外周血单个核细胞介导乙型肝炎病毒感染的transwell小室体外模型研究

Study on in vitro model of hepatitis B virus-infected transwell chambers mediated by peripheral blood mononuclear cells

目的观察感染HBV的PBMC经人绒毛膜癌滋养层细胞（Bewo细胞）构建的胎盘屏障迁移情况，探讨PBMC作为载体转运HBV的生物学作用。方法培养并用细胞计数试剂盒-8检测培养Bewo细胞和PBMC的增殖和活性。用HBVDNA含量为5×10^6拷贝／mL的乙型肝炎患者血清100μL感染PBMC后，用羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）荧光染料标记感染的细胞，transwell小室建立Bewo细胞和感染HBV的PBMC共培养模型。用流式细胞仪检测感染HBV的PBMC迁移情况，荧光定量PCR法检测transwell下室中PBMCHBVDNA含量。结果PBMC、Bewo细胞均在接种24h左右开始增殖，120h左右开始进入生长停滞期。transwelt小室共培养0、12、24和48h，下室中绿色荧光标记的PBMC量分别为（0．445±0．021）％、（21．180±4．653）％、（34．830±7．156）％和（64．185±3．161）％，随着培养时间的延长，下室中检测到的标有绿色荧光的PBMC量越多（F=68．983，P=0．001）。共培养24、48h时，下室PBMCHBVDNA分别为（1．925±0．431）×10^3、（2．565±0．361）×10^3拷贝／mL，即下室中的PBMc发生感染。结论PBMC可以作为HBV肝外感染的靶细胞，利用transwell小室构建胎盘滋养层屏障可以为体外研究HBV跨胎盘传播提供新思路。

Objective To observe the transport of hepatitis B virus （HBV）-infected peripheral blood mononuelear 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/IL 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 diaeetate 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 eytometry. 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. 18± 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）×10^3 eopy/mL and （2. 565±0. 361）×10^ 3copy/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.