Establishment and Comparative Analysis of Yak Skin and Muscle Fibroblast Cell Lines

doi:10.7668/hbnxb.20194332

Abstract

Abstract:

The aim of this study was to establish yak muscle and skin fibroblast cell lines,analyze and compare the differences in morphology,growth,marker genes and proteins between two cell lines to provide materials for a better understanding of yak properties.Firstly,the skin and muscle tissue blocks were isolated from the yak fetus at 3 months of gestation,and the primary cells were cultured by tissue block method and enzymatic hydrolysis method respectively,and their growth status and morphological characteristics were observed.Following,the CCK8 method to detect the proliferation viability of skin and muscle fibroblasts,and immunofluorescence staining analysis was performed on the two cells.Finally,RT-qPCR was used to analyze the expression of fibroblast marker genes in the two kinds of cells.It was found that the enzymatic method was easier to obtain fibroblasts than the tissue method,and the viability of skin fibroblasts was significantly higher than that of muscle fibroblasts(P<0.05).The fluorescence intensity of the marker protein VIMENTIN in skin fibroblasts was significantly higher than that of muscle fibroblasts,but the histone-modified H3K27me3 was not significant between the two kinds of cells.Furthermore,VIMENTIN and S100A4 genes were significantly highly expressed in skin fibroblasts,while FAP and SMA genes were significantly high expressed in muscle fibroblasts,consisting with immunofluorescent staining results.In conclusion,it successfully established yak fetal skin and muscle fibroblast cell lines,and compared their physiological properties of two kinds of cells by using CCK8,immunofluorescence staining and RT-qPCR.

Key words: Yak, Fetal fibroblast, Isolation and culture, Cell viability, Immunofluorescence staining

XU Hongmei, HAI Xinyi, LI Yueyue, LI Qiao, MA Zifeng, XIONG Yan, XIONG Xianrong, LAN Daoliang, LI Jian, FU Wei. Establishment and Comparative Analysis of Yak Skin and Muscle Fibroblast Cell Lines[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 274-280. doi: 10.7668/hbnxb.20194332.

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http://www.hbnxb.net/EN/Y2024/V39/IS1/274

Figures/Tables 10

Tab.1 RT-qPCR primer information

基因 Genes	引物序列(5'-3') Primer sequences(5'-3')	产物长度/bp Product size
VIMENTIN	F: CGCTCAAAGGGACTAACGAG R: TCCAGCAGCTTCCTGTAGGT	220
α-SMA	F: GGTGATGAAGCACAAAGCAA R: TGAGAAGGGTTGGATGCTCT	154
S100A4	F: AAAGTGAAGGCTCCTCAGGTGT R: GTTCAGTATGGTGCTCACGTCT	115
FAP	F: GATTCATGGGCCTCCCAACA R: TGGTCCGAGTACCACATTGC	208
GAPDH	F:ACAGTCAAGGCAGAGAACGG R: GGTTCACGCCCATCACAAAC	235

Fig.1 Isolation of yak fetal fibroblasts by tissue method

Fig.2 Isolation of yak fetal fibroblasts by enzymatic method

Fig.3 Yak skin and muscle fibroblasts

Fig.4 CCK8 detects fibroblastic proliferation Different small letters indicate significant differences(P<0.05).The same as Fig.6,8,9.

Fig.5 Immunofluorescence staining of VIMENTIN protein

Fig.6 Fluorescence intensity analysis of VIMENTIN protein in the fibroblasts

Fig.7 Immunofluorescence staining of histone H3K27me3

Fig.8 Fluorescence intensity analysis of fibroblast H3K27me3

Fig.9 Relative expression of marker genes in skin and muscle fibroblasts detected by RT-qPCR

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