摘要
目的筛选神经生长因子(nerve growth factor,NGF)依赖的TF-1亚克隆细胞株,用于重组人NGF(rhNGF)生物学活性的测定。方法逐步将含重组人粒细胞巨噬细胞集落刺激因子(recombinant human granulocyte macrophagecolony stimulating factor,rhGM-CSF)的培养基替换为含鼠神经生长因子(mouse nerve growth factor,mNGF)的培养基,将rhGM-CSF依赖的TF-1细胞驯化为mNGF依赖的TF-1细胞;绘制mNGF依赖的TF-1细胞的剂量-效应反应曲线,计算信噪比(S/N)和细胞最大效应(maximal effect,Emax)区间;用甲基纤维素半固体培养基对mNGF依赖的TF-1细胞进行克隆化,镜下挑选单克隆细胞,扩大培养,绘制各单克隆细胞株对NGF的剂量-效应反应曲线,并对其中反应性最好的单克隆细胞株进行STR图谱鉴定;对细胞接种密度和培养时间进行优化;用筛选出的细胞检测rhNGF的生物学活性。结果经驯化,rhGM-CSF依赖的TF-1细胞可在只含mNGF的培养基中正常生长,并对NGF的刺激呈良好的剂量-效应反应关系;驯化后的mNGF依赖的TF-1细胞对mNGF的剂量-效应曲线的信噪比为2.0,Emax为0.43;共筛选出15株细胞形态良好、倍增时间稳定的单克隆细胞株,其中A2亚克隆细胞株(TF-1-A2)反应性最好;该细胞中未发现人类细胞交叉污染现象,仅在CSF1PO位点与ATCC TF-1细胞数据有差异,可判定为TF-1细胞;细胞接种密度为5×104个/ml、培养时间为72 h时对rhNGF有良好的反应性,可用于rhNGF生物学活性的检测。结论成功筛选出1株对NGF具有良好反应性的TF-1-A2亚克隆细胞株,可用于定量检测NGF的生物学活性。
Objective To screen a nerve growth factor（NGF）-dependent TF-1 subclone cell line suitable for determina- tion of bioactivity of recombinant human NGF（rhNGF）. Methods The rhNGF-dependent TF-1 cells were habituated to mNGF-dependent ones by substitution of medium containing recombinant human granulocyte macrophage-colony stimula- tion factor（rhGM-CSF）with that containing mouse NGF（mNGF）. The dose-response curve of mNGF-dependent TF-1 cells was plotted,based on which the ratio of signal to noise（S / N）and maximal effect（Emax）interval were calculated. The mNGF-dependent TF-1 cells were subcloned with semi-solid methyl cellulose medium,and the single clones were selected under microscope for amplification. The dose-response curve of various single clones against NGF was plotted,and the single clone with the highest reactivity was subjected to STR profiling. The density of cells inoculated and the time for culture were optimized,and the screened cells were used for determination of bioactivity of rhNGF. Results After habit- uation,rhGM-CSF-dependent TF-1 cells grew normally in the medium containing only mNGF,and showed good dose-re- sponse to the stimulation with NGF. The S / N and Emax of dose-response curve of mNGF-dependent TF-1 cells against mNGF were 2. 0 and 0. 43 respectively. Fifteen single clones with good morphology and stable amplification time were screened,of which TF-1-A2 showed the highest reactivity. TF-1-A2 showed no cross contamination with other human-de- rived cells but only a difference in CSF1PO loci from that of TF-1 cells deposited in ATCC,which was judged as TF-1 cells. The optimal density for inoculation and time for culture were 5 × 104cells / ml and 72 h respectively. Under the optimal condition,the cells showed good reactivity and might be used for determination of bioactivity of rhNGF. Conclusion TF-1-A2 cell strain with good reactivity to NGF was successfully screened,which was suitable for the quantitative determination of bioactivity of NGF.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第2期235-240,244,共7页
Chinese Journal of Biologicals
基金
海淀区科技专项计划资助(K20100115C)
