摘要
本试验以谷氨酰胺作为热休克蛋白70(HSP70)诱导剂,从细胞凋亡线粒体信号转导途径探讨HSP70对热应激猪小肠上皮细胞(IPEC-J2)凋亡相关因子的影响。试验以体外培养的猪小肠上皮细胞为模型,分别加入不同浓度(1、2、4、6、8、10 mmol/L)谷氨酰胺的培养基中培养24 h,提取总RNA和总蛋白,用实时荧光定量PCR和Western blot检测HSP70 mRNA和蛋白的相对表达量,筛选出谷氨酰胺诱导HSP70表达的最佳浓度。取对数生长期的细胞分为3组,分别为对照组(Con组,37℃培养12 h)、热应激组(Hs组,42℃培养12 h)、谷氨酰胺组(Gln组,6 mmol/L谷氨酰胺、42℃培养12 h)。采用Annexin V/PI双染法检测细胞凋亡水平,实时荧光定量PCR和Western blot分别检测HSP70、凋亡酶激活因子-1(Apaf-1)、半胱氨酸天冬氨酸蛋白水解酶-9(Caspase-9)、半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)、B淋巴细胞瘤-2(Bcl-2)的mRNA和蛋白的相对表达量。结果表明:谷氨酰胺对HSP70上调表达呈先上升后下降的趋势,6 mmol/L的谷氨酰胺诱导时HSP70 mRNA和蛋白相对表达量最高,显著高于其他浓度(P<0.05),作为后续试验中Gln组的诱导条件。与Con组相比,Hs组细胞凋亡率极显著增加(P<0.01),HSP70、Apaf-1、Caspase-3、Caspase-9 mRNA和蛋白的相对表达量极显著升高(P<0.01),而Bcl-2 mRNA和蛋白的相对表达量极显著降低(P<0.01)。与Hs组相比,Gln组细胞凋亡率极显著降低(P<0.01),但仍极显著高于Con组(P<0.01);Gln组HSP70 mRNA和蛋白的相对表达量极显著升高(P<0.01);Gln组Apaf-1、Caspase-3、Caspase-9 mRNA和蛋白的相对表达量极显著降低(P<0.01),但极显著高于Con组(P<0.01);Gln组Bcl-2 mRNA和蛋白的相对表达量极显著升高(P<0.01),但极显著低于Con组(P<0.01)。由此可见,上调HSP70的表达可有效缓解热应激导致的猪小肠上皮细胞凋亡。
This experiment was used glutamine as a heat shock protein 70(HSP70)inducer,to investigate the effects of HSP70 on intestinal epithelial cells(IPEC-J2)of heat-stressed piglets through mitochondrial signal transduction pathway of apoptosis.In this experiment,IPEC-J2 cells cultured in vitro were used as the model,and different concentrations(1,2,4,6,8,10 mmol/L)of glutamine were added to the culture medium for 24 h,the total RNA and total protein were extracted.The relative expressions of HSP70 mRNA and protein were detected using real-time fluorescent quantitative PCR and Western blot,and the optimal concentration of glutamine induced HSP70 expression was screened.The cells in logarithmic growth period were divided into three groups:control group(Con group,37℃cultured 12 h),heat stress group(Hs group,42℃cultured 12 h)and glutamine group(Gln group,6 mmol/L glutamine and 42℃cultured 12 h).Annexin V/PI double staining was used to detect the level of apoptosis,real-time fluorescence quantitative PCR and Western blot were used to detect the relative expressions of HSP70,apoptotic protease activating facter-1(Apaf-1),cysteinyl aspartate specific proteinase-9(Caspase-9),cysteinyl aspartate specific proteinase-3(Caspase-3),B-cell lymphoma-2(Bcl-2)mRNA and protein.The results showed that the up-regulated expression of HSP70 by glutamine firstly increased and then decreased,the relative expression of HSP70 mRNA and protein induced by 6 mmol/L glutamine was the highest,and significantly higher than that of other concentration(P<0.05),which served as the induction condition of Gln group in the follow-up experiment.Compared with the Con group,the apoptosis rate of Hs group was significantly increased(P<0.01),the relative expressions of HSP70,Apaf-1,Caspase-3,Caspase-9 mRNA and protein were significantly increased(P<0.01),while the relative expression of Bcl-2 mRNA and protein was significantly decreased(P<0.01).Compared with the Hs group,the apoptosis rate of Gln group was significantly decreased(P<0.01),and still significantly higher than that of the Con group(P<0.01);the relative expression of HSP70 mRNA and protein of Gln group was significantly increased(P<0.01);the relative expressions of Apaf-1,Caspase-3,Caspase-9 mRNA and protein of Gln group were significantly decreased(P<0.01),but significantly higher than those of the Con group(P<0.01);the relative expression of Bcl-2 mRNA and protein of Gln group was significantly increased(P<0.01),but significantly lower than that of the Con group(P<0.01).In conclusion,the up-regulated expression of HSP70 can effectively alleviate the apoptosis of intestinal epithelial cells of piglets induced by heat stress.
作者
孙汇蕾
仲庆振
李明晔
MOLAWA M N
SUN Huilei;ZHONG Qingzhen;LI Mingye;MOLAWA M N(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;Key Laboratory of Animal Production and Product Quality and Safety,Ministry of Education,Changchun 130118,China;Jilin Province Key Laboratory of Animal Nutrition and Feed Science,Changchun 130118,China)
出处
《动物营养学报》
CAS
CSCD
北大核心
2020年第4期1867-1874,共8页
CHINESE JOURNAL OF ANIMAL NUTRITION
基金
国家自然科学基金青年基金项目(31601982)。
