HBsAg重组腺病毒转染的树突状细胞诱导BALB／c小鼠抗HBV免疫的研究

Immune response against hepatitis B virus induced by HBsAg recombinant adenovirus-transduced dendritic cells in BALB/e mice

目的研究HBsAg重组腺病毒转染的小鼠树突状细胞(DC)体内外免疫刺激活性和诱导小鼠抗HBV免疫的特点。方法BALB／c小鼠的骨髓细胞体外扩增为DC,转染HBsAg重组腺病毒Ad-S或被HBsAg蛋白冲击后,流式细胞术分析DC的表型,混合淋巴细胞反应(MLR)检测DC的刺激活性;或与pcDNA3．1(+)-S质粒分别免疫小鼠后,LDH法测定脾细胞CTL活性,放免法检测血清抗- HBs;流式细胞术分析体外自体MLR中及免疫后小鼠脾脏T细胞内细胞因子。结果DC／Ad-S、DC／HBsAg和各对照组DC之间的表型和MLR差异无统计学意义,并均刺激TH和Tc分泌IFN-γ。DC／Ad-S免疫后2周能诱导比DNA疫苗更强产生IFN-γ的TH(P<0．05),以及比DC／HBsAg和DNA疫苗更强分泌IFN-γ的Tc(均P<0．01)和特异性CTL(P<0．05,P<0．01)。但DC／Ad-S和DC/HBsAg诱导的CTL反应及Tc分泌IFN-γ在免疫后4周均明显减弱,且诱导抗-HBs作用弱于DNA疫苗。结论HBsAg重组腺病毒转染的DC比HBsAg冲击的DC及DNA疫苗能诱导更强的TH1／Tc1(Ⅰ型)细胞免疫和特异性CTL反应,是诱导抗HBV细胞免疫的有效刺激细胞。

Objective To study the immune response against hepatitis B virus induced by HBsAg recombinant adenovirus-transduced dendritic cells （DC） in vivo and in vitro. Methods DC were isolated from bone marrow cells of BALB/c mice, transfected with HBsAg recombinant adenovirus Ad-S （DC/Ad-S） or pulsed with HBsAg （DC/HBsAg） and used for analysis of the phenotypic markers by flow cytometry and the stimulatory capacity in mixed leukocyte reaction （MLR）. HBsAg-speciic activity of splenic cytotoxic T lymphocyte （CTL） of BALB/c mice was measured by LDH release assay and the anti-HBs titers in sera was examined by RIA after being immunized with DC/Ad-S （12）, DC/HBsAg （12） or pcDNA3.1 （ ＋ ）-S plsmid （8）. Intracellular cytokines of proliferative T cells in autologons MLR or of splenic T cells after immunization was detected by flow cytometry. Results Either the Ad-S- or Ad-lacZ-transduced DC or HBsAg-pulsed DC or untreated DC expressed similar levels of phenotypic markers, and showed similar stimulatory capacity in either allogeneic or autologous MLR. DC/Ad-S and DC/HBsAg induced more TH or Tc secreting IFN-γ rather than IL-4 in autologous MLR. DC/Ad-S more efficiently stimulated splenic TH to secrete IFN-γ than pcDNA3.1（ ＋ ）-S （P 〈 0.05） and splenic Tc to product IFN-γ than DC/HBsAg （P〈0.01） and plasmid DNA （P 〈 0.01）, and induced stronger splenic HBsAg-specific CFL responses than that induced by DC/HBsAg. However, DC/Ad-S and DC/HBsAg primed splenic Tc to secret IFN-γ and splenic HBsAg-specific CTL, more reductively than plasmid DNA at 4 weeks after immunization. Anti-HBs response elicited by DC vaccination was much weaker than that induced by pcDNA3.1 （ ＋ ）-S at either 2 or 4 weeks after immunization. Conclusion HBsAg recombinant adenovirus-transduced DC can induce more enhanced Tn 1/ Tel （type Ⅰ ） cell-mediated immunity and HBsAg-speeific CTL than HBsAg-pulsed DC or plasmid DNA, and may be an efficient inducer in priming HBV-specific T cell response.