栉孔扇贝消化盲囊亚细胞组分分离与鉴定技术

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(Chlamys farreri)消化盲囊亚细胞组分分离与鉴定技术。结果显示,经Hoechst33258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,荧光强度较高;在匀浆3、4、5 min实验组中,完整细胞数目逐渐减少,且出现许多形态较小、荧光强度弱、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(2~5 min)的增加,细胞破碎率升高,当匀浆时间达到5 min时,细胞破碎率升至94.24%。利用Hoechst 33258染色栉孔扇贝消化盲囊亚细胞分离组分(S2、C2、C4、S5和C5),镜检发现,C2组分荧光强度最强,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)标志酶活力在其他亚细胞组分中有少量检出,但它们在S2、C4、S5和C5组分中的的标志酶活性比例较高(分别为63.90%、64.89%、77.82%和67.55%)。由此推测,S2、C2、C4、S5和C5分离组分分别为细胞膜、细胞核、细胞质、线粒体和微粒体。本研究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,为贝类生理机制研究提供技术支持。

Isolation and Identification of Subcellular Fractions from Digestive Glands of Chlamys farreri

In this study,homogenization,centrifugation,microscopy,and marker enzyme assays were used to study the separation and identification techniques for subcellular fractions of the digestive gland tissues of Chlamys farreri.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,and 5 min homogenization group,the number of intact cells gradually decreased,and many cell fragments with small morphology,weak fluorescence,and blurred outline appeared.Additionally,the blood cell counting results were consistent with the Hoechst staining results.With the increase in homogenization time(2~5 min),the broken cell rate was increased under an optical microscope and when the 2 min homogenization group was used as the control group,the broken cell rate reached 94.24%in the 5 min 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,and C5)of the digestive gland tissues of C.farreri were stained with Hoechst 33258.Therefore,the nucleus mainly presented in the C2 fraction.At the same time,the marker enzyme activity of the cell membrane(5’-nucleotidase),mitochondria(succinate dehydrogenase),cytoplasmic(lactate dehydrogenase),and microsomes(glucose-6-phosphatase)accounted for a high proportion(63.90%,64.89%,77.82%,and 67.55%)in the S2,C4,S5,and C5 fractions,respectively;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 were cell membrane,nucleus,mitochondria,cytoplasm,and microsomes,respectively,and the technical methods for separation and identification of subcellular fractions of the digestive gland tissues of C.farreri were successfully determined.