Study On Epstein-Barr Virus Latent Membrane Protein 1 And Heat Shock Protein 90 β In Nasopharyngeal Carcinoma

1. A study on the expression of HSP90 3 in nasopharyngeal carcinoma and clinical significanceObjective Heat shock protein (HSP) is one of the tumor-associated antigens (TAAs) and is highly expressed in tumor tissues, and HSP70, HSP90 and gp96 are found to be most closely related to tumor genesis. Recent investigations demonstrated that HSP90β was associated with tumor cells differentiating phases. Considering that NPC is mainly malignant oriented and poorly differentiated, HSP90βis supposed to express highly in NPC tissue cells, however the expression level of HSP90βin NPC tissue cells has not been reported yet, thus our experiment intends to evaluate the expression level of HSP90 beta in human NPC cells and analyze its application the grading system and prognosis of human NPC, identifying reliable biological indicator for NPC prognosis assessment. Methods Immunohistochemical SP method was used to detect the expression of HSP90β in 50 cases NPC, and its clinical significance was studied by analyzing data with X~2 test.Results The positive rate of HSP90β in human nasopharyngeal carcinoma was 56.0%, higher than infected tissue ofnasopharynx; The lower the differentiated level of NPC is, the higher theexpression level would be. Conclusion The over expression of HSP9O/3 wasprobably related to the occurrence, development and prognosis ofNasopharyngeal carcinoma; HSP90/3 was probably one of NPC prognosticindexes.2. AstudyonEBV-LMPlandHSP9O0 in NPCObjective NPC is distinctive among the head and neck carcinomas for its marked tendency to metastasis and invasion. Although the primary tumor is sensitive to radiotherapy, most deaths are due to the spread of tumor cells. It has been shown that at the time of diagnosis, 60%-85% of NPC patients already have clinically detectable aggressive metastasis in the regional lymph nodes, in distant organs such as the lungs and in bone. So far there is still no effective treatment for NPC at the stage of metastasis. As a result, prognosis is poor and the 5-year survival rate is less than 50%. So supplementary therapy is required. For the past few years, tumorous immunotherapy displays all-right application prospect and upstanding effect in multi-tumor. At present, the researches associated nasopharyngeal carcinoma antigens have still focused on the relationship between EBV and nasopharyngeal carcinoma, and up to now, the researches about autoantigens of nasopharyngeal carcinoma cells are almost empty. NPC is closely associated with EBV, LMPl is irritation source and target antigen of EBV inducing organism specific cellular immunity, and existing advanced stage LMP gene in NPC cell line, therefore EBV LMPl has pacing factor oftumor specific antigen of NPC. Recent investigations have confirmed that LMP1 possesses specific antigen epitope, it may trigger specific cytoimmunity as a target antigen of CTL, and thus LMP1 possesses rationale for EBV relative tumor vaccine. Otherwise, more and more studies have indicated that HSP can strengthen tumorous immunogenicity, participate submit tumour antigen, arouse T cell mediated tumouspecific immunity. The experimental primary part discovered HSP90 ￡ is highly expression in NPC. Therefore the experimental intention is to construct tumor vaccine of LMP1 and HSP90P, namely amplifying Epstein-Barr virus latent membrane protein 1(LMP1) and heat shock protein 90 ft (HSP90 ￡) from nasopharyngeal carcinoma (NPC) and constructing the mammalian co-expression plasmid pERES-LMPl-HSP90 {3 and detecting the expression of the plasmid in vitro.This will provide the basic material for next running animal experiment and understanding vaccination against EBV. Methods Using cloning technique , total RNA was isolated from human NPC and amplified EBV-LMP1 cDNA and HSP90 ￡ cDNA by RT-PCR were cloned into pDrive Cloning Vector and PGEM-T Easy Vector respectively and sequenced. Theirs cDNA fragments were then subcloned into the mammalian co-expression plasmid vector pIRES. The inserted target genes in the mammalian co-expression plasmid were verified by nucleotide sequencing. COS cell line was transfected with this mammalian co-expression plasmid using lipofectin reagent. The expression of LMP1 and HSP90 P molecules were detected by RT-PCR and Western blot technique. Results The mammalian expression plasmid pIRES-LMPl-HSP90￡ was obtained by cloning technique. The nucleotidesequences of LMP1 gene and HSP90 P gene in this mammalian co-expression plasmid had high homology with LMP1 B95-8(100%) and human HSP90 P (100%) respectively. After transfection with this mammalian co-expression plasmid, the LMP1 and HSP90 P molecules were expressed in COS cells.Conclusion The LMP1 gene and HSP90P gene were successfully amplified and cloned into pIRES mammalian co-expression vector. The constructed plasmid pIRES-LMPl-HSP90P can express LMP1 and HSP90 P molecules in vitro at the same time. The expression of LMP1 and HSP90 P in COS cells provided the basic material for next running in vivo experiment, studying interaction of LMP1 and HSP90 P on anti-tumor aspect and vaccination against EBV.