Vitro Study On The Role Of Placental Cells Apoptosis In Translocation Of HBV-infected PBMC

OBJECTIVE①To establish co-culture model in vitro to simulate transcytosis of the placental barrier.②To explore the role of placental cells apoptosis in translocation of HBV-infected PBMC.METHODS①PBMC were co-cultured with HBV DNA positive serum at 12h, 24h,48h,72h.Cell CountingKit-8 was used to detect cell growth and FQ-PCR was used to detect HBV DNA expression ofPBMC after be washed four times by PBS.②The fusion between HBV-infected PBMC and BeWo cells in the apical chamber(24-mm-diameter Transwell polyethylene terephthalate filters, 1-μm porosity)and PBMC loaded with a fluorescent dye in the basolateral chamber (24-mm-diameterTranswell polyethylene terephthalate filters,8-μm porosity) was assessed by fluorescenceactivated cells sorting(FACS).③T his study has three groups:control BeWo group, HBV and BeWo cells group, HBV-infectedPBMC and BeWo group. These cell groups were cultured for 0h,12h,24h,48h. FACS were usedto detect apoptosis rate of BeWo cells.④Caspase3 mRNAexpression of BeWo cells was detected by RT-PCR in HBV-infected PBMCand BeWo group.⑤In the HBV-infected PBMC and BeWo group FQ-PCR was used to detect HBV DNA andHBV cccDNAexpression of PBMC in the basolateral chamber.RESULTS①The cell counts cultured at 12h, 24h and 48h with positive serum were increasing,butdeclining at 72h.HBV DNA can be detected in PBMC co-cultured with HBV DNA positiveserum for 48 hours and can’t be detected in washing liquid after be washed four times by PBS.②A redistribution of the fluorescent dye was observed from the apical HBV-infected PBMC toBeWo cells,indicating a fusion between HBV-infected PBMC and BeWo cells in the Transwellmodel (1-μm porosity). In the Transwell model (8-μm porosity) ,PBMC loaded with afluorescent dye were detected in the basolateral chamber ,which demonstrated that co-culturemodel in vitro to simulate transcytosis of the placental barrier was successfully established.③When HBV and BeWo cells group, HBV-infected PBMC and BeWo group and controlBeWo group were cultured for 0h,12h, 24h,48h. The difference of early apoptotic rates betweenHBV and BeWo cells group, HBV-infected PBMC and BeWo group and control BeWo group was not statistically significant with all the value of P more than 0.05. However, whilecocultured time was 24h or 48h, total apoptotic rates had a statistically significant difference inthree groups(F= 15.678、14.892，P<0.05). Early apoptotic rates except control group and totalapoptotic rates were all statistically significant(P<0.05) compared to respectively group at thetime 0h,12h,24h or 48h.when the cocultured time was 48h, total apoptotic rates were higher inthe HBV and BeWo cells group（68.90±0.33%）.④Compare Caspase3 mRNA of BeWo cells at different times in HBV~+PBMC and BeWo cellgroups.There was statistically significant difference (F=38.114,P=0.002). The relativeexpression of Caspase3 mRNA in 48h group was higher than 0h、12h and 24h group.(P<0.05),but there was also not significant difference between 12h group and 0h group(P﹥0.05).⑤In HBV~+PBMC and BeWo cell groups,there were positive correlation between translocationrate of PBMC and early apoptotic rates,total apoptotic rates and Caspase3 mRNAexpression ofBeWo cells (r=0.908, 0.969, 0.950 ,p=0.002, 0.001, 0.001).⑥HBV DNA and HBV cccDNA expression of PBMC cocultured 24h and 48h group in thebasolateral chamber were detected.HBV DNA content were (1.925±0.431)×10~3copies/ml and(2.565±0.361)×10~3copies/ml.HBV cccDNA content were (7.560±1.513)×10~2copies/ml and(1.3550±2.473)×10~3copies/ml .These results demonstrated that HBV can infected PBMC andreproducted in them.CONCLUSION①PBMC and BeWo cell can be used as a good model for co-culture model in vitro to simulatetranscytosis of the placental barrier, hepatitis B virus can replicate in PBMC.②The positive correlation of apoptosis rate of BeWo cells and translocation rate of PBMC hasfound . This demonstrated that apoptosis was related to translocation.③P BMCin the basolateral chamber can be infected by HBV-infected PBMC locating in theapical chamber. It can be inferred that maternal PBMC infected with HBV may result in fetalPBMC infection though translocation.