乙型肝炎病毒感染正常人肾小管上皮细胞的实验研究

Infection of hepatitis B virus on human tubular kidney cells in vitro

目的探讨正常人肾小管上皮细胞（HKC）对乙型肝炎病毒（HBV）的易感性。方法（1）体外培养的HKC传至6孔板中，接种密度为1×10^6个／ml，孵育24h后分受感染和阴性对照两组。受感染组加入0．5mlHepG2．2．15细胞上清液（已知该体外培养的细胞系可随细胞传代释放HBV病毒颗粒）；阴性对照组加入015mI10％FCS—DMEM／F-12培养液。两组HKC继续孵育72h，消化、离心沉淀，PBS清洗，实时荧光定量聚合酶链反应（FQ—PCR）检测两组HKC中的HBVDNA。（2）将HKC细胞传代并接种至2个6孔板中盖玻片上制作细胞爬片，接种密度为1×10^6个／ml，24h后行HBV感染实验。然后将两个6孔板分别作为：（1）受感染组：往六孔板中加入制备好的感染用HepG2．2．15细胞上清液2ml；（2）阴性对照组：往六孔板中加入2mI10％FCS—DMEM／F－12培养液。将HepG2．2．15细胞分别传代并接种至6孔板中盖玻片上制作细胞爬片，接种密度为1×10^6个／ml，24h后作为阳性对照组：往六孔板中加入2ml10％FCS—DMEM培养液继续孵育72h，原位杂交检测各组中的HBVDNA。结果实时荧光定量聚合酶链反应检测到受感染组HKC的基因组中HBVDNA的存在，受感染组HKC原位杂交检测HBVDNA阳性，呈核型。结论体外培养的正常HKC对HBV易感。

Objective To infect human tubular kidney cells （HKCs） with hepatitis B virus （HBV）in vitro. Methods HKCs were seeded into six well cluster dishes at 1 × 10^6 cells per ml. After cultured for 24 h, HKCs were divided into control group and infected group. Cells in infected group were cultured with 0.5 ml supernatant of HepG2.2.15 cells, while 0.5 ml 10% FCS-DMEM/F-12 was used in control group. After 72 h, HKCs of both groups were removed and washed with PBS. HBV DNA was detected by fluorescent quantitation polymerase chain reaction （FQ-PCR）. HKCs and HepG2.2.15 cells were seeded on cover slips of six well cluster dishes at 1 × 10^6 cells per ml; 24 hours later the coverslips were divided into control groups and infected groups. Cells of infected groups were cultured with 2 ml supernatant of HepG2.2.15 cells, while 2 ml 10% FCS-DMEM/F-12 was used in negative control group and 2 m110% FCS-DMEM was used in positive control group （HepG2.2.15 cells） .72 h later, HBV DNA was detected by in-situ hybridization （ISH）. Results HBV DNA was observed in the genome of HKC of infected group. Conclusion Human tubular epithelial cell is susceptible to HBV.