一种腺病毒载体疫苗的体外生物活性和细胞毒性研究

目的探讨一个以腺病毒为载体的人用疫苗(代号LMP-Ad)在传代动物细胞株上的表达能力和细胞毒性,为动物试验提供依据。方法受试物LMP-Ad以不同感染复数(MOI=3000、1000、300、100和30)感染非洲绿猴肾细胞(Vero)、幼仓鼠肾细胞(BHK)、小鼠成纤维细胞(L929)和人胚肺二倍体细胞(KMB17)4种传代细胞株后,用免疫细胞化学染色法检测目的蛋白LMP在细胞膜上的表达情况;在MOI=3×105、3×104、3×103、3×102和30时,用MTT法和FCM检测细胞相对增殖率(RGR)和细胞死亡率。结果在4种细胞中均检测到目的蛋白的表达,其中BHK细胞呈弱阳性,而Vero和KMB17在MOI低至30时仍较好地表达目的蛋白;随着感染剂量的提高,4种细胞的RGR下降而死亡率上升,其中以Vero细胞的变化最为明显。结论受试物对上述细胞均具生物活性,并在高感染剂量时表现出对细胞的生长抑制和一定程度的损伤,4种细胞以Vero和KMB17对受试物最为敏感,更适于作为该类新药的体外研究模型。

In vitro study of bioactivity and cytotoxicity of an adenovirus vaccine

Aim To estimate the ability of expression and cytotoxicity of an adenovirus vaccine(labeled as LMP-Ad)on cell lines so as to offer useful information for animal in vivo test.Methods Cell lines such as Vero,BHK,L929 and KMB17,derived from kidney of African green monkey,kidney of baby hamster,mouse fibroblast and lung of human fetus,respectively,were infected with different doses(MOI=3000,1000,300,100 and 30)of LMP-Ad and detected for target protein LMP by immunocytochemistry.Effect on relative growth rate(RGR)and death rate induced by LMP-Ad were quantified by MTT and FMC.Results LMP protein was identified to be expressed on the above cells.Positive signal on infected Vero and KMB17 was clear even the MOI was as low as 30,however,the BHK cell only appeared weak signal.On these 4 cell lines,RGR declined and death rate was enhanced as the infection dose increased,and the most dramatic change appeared on Vero cell,either on doses(P<0.01)or on cell variety.Conclusions LMP-Ad can express the target protein on above cell lines,and can also inhibit growth and make lesion on them especially when the dose is high enough.The Vero and KMB17 cell seem to be the most appropriate model for this kind of new drug since they are most sensitive to LMP-Ad.