| 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. |
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