摘要
12.1 Hepatitis 2006375 Identification of hepatitis B virus integration sites in hepatocellular carcinoma tissues from patients with chronic hepatitis B. TU Hong, et al. Shanghai Can Instit, Shanghai Jiao-Tong Univ, Shanghai 200032. Natl Med J China 2006;86(18):1249-1252. Objective:To carry out a large-scale screening for the HBV integrations sites in HCC samples from Chinese patients. Methods: Cellular DNA was extracted from 40 HBV-related HCC by proteinase K digestion/phenol extraction method. One primer specific to HBV sequence and another primer directed to human Alu repeat were used to amplify the virus/cellular DNA junction. To avoid undesirable amplifications between Alu sequences, primers were constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 15 initial cycles of amplification. Only desirable fragments were then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-Specific primer. The PCR product was purified and subject to direct sequencing by ABI 3700 Auto sequencer. NCBI (national center for biotechnology information) BLAST and MapViewer search were used for identification of HBV location on human genomes. Results:In 40 HBsAg positive HCC samples, 34 (85%) were showed to have at least one copy of HBV fragment in host genome, indicating HBV-Alu-PCR is a rapid way for identification of new cellular DNA sequences adjacent to HBV. Analysis from the 68 isolated viral-cellular junctions, X gene was found to be interrupted at any length, not specifically at DR1 and DR2 regions. Three-prime-deleted X gene was observed in 65 (96%) cases. HBV preferred to integrate into the intron and the up-stream regulatory region of the cellular genes. In no case HBV inserted into the exon. Our results also demonstrated that the cellular genes targeted by HBV are usually key regulators of cell proliferation and cell death.
12.1 Hepatitis 2006375 Identification of hepatitis B virus integration sites in hepatocellular carcinoma tissues from patients with chronic hepatitis B. TU Hong, et al. Shanghai Can Instit, Shanghai Jiao-Tong Univ, Shanghai 200032. Natl Med J China 2006;86(18):1249-1252. Objective:To carry out a large-scale screening for the HBV integrations sites in HCC samples from Chinese patients. Methods: Cellular DNA was extracted from 40 HBV-related HCC by proteinase K digestion/phenol extraction method. One primer specific to HBV sequence and another primer directed to human Alu repeat were used to amplify the virus/cellular DNA junction. To avoid undesirable amplifications between Alu sequences, primers were constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 15 initial cycles of amplification. Only desirable fragments were then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-Specific primer. The PCR product was purified and subject to direct sequencing by ABI 3700 Auto sequencer. NCBI (national center for biotechnology information) BLAST and MapViewer search were used for identification of HBV location on human genomes. Results:In 40 HBsAg positive HCC samples, 34 (85%) were showed to have at least one copy of HBV fragment in host genome, indicating HBV-Alu-PCR is a rapid way for identification of new cellular DNA sequences adjacent to HBV. Analysis from the 68 isolated viral-cellular junctions, X gene was found to be interrupted at any length, not specifically at DR1 and DR2 regions. Three-prime-deleted X gene was observed in 65 (96%) cases. HBV preferred to integrate into the intron and the up-stream regulatory region of the cellular genes. In no case HBV inserted into the exon. Our results also demonstrated that the cellular genes targeted by HBV are usually key regulators of cell proliferation and cell death.
