水牛NONO和PSPC1基因对成纤维细胞抗衰老的作用研究

A Preliminary Study of the Role of Buffalo NONO and PSPC1 Genes on Fibroblast Anti-Aging

旁粒相关基因NONO和PSPC1在DNA损伤修复、转录调控、细胞增殖等方面发挥重要作用.该研究旨在克隆水牛NONO,PSPC1基因并对其进行序列分析,探讨其对水牛胎儿成纤维细胞增殖及衰老的影响.首先扩增水牛NONO,PSPC1编码区序列并构建其过表达载体,对不同代数水牛胎儿成纤维细胞中旁粒相关基因的表达进行检测.构建细胞衰老模型,将过表达NONO,PSPC1载体转染第10代成纤维细胞,培养至15代进行细胞增殖、 β-半乳糖苷酶、细胞衰老和凋亡相关基因表达分析.分别得到1 424 bp和1 574 bp的水牛NONO和PSPC1编码区序列,生物信息学分析发现水牛NONO基因与黄牛和人的序列相似度分别为98.9%和91.2%;水牛PSPC1基因与黄牛、绵羊、山羊和人序列相似度分别为99.5%,86.0%,98.8%,89.3%.随着细胞培养代数增加,旁粒相关基因表达降低.过表达转染试验结果发现:与空白组和阴性对照组相比,2个试验组细胞增殖速度变快,β-Gal细胞阳性率和P21,P16,Bax表达均显著下降(p<0.05);2个试验组间相比,上调PSPC1表达后细胞增殖速度更快,β-Gal细胞阳性率和P21,P16,Bax表达均显著降低(p<0.05).上调NONO和PSPC1基因表达能够促进细胞增殖,降低凋亡相关基因的表达进而延缓细胞衰老.

The paragranules-related genes NONO and PSPC1 play important roles in DNA damage repair,transcriptional regulation,and cell proliferation.This study aimed to clone and sequence the buffalo NONO and PSPC1 genes,and explore their effects on the proliferation and aging of buffalo fetal fibroblasts.Firstly,the coding region sequences of buffalo NONO and PSPC1 were amplified and their overexpression vectors were constructed.The expressions of paragranules-related genes in fetal fibroblasts of buffalo of different generations were detected.A cell senescence model was constructed.The NONO and PSPC1 overexpression vectors were transfected into 10th generation fibroblasts,and cultured to 15 passages for analyzing cell proliferation,β-galactosidase,cell senescence and apoptosis-related gene expressions.The results showed that the 1 424 bp and 1 574 bp of buffalo NONO and PSPC1 coding region sequences were obtained,respectively.Bioinformatic analysis showed that the sequence similarities of buffalo NONO gene with cattle and humans were 98.9% and 91.2%,respectively.The similarities of PSPC1 gene with cattle,sheep,goats and humans were 96%,98%,82.8% and 68.5%,respectively.With the increase of cell culture generations,paragranule-associated gene expression was decreased.The results of overexpression transfection experiments showed that compared with the blank and the negative control groups,the cell proliferation rates of the two experimental groups were faster,and the positive rate of β-Gal cells and the expressions of P21,P16 and Bax decreased significantly(p<0.05).Compared between two experimental groups,the cell proliferation rate was faster after upregulating the PSPC1 expression,and the positive rate of β-Gal cells and the expressions of P21,P16 and Bax were significantly reduced(p<0.05).These results reveal that upregulation of NONO and PSPC1 genes expression can promote cell proliferation,reduce the expression of apoptosis-related genes and delay cell aging.