瓦氏雅罗鱼鳃细胞系的建立及其耐碱生长特性初探

Establishment of the gill cell line of Leuciscus waleckii and alkaline-tolerant growth characteristics

摘要:

为探究碱水环境中自然优势种瓦氏雅罗鱼(Leuciscus waleckii)的耐碱离子调节机制,采用胰蛋白酶消化法对其鳃细胞(Gill cells of Leuciscus waleckii, LWG)进行体外培养,确定该类原代和传代细胞培养的适宜条件并进行耐受实验,用以对瓦氏雅罗鱼在碱生境中的生长特性进行论证。结果表明:瓦氏雅罗鱼原代鳃细胞培养在15 ℃、含有10%胎牛血清(Fetal bovine serum,FBS)的DMEM培养基(Dulbecco’s modified eagle medium)中可获得稳定、良好的培养效果;36～72 h即可传代,稳定传代后的细胞命名为LWG细胞。传代培养过程中,36 h时LWG细胞生长代谢旺盛可用于后续实验。碱水生境中,为缓解碱离子对鱼体造成的损伤,鳃细胞将启动渗透压胁迫应答机制。对LWG细胞进行碱胁迫实验并检测其细胞活性和功能后发现:LWG细胞在25~50 mmol/L NaHCO₃浓度下耐受3 h时,增殖率约60%并初现细胞凋亡。由此推断,在25~50 mmol/L的碳酸盐碱度胁迫下,LWG细胞具有强渗透压的碱度耐受性并促使细胞发生凋亡现象。对LWG细胞原代培养条件的摸索,为探索瓦氏雅罗鱼碱胁迫下渗透压应激响应机制和相关基因功能的验证提供相对稳定的实验材料。

Abstract:

In order to explore the regulation mechanism of alkali resistance of Leuciscus waleckii, a dominant natural species in an alkaline water environment, gill cells of Leuciscus waleckii were cultured by trypsin digestion in vitro, and the suitable conditions for primary and subculture cell culture were determined. The tolerance experiment was carried out to demonstrate its growth characteristics in an alkaline environment. The experimental results show that the primary gill cells of Leuciscus waleckii can be cultured in a DMEM medium containing 10% Fetal bovine serum(FBS) at 15 ℃, which can obtain a stable and good culture effect. It can be passaged within 36-72 h, and the stably passaged cells are named LWG cells. During subculture, LWG cells grew and metabolized vigorously at 36 h, which can be used for subsequent experiments. In alkaline water habitat, gill cells will start the osmotic pressure stress response mechanism to alleviate the damage caused by alkali ions to fish. Therefore, an alkali stress experiment on LWG cells was carried out and their cell activity and function were tasted. It was found that when LWG cells tolerated for three hours the concentration of 25-50 mmol/L NaHCO₃, proliferation rate was about 60% and apoptosis occurred. It can be inferred that LWG cells have strong osmotic alkalinity tolerance and promote apoptosis under carbonate alkalinity stress of 25-50 mmol/L. This will provide stable experimental materials for exploring osmotic stress response mechanisms and verifying related gene functions under alkali stress.