乙型肝炎病毒在造血干细胞内复制规律的研究

Study on the replication of hepatitis B virus in hematopoietic stem cells

目的探讨HBV感染脐血来源造血干细胞后的复制规律。方法从健康产妇脐带血单个核细胞中分离纯化CD34^+HSCs,与HBV阳性血清(107IU/m L)孵育24 h后,反复洗涤并培养,连续测定细胞内HBVDNA。另以不同滴度HBV阳性血清(A组104IU/m L、B组106IU/m L、C组108IU/m L)与CD34+细胞共培养,测定第0、1、6、12天上清中HBVDNA、细胞内HBVDNA和HBsAg水平,并观察细胞形态及进行细胞计数。结果连续测定细胞内HBVDNA,第12天较第0天明显升高(P<0.01)。不同滴度HBV感染CD34^+HSCs的细胞内HBV DNA,A组基本没有增加,B组(P<0.001)和C组呈现逐渐递增趋势(P<0.05)。上清中HBVDNA定量3组均呈上升趋势,第12天与第0天相比,B组明显增加(P<0.01),C组无显著差异(P>0.05),A组有所增加(P<0.05),但病毒载量呈现低水平复制状态。细胞内HBsAg定量A组略增高,B组和C组呈明显增加趋势。各组间细胞形态和数量未见明显差异。结论脐血来源CD34^+HSCs支持HBV复制,暴露于越高载量的HBV,越容易受到感染,感染后未发现对HSCs生长的抑制作用。

Objective To investigate the pattern of the replication of hepatitis B virus（ HBV）in cord blood derived stem cells. Methods CD34~＋hematopoietic stem cells（ HSCs） were purified from healthy volunteers cord blood by using human CD34 positive selection kit. The CD34~＋HSCs were incubated in high load HBV positive serum（ 107 IU / m L） for 24 hours. The cells were cultured after repeated washing. Intracellular HBV DNA was measured every day. In addition,the CD34~＋cells were challenged by different viral titers HBV sera（ HBV DNA of sera： group A 104 IU / m L,group B 106 IU / m L,group C 108 IU / m L）. HBVDNA levels and hepatitis B surface antigen（ HBsAg） quantification of cultures were measured on day 0,1,6and 12. The morphology of cells were observed and the cells were counted. Results Intracellular HBV DNA increased gradually from the starting day to the 12 th day and there were significant difference（ P〈0. 01）. CD34~＋HSCs were challenged by different load HBV,The intracellular HBVDNA were under the limit of detection at different time points of group A,the level of HBVDNA in group B（ P〈0. 001）,and group C gradually increased（ P〈0. 05）. The similar increasing trend of HBV DNA could also be found in the supernatant,group B was significantly increased（ P〈0. 01）,group C had no significant difference（ P〉0. 05）,and group A increased（ P〈0. 05）. The HBsAg of both group B and C were significantly higher on day12 than day 0. There was no obvious difference in the morphology and number of cells in each group. Conclusion HBV might sustain a productive infection in cord blood CD34~＋HSCs,the CD34~＋HSCs are more susceptible to HBV when exposed to higher load HBV. There is not obvious inhibition of CD34~＋HSCs.