塔里木裂腹鱼尾鳍细胞系的建立及盐碱度对其增殖的影响

Establishment and the effect of salinity of cell lines derived from tail fin of Schizothorax biddulphi

中文关键词: 塔里木裂腹鱼  尾鳍组织  细胞培养

英文关键词: Schizothorax biddulphi  Tail fin  Cell culture

中文摘要:

      为了建立塔里木裂腹鱼(Schizothorax biddulphi)尾鳍细胞系，本研究采用组织块法，分别用含胎牛血清(FBS)的DME/F12培养基体外培养塔里木裂腹鱼尾鳍组织，初步建立了塔里木裂腹鱼尾鳍细胞系(BICF1)。采用MTT法测定分析盐度(NaCl)和碱度(NaHCO3)对其增殖的影响。结果显示，BICF1悬浮培养传至45代，最适培养液为DME/F12，最适FBS浓度为20%，最适温度为25℃。第10代BICF1的群体倍增时间为28.11 h，呈“S”型生长。第6代BICF1液氮冻存180 d后复苏，经台盼蓝染色计数，BICF1有(87.85±0.66)%的细胞具有活性，复苏后可增殖并传代。BICF1无细菌、真菌、支原体污染。第10代BICF1线粒体16S rRNA测序结果与GenBank基因序列进行一致性对比，结果显示，BICF1与JQ844133.1的一致率为100%，表明BICF1来自于塔里木裂腹鱼。BICF1增殖数量在盐度为1、2、4、6时呈上升趋势，在盐度为6、8、10时呈下降趋势；盐度为6时，BICF1增殖数量最高。BICF1增殖数量在碱度为2、3、4、5、6 g/L时呈上升趋势，在碱度为6、7、8、10 g/L时呈下降趋势；碱度为6 g/L时，BICF1增殖数量最高。细胞增殖数量随盐碱度增加呈现先升高后下降的趋势。本研究旨在为合理开发和利用塔里木裂腹鱼遗传资源以及建立和保护其种质资源提供一定的科学依据。

英文摘要:

      To establish the caudal fin cell line of Schizothorax biddulphi, this study employed the tissue block method with fetal bovine serum (FBS) DME/F12 medium for in vitro cultivation of the tail fin, using previously established tail-fin cell lines (BICF1) of S. biddulphi. In this study, the effect of NaCl salinity and NaHCO3 alkalinity on the proliferation of tail fin cells was investigated using the MTT method. The main results are as follows: the BICF1 suspension culture was propagated to 45 generations. The optimum medium was DME/F12. The optimal FBS concentration was determined to be 20%. The optimum temperature was 25℃. The population doubling time of the 10th generation BICF1 was 28.11 h, showing an "S" type growth. The 6th generation BICF1 in liquid nitrogen was recovered after freezing for 180 days. Trypan blue staining showed that after recovery, (87.85±0.66)% of BICF1 cells were active, and the cells could proliferate and pass to next generation. The BICF1 cell line is free of contamination by bacteria, fungi, and mycoplasma. The sequencing results of mitochondrial 16S rRNA of the 10th generation BICF1 were consistent with the GenBank gene sequence, and the consistency rate of BICF1 and JQ844133.1 was 100%, proving that BICF1 was from S. biddulphi. BICF1 cell proliferation increased with salinity of 1, 2, 4, and 6, but decreased with salinity of 6, 8, and 10; BICF1 cell proliferation was the highest at 6. The NaHCO3 alkalinity of BICF1 increased at 2, 3, 4, 5, and 6 g/L, whereas the proliferation of BICF1 cells decreased at 6, 7, 8, and 10 g/L, respectively. BICF1 cell proliferation was the highest at 6 g/L. Cell proliferation first increased and then decreased with increasing salinity and alkalinity. This study aimed to provide a scientific basis for the rational development and utilization of genetic resources of S. biddulphi, including the establishment and protection of the germplasm.