乙型肝炎病毒在造血干细胞内复制及对其免疫活性的影响

Study on hepatitis B virus replication in haemopoietic stem cell and its effect on the immune activity of haemopoietic stem cell

目的研究不同载量HBV感染脐血来源的CD34＋造血干细胞（HSC）的一般规律，以及探讨HBV感染HSC后诱生的树突状细胞（DC）的功能状态。方法无菌条件下采集脐血，磁珠分离仪分离纯化CD34＋HSC。把CD34＋HSC分为HBV感染组及对照组，用免疫组织化学及原位杂交方法检测HSC内病毒感染情况。分3组将HSC接种于24孔板连续培养12d，各组分别加入细胞因子组合及不同病毒载量HBV（A组1×10^7拷贝／mL，B组1×10^5拷贝／mL，c组1×10^3拷贝／mL）的血清，于培养的0、1、6、12d检测各组细胞数，上清液及细胞中HBVDNA拷贝数。用两步法诱生的DC作为刺激细胞，以异基因健康者外周血T淋巴细胞作为反应细胞，噻唑蓝比色法检测DC对同种异体T淋巴细胞的刺激增殖作用。组间比较采用t检验。结果免疫组织化学检测结果显示，HBV感染12d的HSC中可检测到HBsAg的表达，而未感染的HSC中无HBsAg阳性细胞。原位杂交检测结果显示，HBV感染12d的HSC中可检测到HBVDNA，未感染的HSC中无阳性细胞。C组第1天的细胞增殖与A、B组比较无明显差异，第6、12天细胞增殖高于A、B组，且第6天的细胞计数差异有统计学意义（t=5．125，t=6．327；均P〈0．05）。在第12天A组细胞内可检出1×10。拷贝／mLHBVDNA，B组可检出1×10^3拷贝／mL，C组则12d内均不能检出。上清液检出情况与细胞内相符。混合淋巴细胞反应显示HBV感染组诱生的DC对同种异体T淋巴细胞刺激增殖作用明显低于对照组，且差异有统计学意义（在DC与T淋巴细胞的比例达到0．05时，t=3．156，P〈0．05；在比例达到0．1、0．5时，t=4．873，t=5．103，均P〈0．01）。结论HBV可以在CD34’HSC内复制并随HSC扩增而同步增加。HBV感染的HSC诱生的DC对同种异体T淋巴细胞刺激增殖能力减低，即HBV感染HSC后其分化的免疫细胞的生物活性降低。

Objective To understand infections with different hepatitis B virus （HBV） loads in human cord blood derived CD34＋ haemopoietic stem cells （HSCs） and to investigate the function of dendritic cells （DCs） derived from HBV-infected HSC. Methods Human cord blood was collected under sterile conditions. CD34＋ HSCs were isolated using mini-MACS and divided into HBV infected group and control group. Intracellular HBV were examined by immunohistochemistry and in situ hybridization. HBV-infected HSCs were divided into 3 subgroups, and inoculated into 24-well plates and cultured for 12 days. Cytokines and sera with different HBV loads were added in each subgroup （subgroup A： 1 ×10^7 copy/mL; subgroup B： 1 ×10^5 copy/mL; subgroup C： 1×10^3 copy/mL）. Cells were counted and intracellular and supernatant HBV DNA levels were examined at day 0, 1 , 6 and 12 after HBV infection. DCs induced by twostep approach were used as stimulation cells and allogenie peripheral blood T lymphocytes from healthy volunteers were used as responder cells. T cell proliferation was detected by methyl thiazolyl tetrazoliym （MTT） chromatometry. Comparison between groups was done using t test. Results Immunohistochemistry showed that HBsAg expression in HSCs could be detected at day 12 after HBV infection, while there were no HBsAg positive cells in control group. In situ hybridization showed that HBV DNA could be detected at day 12 after HBV infection, while there were no HBV DNA positive cells in control group. T cell proliferation at day 1 in subgroup C was not significantly different from those of subgroups A and B, while those at day 6, 12 in subgroup C were both higher than subgroups A and B and the differences at day 6 were significant （t=5. 125 and t=6. 327, respectively, both P〈0.05）. Intracellular HBV DNA levels at day 12 were 1 ×10^6 , 1 ×10^3 copy/mL and undetectable, respectively in subgroups A, B and C. HBV DNA levels in the supernatant were similar with those in HSCs. The mixed lymphocyte reaction showed that the stimulation of DCs induced in HBV infected group on the proliferation of allogenic T cells was weaker than that in control group （DC/T=0.05, t=3. 156, P〈0.05; DC/T=0. 1 and 0.5, t=4. 873 and t 5. 103, respectively, both P〈0. 01）. Conclusions HBV could replicate in CD34＋ HSCs and increase synchronously with HSC proliferation. The stimulation effect of DCs induced by HBV infection on the proliferation of allogenic T cells is reduced, which means that the biological activities of immune cells differentiated from HBV infected HSCs are reduced.