背景:Labeorohita代表了印度水产养殖中最主要的鱼类,鱼类细胞系已被用作进行各种生物学研究的出色体外平台。
结果:从L.rohita的肌肉组织开发的LRM细胞培养物用于研究体外应用。在28℃的温度下,将发育的肌肉细胞维持在补充有10%FBS(胎牛血清)和10ng/mlbFGF的Leibovitzs-15(L-15)中。LRM细胞显示成纤维细胞样形态,并通过测序线粒体基因16SrRNA进行鉴定。在不同阶段的LRM细胞中研究了生肌调节因子(MRFs)的表达;然而,表达方式在不同的段落有所不同。MEF2A,Mrf-4和Myogenin在第25代表达较高,而MyoD在第15代表达最大,Myf-5在第1代表达最高。LRM细胞的转染效率揭示了使用pmaxGFP载体DNA的GFP表达的14%。LRM细胞对由嗜水气单胞菌和爱德华氏杆菌制备的细胞外产物敏感。六种重金属(Hg,Cd,Zn,Cu,Pb,与从MTT和NR测定获得的IC50值相比,通过剂量依赖性方式在LRM细胞中评估Ni)。当使用液氮在-196°C下冷冻保存LRM细胞时,达到70-75%的恢复率。
结论:开发的肌肉细胞可作为毒理学和生物技术研究的功能性体外工具。
BACKGROUND: Labeo rohita represents the most dominant fish species in Indian aquaculture and the fish cell lines have been used as an excellent in vitro platform for performing various biological research.
RESULTS: The LRM cell culture developed from the muscle tissue of L. rohita was used to study the in vitro applications. The developed muscle cells were maintained in a Leibovitz\'s-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF at 28 oC temperature. The LRM cells showed fibroblastic-like morphology and was authenticated by sequencing mitochondrial gene 16S rRNA. The expression of myogenic regulatory factors (MRFs) was studied in different stages of LRM cells; however, the expression patterns varied at different passages. The MEF2A, Mrf-4, and Myogenin expressions were higher in passage 25, while the expression of MyoD was maximum in passage 15, and the expression of Myf-5 was highest in passage 1. The transfection efficiency of LRM cells revealed 14 % of the GFP expression with a pmaxGFP vector DNA. The LRM cells were susceptible to the extracellular products prepared from Aeromonas hydrophilla and Edwardsiella tarda. The acute cytotoxicity of six heavy metals (Hg, Cd, Zn, Cu, Pb, Ni) was assessed in LRM cells by a dose-dependent manner in comparison to IC50 values obtained from MTT and NR assays. A revival rate of 70-75% was achieved when the LRM cells were cryopreserved at - 196 °C using liquid nitrogen.
CONCLUSIONS: The developed muscle cells serve as an functional in vitro tool for toxicological and biotechnological studies.