TF-1细胞增殖法测定神经生长因子生物学活性的建立与应用

目的：建立并优化用于检测神经生长因子（NGF）生物学活性的TF-1细胞增殖法，并应用于重组人NGF和鼠NGF制品的活性检测。方法：将梯度稀释的人NGF国际参考品与TF-1细胞相作用，通过MTT法测定细胞增殖情况，建立测定NGF活性的TF-1细胞增殖法；从粒细胞巨噬细胞集落刺激因子（CM-CSF）残留、NGF加样稀释率、TF-1细胞接种浓度、培养时间等多个环节对实验条件进行优化；将建立的TF-1细胞增殖法应用于重组人NGF和鼠NGF的活性检测。结果：建立了用于NGF活性检测的TF-1细胞增殖法；实验条件优化结果表明，在无GM-CSF残留的情况下，将稀释至1／4的NGF样品与4×10^5／mL的细胞相互作用，培养48h，可以得到更为典型的的四参数曲线；利用条件优化后建立的检测方法对重组入NGF和鼠NGF各2批样品分别进行4次测定，结果平均值分别为10．01×10^5、10．81×10^5和3．55×10^5、3．30×10^5U／mg，变异系数分别为7．5％、7．2％和7．2％、9．1％，检测结果稳定均一，表明检测方法具有很好的重复性。结论：建立并优化了用于NGF活性检测的TF-1细胞增殖法，该方法操作简便、定量准确、重复性好、稳定可靠，可有效应用于人NGF和鼠NGF制品的活性检测。

Development and Application of TF-1 Cell Proliferation Assay Method for Estimating Bioactivity of Nerve Gowth Factor

Objective： To develop an optimized TF-1 cell proliferation assay method for estimating the bioactivi- ty of recombinant human nerve growth faetor（rhNGF） and murine NGF（mNGF）. Methods： TF-1 cell proliferation assay for estimation of NGF-bioactivity was established by MTT method following interaction between proportion- al-diluted international reference preparation of human NGF and TF-1 cells. Several factors including the pres- ence of residual GM-CSF or not, dilution rate of NGF preparations, concentration of TF-1 cells seeded and cul- ture time were considered to optimize the experimental conditions. The bioactivity of rhNGF and mNGF products were detected based on the TF-1 cell proliferation assay method. Results： TF-1 cell proliferation assay for mea- surement of NGF-bioactivity was established and the data from optimization of experimental conditions showed that most classical 4-parameter curve would be fitted on the condition that 4-fold diluted NGF preparations act on TF-1 ceils at the concentration of 4×10^5/mL and culture for 48 hours, absent from residual GM-CSF. The average of two batches of rhNGF and mNGF respectively detected in quadruplicate were 10.01×10^5, 10.81×10^5, 3.55×10^5 and 3.30×10^5 U/mg, and the corresponding coefficient of variation（CV） were 7.5%, 7.2%, 7.2% and 9.1%, suggesting the stable and uniform results from established method with good reproducibility. Conclusion： A TF-1 cell proliferation assay method was successfully developed following optimizations, characterized by simple operations, ac- curate quantification, good reproducibility, stability and reliability, and this method is applicable to estimating the bioactivity of human and murine NGF products.