Preliminary Study In Mechanim Of Phosphorylated STAT3in Inhibiting DCs’s Maturation From PBMC(Peripheral Blood Mononuclear Cell)

Background and objectives:Gastric cancer (GC) is the most common malignant cancer of digestive system, and itshigh mortality and poor prognosis, harming to human health seriously. Escape from thebody’s immunosurveillance is an important mechanism in gastric cancer’s occurrence,development and metastasis. Dendritic cells (DCs) as the main antigen-presenting cells ofbody, regulating the body’s immune balance and immune response, are the most criticalimmune surveillance cells. Researchs have manifested that there was a close relationshipbetween functional defect of immature DCs and gastric cancer’s escaping from immunesysterm. It is need to go through a series of process of differentiation for DCs from the bonemarrow hematopoietic stem cells to perfect immune stimulation function immatureDCs(imDCs). Cancer cell can reduce the number of DCs in patient, and limit the process ofDCs’ mature, and impede the function of antigen presenting. Thus, the body’s immunesurveillance of tumor become weakly and inhibited. Now, it has been confirmed thatcytokines from tumor, such as IL-6、IL-10、VEGF、TGF-β, especially IL-6、IL-10, allcan prevent DCs from maturing and developing. However, specific molecular mechanism isnot fully clear. JAk2/STAT3signaling pathway is IL-6, IL-10common signal path, whichis a classic way of signal transduction and transcriptional activation. Recent studies haveshown that, if given JAK2specific inhibitors, could be reversed IL-6mediated mature DCsobstacles. In addition we found that the STAT3phosphorylation level significantlyincreased in immature DCs model from rat in our preliminary experiments. Based on thesestudies, we infer that the excessive phosphorylated STAT3of cell signaling pathways is thekey point for DC dysmaturity, which may mainly participate in inhibiting DCs’sdifferentiation and maturation. First, this research project detection the phosphorylationprotein expression levels of STAT3and the relationship with differentiation of mature intwo established model mDCs and imDCs from peripheral blood mononuclear cells; Then constructing recombinant adenovirus of hSTAT3and recombinant adenovirus of hSTAT3’ssmall interfering RNA, regulate the expression of STAT3protein by using two kinds ofadenovirus to further verify the above theories, lay the foundation for the researching therelationship between DCs differentiation of mature and gastric cancer’s immune escape.Method:1. Constructing recombinant adenovirus of hSTAT3and recombinant adenovirus ofhSTAT3’s Small interfering RNA. Firstly, completely STAT3code sequence was abstainedby PCR, And then build, packaging, augmentation of STAT3recombinant adenovirus.Using pSOS-HUS vector to select the best STAT3i from the four pairs of siRNA, build theinterference adenovirus and amplification the recombinant adenovirus. Both viruses werenamed AdV-STAT3and AdV-STAT3i.2. PBMC(Peripheral Blood Mononuclear Cell) are used to build mature DCs modelInducted by LPS and imDC model inducted normally. Using FACS(Fluorescence ActivatedCell Sorter) to test the positive rate of two groups of DCs’s surface molecular expression,and using Western-blot to test the phosphorylated STAT3of two models.3. Using recombinant adenoviruses AdV-STAT3and AdV-STAT3i4to regulate STAT3of DCs models, using imDC infected virus AdV-GFP as control at the same time. UsingFACS to test the positive rate of two groups of DCs’s Surface molecular expression, andusing Western-blot to test the total STAT3protein and phosphorylated STAT3protein ofmodels.Result:1. We get people’s STAT3gene sequences and successfully bulid recombinantadenovirus vector AdV-STAT3, we also successfully constructed recombinant adenovirusAdV-STAT3i4use the best STAT3i4, high degrees of the virus were obtained byamplification.2. Flow cytometry Results show that the imDCs group only induced normally, the ratesof positive cells of its cell phenotype molecules (CD83、CD86、HLA-DR)are obviouslylower than group induced by LPS (P＜0.01). Nevertheless, western-blot detects itsphosphorylated STAT3protein levels is much higher than matured model induced by LPS.Thus the protein express of phosphorylated STAT3from immature DCs to mature DCs isfrom high to low. 3. For phosphorylated STAT3protein and the rates of positive cells of its cellphenotype molecules (CD83、CD86、HLA-DR)： Compared with AdV-STAT3group andAdV-GFP group, AdV-STAT3i4group have significant differences(P＜0.01). Comparedwith AdV-STAT3group and AdV-GFP group, group induced by LPS have significantdifferences(P＜0.01). AdV-STAT3i group and AdV-GFP group have no significantdifferences (P＞0.05). AdV-STAT3i4group and mature DCs group induced by LPS have noclear differences (p＞0.05). However, the total STAT3protein of each DCs of the groupsand positive cell rates of their three surface molecular have no such similar results.Conclusion:1. Recombinant adenovirus of AdV-STAT3and AdV-STAT3i4were constructedsuccessfully. The two adenovirus can increase and reduce the STAT3protein expressionobviously, which indicates that we obtain the efficient means of regulating STAT3protein.2. Phosphorylated STAT3protein in mature DCs model induced from peripheral bloodmononuclear cell by LPS was in low expression, yet phosphorylated STAT3protein inimmature DCs model was in high expression. It is indicates that the level of phosphorylatedSTAT3is associated with DCs’s differentiation and maturation.3. Using recombinant adenovirus AdV-STAT3and AdV-STAT3i4to regulate the cellmodels, the results show: The differentiation and maturation of DCs is negatively correlatewith phosohorylation expression of STAT3, regardless with the total STAT3. The resultfurther verified the speculation that phosphorylated STAT3play a important role in processof DCs’s differentiation and maturation, whicn lay the foundation for researching therelationship between DC’s differentiation of mature and gastric cancer’s immune escape.