| Angiogenesis is the formation of new blood vessels from existing ones. It plays a key role in reproduction,wound repair and inflammation. This process occurs a tightly controlled manner during normal wound healing and the reproductive cycle.When this tight regulation is breached,the unregulated angiogenesis occurs and results in different pathological conditions such as rheumatoid arthritis,psoriasis and cancer and so on. Tumor angiogenesis plays an important role in proliferation and transfer , angiogenesis by inhibiting the formation and development of anti-tumor cells to achieve the purpose , is a really effective"hunger"therapy.The recent report showed that , pro-EMAPⅡis expected to have a therapeutic potential as an anti-angiogenic factor. pro-EMAPⅡand is an auxiliary factor of mammalian multi-aminoacyltRNA synthetase which consists of 312 aa, The molecular weight is 34 kD. The experiment data shows its mechanism of anti-tumor is mainly anti-angiogenic activity, not killing to harm a function obviously to the cancer cell.The results show that, pro-EMAPⅡprotein in vitro chick chorioallantoic membrane and in vivo experimental xenograft models (xenograft model) experiments show that a good inhibition of neovascularization, the original cancer and metastatic cancer with a good inhibition role, is expected to become the treatment of solid tumors, such as nasopharyngeal cancer, prostate cancer, breast cancer and lung cancer drug candidates. Most studies in recent years aimed at pro-EMAPⅡprotein caused inhibition of cancer development and the role of inflammatory response of the macro, the micro-fields, such as cell signaling pathway study less, this kind of research for the understanding of pro-EMAPⅡprotein specific mechanism will be of great significance.This experiment from the microscopic point of view, through the PCR amplification has been pro-EMAPⅡgene expression can be connected to a 6×His tag fusion protein of the prokaryotic expression vector, and achieved in Escherichia coli. After Ni-agarose affinity chromatography and gel filtration chromatography for the purpose of protein purification, protein purity is over 98%. The purpose of purified protein, the role of activated endothelial cells in the determination of the main activity for the determination of endothelial cell migration and lumen formation assay experiments. The results show that the activity assay, pro-EMAPⅡprotein for endothelial cell migration and lumen formation are inhibited. In order to study the pro-EMAPⅡprotein in human osteosarcoma cells Saos-2 gene expression, we make use of gene chip technology, the overall level of research from the Saos-2 cells were 50μg / mL pro-EMAPⅡprotein 8 h after stimulation, gene expression profiles differences. The results showed that Saos-2 cells in a total of 39 genes up-regulated and 29 down-regulated genes, 68 genes in which there are 12 differences in a number of genes involved in signal transduction pathway. The results from the microarray analysis, we found that pro-EMAPⅡprotein can increase in Saos-2 cells, APP gene, RFX5 genes and CTSB gene expression. We used reverse transcription - polymerase chain reaction method to verify the results of the chip, the results show that the detection of trends in gene regulation by DNA microarray analysis with the results.In summary, we have a purity of over 98% of pro-EMAPⅡprotein, and endothelial cells through the cell migration experiments and experiments on the formation of luminal pro-EMAPⅡprotein activity were measured and then used gene chip technology, analysis the pro-EMAPⅡprotein in Saos-2 cells on the regulation of gene expression and by RT-PCR method to verify the results of the chip.From the cell activity can be seen the results of the experiment, we cloned the expression of purified pro-EMAPⅡprotein as a new angiogenesis inhibitor, can effectively inhibit endothelial cell migration and vascular lumen formation, from the quantitative analysis and qualitative analysis to prove both its inhibitory effect, indicating that we have with the target protein biological activity due with angiogenesis by inhibiting the tumor growth inhibitory function.Cell gene expression changes in cell activity may reflect the inherent law, gene chip technology is to study an important means of this change, it is the methodology of molecular biology of a major breakthrough, but also tumor-associated genes present study the most effective means. In this study, biological Ltd. Beijing CapitalBio V2.0 human genome-wide oligonucleotide microarray chip to detect the gene expression profiles, results show that, after pro-EMAPⅡstimulated protein, Saos-2 cells the vast majority of gene expression and no obvious change, but there are also a small number of gene expression resulted in differences in these differentially expressed genes, 12 genes involved in multiple signal transduction pathway, these 12 genes are may be involved in the pro-EMAPⅡprotein play a role in inhibiting angiogenesis in the signaling pathway.Through RT-PCR for the gene chip results were verified, shows that Saos-2 cells after pro-EMAPⅡstimulated protein, the APP gene, RFX5 genes and CTSB gene expression is indeed a certain degree of increase. APP, also known asβamyloid precursor protein, is a receptor-like structure of the integration of macromolecular membrane glycoprotein, APP may be the majority in the body cells, mainly related to nervous system damage; RFX5 regulatory factor, also known as X5 is a complex RFX subunits, RFX with other transcription factors to promote MHC II and the combination of promoter and promoter to enhance the specificity of binding to and regulating CIITA with MHC II molecule expression; CTSB cathepsin B, also known as is a cysteine protease in lysosomes, when under certain conditions of its release into the cytoplasm can lead to the activation of NF-κB, activated inflammatory cytokines such as TNF-α, leading to apoptosis.In short, pro-EMAPⅡprotein in Saos-2 cells on gene expression has a certain role in the regulation and control, these differences in gene expression may be involved in the pro-EMAPⅡprotein play a role in inhibiting angiogenesis in the signaling pathway. Expression and regulation of the process may activate or inhibit the adoption of certain transcription factors to achieve, and the differences in the expression of these genes and their corresponding function, or weakened, then the state of the entire cell to the different direction. |
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