翘嘴鳜肝脏组织原代细胞培养技术的建立

Establishment of Primary Cell Culture Technique for Liver Tissue of Mandarin Fish Siniperca chuatsi

运用组织块贴壁法、酶消化法、组织块联合酶消化法探究体质量(33.2±0.2)g翘嘴鳜肝脏组织原代细胞最适培养方法,采用3种培养基(M199培养基、L15培养基、M199-L15)、3种培养容器(12孔板、6孔板、T25培养瓶)及有无CO_(2)探究翘嘴鳜肝脏组织原代细胞最适培养条件,采用线粒体16S rRNA基因序列和染色体数目鉴定细胞,研究氯化铵(0、1、5、10mmol/L)对细胞形态和增殖的影响。试验结果显示:0.25%胰蛋白酶-EDTA消化翘嘴鳜肝脏组织获得细胞,接种到T25培养瓶,生长培养基为M199-L15(体积比1∶1)混合培养基并添加20%体积胎牛血清,在28℃含5%CO_(2)的培养箱中培养,第2~3天贴壁细胞占瓶底的25%~30%,第4~5天细胞长满瓶底的70%~80%,第6~7天得到可传代的原代细胞;传代后细胞饱满、增殖能力强,为上皮样和成纤维样的混合,以成纤维样细胞为主,已传到10代;细胞线粒体16SrRNA基因测序结果与GenBank中基因序列(HQ731435.1)的一致率达99.05%,染色体数目为48条,证明此细胞来自翘嘴鳜;5mmol/L和10mmol/L氯化铵暴露对细胞形态和活力有显著影响(P<0.05)。本试验建立了翘嘴鳜肝脏组织原代细胞培养技术,为开展翘嘴鳜生物学、疾病学、营养学及遗传学等提供重要基础,同时为鱼类肝脏组织细胞原代培养和建系提供重要参考。

The primary cell culture of liver tissue of mandarin fish Siniperca chuatsi with body weight of(33.2±0.2)g was carried out by tissue explants adherent method,enzyme digestion method,and tissue explants adherent combined with enzyme digestion method for screening of the optimal method.The primary cell culture of liver tissue of mandarin fish was in 3 kinds of culture media(M199 culture medium,L15 culture medium,and M199-L15 culture medium),three kinds of culture containers(12 well plate,6 well plate,and T25 culture bottle)and with or without CO_(2)were compared to determine the optimal culture conditions of primary cells from liver tissue of mandarin fish.The cells were identified using mitochondrial 16S rRNA gene sequences and chromosome count.The effects of ammonium chloride(0 mmol/L,1 mmol/L,5 mmol/L,and 10 mmol/L)on cell morphology and proliferation were also investigated.The results showed that the good cells of the liver tissue of mandarin fish was obtained by 0.25%trypsin-EDTA digested cells under conditions of M199-L15(volume ratio 1∶1)mixed medium containing 20%fetal bovine serum,which were inoculated into T25 culture flask,and culture at 28℃in an incubator containing 5%CO_(2).The attached cells accounted for 25%—30%of the bottom of culture flask at 2—3 days,the cells grew 70%—80%of the bottom of culture flask at 4—5 days,and primary monolayer cells were obtained at 6—7 days.The cells were plump and had strong proliferative ability after subculturing,appearing in a mixture of epithelioid and fibroblast-like cells,with fibroblast-like cells being the majority.The liver cells had grown up with 10 passages.The result of mitochondrial 16S rRNA gene sequencing was 99.05%consistent with the gene sequence in GenBank(HQ731435.1),and chromosome count was found to be 48,proving that these cells were from mandarin fish.The significant effects on cell morphology and vitality(P<0.05)were observed at 5 mmol/L and 10 mmol/L ammonium chloride exposure.The primary cell culture technology of liver tissue of mandarin fish was established,and provided an important basis with the development of biology,disease,nutrition and genetics of mandarin fish,and primary culture and establishment of other fish liver cell lines.