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目的:建立碱性成纤维细胞生长因子(bFGF)生物蛋白海绵体外生物学活性测定方法,观察bFGF生物蛋白海绵常温放置的稳定性。方法:bFGF生物蛋白海绵供试品融于稀释液中,离心后获得浸提液,以bFGF标准品为对照,与NIH3T3细胞共培养,MTT法检测bFGF生物蛋白海绵浸提液对NIH3T3细胞的促增值作用,用bFGF标准品计算bFGF生物蛋白海绵的相对百分效价,对该方法进行验证;以常温储存2、3和4年的bFGF生物蛋白海绵为供试品,以低温储存30d的bFGF生物蛋白海绵为标准对照,以不含bFGF生物蛋白海绵和bFGF的培养液为空白对照,测定各组生物学活性效价,比较不同储存时间bFGF生物蛋白海绵的体外生物学活性效价的稳定性。结果:bFGF生物蛋白海绵供试品浸提液随浓度增加其吸光度(A值)增高,即NIH3T3细胞增殖作用增加,与空白组比较差异有统计学意义(P<0.01),与bFGF标准品比较差异无统计学意义(P>0.05);bFGF标准品和bFGF生物蛋白海绵在该方法中均存在量效关系,且符合四参数方程式:y=(A-D)∕[1+(X/C)B]+D;常温条件下储存2、3和4年生物蛋白海绵体与低温储存30d比较,其生物学活性效价(U.mL-1)下降率分别为0.5%、0.6%和0.8%,均低于15%的国家标准。结论:bFGF生物蛋白海绵在体外能够促进NIH3T3细胞增殖,具有良好生物学活性;应用NIH3T3细胞检测bFGF生物蛋白海绵体外生物学活性可作为常规检测方法;bFGF生物蛋白海绵常温储存2～4年稳定性良好。
OBJECTIVE: To establish a method for the biological activity determination of basic fibroblast growth factor (bFGF) biological protein sponge, and observe the stability of bFGF biological protein sponge at room temperature. Methods: The bFGF biotech sponge was dissolved in diluent and centrifuged to obtain the extract. The bFGF standard was used as a control to co-culture with NIH3T3 cells. MTT assay was used to detect the effect of bFGF bioprotein sponge extract on NIH3T3 cells The bFGF bioprotein sponge relative percentage value was calculated with bFGF standard, and the method was validated. The bFGF bioprotein sponge stored for 2, 3 and 4 years at room temperature was used as the test sample, and stored at low temperature for 30 days Biological protein sponge was used as a standard control. BfGF bioprotein sponge and bFGF-free culture medium were used as blank control. The biological activity titers of each group were measured. The stability of biologically active titers of bFGF bioprotein sponges in different storage time was compared. . Results: The absorbency (A value) of the extract of bFGF biotech sponge increased with the increase of concentration, that is, the proliferation of NIH3T3 cells increased, which was significantly different from that of the blank group (P <0.01). Compared with the bFGF standard (P> 0.05). There was a dose-response relationship between bFGF standard and bFGF bioprotein sponge in this method, and the four-parameter equation y = (AD) / [1+ (X / C) B ] + D; Compared with the low temperature storage for 30 days, the biologically active potency (U.mL-1) of 2,3 and 4-year stored protein sponge at room temperature decreased 0.5%, 0.6% and 0.8% Are lower than 15% of national standards. CONCLUSION: The bFGF bioprotein sponge can promote the proliferation of NIH3T3 cells in vitro and has good biological activity. The biological activity of bFGF bioprotein sponges in vitro can be measured by NIH3T3 cells. The stability of bFGF bioprotein sponges stored at room temperature for 2 to 4 years good.