VEGF对体外培养绒山羊次级毛囊外根鞘细胞的影响

doi:10.11843/j.issn.0366-6964.2018.06.004

摘要/Abstract

摘要：

本研究旨在探讨陕北白绒山羊毛囊外根鞘细胞的培养和VEGF对次级毛囊外根鞘细胞增殖、分化及细胞中增殖细胞核抗原（PCNA）、角蛋白10（K10）和成纤维细胞生长因子5（FGF 5） mRNA表达量的影响。采集绒山羊体侧部皮肤，并利用消化等方法分离次级毛囊外根鞘细胞，置于37℃、5% CO₂、饱和湿度条件下培养；选用第5代次级毛囊外根鞘细胞，添加不同质量浓度血管内皮生长因子（VEGF），分别处理24、48 h，噻唑兰比色法（MTT）检测细胞活力，EdU核酸标记技术检测细胞增殖；实时荧光定量PCR技术检测PCNA、K 10、FGF 5 mRNA的表达情况。结果显示：1）成功培养出次级毛囊外根鞘细胞，CK17和CK15细胞免疫组化呈阳性。2） VEGF可以促进绒山羊次级毛囊外根鞘细胞的增殖。相同浓度VEGF处理48 h组的细胞活力极显著高于处理24 h组（P<0.01）；在48 h处理组中，100 ng·mL^-1组的细胞活力极显著高于对照组、1 ng·mL^-1组、10 ng·mL^-1组（P<0.01），并显著高于50 ng·mL^-1组（P<0.05），50 ng·mL^-1组的细胞活力显著高于对照组（P<0.05）；100 ng·mL^-1组和50 ng·mL^-1组增殖细胞比例均极显著高于对照组、1 ng·mL^-1组、10 ng·mL^-1组（P<0.01），100 ng·mL^-1组显著高于50 ng·mL^-1组（P<0.05）。3）PCNA、K10、FGF 5 mRNA在次级毛囊外根鞘细胞中均有表达。在VEGF处理48 h时，PCNA mRNA的表达量100 ng·mL^-1组最高，极显著高于对照组（P<0.01），显著高于10 ng·mL^-1组（P<0.05）；K10 mRNA的表达量对照组最高，极显著高于10 ng·mL^-1组与100 ng·mL^-1组（P<0.01）；FGF 5 mRNA的表达量对照组最高，极显著高于100 ng·mL^-1组（P<0.01）。综上表明，本研究成功分离培养了陕北白绒山羊次级毛囊外根鞘细胞；VEGF能够促进次级毛囊外根鞘细胞的增殖，抑制细胞的分化；VEGF可能通过抑制次级毛囊外根鞘细胞中FGF 5基因的表达而促进毛囊生长。

关键词: 绒山羊, 血管内皮生长因子, 次级毛囊外根鞘细胞, 增殖, 成纤维细胞生长因子5, mRNA表达

Abstract:

This study was conducted to investigate isolation and cultivation of the hair follicle outer root sheath cells (HFORSCs) in vitro, and to estimate the effects of the vascular endothelial growth factor (VEGF) on the proliferation, differentiation and mRNA expression of proliferating cell nuclear antigen(PCNA), keratin 10 (K10) and fibroblast growth factor 5 (FGF 5) genes in the sHFORSCs of Shaanbei White cashmere goat. Skin tissues of goats were collected, and sHFORSCs were obtained through the methods of digestion and then cultured under the condition of 37℃, 5% CO₂ and 100% relative humidity;The 5^th generation of cultured sHFORSCs were selected and treated with different concentration VEGF for 24 and 48 h, respectively. The cell viability was detected by Methyl thiazolyl tetrazolium (MTT), and the cell proliferation was detected by EdU DNA markers;The mRNA expression levels of PCNA, K 10 and FGF 5 in HFORSCs were detected by the real-time fluorescence quantitative PCR. Results showed that:1) sHFORSCs were successfully isolated and cultured in vitro, and cultured cells were positive in the immunohistochemistry test of CK17 and CK15, which were main biomarkers of outer root sheath cells. 2) VEGF could promote the proliferation of sHFORSCs. Treated with same concentration of VEGF, the cell viability of 48 h group was significantly higher than that of 24 h group (P<0.01). Treated with different concentrations of VEGF for 48 h, the cell viability of 100 ng·mL^-1 group was significantly higher than that of control group, 1 ng·mL^-1 group and 10 ng·mL^-1 group (P<0.01), and also significantly higher than that of 50 ng·mL^-1 group (P<0.05). The cell viability of 50 ng·mL^-1 group was significantly higher than that of control group (P<0.05); The proportion of the cell proliferation of 100 and 50 ng·mL^-1 groups were all significantly higher than that of control group, 1 ng·mL^-1 group and 10 ng·mL^-1 group (P<0.01). The proportion of the cell proliferation of 100 ng·mL^-1 group was significantly higher than that of 50 ng·mL^-1 group (P<0.05). 3) The expressions of mRNA of PCNA, K10 and FGF 5 were all detected in HFORSCs. Treated with VEGF for 48 h, the mRNA expression level of PCNA in 100 ng·mL^-1 group was the highest, and was significantly higher than that in control group (P<0.01) and 10 ng·mL^-1 group (P<0.05); The mRNA expression of K10 in control group was the highest, and was significantly higher compared with that in 10 and 100 ng·mL^-1 groups (P<0.01); The mRNA expression of FGF 5 in control group was the highest, and was significantly higher than that in 100 ng·mL^-1 group (P<0.01). In this study, sHFORSCs were successfully isolated from hair follicle and were successfully cultured in vitro; The proliferation of HFORSCs could be promoted and the cell differentiation could be inhibited by VEGF. It inferred that VEGF might play the role of growth promotion of the hair follicle by inhibiting the mRNA expression of FGF 5 in sHFORSCs.

Key words: cashmere goats, vascular endothelial growth factor, secondary hair follicle outer root sheath cells, proliferation, fibroblast growth factor 5, mRNA expression

中图分类号:

S827.2

张婧婧, 王德光, 周小兵, 高晔, 何晓琳, 陈玉林, 张恩平. VEGF对体外培养绒山羊次级毛囊外根鞘细胞的影响[J]. 畜牧兽医学报, 2018, 49(6): 1124-1133.

ZHANG Jing-jing, WANG De-guang, ZHOU Xiao-bing, GAO Ye, HE Xiao-lin, CHEN Yu-lin, ZHANG En-ping. Effect of VEGF on Secondary Hair Follicle Outer Root Sheath Cells of Cashmere Goat in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1124-1133.

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