人促卵泡激素体外生物学活性测定方法的建立  

Development and Verification of a Novel in vitro Assay for Determination of Biological Activity of Human Follicle Stimulating Hormone

作　　者：贾慧[1,2] 齐连权[2] 梁艳[2] 常翠云[2] 王鑫[2] 徐俊杰[1] 陈薇[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071 [2]北京泰德制药股份有限公司,北京100176

出　　处：《生物技术通讯》2015年第4期556-561,共6页Letters in Biotechnology

摘　　要：目的:建立人促卵泡激素(FSH)体外生物学活性检测方法,并对方法进行验证。方法:以人卵巢颗粒细胞KGN为基质细胞,用不同浓度的FSH与KGN细胞表面的FSH受体结合,刺激细胞产生不同浓度的孕酮,通过测定细胞培养上清中的孕酮含量来评价FSH的生物学活性,结果用国际标准品进行校正;对方法的专属性、线性与范围、回收率、精密度、耐用性进行验证;对市售FSH产品进行检测,并与传统动物体内活性检测结果进行比较。结果:KGN细胞对FSH有很好的剂量反应关系,方法专属性符合要求;线性范围为0.1~200 ng/m L,相关系数R2≥0.99;回收率为80%~120%;经3次重复测定,3批市售样品和3批自制样品的活性分别为(13.4±0.4)^(13.5±0.8)和(12.9±0.7)^(14.3±1.2)IU/μg,变异系数在15%之内;结果显示该方法有很好的耐用性。测定3批市售重组人FSH产品和1批尿源FSH国家标准品,其体外活性测定结果与传统动物体内活性测定结果一致。结论:建立并验证了一种利用KGN细胞体外测定FSH生物学活性的方法,有望代替传统的动物体内活性测定方法,可用于FSH的生物学活性评价和质量控制。Objective：To develop and verify a novel in vitro assay for determination of biological activity of hu-man follicle stimulating hormone（FSH）.Methods：The assay is based on a human ovary granulosa cell line,KGNcell line,which expresses the human FSH receptor on its surface.FSH binding can stimulate KGN cell to expressand secret progesterone,concentration of which is determined by a commercially available ELISA kit.The devel-oped method was verified for specificity,linear range,recovery,precision,and tolerance.Three batches of commer-cially recombinant FSH products and one batch of Urofollitropin were assayed by the method,and the results werecompared with the results of traditional in vivo assay.Results：Highly dose-dependent progesterone secretion wasobserved at 72 h of incubation for KGN cell.The method met the specificity requirement.The linear range was 0.1~200 ng/m L,R2≥0.99.The recovery was within 80%~120%,which met the recovery requirement in activity assay ofbiological products.The results of three batches of commercial FSH products and three bathes of samples were（13.4±0.4）^（13.5±0.8） and（12.9±0.7）^（14.3±1.2） IU/μg respectively,CV%≤15%.The results showed good toler-ance to different progesterone ELASA kits.Furthermore,it was demonstrated that the assay delivered comparable re-sults to the in vivo assay for three batches of commercially recombinant FSH products and one batch of Urofollitrop-in.Conclusion：The new in vitro assay for FSH activity is developed and verified,which is supposed to replacethe in vivo assay in the future and can be used for the biological activity assessment and quality control for FSH.

关 键 词：人促卵泡激素 人卵巢颗粒细胞 体外细胞生物学活性 卵巢增重法 孕酮含量 

分 类 号：Q25[生物学—细胞生物学]