| BACKGROUND AND AIMHepatitis B is caused by hepatitis B virus (HBV) infection, and is a public health problem worldwide. The governments and scientists in the world pay high attention to study on its transmission routes due to its high prevalence and harmful to human health. It has been reported that HBV DNA could integrate into human sperm chromosomes and the sperm-introduced HBV genes were able to replicate themselves and express their functions in early embryonic cells. Hepatotropism, however, is a prominent feature of HBV infection. Preimplantation embryonic cells are non-specialized cells that are different from hepatic cells as well as specialized nonhepatic cells. Why can HBV genes replicate and express in early embryonic cells? Do some host genes contribute to these biological events? It not only has important theoretical significance in study on the molecular mechanism of virus transmission but also has broad application prospect in interruption of HBV transmission, in promotion of human birth quality and in prevention of hepatocellular carcinoma to answer these questions clearly.MATERIALS AND METHODSMaterials: (1) human sperm; (2) zona-free hamster ova; (3) the positive recombinant plasmids amplified through transformation of E. coli DH5α; (4) reagents and media.Methods: (1) PCR; (2) labeling cDNA probes with Cy3 and Cy5; (3) gene chip technique; (4) sequencing; (5) bioinformation analysis; (6) interspecific in vitro fertilization between human sperm and zona-free hamster ova; (7) RT-PCR.RESULTS(1) The subtractive cDNA library by PCR amplification contains 152 positive clones, and the inserted fragments were between 500-1,000 bp in length, and most of them were around 1,000 bp in length.(2) All the purified PCR products, without nonspecific bands, were reached to 7μg. The cDNA microarrays were prepared by robotically spotting purified PCR products, repeating each three times, onto the surface of glass slides.(3) The purified cDNA from the forward and the reverse subtractive library were qualified, and then were labeled with Cy3 and Cy5, respectively by PCR to prepare cDNA probes.(4) The microarray hybridization result was scanned and analysed. The 29 differential expression clones in total were obtained by selecting the products showing a greater than twofold change in average signal intensity.(5) Using sequencing and bioinformation analysis, the six target genes were screened.(6) Two-cell embryos derived from the zona-free hamster ova fertilized by human sperm were collected. The presence and expression of the six target genes were demonstrated in two-cell embryos by RT-PCR.CONCLUSIONThe screened target genes are as follows: (1) NADH (reduced form of nicotinamide-adenine dinucleotid); (2) PCBD2 [pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1)2]; (3) CSH (chorionic somatomammotropin hormone); (4) EIF4G2 (eukaryotic translation initiation factor 4 gamma, 2); (5) PSG4 (pregnancy specific beta-1-glycoprotein 4) and (6) TTN (titin). They may contribute to replication and expression of HBV genes in early embryonic cells. |
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