FOXO1基因调控牛前体脂肪细胞增殖和分化的功能研究

Study on the function of FOXO1 gene in regulating the proliferation and differentiation of bovine preadipocytes

为探究干扰叉头转录因子O1(Forkhead box protein O1, FOXO1)对牛前体脂肪细胞增殖和分化的作用,本研究以固原黄牛的前体脂肪细胞为研究对象,采用组织块法分离牛前体脂肪细胞,并利用表型鉴定和标志基因表达谱2种方法鉴定所分离细胞的纯度和分化潜能。筛选并包装牛FOXO1干扰慢病毒,利用EdU染色、流式细胞术、油红O染色和qPCR方法探究干扰FOXO1对牛脂肪细胞增殖和分化的作用。结果表明:1)成功分离了牛前体脂肪细胞,且其具有较高的纯度和分化潜能。2)EdU染色结果表示,侵染Lv-FOXO1极显著增加了牛脂肪细胞的增殖率(P<0.01)。3)流式周期结果显示,干扰FOXO1显著增加了牛脂肪细胞S期阳性细胞的比例,促进了G1/S期的转化,进而促进了牛脂肪细胞增殖。4)干扰FOXO1后,增殖标志基因PCNA、CDK1和CDK2的表达量均极显著上调(P<0.01)。5)干扰FOXO1后牛前体脂肪细胞脂滴生成明显减少,且成脂标志基因PPARG的表达极显著下调(P<0.01),CEBPB和LPL的表达显著下调(P<0.05)。综上,干扰FOXO1可通过上调增殖标志基因PCNA、CDK1和CDK2的表达促进牛脂肪细胞G1/S期的转化,进而促进牛脂肪细胞增殖,且其可通过降低成脂标志基因PPARG、CEBPB和LPL的表达减少牛脂肪细胞脂滴形成,进而影响脂肪沉积。本研究可为中国地方黄牛的肉质遗传改良提供理论依据。

The aim of the study was to investigate the effects of interfering with Forkhead box protein O1(FOXO1)gene on the proliferation and differentiation of bovine preadipocytes.Taking the Guyuan cattle preadipocytes as the research object,bovine preadipocytes were isolated by tissue block method,and the purity and differentiation potential of the isolated cells were identified by phenotypic identification and marker gene expression profiling.The bovine FOXO1 interfering lentivirus was screened and packaged,and the effects of interfering FOXO1 on the proliferation and differentiation of bovine adipocytes were explored by EdU staining,flow cytometry,oil red O staining and qPCR.The results showed that:1)The bovine preadipocytes were successfully isolated and showed high purity and differentiation potential.2)EdU staining showed that infection of Lv-FOXO1 significantly increased the proliferation rate of bovine adipocytes(P<0.01).3)Flow cytometry results showed that interfering with FOXO1 significantly increased the proportion of S-phase positive cells and promoted the G1/S phase transformation of bovine adipocytes,which in turn promoted the proliferation of bovine adipocytes.4)After interfering with FOXO1,the expression levels of proliferation marker genes PCNA,CDK1 and CDK2 were all extremely significantly upregulated(P<0.01).5)After interfering with FOXO1,the production of lipid droplets in bovine precursor adipocytes was significantly reduced,and the expression of PPARG,a lipid-forming marker gene,was extremely significantly down-regulated(P<0.01),together with the expressions of CEBPB and LPL(P<0.05).In summary,interfering with FOXO1 can promote the G1/S phase transformation of bovine adipocytes by up-regulating the expressions of proliferation marker genes PCNA,CDK1 and CDK2,thereby promoting the proliferation of bovine adipocytes.Moreover,it can reduce the formation of lipid droplets in bovine adipocytes by reducing the expressions of lipogenic marker genes PPARG,CEBPB and LPL,thus affecting fat deposition.This study can provide theoretical basis for genetic improvement of meat quality of Chinese local yellow cattle.