Objective To microencapsulate a genetically engineered cell line which stably secrete human endostatin (hES).Methods Endostatin gene fragment was amplified from plasmid pcDNA3-Endo by polymerase chain reaction, and inserted into mammalian eukaryotic expression vector pEGFPN1, resulting into recombinant plasmid pEGFP-N1-ES.Hek-293 cells were transfected with pEGFP-N1-ES via cationic liposome and selected by G418, and were measured by Western blot for endostatin protein expression.The hES/293 cells were further entrapped by alginate-chitosan-alginate (ACA) microcapsules, and the expression of endostatin in the supernatant of cultured hES/293 cell microcapsules was examined by western blot at different time points.Results Recombinant plasmid pEGFP-N1 endostatin was digested by HindIII and BamHI, and resulted into 2 DNA fragments of 7 kb and 600 bp. The sequence of the 600 bp fragment was identical to human endostatin. Western blot of the supernatant of cultured hES/293 cells or hES/293 cell microcapsules detected a positive band with the relative molecular mass of 20times;103.Conclusion The hES protein was expressed in HeK-293 transfected with pEGFP-N1-endostatin, and secreted to the culture medium,and can freely diffused outside the micro-capsule.
Objective To observe the altered gene expression of ECV304 cell after treated with endostatin (ES) using cDNA microarray, and to investigate its molecular mechanism.Methods BiostarH40S gene expression DNA microarrays were used to profile changes in gene expression of ECV304 cells after treated with ES for 36 hours. To prepare the probes, mRNA from both control and treated cells were isolated and purified, and then reversely transcribed to cDNA with the incorporation of fluorescentlabeled dUTP: Cy3 and Cy5 respectively. The probes were hybridized with expressed cDNA microarray, the fluorescent signals of Cy3 and Cy5 were scanned and analyzed by a computer system.Results ES inhibited the proliferation of ECV304 cells. The result of electrophoresis indicated that the extracted total RNA had high quality.Approximately 5.96% of all human genes examined on 4096 gene microarrays showed altered expression, with 225 downregulated genes and 19 upregulated genes.Conclusion The expression of some genes of ECV304 cells decrease after treated with ES, which may be related to the mechanism of inhibition of neovascularization.