烟酰胺腺嘌呤二核苷酸与氢氧化铝联合佐剂对HBsAg诱导小鼠体液免疫应答的影响

目的研究烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide,NAD)与氢氧化铝联合佐剂对HBsAg诱导小鼠体液免疫应答的影响。方法将NAD与氢氧化铝按不同剂量联合后,与不同剂量的HBsAg混合,分别经肌肉注射免疫ICR小鼠1针或2针(间隔2周),每只注射0.2 ml,并设阴性对照组(生理盐水)、单纯抗原对照组、单纯铝佐剂对照组和单纯NAD对照组,共23组,每组6只。分别于末次免疫后2、4、6、8、10、12周采血,分离血清,采用ELISA法检测小鼠血清中抗HBsAg IgG水平;试验期内,观察小鼠是否发生异常状况;14周后,取最佳剂量免疫组及阴性对照组小鼠心、肝、脾、肺组织做组织切片,进行病理分析。结果阴性对照组在各时间点均未检测到抗HBsAg IgG抗体,除联合佐剂12组外,其余各组血清抗HBsAg IgG抗体水平均在末次免疫后第6周达峰值,随着时间的延长,抗体水平呈下降趋势;联合佐剂14组(HBsAg 2μg+氢氧化铝100μg+NAD 2.5μg)产生的抗体水平最高,且持续时间较长,其对HBsAg的体液免疫增强效应显著优于单纯抗原对照组、单纯铝佐剂对照组及单纯NAD佐剂对照组(P<0.05)。试验期内,各组小鼠均未出现异常反应;最佳剂量免疫组(14组)及阴性对照组组织切片观察结果显示,均为发生病变。结论 NAD与氢氧化铝联合佐剂能显著增强HBsAg诱导的小鼠特异性体液免疫应答,在免疫剂量范围内,联合佐剂安全性良好。

Effect of nicotinamide adenine dinucleotide combined with aluminum hydroxide on humoral immune response induced by HBsAg in mice

Objective To investigate the effect of nicotinamide adenine dinucleotid(NAD) combined with aluminum hydroxide on humoral immune response induced by HBsAg in mice.Methods NAD and aluminum hydroxide,at various dosages,were combined and further mixed with HBsAg.ICR mice were divided into 23 groups,6 for each.The mice in 16 HBsAg + combined adjuvant groups were immunized i.m.with the above-mentioned mixture once or twice(at an interval of 2 weeks),0.2 ml for each,while those in four HBsAg + aluminum hydroxide adjuvant groups with the mixture of HBsAg and aluminum,at various dosages.However,the mice in negative control,HBsAg control and NAD control groups were immunized with 200 μl physiological saline,2 μg HBsAg and HBsAg + 5 μg NAD,respectively.Serum samples were collected 2,4,6,8,10 and 12 weeks after the last immunization,and determined for IgG levels against HBsAg by ELISA.The mice were observed for abnormality during test.The heart,liver,spleen and lung tissues of mice immunized with HBsAg + combined adjuvant,at optimal dosages,and those in negative control group were prepared into sections 14 weeks after the last immunization for pathological examination.Results No IgG was detected in negative control group at any time points.However,the serum IgG titers in all the HBsAg + combined adjuvant groups,except those induced by 2 doses of 1 μg HBsAg + 100 μg aluminum hydroxide + 10 μg NAD,reached peak values at week 6 after the last immunization and showed decreasing tendencies as time went on,of which that in HBsAg + combined adjuvant group 14(immunized with 2 doses of 2 μg HBsAg + 100 μg aluminum hydroxide + 2.5 μg NAD) was the highest and the most persistent,indicating significant enhancement of humoral immune response as compared with those in negative control,HBsAg control and NAD control groups(P < 0.05).No abnormalities of mice were observed in various groups during the test.No pathological changes were observed in the tissue sections of mice in HBsAg + combined adjuvant group 14(both HBsAg and adjuvant were at optimal dosages) and in negative control group.Conclusion NAD combined with aluminum hydroxide enhanced the specific humoral immune response induced by HBsAg in mice and,within the range of immunizing dose,showed high safety.