【作者】 刘瑶； 何洁； 苏琦； 向姝霖； 袁静萍； 谢锦云； 陈平； 梁宋平；

【Author】 Liu Yao He Jie Su Qi Xiang Zhu-Lin Yuan Jing-Ping Xie Jin-Yun Chen Ping Liang Song-Ping Cancer Research Institute, Nanhua University, Hengyang, Hunan, 421001, P.R.China

【机构】 南华大学肿瘤研究所； 湖南师范大学生命科学学院；

【摘要】 目的:近年来,蛋白质组学技术已广泛应用于肿瘤研究领域,但对胃癌的研究较少。本课题旨在研究二烯丙基二硫(DADS)体内抑制人胃癌MGC803细胞生长作用及探讨DADS诱导人胃癌MGC803细胞蛋白质组差异表达的相关分子机制。方法:采用蛋白质组学相关技术,如二维电泳、图像分析、质谱技术等方法,观察DADS对体外培养的人胃癌 MGC803细胞的生长抑制以及蛋白质组的差异表达。用固相 PH梯度双向凝胶电泳分离人胃癌MGC803细胞和DADS处理的MGC803细胞的总蛋白,凝胶经过银染后用PDQuest软件进行分析,对部分重复性好、边界清晰的差异蛋白质点经胶内原位酶解,用基质辅助激光解吸电离飞行时间质谱 (MALDI-TOF-MS)测定肽质量指纹图(PMF),将所得的数据进行生物信息学处理。结果:在相同条件下,对DADS 处理前后MGC803细胞两种样品的总蛋白质进行双向电泳, 获得重复性较好的双向电泳银染图谱,经扫描成像及PDQuest 软件分析,在相同条件下识别的蛋白质斑点数分别为对照组 576±14个,处理组583±4个,其平均匹配率分别为76％和 70％。结果显示,在两组图谱中有421个蛋白质斑点匹配,有 200个点未被匹配,其中与对照组相比,蛋白质表达量相差2 倍以上的有291个,相差10倍以上的有61个。随机切割部分差异表达的银染蛋白质点,用TPCK处理的胰酶酶解后上质谱分析,获得相关蛋白的肽质量指纹图谱,经MS—FIT软件上相关数据库查询,初步鉴定出其中的24种差异表达的蛋白质。发现了一些与细胞分化、肿瘤转移、细胞凋亡、细胞周期、细胞免疫及代谢等相关的蛋白质,如胃粘蛋白、nM23蛋白、尿激酶纤溶酶原激活剂受体(uPAR)、LIM激酶、X-连锁凋亡抑制蛋白(XIAP)、CDC2、主要组织相容性复合体DR- beta-1链(MHC DR—beta-1)、T细胞受体(TCR)、Toll／ Interleukin—1受体类似蛋白3(Toll／interleukin—1 recep— tor—like protein 3)、结肠癌抗原NY—CO-45(Colon cancer antigen NY—CO-45)、苹果酸脱氢酶前体(malate dehy- drogenase precursor)、异质核核糖核蛋白F(hnRNPF)、磷酸二酯酶(Phosphodiesterase)、钴胺素传递蛋白II等,这些蛋白质可能在胃癌的发生发展中起着潜在的作用。结论: DADS可能通过上调nM23蛋白,下调uPAR、LIM激酶及CDC2的表达而降低胃癌的侵袭能力。DADS可能通过减少XIAP的表达促进胃癌细胞凋亡。DADS可能通过下调 CDC2表达参与G2／M期阻滞。DADS可能通过促进MHC— I类分子以及TCR的表达、Toll／Interleukin—1 receptor—like 蛋白3激活NF-kappaB途径,以及类蛇毒素蛋白酶促进T细胞及其它杀伤细胞对肿瘤细胞的杀伤,引发机体的杀瘤效应。 NY—CO-45抗原可能作为一种新的胃癌潜在诊断及预后标准。DADS可能导致AMP增加,活化苹果酸脱氢酶, 下调 hnRNP F蛋白及钴胺素传递蛋白II的表达,减少磷酸二酯酶以抑制肿瘤血管的生成,抑制胃癌细胞的增殖。更多还原

【Abstract】 Objective: This study was designed to explore the differential proteomic expression inducing effect of diallyl disulfide on human gastric cancer MGC803 cells and its related molecular mechanisms. Methods: A series of methods, including immobilized pH gradient-two dimensional polyacrylamide gel electrophoresis, silver staining, PDQuest 2-DE software analysis, peptide mass fingerpringting based on matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, were used to separate and identify the differential proteomic expressions inducing effect of diallyl disulfide (DADS) on human gastric cancer MGC803 cells. Results: The results showed that the good 2-DE pattern including high resolution and reproducibility was obtained. After silver staining, the 2-DE image analysis by PDQuest 2-DE software detected average (576 ±14) spots in MGC803, and (583 ±4) spots in DADS treated MGC803. And the average matching rate was76% and 70% respectively.The differential proteomic expression analysis found that there were 421 spots matched and 200 spots unmatched between MGC803 and DADS treated MGC803 maps. The spots on treated group, whose quantity of expressed proteins was above two or ten times compared to control, were 291 and 61. Part of the differential spots were cut off from silver staining gel at random, digested in gel with TPCK-trypsin, measured with MAIDI-TOF-MS and searched in related database with MS-FIT software. Twenty-four proteins were preliminarily identified. These proteins were related to cell differentiation , cell metastasis, cell apotosis, cell cycle, cell immunity and metabolism, etc. There was a significant difference at protein level between MGC803 and DADS treated MGC803 cells. Conclusion: Up-regulated expression of nM23 protein or down-regulated of uPAR , LIM kinase and CDC2 by DADS can inhibit the ability of metastasis.Inhibition expression of XIAP by DADS can promote cell apotosis and inhibit the growth of gastric cancer cells. CDC2 was involved in the effect of DADS on G2/ M arrest in human gastric cancer cells. DADS may induce the effect of reduce tumor via up-regulated expression of MHC-I, TCR, Snake venom-like protease and activation NF-kappaB pathways through Toll/Interleukin-1 receptor-like protein 3. NY-CO-45 antigen may treated as a potential marker of diagnose and prognosis. DADS may inhibit the proliferation via increase the expression of AMP, activate malate dehydrogenase precursor and down-regulated hnRNP F protein, transcobalamin II and phosphodiesterase.更多还原