Reasearch Of Using HRE₂.Grp₇₈ Double Promoters To Regulate The Expression Of VP₃ And Have A 5-ALA-PDT/ Gene Therapy On Nasopharyngeal Carcinoma

Objective:Nasopharyngeal carcinoma(NPC)is one of the most malignant tumors in the South China and it ranks the first in head and neck cancer, which damaged the health of the people seriously.Since more than 95% of the NPC are non-cancroids which is moderately radiosensitive,radical radiation therapy is the first choice of treatment in clinic.With the development of radio-physics and radiobiology,the radiation effect is still far from satisfaction.The general 5-years' survival rate is only up to 50%-60%and damage to the tissue companying with the radiation is serious.After radiation therapy,the blood vessels around the pharynx nasalis atrophied and blood-supply to pharynx nasalis is poor which decreased curative effect and sensitivity to the second radiation. Meanwhile the place where NPC originated is concealed and there are lots of important tissues and organs around it.It is hard to be exposured by surgical operation and the effect of surgical operation is not good as well.The treatment of NPC recidivists after radiation therapy is a tough problem in clinic.In this study we combined the photodynamic therapy (PDT)with apoptotic gene therapy together to observe killing effect on NPC in vitro and explore a new NPC treatment strategy to improve the effect of the NPC therapy.Methods:1)To construct four plasmid expression vectors PcDNA3.1(-) Grp78p.VP₃- Myc -CMV,PcDNA3.1(-)Hre₂.Grp78p.VP₃- Myc-CMV, Pc DNA 3.1(-)CMV.VP₃-Myc and Pc DNA 3.1(-)VP₃-Myc -CMV by PCR,enzyme restriction and ligation techniques.2)To transfect the Vectors PcDNA3.1(-)Grp78p.VP₃- Myc -CMV, PcDNA3.1(-)Hre₂.Grp78p.VP₃-Myc-CMV,Pc DNA 3.1(-)CMV.VP₃ -Myc and PcDNA3.1(-)VP₃-Myc-CMV into CNE-2 cell line by liposome. Total RNA and protein were extracted and VP3 gene expression was detected by RT-PCR and Western-blotting;to investigate the survival rate of CNE-2 after gene therapy by CCK-8 assay. 3)To explore the best concentration and the best incubation time of the photosensitizer 5-ALA and the best dose of laser radiation and observe the rate of cytocide after PDT.4)To combined PDT with gene therapy together,study the survival rate and apoptotic rate of CNE-2,the expression of VP3 and the morphosis change among PDT/gene therapy group,PDT group and gene therapy groupResults:1)PcDNA3.1(-)Grp78p.VP₃- Myc -CMV,PcDNA3.1(-) Hre₂.Grp78p.VP₃- Myc-CMV,Pc DNA 3.1(-)CMV.VP₃-Myc and Pc DNA 3.1(-)VP₃-Myc -CMV were constructed successfully and identified by analysis of DNA sequence2)The killing effect of VP₃ gene on NPC:The above four vectors were transfected to CNE-2 successfully.Expression of VP₃ gene in transfected CNE-2 cell line was confirmed from mRNA and protein level. VP3 gene has a certain cytocide effect on CNE-2.The survival rate of CNE-2 induced PcDNA3.1(-)CMV.VP₃ -Myc(50.8±1.6)%is significantly higher than that of CNE-2 induced PcDNA3.1(-)Grp78p.VP₃-Myc -CMV(64.1±1.4)%,PcDNA3.1(-)Hre₂.Grp78p.VP₃-Myc-CMV(62.3±0.8) %and Pc DNA3.1(-)CMV.VP₃-Myc-CMV(95.5±1.0)%(P＜0.05)3)The killing effect of PDT on NPC:5-ALA produced endogenous PpIX in CNE-2 and PpIX was observed evenly in the cytoplasma,but not in the nuclear region.The expression intensity of 5-ALA was highest after 6 hours' incubation in concentration 1.0mmol/L.The survival rates of the CNE-2 which is mediated by 5-ALA PDT decreased with the time passing or the 5-ALA concentration increasing.The survival rate of the cells only treated by 5-ALA or light irradiation had no significant difference compared with that of the blank control group.4)The killing effect of 5-ALA-PDT combined with gene therapy on NPC:Expression of VP3 in CNE-2 which were transfected with PcDNA3.1(-)Grp78p.VP3-Myc -CMV and PcDNA3.1(-)Hre2.Grp78p. VP3-Myc-CMV is higher than that in CNE-2 transfected with PcDNA3.1(-)CMV.VP3-Myc with PDT(P＜0.05).The cell survival rate of CNE-2 treated with 5-ALA-PDT and gene therapy(18.3±2.7)%is lower than that of CNE-2 treated with gene therapy(74.4±0.9)%or 5-ALA-PDT(55.0±2.2)%only(P＜0.05),while the apoptosis rate of CNE-2 treated with 5-ALA-PDT and gene therapy(68.3±2.3)%is higher than that of CNE-2 treated with gene therapy(27.8±1.6)%or 5-ALA-PDT(54.8±+3.6)%only(P＜0.05).Apoptotic body can be obviously observed in gene therapy or gene therapy/PDT groups.Conclusion:1)PcDNA3.1(-)Grp78p.VP3-Myc-CMV and PcDNA3.1(-)Hre2 Grp78p.VP3-Myc-CMV were successfully constructed.2)PcDNA3.1(-)Grp78p.VP3-Myc-CMV,PcDNA3.1(-)Hre2Grp78p VP3-Myc-CMV and PcDNA3.1(-)CMV.VP3-Myc were induced to CNE-2 cells successfully and can kill the CNE-2 cells3)5-ALA-PDT has a good killing effect on nasopharyngeal carcinoma and it is a great treatment strategy to nasopharyngeal carcinoma4)5-ALA-PDT/gene therapy is a much better treatment strategy to NPC comparing with PDT or gene therapy only.And this strategy can promote the effect of cytocide of PDT and gene therapy with each other.