bFGF生物蛋白海绵体外生物学活性检测方法的建立及其稳定性评价

Establishment of method for detection of biological activity of biological protein sponge of basic fibroblast growth factor and stability evaluation

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 the method for detection of biological activity of basic fibroblast growth factor（bFGF）protein sponge in vitro and investigate the stability of bFGF protein sponge stored at room temperature.Methods The bFGF biological protein sponge was fused into the dilution of the test solution,the extract was obtained after centrifugation,the bFGF standard samples were used as control,and the bFGF protein sponge was cultivated with NIH3T3 cells.MTT assay was used to dected the proflieration of NIH3T3 cells induced with bFGF biological protein sponge extract.The bFGF protein sponge stored for 2,3,4 years at room temperature was used as the test materials,the bFGF biological protein sponge stored for 30 d at low temperature as standard control,the culture medium without bFGF protein sponge and bFGF as blank control,the potency of biological activity of each group was detected.The stability of biological activities in vitro of bFGF protein sponge stored for different time were compared.Results Compared with blank control,the A value of bFGF biological protein sponge extract was increased with the increasing of concentration,in other words the NIH3T3 cell proliferation was increased（P0.01）;compared with bFGF standard control,the difference was not significant（P 0.05）,either bFGF standard or bFGF biological protein sponge showed a dose-response relationship by the developed method,and the determination result complied with the following four-parameter equation：y =（A-D）/[1 ＋（X / C）B]＋D.Compared with protein sponge stored for 30 d at low temperature,the descending rate of biological activity titers（U·mL-1） of the protein sponge stored for 2 to 4 years at room temperature were 0.5%,0.6% and 0.8%,lower than the national standard of 15%.Conclusion The NIH3T3 cell proliferation could be applied for detection of the biological activity of bFGF protein sponge in vitro,it could be used as the routine detection method for bFGF protein sponge,the stabilities of bFGF protein sponge stored for 2-4 years at room temperature are good.