摘要
In this study,sterlet( Acipenser ruthenus) was chosen as the model species of sturgeon,different solutions were used to isolate the sturgeon peripheral blood lymphocytes and study their optimal proliferate response condition. Phytohemagglutinin( PHA),concanavalin A( Con A) and lipopolysaccharide( LPS) were used as lymphocyte proliferation mitogen,respectively. According to L25( 56) five-factor five-level orthogonal experimental design,the conditions for sturgeons proliferation response of peripheral blood lymphocytes were optimized using enhanced cell counting Kit-8( enhanced CCK-8 or WST-8). Five factors were selected to explore the optimal response conditions,including culture time,culture temperature,cell concentration,fetal bovine serum( FBS) concentration,and mitogen concentration. The results showed that 70% Percoll( 1. 092 g/ml) used as the sturgeon lymphocyte separation solution had the best separating effect. The optimal proliferation conditions were as follows: 3. 625 × 10~6 initial cells,20 μg/ml PHA or 50 μg/ml Con A or 10 μg/ml LPS as mitogen,10%-20% FBS,the temperature at 20-25 ℃,and the culture time of 2 d.
In this study, sterlet （ Acipenser ruthenus ） was chosen as the model species of sturgeon, different solutions were used to isolate the sturgeon peripheral blood lymphocytes and study their optimal proliferate response condition. Phytohemagglutinin （PHA）, concanavalin A （ConA）and lipopolysaccharide （LPS） were used as lymphocyte proliferation mitogen, respectively. According to L 25 （5^6） five-factor five-level orthogonal experimental design, the conditions for sturgeons proliferation response of peripheral blood lymphocytes were optimized using enhanced cell counting Kit-8 （enhanced CCK-8 or WST-8）. Five factors were selected to explore the optimal response conditions, including culture time, culture temperature, cell concentration, fetal bovine serum （FBS） concentration, and mitogen concentration. The results showed that 70% Percoll （1.092 g/ml） used as the sturgeon lymphocyte separation solution had the best separating effect. The optimal proliferation conditions were as follows： 3.625×10 6 initial cells, 20 μg/ml PHA or 50 μg/ml ConA or 10 μg/ml LPS as mitogen, 10%-20% FBS, the temperature at 20-25 ℃, and the culture time of 2 d.
基金
Supported by Foundation of Beijing Municipal Science and Technology Project(Z161100004516003)
Innovation Team of Sturgeon and Salmonid of Beijing(BAIC08-2018)
Innovation Team of Sturgeon and Salmonid of Baafs(JNKST201611)
