论文标题:大葱油诱导胃癌细胞凋亡作用机制的初步研究
Initial Study of Gastrocancerous Cellular Apoptogenesis Induced by Allium Fistulosum.L Oil
论文作者 罗海滨
论文导师 任锡玲,论文学位 硕士,论文专业 内科学
论文单位 河北医科大学,点击次数 96,论文页数 46页File Size1567k
2002-03-01论文网 http://www.lw23.com/lunwen_55980262/ 大葱油;胃癌细胞;凋亡;Ca~(2+);cAMP
Allium fistulosum.l oil,Gastrocancerous cell,Apoptosis,Ca~(2+),cAMP
目的：流行病学和实验资料表明，经常食用葱属植物可明显降低胃癌的相对危险性。在本系列研究的前期实验中用大葱提取物作用于体外培养的人胃腺癌细胞MGC80-3，能有效抑制胃癌细胞增殖，诱导其凋亡，并证实其有效成分是大葱油。本实验旨在从细胞传导通路的角度探讨大葱油诱导胃癌细胞凋亡可能的作用机制。 方法：(1)不同浓度的大葱油作用于人胃腺癌细胞株MGC80-3，持续24小时，采用噻唑蓝(MTT)法测定不同浓度组OD值，计算增殖抑制率，绘图测定半数抑制浓度IC_(50)。(2)100μg／ml的大葱油作用于胃癌细胞，取不同作用时间点的细胞，进行Hoechst33258荧光染色，在荧光显微镜下观察胃癌细胞凋亡的形态变化。(3)100μg／ml的大葱油作用于胃癌细胞1～12小时，应用PI-Annexin V染色法测定不同时间胃癌细胞的总凋亡率和早期凋亡率。(4)100μg／ml的大葱油作用于胃癌细胞5分钟至12小时，用Fura-2／AM作为Ca~(2+)指示剂，在不同时间用流式细胞仪测定细胞内游离Ca~(2+)浓度。(5)100μg／ml的大葱油作用于胃癌细胞5分钟至12小时，用放免法测定不同作用时间的胃癌细胞内cAMP浓度。 结果：(1)大葱油对胃癌细胞增殖的影响：随大葱油浓度的增加，实验组细胞OD值下降，对照组OD值无明显变化，细胞增殖抑制率相应升高，大葱油浓度为12.5μg／ 中文摘要ml． 25 u g／ml． 50 u g／ml． 75 u g／il． 00 u g／llil时，作用24小时，细胞增殖抑制率分别达到5．71％、3 6．33％、44．34％、54．52％和 74．15％，测得 IC。。一65Vg／Inl。大葱油对胃癌细胞增殖的抑制作用具有剂量依赖性。o渭癌细胞凋亡形态观察：100 p g／il的大葱油作用于胃癌细胞 ld ．J’时后，取不同时间点的细胞做 Hoech悦33258荧光染色。在荧光显微镜下观察，可见实验组部分细胞的细胞核或细胞质内出现浓染致密的块状或颗粒状荧光，提示细胞核固缩，染色质凝集，DNA片段化，细胞出现凋亡，而对照组细胞核呈弥散均匀的荧光，提示为正常活细胞。＊）大葱油对胃癌细胞凋亡的影响：PI Annekin V双染后流式细胞仪检测显示 100Hg／ttil大葱油作用于胃癌细胞lh、Zh、3h、6h和 12h，总凋亡率分别为 53．48％，50．16％，77．84％，83．71％，41．34％。早期凋亡率分别为50．15％，33．25O，11．69O，4．65o，0．84O，峰值出现在＊。（4）10Vg／ffil的大葱油作用于胃癌细胞 5”、15”、30”、lh、Zh。3h、6h和12h，各相应时间细胞内游离Cah含量，依次为71．co士6．08，79．67士1．53，77石7士1．53，83．33土5刀3，76．0016．06，72．33创．24，75＊7a．52，65＊ort．00；对照组相应 C／”含量分别为 48．67幻．06，sl．00rt．00，49．67土1．53，48＊6士1．53，49．00D＊5，sl．67士3．57 和48．33rt．52。实验组1小时Cf”浓度达到峰值，以后逐渐下降，但仍然高于对照组。Ov 的大葱油作用于胃癌细胞 5”、15”、30”、lh、Zh、3h、6h和 12h，各相应时间点细胞内cAIAI，浓度中mol／mg·pTotein）依次为5．76土o．36，18．51H刀6，20．40d石5，16刀9土1．46， 2 中文摘要 12．16to．78，9＊8t0．53，9．47土0．78，8．82士1＊6；对照组相 应时间。AMP浓度分别为5．8则．38，6＊8切．76，5．93土＊＊7， 5＊土0．41，5＊士0．42，5．89士0．ZI，5．87士0．66，5．45土1＊5。实 验组细胞内。AMP浓度30分钟达到峰值，以后逐渐下降， 也明显高于对照组。 结论：以上实验结果说明大葱油可明显抑制人胃腺癌 细胞株MGC80上增殖，并诱导其凋亡。在细胞凋亡发生 的同时细胞内游离 Ca》和。AMP 7k平升高，且＊”含量 峰值时间与早期凋亡峰值时间一致，CAMP峰值时间则提 前出现，再次证实ca》和c…是胃癌细胞凋亡信号传 导过程中的重要信号分于，提示大葱油诱导胃癌细胞凋亡 的作用可能是通过上调细胞内第二信使CaD，cAMP水平 来实现的。
Objective: Results from epidemic studies and laboratory tests indicated that consumption of allium vegetables may considerably reduce the relative risk of gastric carcinoma. Our previous experiments already proved that the extracts of Allium fistulosum.L can induce apoptosis of gastrocancerous cell and inhibit cell growth, especially lipid portion of extracts did. Our aim to study is further explore the possible signal molecule mechanism in apoptotic process of gastrocancerous cell MGC80-3 induced by Allium fistulosum.L oil (AFLO).Methods: (1) MGC80-3 cells were treated with AFLO in different concentrations for 24 hour. The optical density values (OD) were measured by MTT test. Then the growth inhibitory rates were counted and IC50 was measured. (2) MGC80-3 cells were incubated in nutritious fluid with AFLO (100u g/ml) for one hour to twelve hours. The treated cells were stained with Hoechst33258. Apoptotic shape was investigated with fluorescence microscope. (3) MGC80-3 cells were incubated in nutritious fluid, with AFLO (100u g/ml) for one hour to twelve hours. Total apoptotic rates andearly apoptotic rates were detected by measuring cellular PI and Annixin V fluorescence values on flow cytometry. (4) MGC80-3 cells were incubated in nutritious fluid with AFLO (100 u g/ml) for five minutes to twelve hours. Fura-2/AM was used as an intracellular-free calcium indicator. Intracellular-free calcium content at different time was determined by flow cytometry. (5) MGC80-3 cells were incubated in nutritious fluid with AFLO (100ug/ml) for five minutes to twelve hours. Intracellular cAMP levels were determined by radioimmunoassay (RIA).Results: (1) Effect of AFLO in different concentrations on gastrocancerous cellular growth; MGC80-3 cells were treated with 12.5 u g/ml, 25 u g/ml, 50 u g/ml, 75 u g/ml and 100 U g/ml of AFLO for 24 hours. With the concentration of AFLO increased, the optical density of test groups declined, but cellular growth inhibitory rates went up correspondingly and reached to 5.71%, 36.33%, 44.34%, 54.52% and74.15% respectively. IC50 65ug/ml. Effect of inhibition on gastrocancerous cellular growth depended on concentration of AFLO. There were not any like these changes in control group. (2) Observation for apoptotic gastrocancerous cellular shape: Hoechst 33258 fluorescent stain showed that block or article-liked fluorescence light appeared in nuclei or cytoplasm of some cells treated by AFLO (100u g/ml) for one hour to twelve hours. These imaginations indicted condensation and fragmentation of chromosome. Apoptosistook place in cells treated by AFLO , but fluorescence light in cells of control groups was scattered and well-distributed. (3) Changes of the apoptotic rates at different times: MGC80-3 cells were incubated in nutritious fluid with AFLO (100u g/ml) for Ih, 2h, 3h, 6h and 12h. The total apoptotic rates were 53.48%, 50.16%, 77.84%, 83.71% and 41.34% respectively. The early apoptotic rates were 50.15%, 33.25%, 11.69%, 4.65% and 0.84% respectively. The peak time of early apoptotic rate was at the first hour. (4) Changes of intracellular-free calcium content: MGC80-3 cells were treated by AFLO (100 u g/ml) for 5", 15", 30", Ih, 2h, 3h, 6h and 12h. Measured intracellular-free calcium content of test groups were 71.00+6.08, 79.67+1.53, 77.67+1.53, 83.33+5.03, 76.00+6.06, 72.33+4.24, 75.67+2.52, 65.00+2.00 respectively. Those in control groups were 48.67+3.06, 51.00+2.00, 49.6711.53, 48.06+1.53, 49.0012.65, 51.67+3.57, 47.6713.79 and 48.3312.52 respectively. The peak content of Ca2+ was reached at the first hour, then contents of Ca2+ declined gradually, but were still higher than in the control groups. (5) Changes of cAMP concentration: The gastrocancerous cells treated with AFLO(100 u g/ml) for 5", 15", 30", Ih, 2h, 3h, 6h and 12h, measured intracellular cAMP concentrations were 5.7610.36, 18.51+2.06, 20.40+1.65, 16.0911.46, 12.1610.78,9.8810.53,9.4710.78, 9.8211.06 (pmol/mg protein) respectively. Those in control groups were 5.8910.38, 6.0810.76, 5.9310.67, 5.1210.41,5.17
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