斑点叉尾肾脏组织细胞系的建立及其生物学特性

Establishment and characterization of a cell line derived from kidney of channel catfish,Ictaluris punctatus

采用组织块移植培养技术,对来源于斑点叉尾(Ictalurus punctatus Rafinesque)肾脏组织的细胞进行原代培养,建立了斑点叉尾肾脏组织细胞系,定名为CCK,目前已稳定传代培养70多次。斑点叉尾肾脏组织细胞为成纤维样细胞,最佳培养基为M199,最适培养温度范围为28～32℃,培养基血清体积分数为10%。在此条件下,CCK细胞的倍增时间为38.9～41.0h。斑点叉尾肾脏细胞的集落形成效率为(74.16±3.54)%,第9代传代细胞的染色体数目为正常二倍体2n=58,第33代传代细胞的染色体众数为60。液氮冷冻保存6个月后复苏的细胞经台盼兰染色检验,(86.69±1.04)%的细胞仍保持活性,细胞复苏后培养生长旺盛。通过对第17代离体培养细胞的28SrRNA基因进行PCR扩增及序列分析与比对,表明该细胞系来源于斑点叉尾。

Channel catfish, Ictalurus punctatus Rafinesque, is one of the most popular cultured species all over the world. With the rapid development of channel catfish aquaculture in China, disease problems of the fish have become more and more serious and caused great economic losses. Therefore, cell lines derived from channel catfish are urgently needed for the isolation and identification of viral pathogens and for the generation of disease control protocols. Herein, a cell line, designated as CCK, has been established from the kidney of channel catfish by using tissue explant techniques. CCK cell line has been continuously subcultured over 70 times and has been fully characterized in the aspects of morphological observation, optimal growth kinetics, plating efficiency, karyotyping, cryopreservation and 28S ribosome RNA genotyping, etc. The CCK cell monolayer consists of fibroblast-like cells and the optimal growth condition for CCK cells is： in M199 medium, under 28-32 ℃ temperature; with 10% FBS supplemented. Under such optimized condition the doubling time of CCK cell numbers in culture is about 38.9-41.0 h. The plating efficiency of CCK cells is about 74%. After cryopreservation in liquid nitrogen for 6 months, thawed cells exhibit a viability of （86.69 ± 1.04）%. Chromosome typing of CCK cell line revealed that at the 9th passage the diploid chromosome numeber was 2n=58, and at the 33th passage the modal number is 60. Polymerase chain reaction amplification of partial 28S ribosome RNA gene and sequence analysis of CCK cell indicated that it has 94.0% identity to the sequence of 18S rRNA（AF056008）in channel catfish, which confirms that the cell line was of channel catfish origin.