中国农业科学Issue(8)：1624-1631,8.DOI:10.3864/j.issn.0578-1752.2015.08.17
HOPX基因过表达对鸡前脂肪细胞增殖的影响
Effect of HOPX Gene Overexpression on Chicken Preadipocyte Proliferation
摘要
Abstract
[Objective]The objective of this study was to construct the eukaryotic expression vector of chicken full-length HOPX gene and investigate the effect of HOPX gene overexpression on chicken preadipocytes proliferation.[Method]Using Primer Premier 5.0 software, a pair of primers was designed to amplify the full-length coding sequence (CDS) of chicken HOPX. The full-length coding sequence (CDS) of chicken HOPX gene was PCR amplified from the cDNA from the abdominal fat tissues of AA broiler chickens and cloned into pCMV-HA vector. Chicken preadipocytes were isolated from the abdominal fat tissues of 12-day-old AA broiler chicken by collagenase digestion, cultured and transfected with pCMV-HA-HOPX and pCMV-HA empty vector, respectively. Cell proliferation was assayed by microscopic examination and Cell Counting Kit-8 (CCK-8). Gene expression was measured by Western blotting and quantitative Real-time RT-PCR. [Result] The sequencing results showed that the full-length coding sequence of chicken HOPX gene is 222 bp and identical with NCBI reference sequence (NM_204556). Western blotting analysis showed that pCMV-HA-HOPX could correctly express the HA-tagged HOPX. The microscopic examination showed that the numbers of the preadipocytes transfected with pCMV-HA-HOPX were less than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h and 48 h after of cell adhesion. CCK-8 analysis showed that the OD values of the preadipocytes transfected with pCMV-HA-HOPX were significantly lower than those of the preadipocytes transfected with empty pCMV-HA vector at 24 h, 48 h and 72 h after of cell adhesion (P<0.01). Consistently, at 24 h after of cell adhesion, the mRNA expression of CyclinD1 was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05);at 48 h after of cell adhesion, the mRNA expression of PCNA was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.05);at 72 h after of cell adhension, the mRNA expression of CyclinD1 and PCNA were extremely significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes (P<0.01)[Conclusion]HOPX gene overexpression in vitro inhibits chicken preadipocyte proliferation.关键词
鸡/HOPX/前脂肪细胞/增殖Key words
chicken/HOPX/preadipocyte/proliferation引用本文复制引用
史洪岩,贺綦,程敏,孙婴宁,李辉,王宁..HOPX基因过表达对鸡前脂肪细胞增殖的影响[J].中国农业科学,2015,(8):1624-1631,8.基金项目
国家“973”计划（2009CB941604）、国家肉鸡产业技术体系项目（CARS-42）、黑龙江高校科技创新团队建设项目 （）
