甘露聚糖结合凝集素阻遏人巨细胞病毒感染MD-DC细胞的研究

Mannose-binding Lectin(MBL) Inhibits Human Cytomegalovirus Infection of Human MD-DC

目的探讨不同来源、不同浓度的甘露聚糖结合凝集素（MBL）阻遏人巨细胞病毒（HCMV）感染人外周血单个核细胞衍生出的DC细胞（MD-DC）的作用。方法通过载体构建得到重组MBL（rMBL）,通过血浆分离得到天然MBL（hMBL）,先以不同浓度的hMBL/rMBL预处理MD-DC 0.5 h,再以HCMV感染MD-DC 2 h,比较不同浓度的hMBL/rMBL阻遏MD-DC捕获HCMV的作用差异。经HCMV感染后的MD-DC和MBL共培养,比较不同浓度的hMBL/rMBL阻遏HCMV在MD-DC间扩散的作用差异。荧光定量PCR法检测MD-DC内HCMV-DNA的拷贝数,流式细胞仪检测MD-DC内HCMV-PP65的阳性率,激光共聚焦技术观察MD-DC对HCMV的捕获。结果荧光定量PCR结果显示,与对照组相比,1μg/mL hMBL/rMBL组MD-DC内HCMV-DNA含量均无明显变化（P〉0.05）,而5μg/mL和10μg/mL hMBL/rMBL组MD-DC内HCMV-DNA含量均降低（P〈0.05）。激光共聚焦技术和流式细胞术证实10μg/mL hMBL/rMBL能明显阻遏MD-DC捕获HCMV。经HCMV感染后的MD-DC与MBL共培养,在第72 h,5μg/mL和10μg/mL hMBL/rMBL组开始显现阻遏作用。结论生理浓度范围（5~10μg/mL）内的hMBL/rMBL能显著阻遏HCMV感染。

Objective To explore the inhibitory effect of different sources,different concentrations of Mannose-binding lectin（MBL） on human cytomegalovirus infection of human MD-DC cells.Methods The recombinant MBL was acquired by vector construction,and the natural MBL was purified from human plamsa.MD-DC were pre-exposed to several dilutions of the hMBL/rMBL for 30 min,then HCMV suspensions were added to MD-DC for 2 h to compare the inhibitory effect of hMBL/rMBL on the HCMV infection of MD-DC.MD-DC infected by HCMV co-culture with hMBL/rMBL to compare the inhibitory effect of hMBL/rMBL on the HCMV diffusion between MD-DC.HCMV-DNA in MD-DC was detected by fluorescence quantitative PCR.HCMV-PP65 in MD-DC was analyzed with flow cytometry,the ability of MD-DC to capture HCMV was observed with immunofluorescence confocal microscope.Results In hMBL/rMBL inhibition the ability of MD-DC capture HCMV experiments,the fluorescent quantitative PCR demonstrated that the amount of HCMV-DNA in 1 μg/mL of hMBL/rMBL treated cells was not significantly different from that of control group（P0.05）.But the HCMV-DNA in 5 μg/mL and 10 μg/mL hMBL/rMBL treated group were significantly lower than that of control group（P0.05）.The significant inhibit effects of 10 μg/mL hMBL / rMBL on the ability of MD-DC capture HCMV were observed by immunofluorescence confocal microscopy and flow cytometry.The inhibit effects of hMBL/rMBL on HCMV diffusion between MD-DC were also observed in 5 μg/mL and 10 μg/mL hMBL/rMBL treated groups at 72 hours.Conclusion The hMBL/rMBL in physiological concentration range（5-10 μg/mL） can significantly inhibit human cytomegalovirus infection of human MD-DC cells,and the hMBL is more effective than rMBL.