乙型肝炎病毒体外感染脐血造血干细胞的研究

Study on hepatitis B virus infection in hematopoietic stem cells in vitro

目的探讨脐血来源的CD34+造血干细胞对乙型肝炎病毒(HBV)的易感性。方法留取健康产妇的脐血,分离纯化CD34+造血干细胞,加入细胞因子和高载量HBV阳性血清(107IU/m L)共同培养。于培养12天时提取细胞进行免疫组化、免疫荧光、透射电镜观察和逆转录聚合酶链反应(RT-PCR)检测。结果 HBV感染组CD34+造血干细胞经免疫组化及免疫荧光检测,细胞内可见HBs Ag染色呈阳性表达,与阳性对照组Hep G2.2.15细胞系一致。电镜观察到细胞胞浆内有HBV病毒颗粒。在HBV感染的CD34+造血干细胞中RT-PCR检测到HBV S基因的条带。结论 HBV可以体外感染脐血来源CD34+造血干细胞。这为进一步从干细胞角度研究乙肝慢性化的机制奠定了一定的理论和实验基础。

Objective To investigate the susceptibility of CD34＋ cord blood-derived hemato- poietic stem cells （HSCs） to hepatitis B virus （HBV）. Methods Umbilical cord bloods were obtained from healthy volunteers with no history of HBV exposure. CD34＋ HSCs were obtained from the mononuclear cells fraction by using human CD34 positive selection kit. Primary CD34＋ HSCs were co-cultured with the serum containing high viral load of HBV（ 107 IU/mL） and IL-3, SCF, TPO, Flt-3. On the 12th day of cultivation, immunocytochemistry, transmis- sion electron microscopy and reverse transcription-polymerase chain reaction （RT-PCR） were used to observe the viral related protein and specific viral genomic sequences in HBV-infected CD34＋ HSCs. Results The HBV-infected CD34＋ cells showed positive signals for HBsAg by using immunohistochemistry and immunofluorescenee staining. HBV particles were found in HBV-infected CD34＋ HSCs by applying transmission electron microscopic analysis. RT-PCR results showed that the 403 bp RT-PCR products corresponding to the amplified HBV S gene fragment were found in CD34＋ HSCs infected by HBV. Conclusion HBV ：can infect cord blood-derived CD34 ＋ HSCs, and it provides a theoretical and experimental basis for further study on the mechanism of HBV persistent infection.