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目的建立并优化采用2E8细胞株测定白介素-7生物学活性方法,并应用于重组人白介素-7和鼠白介素-7的活性检测。方法将R&D重组人白介素-7进行梯度稀释后,与2E8细胞相互作用一段时间,通过MTT法测定细胞增殖情况,从而反映重组白介素-7的生物学活性;从细胞和样品稀释培养基类型、细胞密度、样品稀释倍数以及样品和细胞相互时间等多个环节对实验条件进行优化;将建立的2E8细胞增殖法应用于重组人白介素-7和鼠白介素-7的活性检测。结果建立了采用2E8细胞增殖法测定白介素-7生物学活性方法;实验条件优化结果表明,在RPMI1640+20%FBS+0.05m M 2-巯基乙醇的稀释培养基背景下,样品以1μg·m L-1的起始浓度上板,采用5倍梯度稀释,与2×106/m L的2E8细胞相互作用,培养48h,可以得到典型的四因子回归曲线;利用优化后的实验条件对重组人白介素-7和鼠白介素-7各2批样品的活性分别进行3次测定,结果平均值分别为12.09×107、9.00×107、10.19×107、10.23×107U·mg-1,变异系数分别为3.97%、1.40%、3.09%、6.04%,检测结果稳定,表明此方法具有可靠的重复性。结论建立并优化采用2E8细胞株测定白介素-7生物学活性方法,该方法操作简便,定量准确,重复性好,稳定可靠。可有效应用于重组人白介素-7和鼠白介素-7的活性检测。
Objective To establish and optimize the method of measuring the biological activity of interleukin-7 by using 2E8 cell line and to detect the activity of recombinant human interleukin-7 and murine interleukin-7. METHODS: R & D recombinant human interleukin-7 was serially diluted and then allowed to interact with 2E8 cells for a period of time. Cell proliferation was measured by MTT assay to reflect the biological activity of recombinant interleukin-7. Dilution of culture media from cells and samples, Density, dilution of samples and sample and cell time to optimize the experimental conditions; the established 2E8 cell proliferation assay was applied to detect the activity of recombinant human interleukin-7 and murine interleukin-7. Results The method of 2E8 cell proliferation was established to determine the biological activity of interleukin-7. The optimized experimental conditions showed that under the background of diluted medium of RPMI1640 + 20% FBS + 0.05m M 2-mercaptoethanol, 1μg · m L -1 initial concentration of the upper plate, using a 5-fold gradient, and 2 × 106 / m L of 2E8 cells interacting, cultured 48h, you can get a typical four-factor regression curve; using the optimized experimental conditions on recombinant human interleukin -7 and murine interleukin-7 were assayed three times respectively, the average values were 12.09 × 107,9.00 × 107,10.19 × 107,10.23 × 107U · mg-1, respectively, and the coefficients of variation were 3.97% , 1.40%, 3.09% and 6.04% respectively. The test results were stable, indicating that this method is reliable and reproducible. Conclusion The method for determining the biological activity of interleukin-7 by 2E8 cell line is established and optimized. The method is simple, accurate, reproducible and reliable. It can be effectively applied to detect the activity of recombinant human interleukin-7 and murine interleukin-7.