Research On The Expression Inhibition On HBsAg And HBeAg In HepG2.2.25 By 10-23DNAzyme And LANzyme

Hepatis B virus infection is the major cause of acute Hepatitis, chronicHepatitis, liver cirrosis and liver cancer. So far, 350 million people get infectedwith Hepatitis B in the world, 25-40 percent of Hepatitis B patients died fromHepatitis B complications. No specific effective drugs are available at present.Although interferons are utilized for the therapy of HBV, its application is limiteddue to its low efficacy, too much side effects and expensiveness. Nucleotidesanalogs is in hot research nowadays, but this therapy has its own disadvantages oftoo long therapy process, high rate of virus variation and disease reoccurence.Therefore, to develop a new gene therapeutic agents which is cheap, effective, lowside effects, stable and target oriented is the important means for the therapy ofHBV. Gene therapy is a promise of HBV infection. For the gene therapy of viraldiseases, Antisense technique is a hot area. Antisense technique includeantioligonuclitide, ribozyme, DNAzyme. Ribozyme degrades easily in vivo,chemical modification can enhance its stability, but reduce its the activities, eveneliminate its activities. Antioligonucleotide has longer half life time, but can notdestroy target genes, just acting as gene blocking due to lacking of catalyzingactivites. Based on the above discussion, it is hypothesized that DNA derived geneinactivation agents can be established which has the ability to cut RNA andpossesses biological characteristics of antioligoneoclutides.DNAzyme is a kind of DNA molecule with enzyme activities. It can pairswith specific target mRNA and inactivate target mRNA by cutting. At present,most researches focus on 10-23DNAzyme. The structure of 10-23DNAzyme islike hammer ribozyme, containing a highly conserved catalyzing domain and twovariable side arm domains. The catalyzing domain comprises a round catalyzingcenter of 15 deoxynucleotides, two side arms contain substrate binding sites of 7-9deoxynucleotides. Bases in the two combined arms pair with substrate RNAthrough Waston-Crick rules, catalyzing center will cut substrate RNA molecules.The characteristics of 10-23DNAzyme cutting are: a, compared with nucleozyme,cutting side of 10-23DNAzyme is purine-pyrimidine matching sides, these kindsof sides are widely present in RNA molecules, more available cutting sides;b,relatively stable, the stability is one tenth million times of RNA in physiologicalPH, temperature and ions intensity;c, the characteristics of activities centersequence is relatively short, low molecular weight, relative good elasticitydetermines that two dimensional structure of target sequence will have littleeffects on binding to substrate;d, DNA-RNA molecules are easily to ionizecompared with RNA-RNA molecules, so cutting rate is seldom affected byDNAzyme ionization process;e, it is easily prepared in technique and accepted inexpenses. Nowadays, 10-23DNAzyme is applied in the therapy of a variety ofdiseases. Scientists agreed that DNAzyme is a great leap and will play moreimportant roles in gene therapy area.LNA is receiving more and more attention as a new nucleotide ramification.In 1994, LNA was identified by Rodriguez in side products of fermentation. In1998, the syntheses and hybridization of LNA was performed by Koshkin.Koshkin found that the introduction of mono LNA to the binding arm of10-23DNAzyme increased the efficacy of substrate RNA cutting by LNAzyme.LNA possesses powerful hybriding activities with RNA, antisense interferingactivities, better mismatching identifying abilities, antinucleozyme activities, goodDNAzyme is a kind of DNA molecule with enzyme activities. It can pairswith specific target mRNA and inactivate target mRNA by cutting. At present,most researches focus on 10-23DNAzyme. The structure of 10-23DNAzyme islike hammer ribozyme, containing a highly conserved catalyzing domain and twovariable side arm domains. The catalyzing domain comprises a round catalyzingcenter of 15 deoxynucleotides, two side arms contain substrate binding sites of 7-9deoxynucleotides. Bases in the two combined arms pair with substrate RNAthrough Waston-Crick rules, catalyzing center will cut substrate RNA molecules.The characteristics of 10-23DNAzyme cutting are: a, compared with nucleozyme,cutting side of 10-23DNAzyme is purine-pyrimidine matching sides, these kindsof sides are widely present in RNA molecules, more available cutting sides;b,relatively stable, the stability is one tenth million times of RNA in physiologicalPH, temperature and ions intensity;c, the characteristics of activities centersequence is relatively short, low molecular weight, relative good elasticitydetermines that two dimensional structure of target sequence will have littleeffects on binding to substrate;d, DNA-RNA molecules are easily to ionizecompared with RNA-RNA molecules, so cutting rate is seldom affected byDNAzyme ionization process;e, it is easily prepared in technique and accepted inexpenses. Nowadays, 10-23DNAzyme is applied in the therapy of a variety ofdiseases. Scientists agreed that DNAzyme is a great leap and will play moreimportant roles in gene therapy area.LNA is receiving more and more attention as a new nucleotide ramification.In 1994, LNA was identified by Rodriguez in side products of fermentation. In1998, the syntheses and hybridization of LNA was performed by Koshkin.Koshkin found that the introduction of mono LNA to the binding arm of10-23DNAzyme increased the efficacy of substrate RNA cutting by LNAzyme.LNA possesses powerful hybriding activities with RNA, antisense interferingactivities, better mismatching identifying abilities, antinucleozyme activities, goodinterfering target area. C area of HBV genome is highly conserved is closelyassociated with pregenome RNA packaging and virus DNA replication.deoxynucleozyme and LNAzyme designed by targeting at C area will catalyze tocut HBV mRNA and inhibit the replication and expression of HBV.10-23DNAzyme, site sulfur modification 10-23DNAzyme and LNAzymewere designed targetting at HBV. The experiment included experiment groups andcontrols. The experiment groups contain nonmodification 10-23DNAzyme group,site sulfur modification 10-23DNAzyme group, LNAzyme group;The controlscontain blank group, liposome group, 10-23DNAzyme alone, nonrelevent10-23DNAzyme. Nonmodified 10-23DNAzyme, site sulfur modified10-23DNAzyme and LNAzyme transfected HepG2.2.15cells, the expressioninhibition of HBsAg and HBeAg in HepG2.2.15 cells at different10-23DNAzyme concentration of 0.16μmol.L-1, 0.64μmol.L-1, 1.28μmol.L-1,1.60μmol.L-1, 1.92μmol.L-1 were determined at 12h, 24h, 36h, 48h, 60h, 72h, 84hand 96h.The results showed that LNAzyme, site sulfur modification 10-23DNAzymeand nonmodification of 10-23DNAzyme can inhibit the expression of HBsAg andHBeAg in HepG2.2.15cells at. 1.6μmol.L-1-1.92μmol.L-1. At the sameconcentration, the expression inhibition of HBsAg and HBeAg was significantlygreater by LNAzyme than that by site sulfur modification of 10-23DNAzyme(P<0.05), site sulfur modification greater than that by nonmodification10-23DNAzyme (P<0.05). The results also showed that the expression inhibitionis manifested in dose and effects manners. LNAzyme, site sulfur modification andnonmodification of 10-23DNAzyme manifested positive relevence at theconcentration of 0.16μmol.L-1-1.60μmol.L-1, that is more drug, greater expressioninhibition. The HBsAg expression inhibition rate of LNAzyme is 87.4±3.5% atthe concentration of 1.60μmol.L-1,the inhibition rate of site sulfur modification of10-23DNAzyme is 75.8 ± 4.0%;the HBeAg expression inhibition rate ofLNAzyme is 86.1±3.1%, site sulfur modification of 10-23DNAzyme is 73.6±2.5%, increasing the drug concentratin will not result in greater inhibition.To observe the time and effects relationship of expression inhibition onHBsAg and HBeAg in HepG2.2.15cells, the expression inhibition caused byLNAzyme, site sulfur modification 10-23DNAzyme and nonmodification10-23DNAzyme were tested between 12h-96h. The results showed that LNAzyme,site sulfur modification 10-23DNAzyme and nonmodification 10-23DNAzymecan exert inhibition on the expression of HBsAg,HBeAg in HepG2.2.15cellsbetween 12-96 h. At the same time period, the expression inhibition on HBsAgand HBeAg by LNAzyme is significantly greater than that by site sulfurmodification 10-23DNAzyme (P<0.05), site sulfur modification 10-23DNAzymeis greater than that by nonmodification 10-23DNAzym (P<0.05). The resultsshowed that after 12h of administration, the expression inhibition manifested itself,peaked at 48h. At 48h, the expression inhibition rate on HBsAg of LNAzyme is87.4±3.5%,site sulfur modification 10-23DNAzyme is 75.8±4.0%;theexpression inhibition rate on HBeAg of LNAzyme 86.1±3.1%, site sulfurmodification 10-23DNAzyme is 73.6±2.5%. After that, the expression inhibitionrate decreased with time, at 84h LNAzyme still manifested inhibition on HBsAg,HBeAg;site sulfur modification 10-23DNAzyme had inhibiting effects at 72h.Nonmodification 10-23DNAzyme manifested little expression inhibition onHBsAg and HBeAg at 60h.The morphology, growth, viable cell ratio and LDH activities in thesupernatant of cell culture from HepG2.2.15cells were determined to test theeffects of 10-23DNAzyme and LNAzyme on the cells. The results showed that10-23DNAzyme and LNAzyme at the above administration concentration(0.16μmol.L-1-1.92μmol.L-1) and at the observation time (12h-96h) exerted noinfluence on the morphology, growth, viable cells ratio and LDH activities in thesupernatant of cell culture compared with that in controls.Although the present results showed that the expression inhibition rate onHBsAg and HBeAg by 10-23DNAzyme and LNAzyme in HepG2.2.15cells ishigh, compared with theoretical deduction, ideal inhibition rate is not achieved.Several causes accounted for this, such as deoxynucleozyme and LNAzymemodification, that is how to do the modification will maximize the cutting rate;the two dimensional structure of deoxynucleozyme and LNAzyme, target siteselection, two dimensional structure of substrates, etc. So, how to optimize thecutting conditions and achieve the maximum cutting rate is in need. It is deeplybelieved that with the intensive and deep research, deoxynucleozyme andLNAzyme will play more important roles in gene therapy.Creative points in the present research:(1) Two mono LNA were introduced into the two binding arms of LNAzymedesigned by targetting at HBV, the four modification deoxynucleotides are A,modified by adjacent A, the sequence is as following:5′CTCALG GALGA GGC TAG CTA CAA CGA TCT ALALGGC3′(2) LNAzyme was first used in the research on HBV infection control.