RESUMO
Objective To observe the transport of hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC)through placental barrier set up by choriocarcinoma trophoblast cells (Bewo cells),and to explore the biological role of PBMC as a carrier for HBV transport.Methods Bewo cells and PBMC were cultured and their proliferation and activity were detected by cell counting kit (CCK)-8.One hundred μL serum containing 5 ×10 6 copy/mL HBV DNA was used to infect PBMC,and cells infected with HBV were labeled by fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE).A co-culture model of Bewo cells and HBV-infected PBMC was set up by transwell chamber. The migration of HBV-infected PBMC was detected by flow cytometry.Realtime fluorescence quantitative polymerase chain reaction method was used to detect HBV DNA contents of PBMC under transwell chamber.Results PBMC and Bewo cells proliferated at around 24 h and entered into growth stagnation at around 120 h.The contents of PBMC labeled by green fluorescent at 0,12,24 and 48 h during co-culture under chamber were (0.445 ±0.021)%,(21 .180 ± 4.653 )%,(34.830 ± 7.156 )% and (64.185 ± 3.161)%,respectively.The amount of PBMC marked green fluorescence increased over prolonged incubation time (F =68.983,P =0.001 ).PBMC HBV DNA contents at 24 and 48 h of co-culture under chamber were (1.925±0.431)×103 copy/mL and (2.565 ±0.361)×103 copy/mL,respectively,indicating that PBMC under chamber were infected with HBV.Conclusions PBMC may be a target for HBV infection in extrahepatic tissues.Placental trophoblastic barrier built by transwell chambers may provide new ideas to investigate HBV transmission across the placenta in vitro .
RESUMO
Objective To explore the experiment condition and method for the application of in vitro in vasive Transwell chamber and to observe muscarinicreceptor stimulant and muscarinicreceptor antagonist's influence to cholangiocarcinoma's invasiveness.Methods Two hundred microliter cell suspension of various concentrations(0.5×105/mL,1.0×105/mL,1.5×105/mL and 2.0×105/mL)was added into the upper chamber of the Transwell chamber,and the cells were allowed to penetrate the matrigel for 12,18,24and 48 hours respectively.The numbers was gotten as the invasive cells on the under surface of the membrane.After optimal cell concentration and time were gotten,pilocarpine of various concentrations(0 mmol/L,0.1 mmol/L,0.3 mmol/L and 0.5 mmoL/L)was added into the upper chamber of the Transwell chamber,then the cells on the matrigel were stained and counted.So did the cells when atropine of various concentrations(0.01 mmol/L,0.01 mmol/L,0.05 mmoVL and 0.1 mmol/L)were added into the upper chamher of the Transwell chamber in according to pilocarpine of various concentrations(0 mmol/L,0.3 mmol/L,0.3 mmol/L and 0.3mmol/L).Results With the increase of the time and cell concentrations，the cells couts that penetrated the matrigel increased，while the increase tended to he stable when the culture time exceeded 36 hous and the cell concentration Was over 1.0×105/mL.By adding pilocarpine,there were significant differences between the control and experimental groups(P＜0.05)，but there were no significant differences in experimental groups with various concentrations.There were no significant differences in blank group and experimental groups with atropine added(P＞0.05).When added pilocarpine and atropine,there were significant differences between blank and experimental groups(P＜0.05)，but there were no significant differences in experimental groups with various concentrations.Conclusions Thirty-six hours as invasive time,and one cell concentration 1.0 × 105/mL were optimal to test invasion abilities of cholangiocarcingma cells to different medicines or reagents.There is the possibility that museariniereceptor exists in cholangiocarcinoma cells，and may play an important role in cholangiocarcinoma's invasiveness and metastasis.