【作者】 杨帆；

【导师】 陈长兰；

【作者基本信息】 辽宁大学 ， 微生物学， 2012， 硕士

【摘要】 人硒蛋白SelK是哺乳动物体内所含的多种硒蛋白之一,SelK是单跨膜蛋白,前人所作研究已经初步证明其为位于内质网膜和细胞质膜上,但是对其生物功能一直没有阐明。而与SelK基因同源的果蝇硒蛋白dSelK,是果蝇当中功能未知的三种硒蛋白之一。所以本实验的研究可以填补国内外对于这方面研究的空白,得出的一系列试验结果对揭示硒蛋白基因功能具有重要意义。首先,通过将本实验室已构建成功的人硒蛋白SelK在人体细胞中的融合蛋白表达载体pGFP-SelK-secis-C1中SelK-secis的cDNA片段,插入到表达载体pE-Dsred2-C1中,构建了人硒蛋白SelK-secis与红色荧光蛋白DsRed的人体细胞融合表达质粒pDsred2-SelK-secis-Cl,并通过酶切和测序验证其正确性。然后,通过脂质体转染的方法,将pDsred2-SelK-secis-C1质粒转染人胃癌BGC-823细胞中,利用流式细胞仪检测SelK在人胃癌细胞中过表达诱导人胃癌细胞凋亡的情况。研究发现,人体硒蛋白SelK在人胃癌细胞中的过表达能够引起胃癌细胞的凋亡,与对照组(空质粒组)比较差异显著(p<0.05)。随后利用Fluo-3 AM进行对细胞浆中的游离钙离子染色,并通过激光共聚焦显微镜及钙离子荧光强度分析软件比较其转染后细胞浆中游离钙离子浓度的变化。结果显示人硒蛋白SelK-secis和SelK在人胃癌BGC-823细胞中过表达时细胞浆中钙离子浓度分别升高了 1.055倍和0.7421倍,与对照组(转染正常人胚肾HKE293细胞组)比较差异极显著(P<0.01)。接下来通过透射电子显微镜对融合蛋白表达质粒pE-dSelK-Dsred2-N1转染的人胃癌细胞的细胞生长状态进行了研究。研究表明,细胞核内染色质凝集,染色质凝缩于核膜下,细胞出现众多小泡等凋亡早期,中期和晚期的典型特征。另外,用流式细胞仪检测人体硒蛋白在果蝇中的同源基因dSelK在人胃癌细胞中过表达后诱导凋亡的情况和钙浓度变化情况。研究发现,果蝇硒蛋白dSelK在人胃癌细胞中的过表达同样诱导癌细胞凋亡并引起细胞浆中游离钙离子含量的升高,并与各自的对照组比较差异显著(p<0.05)。说明果蝇硒蛋白dSelK能够引起人体癌细胞株的凋亡。综上所述,本实验通过对人硒蛋白SelK-secis和其在果蝇中同源基因dSelK在BGC-823细胞中过表达时细胞浆内钙离子浓度变化的测定及对人胃癌BGC-823细胞生长状态的观察,发现人硒蛋白SelK-secis和其同源基因dSelK能够通过控制内质网内钙离子向细胞质中的释放,进而引起细胞发生细胞凋亡等细胞生长状态的变化。在国内外首次阐明了人硒蛋白SelK-secis和果蝇硒蛋白dSelK的生物功能,上述研究对于揭示生物体中其它硒蛋白的生物功能具有重要的指导意义。更多还原

【Abstract】 Selenoprotein SelK is one of the humen and mammals containing selenoproteins.SelK is a seleprotein which contains a single trans-membrane sequence and is located on the endoplasmic reticulum membrane and cytoplasmic membrane.But its biological function has not been clarified.Its homologue in Drosophila is dSelK,which is one of three selenoproteins in Drosophila whose function is also unknown.Therefore,the present study could fill the blank of domestic and international research in this area,and had significant value for revealing the function of other selenoproteins.Firstly,we taked the SelK-secis DNA fragment from the fusion protein expression vector pGFP-SelK-secis-C1 and inserted into the expression vector pE-Dsred2-C1 to construct the recombinant fusion expression vector pDsred2-SelK-secis-C1,and then to verify its correctness by restriction enzyme digestion and sequencing.Then,the influence of human SelK on the groth of human gastric cancer cells was studied by transfecting the plasmid of pDsred2-SelK-secis-C1 into human gastric cancer BGC-823 cells and detecting by flow cytometry method.It was showed that the over expression of human selenoprotein SelK could inducing the apoptosis of human gastric cancer cells,and the influnce was significantly different from the control group(empty plasmid group)(p<0.05).Then the cytoplasmic free Ca2+ of human gastric cancer BGC-823 cells over expressing Dsred2-SelK was stained by Fluo-3 AM,and the changes of cytoplasmic free Ca2+ concentrations were detected by laser scanning confocal microscopy and analysed by Ca2+ fluorescent intensity analysis software.It was showed that when the human selenoprotein SelK-secis and SelK over expressed in human gastric cancer BGC-823 cells,[Ca2+]i concentrations were increased by 1.055 and 0.7421 times respectively compared to the control group(human embryonic Kidney HKE293 cell group also transfected with the same plasmids),and the difference was highly significant(P<0.01).Next,the cell apoptosis was studied by transmission electron microscopy when the the fusion protein expression plasmid pE-dSelK-Dsred2-N1 was ove rexpressed in human gastric cancer BGC-823 cells.It was showed that the cancer cells displayed the typical characteristics of early,mid and late morphologically apoptotic characters,such as condensed chromatin,chromatin condensed near nucleus membrane.a number of apoptosis bodies in the cells,et cetra.Finally,the changes of apoptosis and calcium concentration in human gastric cancer cells over expressing the Drosophila selenoprotein dSelK,the homologue of human selenoprotein SelK,were also studied by flow cytometry detection.It was showed that Drosophila selenoprotein dSelK could also induce the apoptosis of human cancer cells and the increasing the cytoplasmic free Ca2+ intensity.And the influence was significantly(p<0.05)compared to their respective control group.It could be concluded that the Drosophila selenoprotein dSelK could also induce apoptosis in human cancer cell lines.In summary,in the experiment,we further confirmed that the human selenoprotein SelK and its homologue,Drosophila dSelK,could control the releaseing of calcium from endoplasmic reticulum to the cytoplasm in human gastric cancer BGC-823 cells,and lead to the apotosis of the cancer cells as a result.It is the first time at home and abroad to clarify the biological functions of human selenoprotein SelK and Drosophila selenoprotein dSelK.The above study has important significance for revealing the biological functions of the other selenoproteins in organisms.更多还原