Abstract: 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 200 μg·mL^-1, respectively; the concentrations of the Met experimental group were 0, 10, 15, 20, 25, and 30 μg·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 100 μg·mL^-1, respectively; and the concentrations of the Met experimental group were 0, 2, 4, 6, 8, and 10 μg·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, CDK4), apoptosis-related genes (P21, P53, Bax, Caspase-3, Bcl-2), skin cell differentiation-related gene (IVL) and keratin-related genes (K10, K14) in the DPCs incubated with different concentrations of Cys and Met. Compared with the control group, the addition of 80 and 120 μg·mL^-1 Cys and 10, 15, 20, and 25 μg·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 120 μg·mL^-1 Cys and 20 μg·mL^-1 Met groups were the best. The addition of 20, 40, and 60 μg·mL^-1 Cys significantly affected the proliferation of DPCs (P<0.05), and the addition of 40 μg·mL^-1 Cys significantly up-regulated the expressions of PCNA, CCND1 and CDK4, Bcl-2, K10, K14, and IVL genes mRNA (P<0.05); the addition of 6 μg·mL^-1 Met could significantly affect the proliferation of DPCs and significantly up-regulate the expressions of PCNA, CCND1, CDK4, P21, Bcl-2, K10, and K14 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 120 μg·mL^-1 and 20 μg·mL^-1, respectively. The addition of Cys and Met could promote the proliferation of hair dermal papilla cells and inhibit apoptosis through upregulating the expression of genes for cell proliferation, differentiation, keratin, and other genes, and downregulating the expression of mRNA of apoptotic genes, which could promote the growth of secondary hair follicles of Yanshan cashmere goats.

Key words: cysteine, methionine, cashmere goats, secondary hair follicles, hair dermal papilla cells, gene expression