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

作　　者：史洪岩[1] 贺綦[1] 程敏[1] 孙婴宁[1] 李辉[1] 王宁[1] 

机构地区：[1]东北农业大学动物科学技术学院/农业部鸡遗传育种重点实验室/黑龙江省普通高等学校动物遗传育种与繁殖重点实验室,哈尔滨150030

出　　处：《中国农业科学》2015年第8期1624-1631,共8页Scientia Agricultura Sinica

基　　金：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(2010td02)

摘　　要：【目的】构建鸡HOPX基因(homeodomain only protein X)全长编码区(coding region sequence,CDS)的真核表达载体,转染鸡原代前脂肪细胞,探讨HOPX基因过表达对鸡原代前脂肪细胞增殖的影响。【方法】利用Primer Premier 5.0软件设计鸡HOPX基因CDS区上、下游引物,以AA肉鸡腹部脂肪组织的c DNA为模板,采用PCR扩增、克隆鸡HOPX基因全长CDS区,并将其亚克隆至真核表达载体(p CMV-HA vector),获得HOPX基因的真核表达载体p CMV-HA-HOPX。采用双酶切鉴定、测序及Western blotting方法分析鉴定p CMV-HA-HOPX。采用胶原酶法分离培养12日龄AA商品肉仔鸡腹部脂肪组织原代前脂肪细胞,瞬时转染p CMV-HA-HOPX,转染6 h时后消化细胞,按照每孔50 000个细胞数接种12孔培养板,并在细胞贴壁0、24、48和72 h,分别采用显微镜观察和CCK-8细胞增殖检测试剂盒分析HOPX基因过表达对鸡原代前脂肪细胞增殖的影响;同时,利用TRIzol法提取组织和细胞总RNA,并反转录合成c DNA,采用Real-time RT-PCR方法分析细胞增殖标志基因Cyclin D1和PCNA的m RNA表达。【结果】测序结果显示,鸡HOPX基因的全长CDS区大小为222 bp,与NCBI发布的鸡HOPX基因m RNA序列(NM_204556)一致;利用HA标签抗体的Western blotting分析显示,真核表达载体p CMV-HA-HOPX能够表达出预期大小的蛋白分子(约9.5k D),表明鸡HOPX基因的真核表达载体p CMV-HA-HOPX构建成功。显微镜观察发现,转染p CMV-HA-HOPX载体的细胞(HOPX过表达组)在细胞贴壁后培养24和48 h的细胞数量低于转染p CMV-HA vector空载体(空载体对照组)的细胞数量;CCK-8检测分析发现,转染p CMV-HA-HOPX载体细胞的吸光度值(OD值)在细胞贴壁后培养24、48和72 h都极显著低于空载体对照组(P<0.01)。与细胞增殖检测结果相一致,细胞增殖标志基因表达检测分析发现,在细胞贴壁后培养24 h后,HOPX过表达组细胞Cyclin D1基因的m RNA表达量显著低于空载体对照组(P<0.05);在细胞�【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 c DNA from the abdominal fat tissues of AA broiler chickens and cloned into p CMV-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 p CMV-HA-HOPX and p CMV-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 p CMV-HA-HOPX could correctly express the HA-tagged HOPX. The microscopic examination showed that the numbers of the preadipocytes transfected with p CMV-HA-HOPX were less than those of the preadipocytes transfected with empty p CMV-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 p CMV-HA-HOPX were significantly lower than those of the preadipocytes transfected with empty p CMV-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 m RNA expression of Cyclin D1 was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes（P〈0.05）; at 48 h after of cell adhesion, the m RNA expression of PCNA was significantly lower in HOPX-overexpressing preadipocytes than in control preadipocytes（P〈0.05）; at 72 h after o

关 键 词：鸡 HOPX 前脂肪细胞 增殖 

分 类 号：S831[农业科学—畜牧学]