| Cancer is a major health threat to humans. Depth study of the mechanism of tumordevelopment is the new strategy to explore the premise of cancer treatment, one isanti-tumor angiogenesis, and the other is anti-tumor immunity.Tumor antigen can stimulate the body to produce different types of anti-tumorimmune response, and produce anti-tumor humoral and cellular immune response, directlydetermine the prognosis of cancer patients. When tumor volume grows less than2-3mm3,the tumor cells can obtain diffuse nutritional support and slow growth from thesurroundings. When the volume of tumor continues increased, angiogenesis is required tosupport a large number of tumor cell growth.Notch signaling is a classic and conservative growth regulatory pathway. Thesignaling pathway is constituted by four kinds of ligands and five kinds of receptors,intracellular transcription factors regulate downstream molecules and related molecules.Notch receptors and ligands bind to each other. The conjunction is digested by differentprotease cleavage, the Notch intracellular translocation occurs into the core cells and bindto transcription factor RBP-J. The transcriptional co-suppressor become to transcriptionalco-activator and regulates the expression of downstream genes associated in manyimportant events ontogeny. Notch signaling is also involved in regulation of immune celldevelopment, including T cells, B cells, DC, myeloid cells and their subset. Recent studieshave found that the mesenchymal stem cells can not only concentrate to tumor around anddirect kill tumor cells, but also alter the concentration of cytokine to change oncogene andtumor suppressor gene balance to prevent tumor metastasis. Notch signaling has anirreplaceable role in adult individuals and embryonic vascular development. Theregulation plays a regulatory role in arteriovenous selection, Tip/stalk cell fate decisions, vascular endothelial cell proliferation, migration and pseudopodia formation and adultindividuals maintain cardiovascular stability and other aspects. Therefore, members of theNotch signaling pathway has recently become a candidate target of tumor blood vessels.Therefore, depth studies of Notch signaling in vascular differentiation and immune cellfunction of mesenchymal stem cells have great significance in anti-tumor.The main methods to study the effect of cell physiology in stimulate Notch signalingwas expressing DSL ligands in vitro and interact with Notch receptor. Several soluble DSLligands had been purification. Most of which has the ability to inhibit Notch signaling byneutralization. The ligands can apply in antineoplaston, but some problem still remained.We had known that soluble DSL ligands may inhibit Notch signaling, but we do not knowwhich domain has the function. And if the domain can instead of other drags to decreasethe adverse effect in clinical. This can helpful in understand cellular functional changesunder Notch signaling pathway, and can explore the relationship between Notch signalingand immune suppressing allograft rejection. Because of this, we carry out the study asfollows:1. We analyze the significance of Notch signaling for graft-versus-host disease.Allogeneic bone marrow transplantation was used to establish murine model of GVHD.γ-ray irradiate BALB/c mice, BM mononuclear cells derived from C57BL/6were injectedi.v. into lethally irradiated BALB/c mice, with or without BMSCs of different genotypesinjected simultaneously. Irradiated mice survival time was observed, and cell surfacemolecules and cytokine changes also be observed. Then we analyze Notch signaling forBMSCs immune-modulatory function. In a murine lethal acute graft versus host diseasemodel, BMSCs deficient for RBP-J, the critical transcription factor mediating signalingfrom all four mammalian Notch receptors, failed to delay the development of the disease.RBP-J deficient BMSCs can not inhibit the proliferation and activation of allogenic T-cells.Moreover, RBP-J deficient BMSCs could not down-regulate the expression of MHC IIand co-stimulation molecules CD80and CD86on dendritic cells. Furthermore, we showedthat the productions of IL-6and PGE2, two critical molecules mediating theimmuno-suppressive activities of BMSCs, were reduced significantly in RBP-J deficient BMSCs. Both of the two molecules were importantly involved in the regulation ofBMSCs by Notch signaling.2. To express different truncate human Dll4protein in E. coli expression system.Firstly, we cloned the DSL domain, DSL domain and first EGFR after that, DSL domainand two EGFR after that and two EGFR2lonely. Secondly, these fragments wereconstructed into pET32a(+) and construct new vector as hDll4-DSL, hDll4-DSL-EGFR,hDll4-DSL-EGFR2and hDll4-EGFR2. Meanwhile, construct containing the RGDsequence of these four hDll4truncate prokaryotic expression vector. The E. coliexpression system was transformed with these prokaryote gene expression vectors and thesystem was induced by IPTG under25℃. We found that the truncation of hDll4also hadinhibition function of Notch signaling by reporter assay, real-time PCR and FACS. Of this,the function of DSL and two EGFR after DSL domain had no obvious difference.3. The truncation protein of human Dll1, human Dll4and human Jag1wereexpressed in293T cell and we also verified the activity of them in HUVEC. We cloned theDSL domain, DSL and two EGFR after that, including human Dll1, human Dll4andhuman Jag1. These fragments were constructed into reconstructed vectorpEF-BOS-Neo-RGD-His. The new eukaryote expression vector concluded hDll4-DSL,hDll4-DSL-EGFR2, hDll1-DSL, hDll1-DSL-EGFR2, hJag1-DSL, hJag1-DS-EGFR2domain, separately. We founded that these ligands truncation had activity to stimulateNotch signaling. The DSL and two EGFR after that had the more activity than DSLdomain, and the different activity was existed in these ligands. We think it maybe theNotch receptor that expression on HUVEC membranes had different response to DSLligands. |
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