TRIB3与AKT在大鼠睾丸细胞中的相互关联

Interaction between TRIB3 and AKT in rat testicular cells

目的研究假性激酶蛋白3(Tribbles homology protein3,TRIB3)与蛋白激酶B(Protein kinase B,AKT)在大鼠睾丸细胞中是否存在相互作用关系。方法普通PCR扩增,琼脂糖凝胶电泳鉴定。利用大鼠睾丸组织培养细胞通过RT-qPCR对Trib3的表达进行检测验证,对照组为野生型大鼠睾丸细胞,实验组为Trib3基因敲除大鼠睾丸细胞,运用Western blot和RT-qPCR检测两组共培养细胞中AKT蛋白和Akt mRNA的表达。利用两步酶消法消化野生型大鼠睾丸细胞,提取总蛋白,运用免疫共沉淀来验证TRIB3与AKT的蛋白互作关系。结果实验所用大鼠为Trib3基因敲除鼠,Trib3 mRNA在实验组无表达(P<0.05),实验组较对照组AKT蛋白(P<0.05)和Akt mRNA表达升高(P<0.01)。TRIB3抗体免疫沉淀的蛋白复合物中检测到AKT,同时AKT抗体免疫沉淀的蛋白复合物中也检测到TRIB3。结论TRIB3和AKT在大鼠睾丸细胞中存在相互作用。

Objective To analyze the interaction between Tribbles homology protein 3(TRIB3)and protein kinase B(AKT)in rat testicular tissue cells.Methods Use PCR and agarose gel electrophoresis to identify the rat genotype.Use rat testicles to culture cells,the control group was from wild-type rats,the experimental group was from Trib3 knockout rats.The expression of Trib3 was confirmed by RT-qPCR.Western blot and RT-qPCR were used to detect the expression of AKT protein and A kt mRNA in two groups of co-cultured cells.Two-step enzyme digestion method was used to digest wild-type rat testis cells and extract their total protein.Co-immunoprecipitation assay was performed to analyze the interaction between TRIB3 and AKT in rat testicular cells.Results The rats used in the experiment are Trib3 knockout mice.The expression of Trib3 mRNA was not detected in the experimental group.Compared with those in the control group,the expressions of AKT protein(P<0.05)and A kt mRNA in the experimental group were obviously upregulated(P<0.01).The expression of AKT was found in the protein complex from anti-TRIB3 antibody-based Co-IP,and the expression of TRIB3 in the protein complex from anti-AKT antibody-based Co-IP.Conclusion There exists an interaction between TRIB3 and AKT in rat testicular cells.