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目的利用Ad Easy腺病毒载体系统构建A激酶锚定蛋白(AKAP)1重组腺病毒载体,初步探索AKAP1在心肌细胞中的功能。方法以正常小鼠心肌细胞c DNA为模板,利用PCR获取AKAP1基因,构建腺病毒重组质粒p Ad Easy-AKAP1。用293A细胞,包装重组腺病毒Ad-AKAP1颗粒,并用荧光蛋白标记法测定其滴度。用Western blot检测重组腺病毒感染原代小鼠心肌细胞和糖尿病心肌病细胞模型后AKAP1、Caspase-3的表达情况,用流式细胞仪检测原代小鼠心肌细胞内ROS生成水平及AKAP1对高糖状态下心肌细胞凋亡的影响。结果构建了携带AKAP1基因的过表达腺病毒载体,并获得5×10~(10)pfu/ml的高滴度重组腺病毒。重组腺病毒可感染原代心肌细胞和糖尿病心肌病模型H9c2细胞并介导AKAP1过表达(P<0.05)。与阴性对照组相比,过表达AKAP1可抑制高糖高脂状态下心肌细胞内ROS的生成(P<0.05),可抑制高糖状态下心肌细胞的凋亡和Caspase-3活化(P<0.01)。结论过表达AKAP1对高糖高脂状态下的心肌细胞具有潜在保护作用,对心肌功能发挥具有重要作用。其机制可能与AKAP1抑制心肌细胞内ROS生成、抑制Caspase-3活化、抑制细胞凋亡有关。
Objective To construct AKT-1 recombinant adenovirus using Ad Easy adenovirus vector system and to explore the function of AKAP1 in cardiomyocytes. Methods Using normal mouse cardiomyocyte c DNA as a template, the AKAP1 gene was obtained by PCR and the adenovirus recombinant plasmid pAd Easy-AKAP1 was constructed. The 293A cells were used to package the recombinant adenovirus Ad-AKAP1 particles and their titers were determined by fluorescent protein labeling. Western blot was used to detect the expression of AKAP1 and Caspase-3 in primary murine cardiomyocytes and diabetic cardiomyocytes infected with recombinant adenovirus. The level of ROS production in primary mouse cardiomyocytes was measured by flow cytometry and the level of AKAP1 Effect of glucose on cardiomyocyte apoptosis. Results The overexpressed adenovirus vector carrying AKAP1 gene was constructed and a high titer recombinant adenovirus of 5 × 10 ~ (10) pfu / ml was obtained. Recombinant adenovirus infected primary cardiomyocytes and diabetic cardiomyopathy model H9c2 cells and mediated AKAP1 overexpression (P <0.05). Compared with the negative control group, overexpression of AKAP1 inhibited the production of ROS in cardiomyocytes under high glucose and high fat (P <0.05), and inhibited the apoptosis of cardiomyocytes and the activation of Caspase-3 under high glucose (P <0.01) ). Conclusion Overexpression of AKAP1 has a potential protective effect on cardiomyocytes in high glucose and high fat state and plays an important role in myocardial function. The mechanism may be related to AKAP1 inhibition of ROS generation in myocardial cells, inhibition of Caspase-3 activation, inhibition of apoptosis.