| Nasopharyngeal carcinoma (NPC) is a common human malignancy in department of otorhinolaryngology,which has a remarkably geographic distribution in south China and Southeast Asia.Sutdy on etiology indicated that the infection of Epstein-Barr virus(EBV) and hereditary susceptibility, eating habit and some environmental factor play an important role in the development of NPC ,it is an complicated process with multiple gene and multiple procedure.And EBV acts the most important part. Latent membrane protein-1(LMP1) is the only confirmed malignant transforming protein of the EBV encoded protein.As a novel gene therapy method, DNAzyme(DZ) can inhibit the express of target gene effectively.It has used in the therapy of tumor and virus.Our research aimed at EBV-LMP1mRNA, designing and synthesizing DZ with high inhibition capability and specificity, then to observe the inhibition effect of EBV-LMP1mRNA on NPC cell and transplanted tumor of NPC in nude mice.as well as the biology effect of cells,so to offer an new gene therapy strategy of NPC on prevention and treat. Partâ… Inhibition of EBV-LMP1 Gene Expression in Nasopharygeal Carcinoma Cells by DNAzymeObjective: To synthesize DNAzymes(10-23DRz) against the LMP1 mRNA by early groundwork, investigating the inhibition effect on target gene.Methods:Taking EBV-LMP1 gene as target point, synthsize high specificity DZ509(10-23DRz) on the basis of early study(with high performance and specificity through a screening test by in vitro transcription),as well as mutant of DZ509(mutDZ509) Which has a point mutation of the 4th site of active center and oligonucleotide509(ASODN509) which has no active center.Marking the 5'terminal by FITC,and all were modified by thio-modification in terminal 2 bases. Transfecting them into C666-1 cells by lipotectamine, called DZ509 group,mutDZ509 group,ASODN509 group respectively.Taking blank group and lipotectamine group as control.24 hours after transfection, observing the transfect efficiency under the fluorescence microscope. RT-PCR was used to detect the expression of LMP1 and protein level of LMP1 was measured by Western blot.Results: 24 hours after transfection,the tranfect efficience was 64%,60% and65% .RT-PCR showed that comparing to blank control group,the inhibition ratio of lipofectamine group on EBV-LMP1mRNA was 5.94%,which has no significant difference(P>0.05). But ASODN509,mutDZ509,DZ509 can depress the expression of EBV-LMP1 to a certain extent,the inhibition ratio was 23.33%,53.18%,68.76%,the inhibition effect: ASODN509 0.05). inhibition ratio of DZ509,mutDZ509,ASODN509was 31%,16%,14% , the difference was statistically significant (P<0.05).The inhibition effect on cell proliferation: ASODN5090.05).The result of 72 hours was accord with 48 hours.24 hours after transfection ,the cell cycle was detected by FCM.With the ratio of G0-G1 phase, comparing to blank control group of 50.28%, the lipofectamine group was55.23%, which has no significant difference(P>0.05). The ratio of DZ509 was 68.46%, the difference was particularly significant(P<0.05);mutDZ509,ASODN509 group was 63.72% and 62.23%, P>0.05. DZ509 group compared with ASODN509 group and mutDZ509 group, the difference was particularly significant(P<0.05). ASODN509 group compared with mutDZ509 group, the difference was particularly significant(P<0.05). With the ratio of S phase , comparing to blank control group of 39.30%, the lipofectamine group was33.72%, which has no significant difference(P>0.05). The ratio of DZ509 was 12.37%, P<0.05,mutDZ509,ASODN509 group was27.05% and 28.77%,the P>0.05. DZ509 group compared with ASODN509 group and mutDZ509 group, the difference was particularly significant(P<0.05). ASODN509 group compared with mutDZ509 group, the difference has no particularly significant(P>0.05).Though FCM, 24 hours after transfection, comparing to blank control group , the cell apoptosis rate of lipofectamine group was0.48%, which has no significant difference(P>0.05). the cell apoptosis rate of DZ509,mutDZ509,ASODN509 group was about 16.64 %,10.47 %,8.81 %. the effect on apoptosis: ASODN5090.05).Comparing to PBS group, DZ509,mutDZ509 and ASODN509 can inhibit the expression of EBV-LMP1 mRNA by RT-PCR: ASODN509 |
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