| In this study,we studied the effects of methionine,cysteine and melatonin on cell proliferation,keratin and related gene mRNA expression in Inner Mongolia cashmere goat skin fibroblasts,and further explored the energy and nutrient sensing molecule AMPK-mTOR pathway.The law of change.The experimental research of this thesis is divided into five parts.In the first experiment,the skin of the ear of the Inner Mongolian cashmere goat was cultured in vitro by the method of tissue block adherence.It was observed,purified and identified,and the growth curve was plotted.Experiment 2 added cysteine and methionine to fibroblasts in the growth phase,and studied different concentrations of Cys and Met(1X=0.12 mM,5X=0.6 mM,10X=1.2 mM,20X=2.4 mM)for fiber formation.The effects of cell proliferation,keratin and mRNA expression levels of AMPK-mTOR signaling pathway-related genes.Different proportions of Cys and Met(1:1,1:3,1:5,3:1,5:1)were added to the fibroblasts,and the proliferation of fibroblasts by different ratios of two amino acids was studied.The effect of keratin and mRNA expression levels of AMPK-mTOR signaling pathway-related genes.The melatonin was added to the fibroblasts to study the mRNA expression of fibroblast proliferation,keratin and AMPK-mTOR signaling pathways in different concentrations of MeL(0,100,300,500,700 pg/m L).The impact of quantity.On the basis of Test 3 and Test 4,Experiment 5 selected mixed groups with strong concentration of 1:1,3:1,500 pg/ml and 700 pg/ml for fibroblast proliferation,and studied the mixed addition group to fiber.The effects of cell proliferation,keratin and mRNA expression levels of AMPK-mTOR signaling pathway-related genes.This experimental study initially yielded the following results:(1)Morphological observation of the cultured cells revealed that the cells were long-spin type,which was a typical fibroblast morphology;and the cell count and cell growth curve were performed,showing a typical "S" shape.(2)Compared with the control group,the addition of different concentrations of Cys significantly promoted the proliferation of fibroblasts(P<0.05),and the best in the 5X concentration group.The expression levels of PCNA,K10,K14,K15,P53,P21,BMP4,TGF?,1VLmTOR and other genes can be significantly up-regulated.Adding different concentrations of methionine can significantly inhibit the proliferation of fibroblasts,and significantly increase PCNA,CCND1,K14,K15,P21,BMP4,TGF?,mTOR mRNA expression levels;(3)Compared with the control group,the addition of two amino acids at different concentrations significantly promoted the proliferation of fibroblasts(P<0.05).When the concentration of cysteine was constant and the concentration of methionine was gradually increased,the cell proliferation rate was highest in the 1:1 group,and then gradually decreased.The addition of two amino acids at different concentrations significantly increased the expression levels of PCNA,CCND1,CDC42,K15,P53,LOR,IVL,TGF,and mTOR mRNA.When the concentration of methionine is unchanged and the concentration of cysteine is gradually increased,the cell proliferation rate reaches the highest at 3:1,which can significantly increase the gene PCNA,CDC42,K14,K15,P53,IVL,TGF? mRNA.Amount of expression;(4)Compared with the control group,the addition of MeL significantly promoted the proliferation of fibroblasts from Inner Mongolia cashmere goats(P<0.05).The relative proliferation rate was the lowest in the concentration of 100 pg/ml,and the highest in the concentration of 500 pg/ml.Single addition of MeL can significantly increase the expression levels of related genes CCND1,K14,K15,P53,BMP4,LOR,mTOR,and P53 mRNA.(5)Compared with the control group,the mixed-added group significantly promoted the proliferation of fibroblasts(P<0.05).The relative proliferation rate reached the highest in the 1:1+500 pg/ml group and the lowest in the 1:1+700 pg/m L group.The mixed addition group can also significantly increase the expression levels of related genes PCNA,K14,and mTOR mRNA. |
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