pcDNA3-HBV瞬时转染对树突状细胞的影响

Effects of transient transfection of pcDNA3-HBV on dendritic cells

目的:研究HBV感染对树突状细胞功能的影响,探讨慢性乙型肝炎患者免疫耐受发生的机制。方法:利用脂质体转染的方法,以携载HBV全基因的真核表达载体pcDNA3-HBV及对照空载体pcDNA3质粒DNA分别转染小鼠骨髓细胞,以细胞因子GM-CSF、IL-4及TNF-α诱导培养树突状细胞,RT-PCR检测转染后HBVPreS1 mRNA的表达。流式细胞术检测转染前后树突状细胞表面分子CD80的表达,3HTdR掺入法观察其刺激同种异体淋巴细胞增殖的能力,流式细胞术进一步检测NF-κB RelA蛋白的表达。结果:pcDNA3-HBV瞬时转染后树突状细胞CD80表达降低,其刺激同种异体淋巴细胞增殖的能力下降,NF-κB RelA蛋白表达显著减少。结论:HBV可抑制树突状细胞的分化成熟,降低其刺激T淋巴细胞增殖的功能,这可能是慢性乙型肝炎患者免疫耐受的机制之一。

Objective： To study the role of HBV infection on the function of dendritic cells （DCs） and explore the mechanism of immune tolerance in chronic B hepatitis. Methods： Bone marrow cells were isolated, cultured and transiently transfected with the pcDNA3-HBV or pcDNA3 plasmid DNA and then were cultured under the stimulation of GM-CSF, IL-4, TNF-α to be induced to become mature dendritic cells. RT-PCR was performed to detect the expression of PreS1 mRNA. FCM was used to detect the expression of CD80, NF-κB. The 3H-TdR adulteration assay was used to detect the potential for stimulating the proliferation of allogenic lymphocytes. Results： After being transfected with pcDNA3-HBV, mature DCs had an expression of PreS1 mRNA, but had a lower level of superficial marker CD80 and transcriptional factor NF-κB, compared with pcDNA3-tmnsfected control cells. The potential for stimulating the proliferation of allogenie lymphocytes was also decreased. Conclusion： Transient transfection of peDNA3-HBV could decrease the function of DCs directly by inhibiting the maturation of DCs, which may be one of the mechanisms of immune tolerance in chronic B hepatitis.