HOPX基因过表达对鸡前脂肪细胞增殖的影响

doi:10.3864/j.issn.0578-1752.2015.08.17

摘要/Abstract

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cDNA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（pCMV-HA vector），获得HOPX基因的真核表达载体pCMV-HA-HOPX。采用双酶切鉴定、测序及Western blotting方法分析鉴定pCMV-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pCMV-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cDNA，采用Real-time RT-PCR方法分析细胞增殖标志基因Cyclin D1和PCNA的mRNA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mRNA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pCMV-HA-HOPX能够表达出预期大小的蛋白分子（约9.5kD），表明鸡HOPX基因的真核表达载体pCMV-HA-HOPX构建成功。显微镜观察发现，转染pCMV-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pCMV-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pCMV-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mRNA表达量显著低于空载体对照组（P＜0.05）；在细胞贴壁后培养48 h时，HOPX过表达组的PCNA基因的mRNA表达量显著低于空载体对照组（P＜0.05）；在细胞贴壁后培养72 h时，HOPX过表达组的PCNA和Cyclin D1基因的表达量均极显著低于空载体对照组（P＜0.01）。【结论】HOPX基因过表达在体外抑制鸡原代前脂肪细胞的增殖。

关键词: 鸡, HOPX, 前脂肪细胞, 增殖

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 is222bp 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.

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, 48(8): 1624-1631.

SHI Hong-yan, HE Qi, CHENG Min, SUN Ying-ning, LI Hui, WANG Ning. Effect of HOPX Gene Overexpression on Chicken Preadipocyte Proliferation[J]. Scientia Agricultura Sinica, 2015, 48(8): 1624-1631.

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