厚颌鲂幼鱼肠上皮细胞刷状缘膜囊的提取

Study on isolation of enterocytes brush border membrane vesicles from early young fish of Megalobrama pellegrini

根据沉淀方法和离心速度的不同分为4个试验组提取厚颌鲂(Megalobrama pellegrini)70日龄幼鱼的肠上皮细胞刷状缘膜囊(BBMV)。四个试验组如下:A组-钙离子沉淀法,28 000 g离心;B组-钙离子沉淀法,43000 g离心;C组-PEG沉淀法,28 000 g离心;D组-PEG沉淀法,43 000 g离心。采用细胞器和细胞膜指示酶的富集系数、核糖核酸和脱氧核糖核酸含量来评价各试验组提取BBMV的纯度。结果表明:肠上皮细胞刷状缘膜囊提取后,DNA的富集系数各组排列为D<B<C<A,D组的富集系数为0;RNA的富集系数排列为D<B<C<A,D组的富集系数为0.007;各组的蔗糖酶、γ谷氨酰转移酶(γ-GT)及碱性磷酸酶(AKP)富集系数排列为A<C<B<D,D组各酶的富集系数分别为:6.172、12.700、20.185。麦芽糖酶的富集系数排列为A<C<D<B,B组的富集系数为5.789;而Na+-K+-ATP酶和琥珀酸脱氢酶(SDH)在采用43 000 g离心后均为0。上述结果说明采用PEG沉淀法、43 000 g离心试验组提取的BBMV的纯度优于其余各试验组。

The method of isolation of enterocytes brush border membrane vesicles from early young fish （70 days） of Megalobroma pellegrini using precipitation and centrifugal speed was studied. Four experiment groups were as follows ： A group - Ca^2＋ precipitation, 28 000 G centrifugal; B group - Ca^2＋ precipitation, 43 000 G centrifugal; C group - PEG precipitation, 28 000 G centrifugal; D group - PEG precipitation, 43 000 G centrifugal. These preparations were assessed for enrichment of marker enzymes, contamination by subcellular structures, deoxygenrobose nucleic acid （DNA） content and robose nucleic acid （RNA） content. The results of experiment were as follows ： the sequence of enrichment of DAN was D 〈 B 〈 C 〈 A, the enrichment of D group was zero. The sequence of enrichment of RNA was D 〈 B 〈 C 〈 A, the enrichment of D group was 0. 007. The sequence of enrichment of sucrase, γ - GT and AKP was A 〈 C 〈 B 〈 D, the enrichment of D group was 6. 172, 12. 700 and 20. 185 respectively. The sequence of enrichment of maltose is A 〈 C 〈 D 〈 B, the enrichment of B group was 5. 789. The enrichment of Na^＋ - K^＋ - ATP, SDH were zero in the final membrane preparation of B, D group. The results indicated that PEG precipitation and 43 000 G centrifugal was better the others