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Objective@#To investigate the antiviral effect of hepatitis B virus (HBV) S gene-specific anti-gene locked nucleic acid (LNA) in transgenic mice.@*Methods@#A total of 30 HBV transgenic mice were randomly divided into blank control group (5% glucose + liposome), unrelated sequence control group, lamivudine control group, antisense LNA control group, and anti-gene LNA group, with 6 mice in each group. The mice in the lamivudine group were given lamivudine by gavage, and LNA was injected via the caudal vein. Quantitative real-time PCR was used to measure serum HBV DNA, ELISA was used to measure serum HBsAg, RT-PCR was used to measure HBV S mRNA level in the liver, and immunohistochemistry was used to measure the level of HBsAg in hepatocytes.@*Results@#At 3, 5, and 7 days after treatment, there were significant changes in the inhibition rates of HBV DNA (37.18%, 50.27%, and 61.46%, respectively) and HBsAg (30.17%, 44.00%, and 57.76%, respectively) achieved by anti-gene LNA (P < 0.01), and there were significant differences between the anti-gene LNA group and the other four control groups (P < 0.05). In the anti-gene LNA group, the relative mRNA expression of HBV S gene was 0.33 and the percentage of HBsAg-positive hepatocytes was 31%, which were significantly different from these two indices in the control groups (P < 0.05). There were no abnormal changes in liver/renal biochemical parameters and HE staining results.@*Conclusion@#Anti-gene LNA targeting at HBV S gene has a strong antiviral effect in transgenic mice, which provides theoretical and experimental bases for gene therapy for HBV.
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Objective To study the screening of trace amount mutation of BRAF V 600E gene for avoiding the appearance of ineffective treatment in cancer patients .Methods The internal competitive amplification fragments were used to improve the inhibition of wild-type blocking (WTB) probe on wile-type BRAF V600E gene to increase the detection efficiency of BRAF V600E genotype of trace amount mutation occurrence .Re-sults When the template DNA concentration was 50 -200 ng/μL ,the constructed trace amount gene muta-tion real time fluorescence quantitative detection method could completely block the amplification of the wild-type BRAF V600E gene .The sensitivity of this assay reached as high as 0 .1％ ,which was in line with the sen-sitivity requirement for the gene trace amount mutation detection technique .In the colorectal biopsy tissues from 50 cases of suspected colorectal cancer ,8 cases (16 .0％ ) of BRAF V600E gene trace amount mutation were detected by using this constructed method ,which had higher detection rate .Conclusion The constructed gene trace amount mutation detection method can make the rapid ,simple and low cost quantitative analysis for BRAF V600E gene trace amount mutation in clinical samples .
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Objective There is still a lack of effective treatment for hepatitis B. The article aimed to investigate the inhibito-ry and antiviral effects of hepatitis B virus(HBV)S gene expression by anti-gene locked nucleic acid(LNA)in vivo in transgenic mice. Methods The HBV transgenic mice were divided into 5 groups by random number,6 in each group. They were blank group, irrelevant sequence group,lamivudine group,antisense locked nucleic acid group,and anti-gene locked nucleic acid group respective-ly. The lamivudine group was treated by oral gastric lavage,with 2mg/kg dose 2 times per day for continuous 7d,and the rest groups were injected with 500 mL by tail vein injection at 1,3,5 d after ad-ministration. HBV DNA was tested by real-time quantitative polymer-ase chain reaction(qPCR);HBsAg was tested by ELISA;mRNA of HBV S gene was detected by reverse transcription PCR(RT-PCR);the positive rate of HBsAg in liver cells was detected by immunohisto-chemistry. Results After the treatment of anti-gene locked nucleic acid on 3rd,5th,7thday,the inhibition rate of HBV DNA were 37.18%,50.27%,61.46%,and HBsAg were 30.17%,44%,57.76%. The inhibitory effect on 7thday was more obvious than those of the blank group,the unrelated sequence group,the lamivudine group and the antisense lock nucleic acid group,and the difference was statistically significant(P<0.05).The expression of mRNA S gene HBV(0.33)and liver cells HBsAg positive rate(31%)was signif-icantly reduced compared with control group(P<0.05).No abnormal change was found in the function of liver and kidney tests and tis-sue HE staining. Conclusion Anti-gene lock nucleic acid targeting to S gene has strong inhibitory effects on HBV replication and expression in transgenic mice,which provides theoretical and experimental knowledge for HBV gene therapy.
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AIM:To observe the effect of antisense locked nucleic acid (anti-LNA) blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells.METHODS:The antiLNA that was complementary to the translation initiation region of c-myc exon 2 was designed,synthesized,and introduced into the HepG2 cells by cationic liposome-mediated transfection.The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot.The change of cell apoptosis was analyzed by flow cytometry,and the toxicity of anti-LNA to the cells was detected by MTT assay.RESULTS:Five days after transfection,the mRNA level of c-Myc in anti-LNA group was 0.335 ±0.016,and the protein level was 0.448 ± 0.037,significantly lower than those in control group (both P ＜ 0.05).The ratio of apoptotic cells in anti-LNA group was 32％ ±-6％,which was higher than that in control group (P ＜ 0.05).CONCLUSION:Antisense locked nucleic acid targeting at the translation initiation region of cmyc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells.
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AIM:To observe the effect of antisense locked nucleic acid (anti-LNA) blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells.METHODS:The antiLNA that was complementary to the translation initiation region of c-myc exon 2 was designed,synthesized,and introduced into the HepG2 cells by cationic liposome-mediated transfection.The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot.The change of cell apoptosis was analyzed by flow cytometry,and the toxicity of anti-LNA to the cells was detected by MTT assay.RESULTS:Five days after transfection,the mRNA level of c-Myc in anti-LNA group was 0.335 ±0.016,and the protein level was 0.448 ± 0.037,significantly lower than those in control group (both P ＜ 0.05).The ratio of apoptotic cells in anti-LNA group was 32％ ±-6％,which was higher than that in control group (P ＜ 0.05).CONCLUSION:Antisense locked nucleic acid targeting at the translation initiation region of cmyc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells.
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Oligonucleotide microarray has been one of the most powerful tools in the ‘Post-Genome Era’ for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.
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Objective To investigate the inhibitory effects of hepatitis B virus (HBV) S gene-specific antisense locked nucleic acid (LNA) on HBV replication and expression in HepG_22.2.15 cells,and to screen the effective short sequence of LNA.Methods Four different lengths of short sequence of antisense locked nucleic acid which were complementary to the initiator of HBV S gene were designed,synthesized and transfected by cationic liposomes into HepG_22.2.15 cells.The HBsAg and HBV DNA of supematant was tested by enzyme linked immunoadsorbent assay(ELISA) and real-time fluorescent quantitative PCR(FQ-PCR) at 24,48 and 72 hours after treatment.LNA's cyto-toxicity on cell was evaluated by MTT method.Results Four different lengths of short sequence of LNA inhibi-ted the expression of HBsAg and the replication of HBV DNA with the inhibition rates of 46.58%,54.38%,72.43% ,69.92% and 27.09% ,28.77% ,34.71% ,32.68% respectively after 72 hours.There's no obvious tox-icity on cell.Conclusion Antisense LNA that targeting at HBV S gene has strong inhibition on HBV in vitro,and the optimal length of LNA sequence might be in the range of 15 base to 25 base.It has a therapeutic potential in the treatment of patients infected with HBV.