双峰母驼肌肉中TPM2与TPM3表达模式及对肌纤维细胞凋亡的影响

Expression Mode of TPM2 and TPM3 in Muscles of Female Bactrian Camels and Effect on Apoptosis of Muscle Fibroblasts

为了验证原肌球蛋白2(TPM2)与原肌球蛋白3(TPM3)在繁殖季节和非繁殖季节双峰母驼不同肌肉组织(平滑肌、骨骼肌、心肌)中的具体差异表达情况,揭示TPM2肌纤维细胞中的分布以及敲低后对其凋亡的影响,采用实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹,分别对TPM2和TPM3对应的mRNA及蛋白在繁殖季节和非繁殖季节双峰母驼不同肌肉组织的表达进行分析,结合免疫荧光技术检测TPM2在细胞中定位情况,设计TPM2-siRNA序列瞬时转染双峰驼肌纤维细胞,检测0 h、24 h、48 h时促凋亡蛋白(Bax)和细胞淋巴瘤2(Bcl-2)的表达情况。结果显示,繁殖季节双峰驼肌肉组织中TPM2与TPM3对应的mRNA的表达极显著高于非繁殖季节(P<0.01),蛋白的表达显著高于非繁殖季节(P<0.05);TPM2与TPM3对应的mRNA和蛋白在繁殖季节与非繁殖季节骨骼肌肌肉组织中的表达量均极显著高于同期其他组织(P<0.01);肌纤维细胞敲低TPM2基因后,Bax基因表达上调,Bcl-2基因表达下调。TPM2与TPM3在繁殖季节各肌肉中的表达高于非繁殖季节肌肉组织,且在同期中表现出它们在骨骼肌中表达量较高的规律,敲低TPM2基因,能促进肌纤维细胞凋亡。

In order to verify the differential expressions of TPM2 and TPM3 in variety of muscle tissues(smooth,skeletal,cardiac)of Bactrian camels in reproductve season and non-reproductve season,reveal the apoptosis influences of muscle fiber cells after TPM2 knockdown,in this study,the RT-qPCR and Western blotting were used to analyze the mRNA and protein expressions of TPM2 and TPM3 in different muscle tissues of Bactrian camels in diferent reproductive seasons.Combined with immunofluorescence technique to detect the localization of TPM2 in cells,we designed TPM2-siRNA sequence to transfect Bactrian camel muscle fiber cells,detected expressions of Bax and Bcl-2 genes at 0 h,24 h,and 48 h.The results showed that the expression levels of TPM2 and TPM3 mRNA in muscle of Bactrian camels during reproductive season was significantly higher than that during non-reproductive season(P<0.01),and the expression level of protein during reproductive season was significantly higher than that during non-reproductive season(P<0.05);The expression levels of TPM2 and TPM3 mRNA and protein in skeletal muscle during reproductive and non-reproductive season were significantly higher than those in other muscle tissues during the same period(P<0.01);After the TPM2 was knocked down in fibroblasts,the expression level of Bax mRNA was up-regulated,whereas the expression level of Bcl-2 mRNA was down-regulated.It is concluded that the expression levels of TPM2 and TPM3 in each muscle tissues of the reproductive season are higher than those of non-reproductive season,and during the same period,they show a higher pattern of expression in skeletal muscle.Knocking down the TPM2 gene promotes fibroblast apoptosis.