摘要
【目的】构建无量山乌骨鸡血红素加氧酶1(heme oxygenase 1,HMOX1)基因过表达载体,并转染鸡原代前脂肪细胞,探究过表达HMOX1基因对鸡原代前脂肪细胞增殖的影响。【方法】取1日龄无量山乌骨鸡雏鸡皮下脂肪组织,提取总RNA并反转录成cDNA,扩增HMOX1基因CDS区,使用质粒pcDNA3.1-mRFP构建过表达载体pcDNA3.1-HMOX1-mRFP,并进行酶切和测序鉴定;通过转染试剂将pcDNA3.1-HMOX1过表达载体转染鸡原代前脂肪细胞中并检测过表达效率;利用CCK-8法检测细胞活力,流式细胞术检测细胞周期;利用实时荧光定量PCR和Western blotting检测增殖相关基因P21、CDK1、PCNA mRNA和蛋白表达量。【结果】酶切和测序结果表明,pcDNA3.1-HMOX1-mRFP过表达载体构建成功。实时荧光定量PCR和Western blotting检测结果显示,与对照组相比,转染pcDNA3.1-HMOX1-mRFP后细胞中HMOX1基因mRNA和蛋白表达量均极显著上调(P<0.01),表明鸡原代前脂肪细胞中成功过表达HMOX1。CCK-8检测结果显示,与对照组相比,转染12 h时过表达HMOX1组细胞活力显著上升(P<0.05),转染36和48 h时细胞活力极显著下降(P<0.01)。流式细胞术检测结果显示,与对照组相比,12 h时过表达HMOX1组S期的细胞数量极显著增加(P<0.01),G1期的细胞数量极显著降低(P<0.01);48 h时S期的细胞数量显著降低(P<0.05)。增殖相关基因检测结果显示,两组间细胞中P21、CDK1基因mRNA和蛋白表达量无显著差异(P>0.05);过表达HMOX1组细胞中PCNA基因mRNA和蛋白表达量均显著低于对照组(P<0.05)。【结论】本研究成功构建HMOX1基因过表达载体,转染无量山乌骨鸡原代前脂肪细胞后抑制了细胞增殖。试验结果可为进一步研究HMOX1对无量山乌骨鸡原代前脂肪细胞的分化作用提供理论指导。
【Objective】This experiment aimed to construct an overexpression vector of heme oxygenase 1(HMOX1)gene in Wuliangshan Black-boned chickens and transfect it into chicken primary preadipocytes,and investigate the effect of HMOX1 gene overexpression on the proliferation of chicken primary preadipocyte.【Method】Collect the subcutaneous fat tissue from 1-day-old chicks of Wuliangshan Black-boned chicken,extract total RNA and reverse transcribe it into cDNA,and amplify the CDS region of HMOX1 gene.The overexpression vector pcDNA3.1-HMOX1-mRFP was constructed using the plasmid pcDNA3.1-mRFP,followed by restriction enzyme digestion and sequencing for identification.The pcDNA3.1-HMOX1 overexpression vector was transfected into chicken primary preadipocytes by using transfection reagents,and the overexpression efficiency was detected.Cell viability was measured using CCK-8 method,and the cell cycle was analyzed by flow cytometry.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of proliferation-related genes(P21,CDK1 and PCNA).【Result】Restriction enzyme digestion and sequencing indicated that the pcDNA3.1-HMOX1-mRFP overexpression vector was successfully constructed.Real-time quantitative PCR and Western blotting test results showed that,compared with control group,the mRNA and protein expression of HMOX1 gene in cells transfected with pcDNA3.1-HMOX1-mRFP were extremely significantly upregulated(P<0.01),demonstrated that HMOX1 was successfully overexpressed in chicken primary preadipocytes.CCK-8 assay results showed that compared with control group,the cell viability of HMOX1 overexpression group significantly increased at 12 hours after transfection(P<0.05),and decreased extremely significantly at 36 and 48 hours after transfection(P<0.01).The results of flow cytometry test showed that,compared with control group,the number of cells in the S phase in HMOX1 overexpression group increased extremely significantly at 12 hours(P<0.01),while the number of cells in the G1 phase decreased extremely significantly(P<0.01).The number of cells in the S phase at 48 hours was significantly reduced(P<0.05).Detection results of proliferationrelated genes showed that there was no significant difference in the mRNA and protein expression of P21 and CDK1 genes in the cells between the two groups(P>0.05).The mRNA and protein expression of PCNA gene in overexpression group were significantly lower than those in control group(P<0.05).【Conclusion】The HMOX1 gene overexpression vector was successfully constructed.After transfection of the original preadipocytes of Wuliangshan Black-boned chicken,cell proliferation was inhibited.The experimental results could provide theoretical guidance for further study of the effect of HMXO1 on the differentiation of primary preadipocytes of Wuliangshan Black-boned chicken.
作者
杨平远
殴小嫚
殴正淼
陈粉粉
YANG Pingyuan;OU Xiaoman;OU Zhengmiao;CHEN Fenfen(College of Biological and Food Engineering,Southwest Forestry University,Kunming 650224,China)
出处
《中国畜牧兽医》
北大核心
2026年第1期359-367,共9页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金项目(32060739)。
