干扰MAP3K1基因对山羊毛囊干细胞增殖和凋亡的影响

Effect of interference with MAP3K1 gene on proliferation and apoptosis of goat hair follicle stem cells

研究旨在探讨促分裂原活化蛋白激酶激酶激酶(Mitogen-activated protein kinase kinase kinase 1,MAP3K1)基因对体外培养长江三角洲白山羊毛囊干细胞(HFSCs)增殖和凋亡的影响。针对MAP3K1基因设计3个干扰靶点,构建pDNA316-ZsGreen-ShRNA腺病毒干扰载体。转染HFSCs,以空载体细胞为对照,RT-qPCR技术检测干扰MAP3K1效果及增殖相关基因(PCNA、CDK1、CCND2)、凋亡相关基因(Bax、Bcl-2)表达水平,CCK-8和EdU法检测细胞增殖,流式细胞仪检测细胞周期和细胞凋亡。结果表明,shRNA(MAP3K1-sh)转染HFSCs后,MAP3K1基因相当表达量极显著下降(P<0.01);增殖相关基因mRNA表达水平显著下降,促凋亡基因Bax的mRNA表达水平显著提高,相反,抗凋亡基因Bcl-2的mRNA表达显著降低;CCK-8和EdU结果发现细胞增殖受抑制,S期细胞数量显著上升;细胞总凋亡量显著上升。综上,干扰MAP3K1基因表达显著抑制HFSCs增殖并促进其凋亡,研究为阐明调控长江三角洲白山羊优质笔料毛分子形成机制提供科学参考。

This study aimed to investigate the effect of mitogen-activated protein kinase kinase kinase 1(MAP3K1)gene on the proliferation and apoptosis of HFSCs from Yangtze River Delta White Goats in vitro.Designed three interference targets for MAP3K1 gene and constructed pDNA316-ZsGreen-ShRNA adenovirus interference vector.HFSCs were transfected with empty vector cells as the blank control.RT-qPCR was used to detect the interference effect of MAP3K1 and the expression levels of proliferation related genes(PCNA,CDK1 and CCND2)and apoptosis related genes(Bax and Bcl-2),CCK-8 and EdU methods were used to detect cell proliferation,and flow cytometry was used to detect cell cycle and apoptosis.The results showed that after shRNA(MAP3K1-sh)was transfected into hair follicle stem cells,the equivalent expression of MAP3K1 gene decreased extremely significant(P<0.01);the mRNA expression level of proliferation-related genes decreased significantly,and the mRNA expression level of the pro-apoptotic gene Bax increased significantly.The mRNA expression of antiapoptotic gene Bcl-2 was significantly reduced;CCK-8 and EdU results showed that cell proliferation was significantly inhibited,and the number of S-phase cells increased significantly;the total amount of cell apoptosis increased significantly.In summary,interfering with the expression of MAP3K1 gene significantly inhibited the proliferation of HFSCs and promoted their apoptosis,which provided a scientific reference for elucidating the molecular formation mechanism that regulated the superiorquality brush hair in the Yangtze River Delta White Goats.