Construction For The Mouse Cell Line Models Of SLPI Gene Knockout Or Knockin And The Molecular Mechanism Of Migration Of U2OS Cell Induced By SLPI

Part oneConstruction for the mouse cell line models of SLPI gene knockout or knockinTooth development is a complex process for a long time and it is the result of epithelial-mesenchymal reciprocal induction, including several stages of the occurrence and formation of tooth germ, cellular differentiation, matrix secretion and mineralization. The morphological development of teeth has been studied clearly now, but there are still a lot for us to explore in the matter of molecular mechanism.SLPI is a non glycosylated single stranded polypeptide protein secreted by epithelial cells and is a single-chain polypeptide consists of 107 amino acids. SLPI belongs to the family of whey acidic proteins. SLPI is composed of two highly homologous domain, one is cysteine rich region, the other is C terminal region, C terminal region can inhibit the activity of elastase and N terminal area has anti-bacterial, anti-retroviral and anti-inflammatory activity. Although teeth injury induces odontoblasts to express SLPI, it has not been reported that how to SLPI participate in tooth development. What’s the role of it in the development of dentin? Through what kind of molecular mechanism does it realize? It needs animal and cell models for the research on mechanism. The first part of this paper is to establish SLPI silence and overexpression models to pave the way for further research on the role and mechanism of SLPI in tooth development. Experimental contents and results:1. Through the experimental method of molecular biology, the SLPI over expression vector of pCMV6-SLPI (+)-GFP was constructed;2. Through the combination of classical breeding and PCR method, the homozygous mice of SLPI gene over expression of was established;3. The primary culture of germ cells of SLPI gene wild type and SLPI gene knockout.Part twoThe molecular mechanism of migration of U2OS cell induced by SLPIWith the deteriorative ecological environment and the unhealthy lifestyle, the cancer rates rising year by year, now it has became the first killer that threat to human health. Now there are mainly three cancer treatments including surgical resection, radiotherapy and chemotherapy. But these treatments are still unable to overcome the tumor cells infinite proliferation, relapse and metastasis.We found that SLPI can promote the odontoblast migration through the preliminary experiments, and inferred the feasible pathways. Others reported that when the most tumor migration the SLPI expressed highly, but the mechanism was not clearly. So we speculate that the SLPI who can promote the odontoblast migration also plays an important role in the tumor metastasis, and we can confirm that SLPI promote the tumor metastasis through the SLPI overexpression, and inhibit it to confirm whether the SLPI can reduce the tumor metastasis.In this study we selected the osteosarcoma that relatively high incidence in adolescents as the research object, by constructing the SLPI gene over expression cell line and the SLPI gene silencing model and with ERK1/2 inhibitor U0126 processing, to explore the SLPI gene whether the activation of ERK1/2 signal path to promote the migration of tumor cells, SLPI gene silencing through inhibition of the ERK1/2 pathway to reduce the migration of tumor cells. Experimental contents and results:1. Construction of human SLPI gene over expression vector pCMV6-SLPI-GFP. The vector of pCMV6-SLPI-GFP and pCMV6-AC-GFP were transfected into U2OS cells. Construct the stable expression monoclonal cell line of U2OS;2. Through PCR and blot Western experiments, the optimal conditions for siRNA interference SLP expression in U2OS cells were determined;3. Through the cell migration experiments, confirmed the SLPI gene over expression to promote the migration of U2OS cells, SLPI gene silencing reduced U2OS cell migration;4. Western blot and zymography confirmed the hypothetical pathway.