半胱氨酸、蛋氨酸对体外培养绒山羊次级毛囊生长及毛乳头细胞增殖的影响

Effects of Cysteine and Methionine on Secondary Hair Follicle Growth and Hair Dermal Papilla Cell Proliferation in vitro in Cashmere Goats

旨在探讨半胱氨酸(Cys)、蛋氨酸(Met)对体外培养燕山绒山羊次级毛囊(SF)生长及毛乳头细胞(DPCs)增殖凋亡的影响。本研究选取1只1周岁左右健康雄性燕山绒山羊皮肤若干,完整分离的次级毛囊随机分成Cys试验组和Met试验组,每组各浓度3个重复,每个重复8根毛囊,Cys试验组添加浓度分别为0、40、80、120、160和200μg·mL^(-1);Met试验组添加浓度分别为0、10、15、20、25和30μg·mL^(-1)。毛囊培养7 d,每隔24 h观察毛囊生长形态并测量毛囊生长长度。从燕山绒山羊次级毛囊中提取、纯化DPCs,细胞计数并绘制DPCs生长曲线,将DPCs随机分为Cys试验组和Met试验组,每组各浓度6个重复,Cys试验组添加浓度分别为0、20、40、60、80、100μg·mL^(-1);Met试验组添加浓度分别为0、2、4、6、8、10μg·mL^(-1)。培养2 h后用CCK-8试剂盒进行细胞增殖活力测定,确定燕山绒山羊DPCs中Cys和Met的最适培养浓度,利用qRT-PCR技术检测不同浓度Cys和Met的DPCs增殖相关基因(PCNA、CCND1、CDC42、CDK 4)、凋亡相关基因(P21、P53、Bax、Caspase-3、Bcl-2)、皮肤细胞分化相关基因(IVL)及角蛋白相关基因(K10、K 14)mRNA表达水平。结果表明,与对照组相比,添加80和120μg·mL^(-1) Cys与10、15、20和25μg·mL^(-1) Met均可显著影响次级毛囊生长速率和累计生长长度(P<0.01),且120μg·mL^(-1) Cys和20μg·mL^(-1) Met促生长效果最好;添加20、40和60μg·mL^(-1) Cys可以显著影响DPCs增殖(P<0.05),添加40μg·mL^(-1) Cys显著上调PCNA、CCND1、CDK4、Bcl-2、K10、K 14和IVL基因mRNA表达量(P<0.05);添加6μg·mL^(-1) Met可以显著影响DPCs增殖(P<0.05),并显著上调PCNA、CCND1、CDK4、P21、Bcl-2、K 10和K 14基因mRNA表达量(P<0.05)。综上,添加Cys和Met可以显著促进长绒期燕山绒山羊次级毛囊生长,最适添加量分别为120μg·mL^(-1)和20μg·mL^(-1);添加Cys和Met可通过上调细胞增殖、分化、角蛋白等基因mRNA、下调细胞凋亡基因mRNA表达,促进乳头细胞增殖、抑制细胞凋亡进而促进燕山绒山羊次级毛囊生长。

The aim of this study was to investigate the effects of cysteine(Cys)and methionine(Met)on the growth of secondary hair follicles(SF),and the proliferation and apoptosis of hair dermal papilla cells(DPCs)cultured in vitro of Yanshan cashmere goats.The skins of one healthy male Yanshan cashmere goat at one year old was collected,and the completely isolated secondary hair follicles were randomly divided into the Cys experimental group and the Met experimental group,with 3 replicates for each concentration in each group and 8 hair follicles for each replicate.The concentrations of the Cys experimental group were 0,40,80,120,160,and 200μg·mL^(-1),respectively;the concentrations of the Met experimental group were 0,10,15,20,25,and 30μg·mL^(-1),respectively.Hair follicles were cultured for 7 days,the hair follicle growth pattern was observed and hair follicle length was measured every 24 h.DPCs were extracted and purified from the secondary hair follicles of Yanshan cashmere goats,the cells were counted,and the growth curves of DPCs were plotted.The DPCs were randomly divided into the Cys experimental group and the Met experimental group,with 6 replicates of each concentration in each group.The concentrations of the Cys experimental group were 0,20,40,60,80,and 100μg·mL^(-1),respectively;and the concentrations of the Met experimental group were 0,2,4,6,8,and 10μg·mL^(-1),respectively.After 2 h of incubation,cell proliferation viability was detected using the CCK-8 kit to determine the optimal culture concentration of Cys and Met in Yanshan cashmere goat DPCs.The qRT-PCR was used to detect the mRNA expression levels of proliferation-related genes(PCNA,CCND1,CDC42,CDK 4),apoptosis-related genes(P21,P53,Bax,Caspase-3,Bcl-2),skin cell differentiation-related gene(IVL)and keratin-related genes(K10,K 14)in the DPCs incubated with different concentrations of Cys and Met.Compared with the control group,the addition of 80 and 120μg·mL^(-1) Cys and 10,15,20,and 25μg·mL^(-1) Met significantly affected the growth rate and cumulative growth length of secondary hair follicles(P<0.01),and the growth-promoting effects of 120μg·mL^(-1) Cys and 20μg·mL^(-1) Met groups were the best.The addition of 20,40,and 60μg·mL^(-1) Cys significantly affected the proliferation of DPCs(P<0.05),and the addition of 40μg·mL^(-1) Cys significantly up-regulated the expressions of PCNA,CCND 1 and CDK4,Bcl-2,K10,K 14,and IVL genes mRNA(P<0.05);the addition of 6μg·mL^(-1) Met group could significantly affect the proliferation of DPCs and significantly up-regulate the expressions of PCNA,CCND1,CDK4,P21,Bcl-2,K 10,and K 14 genes mRNA(P<0.05).In conclusion,the addition of Cys and Met could significantly promote the growth of secondary hair follicles in Yanshan cashmere goats during the growing-fleece period,and the optimal addition concentrations were 120μg·mL^(-1) and 20μg·mL^(-1),respectively.The addition of Cys and Met could promote the proliferation of hair dermal papilla cells and inhibit apoptosis through the upregulation of mRNA upregulating the expression of genes for cell proliferation,differentiation,keratin,and other genes,and the downregulation downregulating the expression of mRNA of apoptotic genes,which could promote the growth of secondary hair follicles of Yanshan cashmere goats.