Establishment and characterization of Yellow River carp (Cyprinus carpio haematopterus) muscle cell line and its application to fish virology and immunology

by Selleckchem | Sep 09 2023

The Yellow River carp (Cyprinus carpio haematopterus) is a vital economically farmed fish of the Cyprinidae family. With the development of intensive aquaculture, carp production has increased dramatically, leading to the frequent occurrence of various diseases. Cell lines are considered the most cost-effective resource for in vitro studies and are widely used for physiological and pathological studies because of accessibility and convenience. This research established a novel immortal cell line CCM (Yellow River carp muscle cells) derived from the carp muscle. CCM has been passed over 71 generations for 1 year. The morphology of CCM and the adhesion and extension processes were captured by light and electron microscopy. CCM were passaged every 3 days with 20% FBS DMEM/F12 at 1:3. The optimum conditions for CCM growth were 28 °C and 20% FBS concentration. DNA sequencing of 16S rRNA and COI showed that CCM was derived from carp. CCM positively reacts to anti-PAX7 and anti-MyoD antibodies of carp. Analysis of chromosomes revealed that the chromosomal pattern number of CCM was 100. Transfection experiment demonstrated that CCM might be utilized to express foreign genes. Furthermore, cytotoxicity testing showed that CCM was susceptible to Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii, and Staphylococcus Aureus. The organophosphate pesticides (chlorpyrifos and glyphosate) or heavy metals (Hg, Cd, and Cu) exhibited dose-dependent cytotoxicity against CCM. After LPS treatment, the MyD88-IRAKs-NFκB pathway stimulates inflammatory-related factor il1β, il8, il10, and nfκb expression. LPS did not seem to cause oxidative stress in CCM, and the expression of cat and sod was not affected. Poly (I:C) through TLR3-TRIF-MyD88-TRAF6-NFκB and TRIF-TRAF3-TBK1-IRF3 activated the transcription of related factors, increased expression of anti-viral protein, but no changes in apoptosis-related genes. To our knowledge, this is the first muscle cell line in Yellow River carp and the first study on the immune response signal pathways of Yellow River carp based on the muscle cell line. CCM cell line provides a more rapid and efficient experimental material for fish immunology research, and this study preliminarily elucidated its immune response strategy to LPS and poly (I:C).

Comments:

The research described in your statement establishes a novel immortal cell line called CCM (Yellow River carp muscle cells) derived from the muscle of Yellow River carp (Cyprinus carpio haematopterus). This cell line has been maintained for 1 year and passed over 71 generations. The researchers used light and electron microscopy to observe the morphology of CCM and the adhesion and extension processes. CCM cells were cultured in DMEM/F12 medium supplemented with 20% fetal bovine serum (FBS) and passaged every 3 days.

The optimal conditions for CCM growth were found to be a temperature of 28 °C and a 20% concentration of FBS. DNA sequencing of 16S rRNA and COI confirmed that CCM was derived from carp. Immunostaining with anti-PAX7 and anti-MyoD antibodies showed positive reactions in CCM, indicating their origin from carp muscle cells. Chromosome analysis revealed that CCM cells had a chromosomal pattern number of 100. Additionally, transfection experiments demonstrated that CCM cells could be used to express foreign genes.

Cytotoxicity testing showed that CCM cells were susceptible to certain bacterial strains such as Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii, and Staphylococcus aureus. Moreover, exposure to organophosphate pesticides (chlorpyrifos and glyphosate) or heavy metals (Hg, Cd, and Cu) resulted in dose-dependent cytotoxicity in CCM cells.

In terms of immune response, the researchers investigated the effects of lipopolysaccharide (LPS) and poly (I:C) on CCM cells. After LPS treatment, the MyD88-IRAKs-NFκB pathway was activated, leading to the upregulation of inflammatory-related factors such as il1β, il8, il10, and nfκb expression. However, LPS did not induce oxidative stress in CCM cells, as the expression of cat (catalase) and sod (superoxide dismutase) was not affected.

Poly (I:C) stimulation activated two distinct pathways: TLR3-TRIF-MyD88-TRAF6-NFκB and TRIF-TRAF3-TBK1-IRF3. This resulted in the transcriptional upregulation of related factors and increased expression of antiviral proteins. However, there were no changes observed in apoptosis-related genes.

Overall, this research establishes the CCM cell line as a valuable tool for studying fish immunology, specifically in the context of Yellow River carp. It provides a more efficient and rapid experimental material for investigating the immune response strategies of this fish species, as demonstrated by the findings related to LPS and poly (I:C) stimulation. This study represents the first muscle cell line established in Yellow River carp and the first investigation of immune response signal pathways based on a muscle cell line in this species.