hoechst/PI细胞凋亡检测试验组分别加入80、50、40、30μg/ml的毛叶香茶菜素F样品,阴性对照组加相同体积的完全培养基,在37℃5%的CO2培养箱中分别培养24h、48h、72h后加入荧光染料,然后用流式细胞仪检测细胞凋亡率。5.细胞周期检测试验组分别加入50、40、30μg/ml的毛叶香茶菜素F样品,阴性对照组加相同体积的完全培养基,在37℃5%的CO2培养箱中分别培养24h、48h、72h后加入荧光染料,然后用流式细胞仪检测细胞周期分布情况。6.Western blot检测样品对EC9706细胞中5种蛋白表达的影响试验组按照50、40、30μg/ml的浓度加入毛叶香茶菜素F样品,阴性对照组加相同体积的完全培养基,在37℃5%的CO2培养箱中分别培养72h后,提取相关蛋白,用Western blot方法检测毛叶香茶菜素F作用后Bax、Bcl-2、Caspase-3、m TOR及P53五种蛋白表达水平的变化。7.SPSS统计学处理采用统计学处理软件SPSS17.0,数据资料采用x±s的表示形式,每组试验均重复三次以上。结果1.MTT法检测结果显示,毛叶香茶菜素F作用24h、48h和72h后对EC9706细胞的生长均表现出明显的抑制作用,其IC50分别为46.7μg/ml、25.4μg/ml和20.6μg/ml,且随着浓度的增大和时间的延长抑制作用也呈增强的趋势。2.试验组在不同浓度的毛叶香茶菜素F样品作用72h后与对照组相比,细胞体积变小,细胞质皱缩,呈较小的颗粒状。3.在细胞划痕试验中,毛叶香茶菜素F终浓度为50μg/ml组细胞培养24h后划痕面积约为3.10×105,与对照组2.52×105相比明显增大,并且差异具有统计学意义(P0.05)。4.当浓度为50μg/ml时,毛叶香茶菜素F作用24h、48h和72h后细胞凋亡率约分别为20.20%、21.65%和53.21%,当作用时间为72h,随着浓度的增大细胞凋亡率分别为10.37%、17.84%、53.21%和86.63%,;当浓度为50μg/ml时,毛叶香茶菜素F作用24h、48h和72h后细胞周期G1期细胞比例分别为58.53%、57.37%和52.37%,S期细胞比例分别为40.06%、41.16%和45.62%,当作用时间为72h,随着浓度的增大G1期细胞比例分别为59.65%、56.43%和52.37%,S期细胞比例分别为39.71%、42.35%和45.62%,均呈时间和浓度依赖性,且组间差异具有统计学意义(P0.05),G2期细胞无明显变化。5.毛叶香茶菜素F对EC9706细胞5种蛋白表达水平的影响毛叶香茶菜素F作用72h后,随着浓度的增大,Bax蛋白灰度值分别为0.91、1.20和1.26、Caspase-3蛋白灰度值分别为0.77、0.83和1.11,而Bcl-2蛋白灰度值分别为1.01、0.95和0.92,m TOR蛋白灰度值分别为1.08、0.84和0.71,P53(突变型)蛋白的灰度值分别为0.94、0.72和0.58,与对照组相比发现其可以上调Bax和Caspase-3蛋白的表达、下调Bcl-2、m TOR及P53(突变型)蛋白的表达。结论毛叶香茶菜素F可以上调Bax、Caspase-3蛋白的表达水平,下调Bcl-2、m TOR及P53(突变型)蛋白的表达水平,能够抑制食管癌EC9706细胞的增殖、迁移,将细胞生长阻滞于S期,促进细胞的凋亡,这些可能是毛叶香茶菜素F产生抗肿瘤作用的机制之一。
[Abstract]:Background and objective: esophageal cancer is one of the most common gastrointestinal disease, the mortality rate of gastric cancer and almost equivalent to that of poor treatment, 5 year survival rate is less than 10%, a serious threat to human health. Esophageal cancer is divided into squamous cell carcinoma of the esophagus and esophageal adenocarcinoma, squamous cell carcinoma of the esophagus in our country with the majority, food tube adenocarcinoma is less, especially Henan Anyang, Linzhou and other places is the high incidence of esophageal cancer. At present there is no effective drugs for the treatment of esophageal cancer, and the treatment options are also associated with the incidence of early onset period, in the middle part and can be used in patients with surgical treatment, and patients can only use chemotherapy, and the effect is not ideal. Their hair tea dish is Labiatae, mainly containing two terpenes, three terpenes, lignin and flavonoid compounds, existing research shows that in vivo, Mao Yexiang Nervosin A, G and E in vitro on Ehrlich ascites carcinoma (ECA). Cloning has obvious inhibitory effect, in vivo showed the same anti-tumor effect, life Ye Xiang Mao Nervosin B on L1210 leukemia mouse model extension showed considerable effect, Mao Ye Xiang Nervosin C and D have no antitumor activity. The Ye Xiang Mao tea dish less research in F. On the role of esophageal cancer has not been reported in the literature, this study aims to investigate the hair Ye Xiang Nervosin inhibitory effect of F on proliferation of human esophageal carcinoma EC9706 cells and its mechanism. Materials and methods 1.MTT crude sample activity in esophageal carcinoma EC9706 cells as the model, the experimental groups were g/ml and 100,80,50,40,30 the hair Ye Xiang Nervosin F samples, negative control group and the same volume of complete medium, incubator were cultured in 24h, 48h at 37 DEG C, 5% CO2, 72h 20 l per hole adding MTT solution to 4h after culture and absorb the supernatant per hole adding 160 L DM SO, 80,50,40,30 were added to g/ml by ELISA test group were detected.2. observation sample absorbance effect on cell growth of hair Ye Xiang Nervosin F samples, negative control group and the same volume of culture medium, 72h culture box under a microscope to observe cell growth state and cell scratch test camera.3. the negative control group without dosing at 37 C for 5% CO2, the test group were added with 50,40,30 g/ml hair Ye Xiang Nervosin F samples, 50 g/ml of cisplatin with the positive control group, 24h, under the microscope observation, sampling, photo and test group of apoptosis.4.Hoechst/PI cells to detect the scratch area area were added 80,50,40,30 g/ml Ye Xiang Nervosin F samples, the negative control group and the same volume of complete medium, incubator were cultured in 24h, 48h at 37 DEG C, 5% CO2, 72h after adding fluorescent dye, followed by flow cytometry The apoptosis rate of.5. cell cycle detector test group were added to 50,40,30 g/ml their hair Nervosin F samples, negative control group and the same volume of complete medium, incubator were cultured in 24h, 48h at 37 DEG C, 5% CO2, 72h after adding fluorescent dye, and then detected by flow cytometry samples cell cycle distribution was detected by.6.Western blot in 5 EC9706 cell protein expression in the experimental group were in accordance with the concentration of 50,40,30 g/ml with their hair Nervosin F samples, negative control group and the same volume of culture medium, CO2 culture in 37 C 5% a 72h box were cultured after extraction related protein using Western, blot method to detect their hair Nervosin after F Bax, Bcl-2, Caspase-3,.7.SPSS were analyzed by statistical software SPSS17.0 m and P53 TOR expression of five proteins, said the data was analyzed using X + S Form, each test was repeated three times or more. The results of 1.MTT assay showed that their hair Nervosin F role of 24h, 48h and 72h on the growth of EC9706 cells significantly inhibited, the IC50 were 46.7 g/ml, 25.4 g/ml and 20.6 g/ml, and with the increase of concentration and prolonged inhibition also showed the trend of the.2. test group increased in different concentrations of their hair Nervosin F samples after 72h compared with the control group, the cell size, cytoplasmic shrinkage, a small granular.3. in cell scratch test, their hair Nervosin F final concentration of 50 mu g/ml group cell culture after 24h scratch area of about 3.10 x 105, 2.52 x 105 compared with the control group increased significantly, and the difference was statistically significant (P0.05) when the.4. concentration is 50 g/ml, their gross Nervosin F 24h, 48h 72h and apoptosis rate were 20.20%, 21.65% and 5 3.21%, when the time is 72h, with the increase of the concentration of cell apoptosis rate were 10.37%, 17.84%, 53.21% and 86.63%; when the concentration is 50 g/ml, their hair Nervosin F role of 24h, 48h and 72h after the proportion of cells in the G1 phase of the cell cycle were 58.53%, 57.37% and 52.37%, the proportion of S cells respectively 40.06%, 41.16% and 45.62%, when the time is 72h, with the increase of the concentration, the proportion of cells in G1 phase were 59.65%, 56.43% and 52.37%, the proportion of cells in S phase were 39.71%, 42.35% and 45.62%, were in a time and concentration dependent, and the difference between groups was statistically significant (P0.05). The cells in G2 phase of.5. had no obvious change in their gross Nervosin F of 5 EC9706 cell protein expression level influence their hair Nervosin F 72h, with the increase of the concentration of Bax protein, the gray value of 0.91,1.20 and 1.26 respectively, the gray value of Caspase-3 protein were 0.77,0.83 and 1.11, Bcl-2 protein The gray value of 1.01,0.95 and 0.92 respectively, m TOR protein gray values were 1.08,0.84 and 0.71, P53 (mutant) protein gray values were 0.94,0.72 and 0.58 respectively, compared with the control group found its expression, up regulation of Bax and down-regulation of Caspase-3 protein Bcl-2, m TOR and P53 (mutant) protein expression. Conclusion their hair Nervosin F can upregulate Bax expression, down-regulation of Caspase-3 protein Bcl-2, m TOR and P53 (mutant) protein expression, can inhibit EC9706 esophageal cancer cell proliferation, migration, cell growth arrest in S phase, promote cell apoptosis, which may be one of the mechanisms of their hair Nervosin F has antitumor effect.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285

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