亚甲兰光化学法对血浆乙肝病毒的灭活

Methylene blue photochemical technique inactivation activity on hepatitis B virus in human plasma

目的:研究灭活病毒对乙肝病毒和血浆成分的影响。方法:在已知HBV-DNA阳性血浆中加入MB,调整MB终浓度分别为1.0μmol/l、5.0μmol/l、10.0μmol/l,光照度为40000LUX,照射时间分别为0min、20min、40min、60min,在各点分别取样抽提基因组DNA用于荧光定量分析,测定HBV-DNA的浓度,推断在不同时间和浓度下的灭活效果。在灭活效果最好的点取样检测血浆正常成分有无显著变化,其中包括生化指标,酶学指标,凝血因子FⅧ:C活性,SDS聚丙烯酰胺凝胶电泳观察蛋白亚基的变化,蛋白免疫印迹观察血浆的抗体蛋白活性变化。结果:当MB为10.0μmol/L,光照60min时,灭活乙肝病毒的效果最好,对于高拷贝数的病毒浓度,在本实验的条件下,尚无法完全灭活病毒,但可以使HBV-DNA浓度明显下降;从病毒灭活的表格看,灭活效果与时间和MB浓度呈正相关,作用60min时血浆中主要成分的生化指标无显著变化及凝血因子FⅧ:C活性无明显改变,部分血浆酶活性有明显下降(P<0.05)。血浆蛋白的亚基数目和迁移速度未见明显变化,血浆的抗体蛋白也具有正常的生物学活性。结论:MB10μmol/L辅助荧光光照60min可使血浆中乙肝病毒量明显降低,血浆的大多数成分能保持较高活性。

Objective：To investigate the effect of the methylene blue on Hepatitis B virus and compositions of plasma. Methods： 1.0μmol/L, 5.0μ mol/L, 10.0μ mol/L Methylene blue （MB） were added to Hepatitis B Virus （HBV） DNA-positive human blood plasma. Each concentration solutions was subjected to irradiation intensity of 40000LUX within 0,20, 40 and 60 minutes. DNA concentration of each concentration solutions, which were subjected to irradiation intensity of 40000LUX within 0 , 20, 40, and 60 minutes, was determined by FQ-PCR assay. The effects of the MB and time in subjecting to light on the HBV DNA-positive human blood plasma were detected. The plasma fraction, the biochemical index, the enzymology index, and the coagulation factor F Ⅷ ：C activity were determined. The sub-unit were observed by SDS-PAGE, and plasma antibodies were also detected by western-blot. Results： The experiment showed that the most effective concentration of MB and length of irradiation time was 10.0？mol/L and 60 minutes, respectively. The result showed that methylene blue could not inactivate high copy viruses, but it could decrease the concentration of viruses. The irradiation time and concentration were positively correlated to the inactivation effect. There was no notable change of the plasma principle constitution when irradiated for 60 minutes. There was no influence for blood coagulation factor FⅧ ： C activity, but the action of some enzymes decreased （P〈0.05）. The subunit and moving speed had no conspicuous change, while the antibodies had normal biological activity. Conclusion： The HBV concentration in human blood plasma decreased when treated by 10？mol/L methylene blue and visible light irradiating for 60 min, and the activities of blood plasma remained normal.