牦牛皮肤和肌肉成纤维细胞系的建立与比较分析

doi:10.7668/hbnxb.20194332

华北农学报 ›› 2024, Vol. 39 ›› Issue (S1): 274-280. doi: 10.7668/hbnxb.20194332

所属专题： 畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

牦牛皮肤和肌肉成纤维细胞系的建立与比较分析

徐红梅¹^,²^,³, 海欣怡¹^,²^,³, 李悦悦¹^,²^,³, 李巧¹^,²^,³, 马梓峰¹^,²^,³, 熊燕¹^,²^,³, 熊显荣¹^,²^,³, 兰道亮¹^,²^,³, 李键¹^,²^,³, 付伟¹^,²^,³

¹ 西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都 610041
² 西南民族大学青藏高原动物遗传资源保护与利用四川省重点实验室,四川成都 610041
³ 西南民族大学动物科学国家民委重点实验室,四川成都 610041

收稿日期:2024-04-25 出版日期:2025-01-24
通讯作者:
付伟(1989-),男,四川资阳人,讲师,博士,主要从事动物繁育与胚胎工程研究。
作者简介:
徐红梅(1999-),女,黑龙江大庆人,在读硕士,主要从事动物遗传育种与繁殖研究。
基金资助:
国家重点研发计划项目(2021YFD1600200); 四川省科技计划项目(2024YFTX0019); 甘孜州“校州合作”项目(横20240054)

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

XU Hongmei¹^,²^,³, HAI Xinyi¹^,²^,³, LI Yueyue¹^,²^,³, LI Qiao¹^,²^,³, MA Zifeng¹^,²^,³, XIONG Yan¹^,²^,³, XIONG Xianrong¹^,²^,³, LAN Daoliang¹^,²^,³, LI Jian¹^,²^,³, FU Wei¹^,²^,³

¹ Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education,Southwest Minzu University,Chengdu 610041,China
² Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province,Southwest Minzu University,Chengdu 610041,China
³ Key Laboratory for Animal Science of National Ethnic Affairs Commission of China,Southwest Minzu University,Chengdu 610041,China

Received:2024-04-25 Published:2025-01-24

摘要/Abstract

摘要：

旨在建立牦牛肌肉和皮肤成纤维细胞系,并分析比较两者的形态、生长、标志基因和蛋白的差异,为更好地了解和研究牦牛相关特性提供材料。首先,从妊娠3个月的牦牛胎儿分离皮肤和肌肉组织块,分别采用组织块法和酶解法培养其原代细胞,观察其生长状态和形态特点;之后,利用CCK8法检测皮肤成纤维细胞和肌肉成纤维细胞的增殖活力,并对2种细胞进行免疫荧光染色分析;最后,利用RT-qPCR分析2种细胞中成纤维细胞标志基因的表达。结果显示,酶解法比组织块法更容易获得胎牦牛皮肤和肌肉成纤维细胞,胎牦牛皮肤成纤维细胞的活力显著高于肌肉成纤维细胞(P<0.05),且皮肤成纤维细胞中标志蛋白VIMENTIN的荧光强度显著高于肌肉成纤维细胞,但二者组蛋白修饰H3K27me3的差异不显著;VIMENTIN和S100A4基因在皮肤成纤维细胞中显著高表达,而FAP和SMA基因在肌肉成纤维细胞中显著高表达,与免疫荧光染色结果一致。综上,建立了牦牛胎儿皮肤和肌肉成纤维细胞系,并通过CCK8、免疫荧光染色和RT-qPCR等方法比较了2种细胞的生理特性。

关键词: 牦牛, 胎儿成纤维细胞, 分离培养, 细胞活力, 免疫荧光染色

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

徐红梅, 海欣怡, 李悦悦, 李巧, 马梓峰, 熊燕, 熊显荣, 兰道亮, 李键, 付伟. 牦牛皮肤和肌肉成纤维细胞系的建立与比较分析[J]. 华北农学报, 2024, 39(S1): 274-280. doi: 10.7668/hbnxb.20194332.

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.

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

图/表 10

表1 RT-qPCR引物信息

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

图1 组织块法分离牦牛胎儿皮肤成纤维细胞

Fig.1 Isolation of yak fetal fibroblasts by tissue method

图2 酶解法分离牦牛胎儿皮肤成纤维细胞

Fig.2 Isolation of yak fetal fibroblasts by enzymatic method

图3 牦牛皮肤和肌肉成纤维细胞

Fig.3 Yak skin and muscle fibroblasts

图4 CCK8检测成纤维细胞增殖情况不同小写字母表示差异显著(P<0.05)。图6,8,9同。

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

图5 VIMENTIN蛋白的免疫荧光染色

Fig.5 Immunofluorescence staining of VIMENTIN protein

图6 成纤维细胞VIMENTIN蛋白的荧光强度分析

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

图7 组蛋白H3K27me3的免疫荧光染色

Fig.7 Immunofluorescence staining of histone H3K27me3

图8 成纤维细胞H3K27me3的荧光强度分析

Fig.8 Fluorescence intensity analysis of fibroblast H3K27me3

图9 RT-qPCR检测成纤维标志基因在皮肤和肌肉成纤维细胞中的相对表达量

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

参考文献 26

[1]	Plikus M V, Wang X J, Sinha S, Forte E, Thompson S M, Herzog E L, Driskell R R, Rosenthal N, Biernaskie J, Horsley V. Fibroblasts:origins,definitions,and functions in health and disease[J]. Cell, 2021, 184(15):3852-3872.doi:10.1016/j.cell.2021.06.024.
[2]	王玉玲, 张露露, 周珊, 蒋涛, 兰道亮, 吉文汇. 牦牛心脏原代成纤维细胞系的建立及鉴定[J]. 西南民族大学学报(自然科学版), 2022, 48(2):119-124.doi:10.11920/xnmdzk.2022.02.001.
	Wang Y L, Zhang L L, Zhou S, Jiang T, Lan D L, Ji W H. Establishment and identification of yak heart primary fibroblast cell line[J]. Journal of Southwest Minzu University (Natural Science Edition), 2022, 48(2):119-124.
[3]	Machado L S, Borges C M, de Lima M A, Sangalli J R, Therrien J, de Figueiredo Pessôa L V, Fantinato Neto P, Perecin F, Smith L C, Meirelles F V, Bressan F F. Exogenous OCT4 and SOX2 contribution to in vitro reprogramming in cattle[J]. Biomedicines, 2023, 11(9):2577.doi:10.3390/biomedicines11092577.
[4]	Stafa K, Rella A, Eagle W, Dong K, Morris K, Layman D, Corallo K, Trivero J, Maidhof R, Goyarts E, Pernodet N. MiR-146a is a critical target associated with multiple biological pathways of skin aging[J]. Frontiers in Physiology, 2024, 15:1291344.doi:10.3389/fphys.2024.1291344.
[5]	耿尧. 牦牛皮肤结构与功能研究[D]. 兰州: 兰州大学, 2006.doi:10.7666/d.Y917686.
	Geng Y. Study on structure and function of the skin of yaks(Bos Gruniens)[D]. Lanzhou: Lanzhou University, 2006.
[6]	Wang M, Li H J, Zhang X H, Yang L, Liu Y, Liu S Q, Sun Y J, Zhao C J. An analysis of skin thickness in the Dezhou donkey population and identification of candidate genes by RNA-seq[J]. Animal Genetics, 2022, 53(3):368-379.doi:10.1111/age.13196.
[7]	刘通, 王恒, 李运生, 张运海. 克隆山羊成纤维细胞系的建立与生物学特性的研究[J]. 华北农学报, 2015, 30(5):77-82.doi:10.7668/hbnxb.2015.05.013.
	Liu T, Wang H, Li Y S, Zhang Y H. Establishment and biological characteristics of fibroblast cells derived from somatic cloned goat[J]. Acta Agriculturae Boreali-Sinica, 2015, 30(5):77-82. doi: 10.7668/hbnxb.2015.05.013
[8]	Gharbia F Z, Abouhashem A S, Moqidem Y A, Elbaz A A, Abdellatif A, Singh K, Sen C K, Azzazy H M E. Adult skin fibroblast state change in murine wound healing[J]. Scientific Reports, 2023, 13(1):886.doi:10.1038/s41598-022-27152-4. pmid: 36650180
[9]	布嘎. 西藏地区牦牛养殖问题及对策[J]. 畜禽业, 2021, 32(12):102-103.doi:10.19567/j.cnki.1008-0414.2021.12.048.
	Bu G. Problems and countermeasures of yak breeding in Tibet[J]. Livestock and Poultry Industry, 2021, 32(12):102-103.
[10]	Meng G Y, La Y F, Bao Q, Wu X Y, Ma X M, Huang C, Chu M, Liang C N, Yan P. Early growth and development and nonlinear model fitting analysis of ashidan yak[J]. Animals, 2023, 13(9):1545.doi:10.3390/ani13091545.
[11]	Shah A M, Bano I, Qazi I H, Matra M, Wanapat M. The Yak-a remarkable animal living in a harsh environment:an overview of its feeding,growth,production performance,and contribution to food security[J]. Frontiers in Veterinary Science, 2023, 10:1086985.doi:10.3389/fvets.2023.1086985.
[12]	任子利, 赵彦玲, 王建洲, 李瑜鑫, 强巴央宗. 西藏牦牛胎儿成纤维细胞的分离与培养[J]. 江苏农业科学, 2011, 39(3):42-45.doi:10.15889/j.issn.1002-1302.2011.03.208.
	Ren Z L, Zhao Y L, Wang J Z, Li Y X, Qiang B. Isolation and culture of fetal fibroblasts in Tibetan yak[J]. Jiangsu Agricultural Sciences, 2011, 39(3):42-45.
[13]	杨玉莹, 荆海霞, 董宝霞, 张勤文. 牦牛乳腺上皮细胞的体外分离培养及鉴定[J]. 动物医学进展, 2022, 43(2):64-69.doi:10.16437/j.cnki.1007-5038.2022.02.021.
	Yang Y Y, Jing H X, Dong B X, Zhang Q W. Isolation,culture and identification of yak mammary epithelial cells in vitro[J]. Progress in Veterinary Medicine, 2022, 43(2):64-69.
[14]	丁生财, 陈意生, 魏泓, 刘萍. 猪皮肤成纤维细胞的培养与鉴定[J]. 第三军医大学学报, 2004, 26(13):1171—1174.doi:10.3321/j.issn:1000-5404.2004.13.014.
	Ding S C, Chen Y S, Wei H, Liu P. Cultivation and identification of porcine skin fibroblasts[J]. Journal of Third Military Medical University, 2004, 26(13):1171-1174.
[15]	于玲珠, 刘娣, 孙兴参, 张冬杰, 梁洋, 杨秀芹, 于浩, 高文学. 猪耳皮肤成纤维细胞的培养[J]. 细胞生物学杂志, 2003, 25(4):258-260.doi:10.3969/j.issn.1674-7666.2003.04.016.
	Yu L Z, Liu D, Sun X C, Zhang D J, Liang Y, Yang X Q, Yu H, Gao W X. Culture of porcine ear skin fibroblasts[J]. Chinese Journal of Cell Biology, 2003, 25(4):258-260.
[16]	刘赫. 牛成纤维细胞系的建立、体外受精及核移植的研究[D]. 哈尔滨: 东北农业大学, 2008.doi:10.7666/d.Y1403807.
	Liu H. The research on the establishment of bovine fibroblast cell line and in vitro fertilization and nuclear transfer of bovine[D]. Harbin: Northeast Agricultural University, 2008.
[17]	李小英. 简述西藏牦牛胎儿成纤维细胞的分离与培养[J]. 中国科技信息, 2014(7):187-188.doi:10.3969/j.issn.1001-8972.2014.07.065.
	Li X Y. Isolation and culture of fetal fibroblasts from Tibetan yak[J]. China Science and Technology Information, 2014(7):187-188.
[18]	杨素芳, 汪彩珠, 陈凌声, 覃兆鲜, 黄雅琼, 石德顺. 广西巴马香猪耳皮成纤维细胞的分离与培养[J]. 湖北农业科学, 2009, 48(3):547-552.doi:10.3969/j.issn.0439-8114.2009.03.012.
	Yang S F, Wang C Z, Chen L S, Qin Z X, Huang Y Q, Shi D S. Dissociation and culture of ear skin fibroblasts in Guangxi Bama miniature pigs[J]. Hubei Agricultural Sciences, 2009, 48(3):547-552.
[19]	韩瑞, 孙永琨. 大鼠乳鼠皮肤成纤维细胞的分离和体外培养[J]. 新乡医学院学报, 2016, 33(2):96-100.doi:10.7683/xxyxyxb.2016.02.004.
	Han R, Sun Y K. Separation and culture of skin fibroblasts of newborn rat in vitro[J]. Journal of Xinxiang Medical University, 2016, 33(2):96-100.
[20]	Vitali T, Sanchez-Alvarez R, Witkos T M, Bantounas I, Cutiongco M F A, Dudek M, Yan G H, Mironov A A, Swift J, Lowe M. Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex[J]. Journal of Cell Science, 2023, 136(20):jcs260577.doi:10.1242/jcs.260577.
[21]	Babadag S, Altundag-Erdogan Ö, Akkaya-Ulum Y Z, Çelebi-Saltik B. Evaluation of tumorigenic properties of MDA-MB-231 cancer stem cells cocultured with telocytes and telocyte-derived mitochondria following miR-146a inhibition[J]. DNA and Cell Biology, 2024, 43(7):341-352.doi:10.1089/dna.2024.0031.
[22]	罗惠娜, 罗冬章, 詹小舒, 陈胜锋, 陈志胜, 王丙云. 金毛犬睾丸成纤维细胞的分离、培养及鉴定[J]. 黑龙江畜牧兽医, 2020(11):140-143.doi:10.13881/j.cnki.hljxmsy.2019.06.0104.
	Luo H N, Luo D Z, Zhan X S, Chen S F, Chen Z S, Wang B Y. Isolation,culture and identification of testicular fibroblasts in golden retriever[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020(11):140-143.
[23]	Qin H Y, Li M D, Xie G F, Cao W, Xu D X, Zhao H, Fu L. Associations among S100A4,sphingosine-1-phosphate,and pulmonary function in patients with chronic obstructive pulmonary disease[J]. Oxidative Medicine and Cellular Longevity, 2022(1): 6041471.doi:10.1155/2022/6041471.
[24]	Goldsmith E C, Bradshaw A D, Zile M R, Spinale F G. Myocardial fibroblast-matrix interactions and potential therapeutic targets[J]. Journal of Molecular and Cellular Cardiology, 2014, 70:92-99.doi:10.1016/j.yjmcc.2014.01.008. pmid: 24472826
[25]	李亮, 张传山, 毕晓娟, 马洋洋, 王慧, 吐尔干艾力·阿吉, 林仁勇. miR-133a-3p重组腺相关病毒的构建及抑制LX-2细胞α-SMA基因表达[J]. 中国病原生物学杂志, 2019, 14(6):625-628.doi:10.13350/j.cjpb.190602.
	Li L, Zhang C S, Bi X J, Ma Y Y, Wang hui, Tuerganaili A J, Lin R Y. Construction of AAV8 that over expresses miR-133a-3p and its inhibitory effect on α-SMA expression in LX2 cells[J]. Journal of Pathogen Biology, 2019, 14(6):625-628.
[26]	Fearon D T. The carcinoma-associated fibroblast expressing fibroblast activation protein and escape from immune surveillance[J]. Cancer Immunology Research, 2014, 2(3):187-193.doi:10.1158/2326-6066.CIR-14-0002. pmid: 24778314

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