文章摘要
栉孔扇贝消化盲囊亚细胞组分分离与鉴定技术
Isolation and identification of subcellular fractions from scallop Chlamys farreri’s digestive glands
投稿时间:2019-02-14  修订日期:2019-03-04
DOI:
中文关键词: 栉孔扇贝  消化吂囊  亚细胞组分  分离鉴定
英文关键词: Chlamys farreri  Digestive glands tissues  Subcellular fractions  Isolation and identification
基金项目:
作者单位E-mail
陈维 中国海洋大学海水养殖教育部重点实验室 2209236262@qq.com 
魏守祥 中国海洋大学海水养殖教育部重点实验室  
李禹含 中国海洋大学海水养殖教育部重点实验室  
潘鲁青① 中国海洋大学海水养殖教育部重点实验室 青岛 266003 panlq@ouc.edu.cn 
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中文摘要:
      采用匀浆、差速离心、镜检和标志酶测定等方法,研究了栉孔扇贝消化盲囊亚细胞组分分离与鉴定技术。结果表明:经Hoechst 33258染色,在匀浆2min对照组中,观察到大量栉孔扇贝消化盲囊完整细胞,呈圆形或椭圆形,细胞膜完整,荧光亮度较高;在匀浆3、4、5min实验组中,完整细胞数目逐渐减少,且出现许多形态较小,荧光亮度弱,轮廓模糊的细胞碎片。通过血球计数板法得到的细胞破碎结果与上述染色结果一致,随着匀浆时间(2-5min)的增加,细胞破碎率升高,当匀浆时间达到5min时,细胞破碎率升至94.24%。通过Hoechst 33258染色栉孔扇贝消化盲囊亚细胞分离组分(S2、C2、C4、S5、C5),镜检发现C2组分荧光亮度最强,荧光颗粒数目多,而其他组分荧光强度弱,且基本观察不到荧光颗粒,由此推测细胞核主要存在于C2组分中;同时细胞膜(5’-核苷酸酶)、线粒体(琥珀酸脱氢酶)、细胞质(乳酸脱氢酶)和微粒体(葡萄糖-6-磷酸酶)标志酶酶活力虽然在其他亚细胞组分中有少量检出,但它们分别在S2、C4、S5、C5组分中占有高比例(63.90%、64.89%、77.82%、67.55%)的标志酶活性。由此推测S2、C2、C4、S5、C5分离组分分别为细胞膜、细胞核、细胞质、线粒体、微粒体,成功构建了栉孔扇贝消化盲囊亚细胞组分分离与鉴定技术方法。
英文摘要:
      In this research, homogenization, centrifugation, microscopy, and marker enzyme assay were used to study the separation and identification techniques of subcellular fractions of Chlamys farreri’s digestive glands tissues. The results showed that after Hoechst 33258 staining, a large number of intact cells which were round or elliptical, with complete cell membrane and high fluorescence intensity; were observed in the 2 min homogenization group. In the 3, 4, 5 min homogenization group, the number of intact cells gradually decreased, and many cell fragments with small morphology, weak fluorescence, and blurred outline appeared. Besides, the blood cell counting results are consistent with the Hoechst staining results. With the increasing of homogenization time (2-5min), the cell broken rate was increased under an optical microscope and when the 2min homogenization group was used as the control group, the cell broken rate reached 94.24% in 5min homogenization group. There were more fluorescent particles in C2 with appropriate size,, and clear structure, and fewer fluorescent particles were observed in other separated fractions, when fractions (S2, C2, C4, S5, C5) of Chlamys C. farreri’s digestive glands tissues were stained with Hoechst 33258. Therefore the nucleus mainly presented in the C2 fraction. At the same time, the marker enzyme activity of cell membrane (5'-nucleotidase), mitochondria (succinate dehydrogenase), , cytoplasmic (lactate dehydrogenase) and microsomes (glucose-6-phosphatase) and marker enzyme accounts for a high proportion (63.90%, 64.89%, 77.82%, 67.55%) in the S2, C4, S5, and C5 fractions, however, there were also a small amount found in other subcellular fractions. As a result, the separated fractions of S2, C2, C4, S5 and C5 are cell membrane, nucleus, mitochondria, cytoplasm, and microsomes respectively, and the technical methods for separation and identification of subcellular fractions of Chlamys C. farreri’s digestive glands tissues were successfully constructed.
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