重组腺病毒介导的肌浆网钙ATP酶在大鼠心肌细胞的过表达

Overexpression of sarcoplasmic reticulum calcium ATPase in isolated rat myocytes mediated by recombinant adenovirus

目的:为研究和评估SERCA2a在充血性心力衰竭的基因治疗中的价值和SERCA2a转基因对心力衰竭病生理的影响。方法:采用重组腺病毒AdEasy系统,构建了含SERCA2a cDNA的重组病毒载体rAd-trSER-CA2a。感染培养的乳鼠心肌细胞,以RTPCR和Western Blotting检测SERCA2a在心肌细胞中的表达。结果:经PCR法和Southern确定目的基因SERCA2a存在于rAd-trSERCA2a基因组中,且连续传代保持其遗传稳定性。感染组心肌细胞SERCA2a蛋白mRNA和蛋白含量分别为对照的4.6倍和1.5倍。结论:我们成功地构建了含SER-CA2a的重组腺病毒rAd-trSERCA2a,该病毒可有效介导SERCA2a在心肌细胞中的表达。

Objective: To assess value of SERCA2a in the gene therapy of congestive heart failure, furthermore to analysis the effect of transfeetion to pathophysiology. Methods: A recombinant adenovirus, named rAd-trSERCA2a, was constructed with AdEasy system, in which the SERCA2a cDNA was inserted under the control of CMV promoter. The cardiac myoeytes of newborn rat were cultured, and were transfected with rAd-trSERCA2a. RTPCR and Western Blotting were performed to detect the expression of SERCA2a in cardiac myoeytes. Results; the sequence of SERCA2a was stably existed in rAd-trSERCA2a, which was tested by PCR and southern blot. The quantity of mRNA of SERCA2a in cardiac myoeytes increased 3. 6 times compared with the control, and the protein level is 1. 5 times of the control, 72 hours after the transfeetion. Conclusion: This recombinant virus, rAd-trSERCA2a, can effectively delivery the SERCA2a gene into cultured cardiac myoeytes and may became a feasible tool to modulate myoeyte Ca2+ homeostasis in the failure heart.