RESUMEN
The fibroblast growth factor 21 (FGF21) gene plays an important role in the mechanism of glucose and lipid metabolism and is a promising therapeutic target for metabolic disease. Camels display a unique regulation characteristic of glucose and lipid metabolism, endowing them with the ability to adapt to survive drought and chronic hunger. However, the knowledge about the camel FGF21 gene regulation and its differences between humans and mice is still limited. In this study, camel FGF21 gene promoter was obtained for ~2000 bp upstream of the transcriptional start site (TSS). Bioinformatics analysis showed that the proximal promoter region sequences near the TSS between humans and camels have high similarity. Two potential core active regions are located in the -445-612 bp region. In addition, camel FGF21 promoter contains three CpG islands (CGIs), located in the -435~-1168 bp regions, significantly more and longer than in humans and mice. The transcription factor binding prediction showed that most transcription factors, including major functional transcription factors, are the same in different species although the binding site positions in the promoter are different. These results indicated that the signaling pathways involved in FGF21 gene transcription regulation are conservative in mammals. Truncated fragments recombinant vectors and luciferase reporter assay determined that camel FGF21 core promoter is located within the 800 bp region upstream of the TSS and an enhancer may exist between the -1000 and -2000 bp region. Combining molecular docking and in silico ADMET druggability prediction, two compounds were screened as the most promising candidate drugs specifically targeting FGF21. This study expanded the functions of these small molecules and provided a foundation for drug development targeting FGF21.
RESUMEN
Immortalized cell lines with many advantages are widely used in various experimental contexts by many different labs. However, the absence of available cell lines poses difficulties for research in some species, such as camels. To establish an immortalized Bactrian camel fibroblast (iBCF) cell line and understand its biological characteristics, primary fibroblast cells from Bactrian camels were isolated and purified using enzymatic digestion in this study, and telomerase reverse transcriptase (hTERT) vectors were introduced into primary BCF (pBCF) for continuous passage to 80 generations after screening with G418. The cell morphology of different generations was examined under a microscope. Cell cycle and viability were evaluated by flow cytometry and CCK-8 assay, respectively. Cellular genes expression was monitored by qPCR, immunofluorescence, and Western blot, respectively. Chromosomes were determined by karyotyping. The results showed that like most other cells, both pBCF and iBCF were sensitive to nutrient concentrations and adapted to culture in the medium with 4.5 g/L glucose and 10% fetal bovine serum (FBS) concentration. hTERT gene was introduced and stably expressed in iBCF cells, which promoted BCF cell immortalization. The fibroblast specific marker vimentin (VIM) is expressed in both pBCF and iBCF, but epithelial marker cytokeratin18 (CK18) expression is weak in BCF cells. Proliferation and viability detection showed that hTERT-induced iBCF exhibits faster growth rates and higher viability than pBCF. Karyotyping showed that iBCF maintained the same number and morphology of chromosomes as the pBCF. This study demonstrated that we have successfully constructed an immortalized Bactrian camel fibroblast cell line, which was named BCF23. The establishment of the BCF23 cell line provides a foundation for expanding camel-related research.