The Expression Of CXCR2 In Hepatocellular Carcinoma And The Inhibition Of Anti-cxcr2 Monoclonal Antibody E4 To The Combination Of CXCR2 And GROα

HCC (hepatocellular carcinoma, HCC) is one of common malignant tumors and is the secondary cancer in China. HCC has a rising trend year by year.It is difficult to be discovered that early HCC which doesn't have clinical symptoms. Once the typical clinical presentation,it is usually already in the late.Though, looking for a higher sensitivity, greater specificity tumor markers for early diagnosis of hepatocellular carcinoma is essential for clinical treatment and improving survival rate after 5 years.Chemokines have multiple effects for regulating angiogenesis, promoting proliferation of tumor cells and mediating tumor cell invasion and trafficking. Especially the Glu-Leu-Arg (ELR) CXC Chemokines play a functional role in tumor and promot tumor cell proliferation and chemotaxis. The ELR+ members have proven to be significant promoters of angiogenesis associated with a variety of human tumors Growth-related oncogeneα(GROα) is an ELR+ CXC chemokine has been reported in several human cancers such as melanoma and esophageal cancer and plays a major role in inflammation and wound healing.GROαmediate proangiogenic effects through a common receptor CXCR2. Many studies have shown that CXCR2 is related to many kinds of tumor. CXCR2 is a type 2 receptor of chemokines and has senven transmembrane segments. CXCR2 and GROαwith high affinity, CXCR2 plays an important role in GROα-mediated metastasis which is affected by autocine and paracine functions.Aims 1. Preparing a specific monoclonal antiby for the binding site between CXCR2 and GROα, identifying the CXCR2 gene expression in HCC and analysising the clinical data.2. Observing the inhibition of anti-CXCR2 antibody for the tumor cells proliferation by the stimulation with GROαhas important significance for the study of tumor.Methods1. According to the literatures, product synthetic peptide which sequence is the binding site for CXCR2 and GROαin CXCR2. After mice be immunized, preparation monoclonal antibody for CXCR2 by cell fusion, followed by ELISA, Western-blot, Immumohistochemistry and immunoflurescence method on the antibody identification.2. Using RT-PCR and immunohistochemistry identify the expression of CXCR2 in human hepatocellular carcinoma cells and hepatocellular carcinoma tissue.3. Kaplan-Meier (KM) estimator and univariate Cox were used to assess the marginal effect of each factor. The differences between groups were tested by log-rank analyses. Multivariate Cox regression was used to assess the joint effect of 2 or more factors.4. ELISA was used to detective anti-CXCR2 mAb competitive inhibition of GROαbinding with CXCR2 protein.Results1. Obtaining a CXCR2 secreting monoclonal antibody cell line E4, and purified anti-CXCR2 monoclonal antibody, subtype is IgG2a, titer of the antibody is 1:32000.2. RT-PCR and immunohistochemistry (positive rate 63.2%) confirmed that the CXCR2 gene is highly expressed in human hepatocellular carcinoma3. Analysis 128 HCC cases with clinical follow-up data and immunohistochemistry results. The death rate of HCC patients without CXCR2 over expression was remarkably higher than for those without CXCR2 over expression (p=.0002). According to the multivariate analysis, CXCR2 gene expression was the strongest predictors of survival.4. GROαprotein can promote the proliferation of hepatoma cell line, after joining the anti-CXCR2 antibody, the trend of proliferation appears to be inhibited.Conclusions1. Monclonal antibody E4 is the specific antibody for CXCR2.2. CXCR2 gene is highly expressed in human hepatocellular carcinoma can be used as an independent indicator of clinical diagnosis.3. Anti-CXCR2 mAb has competitive inhibition of GROαbinding with CXCR2 protein.