Interruption Of Hepatitis B Virus Replication In Human Placental Trophoblasts By RNA Interference

Objective1. To establish a practical protocol for the harvest of large amount of purified term human placental trophoblast.2. To observe the expression of hepatitis B virus (HBV) antigens and DNA in trophoblasts isolated from gravidae with HBV surface antigen (HBsAg) and e antigen (HBeAg) seropositivity and, to investigate wheather there is HBV replication in HBV affected placental trophoblasts.3. To establish an in vitro model of HBV-affected-trophoblast by transfecting trophoblast-derived cell strains with recombinant HBV expressing plasmid. Comparing with transfected hepatocarcinoma cell strain HepG2, we study the expression of viral nucleic acid and protein in the model.4. Design and construct different short hairpin RNA (shRNA) expressing vectors targeting at HBsAg coding mRNA (SmRNA), as to analyze their gene knock-down efficiency after being co-transfected with recombinant HBV expressing plasmid into the host cells, and to select the most capable siRNA (small interfering RNA) sequence. Methods1.50g of chorion tissue from 20 normal term placentas were isolated respectively, digested by trypsin and DNase I, purified by the combination of modified gradient gravity centrifugation and repeatedly adherence to culture vask. Immunofluo-rescence labeling of intracellular cytokeratin-7 (CK-7) was measured by laser scanning confocal microscopy (LSCM) for the calculation of the purity of trophoblasts.2. Placental trophoblasts from 9 gravidae with HBsAg and HBeAg seropositivity were isolated, purified and cultured in vitro for 72 hours. The culture supernatant were collected at 24h,48h,72h and subjected to HBsAg/HBeAg detection by Microparticle Enzyme Immunoassay (MEIA) as well as to HBV DNA measurement by fluorescence quantitative PCR (FQ-PCR), as to confirm wheather the sample was affected by HBV. HBV covalently closed circular DNA (cccDNA) was detected by FQ-PCR as to study if there was HBV replication in HBV affected trophoblasts.3. Trophoblast derived cell strains JEG-3 and JAR, as well as hepatocarcinoma cell strain HepG2, were used for liposome mediated transfection of recombinant HBV expressing plasmid. After transfection, cell samples and culture supernatant were collected for laboratory examination at 24h,48h and 72h. The intracellular rcDNA (relaxed circular DNA) and cccDNA of HBV were detected by PCR, SmRNA was detected by RT-PCR (reverse transcription PCR). HBV DNA in culture supernatant was determined by FQ-PCR. Intracellular HBsAg/HBcAg were examined by immunofluorescence labeling, HBsAg/HBeAg in culture supernatant were measured by MEIA. Meanwhile, G418 containing culture medium was applied for the selection of transformed choriocarcinoma cell line.4. Design and construct 4 kinds of shRNA expressing vectors targeting at SmRNA, named pS-603,pS-475,pS-66,pS294 respectively. pS-scramble and empty vector pS-0 were used as transfection specificity control and negative control respectively. ALL shRNA expressing plasmids were co-transfected into the host cells with recombinant HBV expressing plasmid by LipofectamineTM 2000 respectively. After transfection, cell samples and culture supernatant were collected for laboratory examination at 48h,72h and 96h. Intracellular HBsAg were measured quantitatively by immunofluorescence staining and Western blotting, HBsAg in culture supernatant were measured by MEIA. The expression of SmRNA was determined by FQ-RT-PCR (fluorescence quantitative reverse transcription PCR). Supernatant HBV DNA titer was measured by FQ-PCR.Results1. After being digested by trypsin and DNase I, purified by the combination of gradient gravity centrifugation and repeatedly adherence to culture vask, the harvest of trophoblast yeilded to about 108 cells per 50g chorion tissue with 85% purity and 88% vitality on average.2.9 trohpoblast samples from the gravidae with HBsAg/HBeAg seropositivity were prooved to be HBV-affected. HBsAg expression (S/N ratio) in the culture supernatant were 3.72±1.41 at 24h,1.99±0.87 at 48h and 1.75±0.99 at 72h respectively. HBeAg (S/CO ratio) were 2.26±1.31 at 24h,0.62±0.36 at 48h and 0.49±0.40 at 72h, respectively. At 24h, the expression of HBsAg and HBeAg were higher than those at 48h and 72h (p<0.05). Supernatant HBV DNA titer were (2.39±1.87)×103 copies/mL at 24h, (1.04±0.94)×103copies/mL at 48h, (0.78±0.68)×103 copies/mL at 72h respectively, but there was no significant differences about the DNA titers among the 3 timepoints. A low expression of HBV cccDNA was detected in 2 samples among the 9 samples, which were 6.75×103 copies/mL and 2.22×103 copies/mL.3. RcDNA, cccDNA and SmRNA were detected in the transfected JEG-3, JAR and HepG2 cells. HBV DNA was detected in the culture supernatant. There was no significant differences about supernatant HBV DNA titer between JEG-3 and JAR, yet HepG2 had higher titer than the two choriocarcinoma cells did (p< 0.05). Intracellular HBsAg/HBcAg were detected by immunofluorescence labeling. There was no significant differences in supernatant HBsAg/HBeAg expression between JEG-3 and JAR, yet HepG2 had higher expression of the two viral antigens than the two choriocarcinoma cells did (p< 0.05). Positive clones of transformed JAR were selected by G418 medium successfully, but JEG-3 extincted gradually during the selection procedure.4. After co-transfection of shRNA expressing vectors and recombinant HBV plasmid, the expression of intracellular and supernatant HBsAg, SmRNA, as well as the supernatant HBV DNA, declined gradually day after day in pS-603, pS-475 and pS-66 groups. There were no significant differences in the expression of HBsAg, SmRNA and HBV DNA among pS-294, pS-scramble and pS-0 groups.Conclusion1. The combination of modified gradient gravity centrifugation and repeatedly adherence to culture vask might be a pratical protocol for the harvest of large amount of purified term human placental trophoblast.2. There might be a low level of viral replication in HBV affected placenta. But the expression of HBsAg/HBeAg and HBV DNA might not result from continuos replication of HBV.3. There were viral replication and protein expression in the in vitro model of HBV-affected-trophoblast constructed by transfecting recombinant HBV expressing plasmid into the host cell.4. pS-603, pS-475 and pS-66 were able to knock down the expression of HBV SmRNA but pS-294 was not.