| Many proteins present in extracellular matrix(ECM)and plasma protein,contain a common tripeptide amino acid sequence Arg-Gly-Asp(RGD)as a recognition site for cellular adhesion,spreading and motility of cells. The RGD motif plays a key role in mediating integrin-matrix interaction and signals transmission,which modulate cell-survival and function. RGD and some synthetic RGD-containing peptides as competitive, reversible inhibitors for adhesive proteins binding have been widely used to study adhesive interaction between cells and suppress tumor metastasis etc. Recently, it has been also reported that RGD can enter the cells directly and induce apoptosis by activating some members of caspase family. It attracts more and more attention of researchers on studying the effect of RGD on inducing apoptosis of tumor cells.Recently, it has been reported that RGD can kill human prostate cancer cells (TSU), glomerular mesangial cells from adult human kidneys (GMC) and human leucocythemia cells (HL-60). This study armed to investigate the inhibitory effect of RGD on growth and proliferation of human poorly differentiated gastric adenocarcinoma cells BGC823 and inducing apoptosis. We test the proliferation of BGC823 cells by the MTT assay and observe cell morphological feature and ultramicroscopic structure of apoptotic cells after treated with RGD. We find RGD can inhibit proliferation of BGC823 cells significantly by inducing apoptosis. To approve it further, flow cytometry analysis and electrophoresis of DNA are done to test apoptosis. From the bar chart we find an apoptotic peak before the diploid peak. With increasing the concentration of RGD, proportion of apoptotic peak increased, while the control group has no apoptotic peak. In the figure of electrophoresis of DNA, there is a typical DNA ladder of apoptotic cells. These results indicate that RGD does induce apoptosis of BGC823 cells and the RGD concentration of 50μg/ml is the best condition. In addition, we display anti-apoptosis protein Survivin in cells by way of immunohistochemical staining and find that RGD can inhibit expression of Survivin. It suggests that it may be a mechanism of RGD inducing apoptosis. We would do more study to approve this later.Though it has been confirmed that RGD can inhibit growth of tumor cells and induce apoptosis, it has many shortages in using, for example it has small molecular weight and can be easily decomposed by enzyme. Its half life in vivo is very short. Some efforts had been made to increase circulating time and enhance their stability by modifiing the structure of RGD. The synthetic are things such as RGD repetitive sequence, RGD conjugates conjugated with polyethylene glycol or CM-chitin, RGD-containing pseudopeptide and peptide-simulacrum. However, the amount of RGD, contained in these linear peptides are limited. In this paper, RGD-loaded chitosan microspheres were designed. Chitosan microspheres are new drug delivery and have many characters: longest effect, target effect, slow release and unfrequently administration. RGD-loaded chitosan microspheres were prepared by emulsify-chemical crosslinking method. The impacts of every factors on the preparation were investigated, and the optimum conditions were determined: the ratio of liquid paraffin and 3% chitosan-acetic acid solution is 8:2, the volum of emulsifier is 4% of the whole, magnetic stirring 800rpm/min, and the volum of cross-linking agent is 2%. The obtained microspheres were uniform and well-dipersed. They were of 60-80μm in size, more than 80% of the whole. With the optimum conditions, we prepared the RGD-loaded chitosan microspheres. The highest encapsulation efficiency is 93.3%, and the loading efficiency is 2.54%. In vitro, the release test showed the release percentage of RGD-loaded chitosan microspheres is only 37.5% in 27 hours. It proved that RGD-loaded chitosan microspheres could release drug slowly. |
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