EGF和FGF-2对猪皮下脂肪神经嵴干细胞增殖和分化的影响

doi:10.16431/j.cnki.1671-7236.2022.12.035

Abstract

Abstract: 【Objective】 The aim of this study was to investigate the effects of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) on proliferation and differentiation of porcine subcutaneous adipose neural crest stem cells (NCSCs), so as to optimize the culture conditions of porcine subcutaneous adipose NCSCs.【Method】 The primary porcine subcutaneous adipose NCSCs were isolated and cultured in vitro, and the NCSCs marker p75 NTR was identified by immunofluorescence staining.The porcine subcutaneous adipose NCSCs were treated with different concentrations of EGF and FGF-2 (0 and 0, 10 and 10, 10 and 20, 20 and 10, 20 and 20, 30 and 30 ng/mL).The cell proliferation rate was tested by CCK-8 kit to determine the optimal concentration of EGF and FGF-2 for cell growth.The experiment was divided into control and optimal concentration groups.The cell growth curve of two groups was drawn, and the lipid droplet production of cells in two groups was compared by oil red O staining after the adipogenic induction.【Result】 The results of immunofluorescence staining showed that the primary porcine subcutaneous adipose NCSCs were positive by p75 NTR.The results of CCK-8 cell proliferation showed that EGF and FGF-2 had the best proliferative effect on porcine subcutaneous adipose NCSCs when the concentration of EGF and FGF-2 was 20 ng/mL, respectively.The results of growth curve showed that the cells in two groups were in logarithmic growth phase from 5th to 9th days, and the cell proliferation slowed down from 10th to 15th days, and gradually reached stagnation stage.The results of oil red O staining showed that the amount of lipid droplets in the cytoplasm of the optimal concentration group was much more than that of control group.【Conclusion】 Adding 20 ng/mL EGF and 20 ng/mL FGF-2 in culture medium could promote the proliferation and differentiation of porcine subcutaneous adipose NCSCs.

Key words: porcine subcutaneous adipose; epidermal growth factor (EGF); fibroblast growth factor 2 (FGF-2); neural crest stem cells (NCSCs)

CLC Number:

DONG Bowen, CUI Xiaozhen, BAI Rui, ZHENG Mingxue, ZHANG Li, LYU Xiaoling, REN Yuhong. Effects of EGF and FGF-2 on Proliferation and Differentiation of Porcine Subcutaneous Adipose Neural Crest Stem Cells[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4878-4886.

References

[1] HUANG X, SAINT-JEANNET J P.Induction of the neural crest and the opportunities of life on the edge[J].Developmental Biology, 2004, 275(1):1-11.
[2] NOCE M L, MELE L, TIRINO V, et al.Neural crest stem cell population in craniomaxillofacial development and tissue repair[J].European Cells & Materials, 2014, 28:348-357.
[3] MORT R L, JACKSON I J, PATTON E E.The melanocyte lineage in development and disease[J].Development, 2015, 142(7):620-632.
[4] STEMPLE D L, ANDERSON D J.Isolation of a stem cell for neurons and glia from the mammalian neural crest[J].Cell, 1992, 71(6):973-985.
[5] YANG S Y, BEAVAN M, CHAU K Y, et al.A human neural crest stem cell-derived dopaminergic neuronal model recapitulates biochemical abnormalities in GBA1 mutation carriers[J].Stem Cell Reports, 2017, 8(3):728-742.
[6] SALEHINEJAD P, ALITHEEN N B, MANDEGARY A, et al.Effect of EGF and FGF on the expansion properties of human umbilical cord mesenchymal cells[J].In Vitro Cellular & Developmental Biology-Animal, 2013, 49(7):515-523.
[7] PARK J, LEE J, SONG K D, et al.Growth factors improve the proliferation of Jeju Black pig muscle cells by regulating myogenic differentiation 1 and growth-related genes[J].Animal Bioscience, 2021, 34(8):1392-1402.
[8] 漆国栋, 漆伟, 江琼, 等.两种培养基对新生SD大鼠神经干细胞增殖的影响[J].医学理论与实践, 2020, 33(7):1037-1039. QI G D, QI W, JIANG Q, et al.Effects of two medium on proliferation of neonatal SD rats neural stem cells[J].Medical Theory & Practice, 2020, 33(7):1037-1039.(in Chinese)
[9] HENNENBERG M, SCHREIBER A, CIOTKOWSKA A, et al.Cooperative effects of EGF, FGF, and TGF-β1 in prostate stromal cells are different from responses to single growth factors[J].Life Sciences, 2015, 123:18-24.
[10] JIA T, JACQUET T, DALONNEAU F, et al.FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1- dependent axis that controls VEGFR1 splicing in endothelial cells[J].BMC Biololgy, 2021, 19(1):173.
[11] LEE T J, JANG J, KANG S, et al.Enhancement of osteogenic and chondrogenic differentiation of human embryonic stem cells by mesodermal lineage induction with BMP-4 and FGF2 treatment[J].Biochemical & Biophysical Research Communications, 2013, 430(2):793-797.
[12] KELLY C M, TYERS P, BORG M T, et al.EGF and FGF-2 responsiveness of rat and mouse neural precursors derived from the embryonic CNS[J].Brain Research Bulletin, 2005, 68(1-2):83-94.
[13] SAVCHENKO E, TEKU G N, BOZA-SERRANO A, et al.FGF family members differentially regulate maturation and proliferation of stem cell-derived astrocytes[J].Scientific Reports, 2019, 9(1):9610.
[14] 杨洁, 张菁华.神经嵴干细胞的研究进展[J].医学理论与实践, 2011, 24(17):2049-2050. YANG J, ZHANG J H.Research progress of neural crest stem cells[J].Journal of Medical Theory & Practice, 2011, 24(17):2049-2050.(in Chinese)
[15] 田霖, 孙筱放, 刘海波, 等.人脂肪干细胞的分离培养与生物学特性[J].中国组织工程研究, 2012, 16(32):5946-5952. TIAN L, SUN X F, LIU H B, et al.Isolation, culture and biological characteristics of human adipose-derived stem cells in vitro[J].Chinese Tissue Engineering Research, 2012, 16(32):5946-5952.(in Chinese)
[16] 赵燕翔, 王劲松, 王松灵.p75(NTR)对人牙髓干细胞神经分化影响的初步研究[J].北京口腔医学, 2018, 26(4):201-204. ZHAO Y X, WANG J S, WANG S L.The preliminary study of p75 neurotrophin receptor on the neural differention of dental pulp stem cells[J]. Beijing Journal of Stomatology, 2018, 26(4):201-204.(in Chinese)
[17] 杨琨, 李骏, 丰奇昊, 等.大鼠牙胚来源外胚间充质干细胞中p75 NTR时钟节律性表达[J].现代医药卫生, 2018, 34(20):3105-3108. YANG K, LI J, FENG Q H, et al.Circadian expression of p75 NTR in ectomesenchymal stem cells originated from rat teeth germ[J].Modern Medicine and Health, 2018, 34(20):3105-3108.(in Chinese)
[18] WISLET S, VANDERVELDEN G, ROGISTER B.From neural crest development to cancer and vice versa:How p75 NTR and (Pro)neurotrophins could act on cell migration and invasion?[J].Frontiers in Molecular Neuroscience, 2018, 11:244.
[19] VENTURI S, VENTURI M.Iodine in evolution of salivary glands and in oral health[J].Nutrition & Health, 2009, 20(2):119-134.
[20] 段步婷, 席柔, 李瑞琪, 等.不同浓度表皮生长因子(EGF)对鸡胚盲肠上皮细胞生长的影响[J].中国兽医杂志, 2019, 55(8):30-33. DUAN B T, XI R, LI R Q, et al.Different concentrations of epidermal growth factor (EGF) on chicken embryo cecum epithelial cell growth[J].Chinese Journal of Veterinary Medicine, 2019, 55(8):30-33.(in Chinese)
[21] 张磊, 纪洵敏, 苏志坚, 等.表皮生长因子对大鼠睾丸间质干细胞增殖和分化的影响研究[J].安徽医科大学学报, 2019, 54(10):1589-1593. ZHANG L, JI X M, SU Z J, et al.Effect of epidermal growth factor on proliferation and differentiation of rat Leydig cells[J].Journal of Anhui Medical University, 2019, 54(10):1589-1593.(in Chinese)
[22] KIM H S.Assignment1 of the human basic fibroblast growth factor gene FGF2 to chromosome 4 band q26 by radiation hybrid mapping[J].Cytogenetics and Cell Genetics, 1998, 83(1-2):73.
[23] LOU Z, LOU Z, JIANG Y, et al.FGF2 and EGF for the regeneration of tympanic membrane:A systematic review[J].Stem Cells International, 2021, 2021:2366291.
[24] 董高宏, 孟安锋, 王小霞, 等.碱性成纤维细胞生长因子对激光烧伤大兔皮肤愈合的影响分析[J].现代生物医学进展, 2021, 21(22):4211-4216. DONG G H, MENG A F, WANG X X, et al.Analysis of the effect of basic fibroblast growth factor on the skin healing of laser burned rabbits[J].Modern Biomedical Progress, 2021, 21(22):4211-4216.(in Chinese)
[25] 王玉聪, 张前法, 付有伟.不同浓度碱性成纤维细胞生长因子对体外培养兔肌腱细胞增殖的影响[J].临床骨科杂志, 2012, 15(4):462-465. WANG Y C, ZHANG Q F, FU Y W.Effect of basic fibroblast growth factor in different concentration on proliferation of tendon cells cultured in vitro[J].Journal of Clinical Orthopedics, 2012, 15(4):462-465.(in Chinese)
[26] 周琦琪, 韩祥祯, 张文静, 等.碱性成纤维细胞生长因子诱导大鼠骨髓间充质干细胞膜片成骨及成血管分化[J].中国组织工程研究, 2021, 25(1):1-5. ZHOU Q Q, HAN X Z, ZHANG W J, et al.Basic fibroblast growth factor induces osteogenic and angiogenic differentiation of rat bone marrow mesenchymal stem cell patches[J].Tissue Engineering Research in China, 2021, 25(1):1-5.(in Chinese)
[27] CHEN S, WANG M, CHEN X, et al.In vitro expression of cytokeratin 19 in adipose-derived stem cells is induced by epidermal growth factor[J].Medical Science Monitor, 2018, 24:4254-4261.
[28] CHOI K H, YOON J W, KIM M, et al.Optimization of culture conditions for maintaining pig muscle stem cells in vitro[J].Food Science of Animal Resources, 2020, 40(4):659-667.
[29] MOSSAHEBI-MOHAMMADI M, QUAN M, ZHANG J S, et al.FGF signaling pathway:A key regulator of stem cell pluripotency[J].Frontiers in Cell and Developmental Biology, 2020, 8:79.
[30] 周化腾, 李长明, 王利祥, 等.人尿液细胞来源的诱导多能干细胞定向分化为毛囊干细胞[J].基础医学与临床, 2020, 40(11):1468-1473. ZHOU H T, LI C M, WANG L X, et al.Differentiation of human urine-derived iPSCs into hair follicle stem cells[J].Basic Medical Sciences and Clinics, 2020, 40(11):1468-1473.(in Chinese)
[31] EOM Y W, OH J E, LEE J I, et al.The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells[J].Biochemical & Biophysical Research Communications, 2014, 445(1):16-22.
[32] LI J, LV Y, WANG H, et al.Cardiomyocyte-like cell differentiation by FGF-2 transfection and induction of rat bone marrow mesenchymal stem cells[J]. Tissue Cell, 2021, 73:101665.
[33] CHEN L, CARLTON M, CHEN X, et al.Effect of fibronectin, FGF-2, and BMP4 in the stemness maintenance of BMSCs and the metabolic and proteomic cues involved[J].Stem Cell Research & Therapy, 2021, 12(1):165.

Effects of EGF and FGF-2 on Proliferation and Differentiation of Porcine Subcutaneous Adipose Neural Crest Stem Cells

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