Determination of active ingredient in canine amniotic membrane and screening of preservation conditions

Zhang Jiaren; Li Hui; Wang Yingqi; Zhang Bei; Xue Zhanmei; Zhao Can; Sheng Shuxin; Wu Ligang

Abstract：The aim of the study was to determine the effective components of canine amniotic membrane and the optimal conditions for short and medium term preservation.Fresh amniotic membrane was collected by caesarean section.Amniotic membrane was treated with different grinding methods(normal temperature grinding,low temperature grinding,dry grinding)and preservation methods(DMEM grinding,DMEM wet recombination,lyophilized powder),and then ELISA kit was used to detect the concentrations of bFGF,TIMP,HGF and TGF-β1 in the supernatant of dog amniotic membrane.The concentrations of protein in amniotic supernatant was determined by BCA method.The results showed that the concentrations of bFGF,TIMP,HGF,TGF-β1 in low-temperature grinding group(group D)were 73.58 ng/L,22.65 μg/L,46.12 ng/L and 108.22 μg/L,which were significantly higher than those in dry grinding group(group G)(P＜0.05).On the 20th day,at 4℃ and-18℃,bFGF concentrations in lyophilized powder group was significantly lower than that in the other two groups(P＜0.05),and HGF concentrations was extremely lower than that in the other two groups(P＜0.01).At the 40th day,the concentrations of bFGF in lyophilized powder group was extremely higher than that in the other two groups at 4℃ and-18℃(P＜0.01).At-18 ℃,TIMP concentrations in lyophilized powder group was significantly higher than that in the other two groups(P＜0.05).At the 80th day,the concentrations of bFGF and TIMP in lyophilized powder group were extremely higher than those in the other two groups at 4 ℃ and-18 ℃(P＜0.01).The TIMP concentrations of lyophilized powder group was extremely higher than that of the other two groups at-18 ℃(P＜0.01).At-18 ℃,the concentrations of bFGF in canine amniotic supernatant in lyophilized powder group was significantly higher than that at 4 ℃ on the 80th day(P＜0.05),and the concentrations of TGF-β1 was significantly higher than that at 4 ℃ on the 40th day(P＜0.05).The study indicates that there are some protein components in canine amniotic membrane,as well as bFGF,TIMP,TGF-β1 and other active components with biological activities.The amniotic membrane products added with DMEM preservation solution are suitable for short-term preservation,and the freeze-dried powder formulation is suitable for medium-term preservation.Low temperature freezing(-18 ℃)can effectively delay the degradation of relevant active components.