不同细胞因子对水牛原始生殖细胞传代培养的影响

为探讨不同细胞因子对水牛原始生殖细胞(PGCs)传代培养的影响,将PGCs分别采用A、B、C、D、E、F和G共7种培养基进行培养,即A组:基础液+10 ng/mL白血病抑制因子(LIF)+10 ng/mL碱性成纤维细胞生长因子(bFGF);B组:基础液+20 ng/mL LIF+20 ng/mL bFGF;C组:基础液+20 ng/mLLIF+10 ng/mL bFGF;D组:基础液+20 ng/mL LIF;E组:基础液+20 ng/mL LIF+20 ng/mL bFGF+20ng/mL干细胞因子(SCF);F组:基础液+20 ng/mL LIF+40 ng/mL bFGF+40 ng/mL SCF;G组(对照组):基础液。将机械法分离的PGCs小集落接种到水牛胎儿成纤维细胞(BEF)饲养层上进行传代培养。结果,原代时,A、B、C、D、E和F组的克隆数目都显著高于对照组(P<0.05),其中E和F组的克隆数目显著高于A组(P<0.05),而与B、C、D组差异均不显著(P>0.05);1~8代时,B、C、D、E、F组的克隆数目显著高于A组(P<0.05);对照组传代数仅为2代,A组为5代,而B、C、D、E和F组均为8代以上。结果表明,在传代过程中,LIF起主要作用,20 ng/mL的LIF浓度可以满足水牛PGCs传代培养的需要。

Effect of different cytokines on subculture of buffalo PGCs

To explore the influence of different cytokines on the subculture of buffalo PGCs,PGCs were cultured with media A(basic culture medium+10 ng/mL LIF+10 ng/mL bFGF),B(basic culture medium+20 ng/mL LIF+20 ng/mL bFGF),C(basic culture medium+20 ng/mL LIF+ 10 ng/mL bFGF),D(basic culture medium+20 ng/mL LIF),E(basic culture medium+20 ng/mL LIF+20 ng/mL bFGF+20 ng/mL SCF),F(basic culture medium+20 ng/mL LIF+ 40 ng/mL bFGF+40 ng/mL SCF) and G(basic culture medium,as control),respectively.Then PGCs were dissociated by mechanical method and plated into BEF feeder for subculture.Groups A,B,C,D,E and F were significantly higher than group G in the number of the primary PGCs clones(P<0.05),and groups E and F were significantly higher than group A in the number of the primary PGCs clones(P<0.05),and no significant difference was observed among groups B,C and D(P>0.05).In 1-8 passage subculture, groups B,C,D and F were significantly higher than group A in the number of the PGCs clones(P<0.05).In the maximal subculture,groups B,C,D,E and F were at least eight passages,group A was five passages,but group G was only two passages.The results show that the main factor affecting buffalo PGCs subculture is LIF,and 20 ng/mL LIF can meet the need of buffalo PGCs subculture.