Effect Of BFGF On Fibroblasts Derived From The Golden Snub-nosed Monkey

Golden snub nosed monkey(Rhinopithecus roxellana)as an endangered wild animal in China is listed in The Chinese red list of endangered animal species and The Convention on International Trade in Endangered Species of Wild Fauna and Flora.In order to golden snub-nosed monkey(Rhinopithecus roxellana)in China,many natural reserves have been set up.However,the number of golden snub-nosed monkeys is declining In addition to macro protection methods,seeking other methods to protect the population number and ecological diversity of Sichuan golden monkey has aroused the interest of researchers at home and abroad.In recent years,the protection of Sichuan snub nosed monkey germplasm resources tends to the macro aspect,but the micro research on cell level is very few,and cell resources are just the important genetic resources to protect endangered animals.In this study,we investigated the effects of basic fibroblast growth factor(bFGF)on the in vitro culture,growth and differentiation,gene expression,mitochondrial membrane potential and transcriptome analysis of the ear edge fibroblasts of Rhinopithecus roxellana.(1)The fibroblasts of Rhinopithecus roxellana were isolated and cultured in vitro.A large number of primary cells were obtained from the dead adult male Sichuan golden monkey by tissue block culture.The fibroblasts of Sichuan golden monkey were cultured for 7 days.At the initial stage of culture,the fibroblasts maintained a typical long fusiform shape,with full cytoplasm and oval nucleus.However,when cultured to Passage 10,the fibroblasts showed a tendency of degeneration and fibrillation The results showed that with the increase of subculture times,the activity of cells in Passage 1 was higher than that in Passage 4 and Passage 10,and the activity of cells in P1 passage was statistically higher than that in Passage 4 and Passage 10 There was no significant difference in cell viability between Passage 4and Passage 10(P > 0.05);(2)BFGF was used to treat fibroblasts from the ear margin of Rhinopithecus roxellana.In this study,we used bFGF,a small molecular compound,to treat Sichuan golden monkey ear edge fibroblasts.After treatment,Sichuan golden monkey fibroblasts continued to maintain long fusiform,and CCK8 kit was used to detect the cell viability.After bFGF treatment,the cell viability of Sichuan golden monkey fibroblasts in the experimental group was significantly higher than that of the control group(P < 0.05).The level of integrin ? 1(ITG ? 1)in the experimental group was significantly increased(P < 0.05)in both m RNA level and protein optical density(P < 0.05).The results of immunofluorescence staining showed that the fluorescence intensity of ITG ? 1 in the experimental group was higher than that in the control group,indicating that the activity of ITG ? 1 was maintained after bFGF treatment;Compared with the control group,1668 genes were up-regulated and1414 genes were down-regulated in the experimental group,and the related signal pathways were enriched in P13 K and MAPK signaling pathways,and WNT signaling pathway was activated synergistically to keep fibroblasts alive and proliferate;(3)Compared with the control group,1668 genes were up-regulated and 1414 genes were down-regulated in the experimental group,and the related signal pathways were enriched in P13 K and MAPK signaling pathways.What's more,Wnt signaling pathway was activated synergistically and fibroblasts maintained ability of proliferation;(4)After bFGF treatment,there were differences in cell viability between Passage 3and Passage 8,but the difference was not significant.At the later stage of cell culture,fibroblasts successfully expressed fibroblast markers prrx1,Twist2,esrrb,vimentin,FSP1 and FBN1.The expression levels of esrrb,FBN1 and prrx1 in Passage 8 were significantly higher than those in Passage 3(P < 0.05).The expression levels of Twist2,vimentin and FSP1 were different,but the differences were not statistically significant(P > 0.05).The expression levels of pnpt1,TIM23 and cyto C in Passage 3 were significantly lower than those in Passage 8(P < 0.05).There was no significant difference in mitochondrial membrane potential between Passage 3 and Passage 8 indicating that fibroblasts treated with bFGF maintained vitality and had a tendency to differentiate into hematopoietic cell lines.In this study,primary fibroblasts were cultured in vitro and bFGF treated with small molecules.After bFGF treatment,the ear edge fibroblasts of Sichuan golden monkey can not only maintain its classic long fusiform morphology,maintain cell morphology,but also increase cell viability.In the experimental group,the genes expressed in hematopoietic stem cells and mesenchymal stem cells were also found in the experimental group,which provides a reference for the potential of fibroblasts to differentiate into pluripotent stem cells.