Study On The Correlation Between EB Virus Infection And The Up Regulation Of S100A8 And S100A9 In Nasopharyngeal Carcinoma Cells

This paper includes three parts:(I) Construction of EB virus infectioned nasopharyngeal carcinoma cell line and detect expression of S100A9 and S100A8 protein; (Ⅱ) The construction of the eukaryotic expression vectors EBV-LMP1 and EBV-LMP2A with the carrying green floprescent report genes; (Ⅲ) Expression of LMP1and LMP2A in nasopharyngeal carcinoma cells and the effect of and S100A9 on S100A8.Parti Construction of EB virus infectioned nasopharyngeal carcinoma cell line and detect expression of S100A9 and S100A8 proteinObjectives:To investigate whether the upregulation of S100A8 and S100A9 protein in nasopharyngeal carcinoma tissues and cells are associated with EBV infection, whether the latent infection of EBV is due to the expression of S100A8, S100A9 protein.Methods:Using the way of co-culture, B95-8 marmoset lymphoma cell (EBV positive) to let EBV infect nasopharyngeal carcinoma cell(EBV negetive). After the infected EBV kill B95-8 cell by immune toxicity, establish carrying EBV cell lines CNE1-E and CNE2-E. Based on the existence of EBV in NPC time, make sure of EBV infection period by our pre-experiment. Detect CNE1-E, CNE2-E periodic by flow cytometry and to observe the morphological changes of cells by HE staining. The cell clone formation assay, cell clone formation ability after infection. Resistance ability of nasopharyngeal carcinoma cells in 5-Fu (IC50 concentration). Detected by CCK-8 after EBV infection, The expression of S100A8, S100A9 and MMPs in nasopharyngeal carcinoma cell line after infected EBV qRT-PCR. The expression of S100A8 and S100A9 protein, detect by Western-blot. Use RT-PCR to detect LMPl expression in high expression of S100A9 and S100A8 in CNE1-E, CNE2-E.Results: ①The cell number (1:1) co-cultured, B lymphoma cell and nasopharyngeal carcinoma cell contact with each other, after killing B95-8 cells, remaining cells like epithelial cells. We use EBV specific gene BamHI-W to detect EBV and marmoset specific gene Caja make sure B95-8 were cleaned up,all those detected by PCR. The results shows that EBV infected nasopharyngeal carcinoma cells, and no residues of B95-8 cells.②Using PCR to detect the presence of EBV in the cells was about 20±5 days after infection. (3)The differentiation of different cell have different cycle changes after EBV infect, CNE1-E first appeared in G0/Gl phase reduce, increase the period of thermotherapy in S phase, increased with time in G0/G1 phase increased gradually, gradually reduce the period of thermotherapy in S phase, showed a trend of first proliferation inhibition. CNE2-E cells appeared G0/G1 phase increase, S +G2 phase period decreased, with the increase of time G0/G1 phase gradually decreased, S +G2 phase gradually increased, the performance of the first inhibition after the proliferation of the trend. In later stages of infection was observed in cells chromatin bold and cell proliferation characteristics in the late stages of infection.④The colony formation ability of CNE1-E and CNE2-E cells was higher than that of CNE1 and CNE2 cells (P<0.01).⑤After infected EBV 72h, compared with CNE1-E and CNE1,CNE1-E resistance to 5-FU treatment strongly (P< 0.05). CNE2-E and CNE2 no significant difference (P> 0.05). ⑥In CNE1-E, CNE2-E cells, S100A8, S100A9 mRNA expression were raised, but varies with the change of time, to show the basic first increased and then decreased trend. The S100A8 and CNE2-E in S100A9 showed the trend of decreasing first and then increasing. ⑦The results of Western-blot and qRT-PCR results are basically the same.⑧During S100A8 and S100A9 expression is upregulated that LMP1 protein mRNA expressed positively, S100A8, S100A9 expression may be associated with EBV latent infection.Conclusions: ①EBV infection of nasopharyngeal carcinoma cells can cause S100A8, S100A9 up regulation expression, the period of infection after EBV and S100A8, S100A9 expression has a closed relationship. ②S100A8, S100A9 protein expression is usually appear in the latent infection period of EBV.Part2 The construction of the eukaryotic expression vectors EBV-LMP1 and EBV-LMP2A with the carrying green floprescent report genesObjectives:To have basis work for studying whether the increase of S100A9 and S100A8 protein asocial with the expression of EBV-LMP1 and EBV-LMP2A.Methods:①Extract total RNA from B95-8 cell and then reverse-transcription as cDNA.②Using specific primers amplification of the mRNA sequence LMP1 and LMP2A, and constructs a subclone containing LMP1, LMP2A sequence, PCR and DNA sequencing verified. ③Sequencing correct sequence by restriction endonuclease shear directionally cloned into carrying green fluorescent eukaryotic expression vector pIRES2-Zs-Greenl, to construct expression plasmid pIRES2-Zs-Greenl-LMP1 and pIRES2-Zs-Greenl-LMP2A.Results:We get the LMP1, LMP2A sequences from B95-8 cells RNA successfully. LMP1, LMP2A DNA sequence alignment compared with the NCBI database found our laboratory B95-8 cell derived LMP1 gene have 6 mutation(136 G> A,202 A> C,253 A> C,317 T> A,691 G>T,703 C>T). But the amino acid sequence of the codon is identical with that of NCBI.③The success of the LMP1 LMP2A and inserted into the eukaryotic expression vector pIRES2-Zs-Greenl, PCR validation and sequencing verified correct.Conclusions:carrying green fluorescent protein gene pIRES2-Zs-Green1-LMP1 and pIRES2-Zs-Green1-LMP2A eukaryotic expression plasmid was constructed successfully.Part3 Expression of LMP1and LMP2A in nasopharyngeal carcinoma cells and the effect of and S100A9 on S100A8.Objectives:To verify S100A8 and S100A9 upregulation of expression and EBV latent infection related, especially of EBV latent infection period of two important protein LMP1 and LMP2A is involved in regulation in nasophary ng-eal carcinoma (NPC) of S100A8 and S100A9 Protein.Methods:Using liposome transfection put pIRES2-Zs-Green1-LMP1, pIRES2-Zs-Greenl-LMP2A plasmid into nasopharyngeal carcinoma cells, the nasopharyngeal carcinoma cells expression of LMP1 protein and protein2A co-expression of LMP1 and LMP2A protein, according to the vector carrying green fluorescence expression strength to determine transfection efficiency. Use WB, ICC and RT-PCR to detect the expression of LMP2A and LMP1. Extraction of LMP1, LMP2A and LMP1 and LMP2A group of nasopharyngeal carcinoma cell total RNA, qRT-PCR detection of S100A8, mRNA protein S100A9, MMP2, MMPs, MMP3, MMP9, mRNA protein expression of MMP7.Results:in CNE1 cells, single transfection LMP1 or LMP2A plasmid does not enable S100A8 and S100A9 expression. Only the transfection of LMP1, LMP2A plasmid S100A8 and S100A9 gene expression upregulated (P< 0.05); CNE2 cells and single transfection LMP1 can increase S100A8 and S100A9 genes of (P< 0.05) and co transfected with LMP1 and LMP2A plasmid can S100A9 upregulated (P< 0.05) and S100A8 gene expression increased but the difference was not significant (P> 0.05). In CNE1, S100A8, S100A9 up-regulated expression at the same time, MMP2, MMP3, MMP9 gene expression up regulation (P< 0.05), CNE2 in S100A8, S100A9 up-regulated expression at the same time, MMP3 and MMP7 gene expression (P< 0.05).Conclusions:the up-regulated expression of S100A9 and EBV was related to the LMP1 and LMP2A proteins encoded by S100A8 latency. Third, S100A8, S100A9 in the rise of matrix metalloproteinases also changes, changes in the differentiation of various cell MMPs also have different low differentiation of CNE1 cells in S100A8 and S100A9 expression is upregulated more likely to express enzyme gel hydrolases:MMP2, MMP9, and highly differentiated CNE2 cells in S100A8 and S100A9 upregulation tend to expression of MMP3, MMP7 and cell matrix enzyme.