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不同浓度的血管紧张素II对心肌细胞钙调磷酸酶Aβ基因表达水平的影响(PDF)

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2007年第5期
页码:
505-509
栏目:
基础研究
出版日期:
2007-10-01

文章信息/Info

Title:
Effect of angiotensin II at different concentrations on Calcineurin Aβ mRNA expression
作者:
栾颖;  曹威;  尹新华
哈尔滨医科大学附属第二医院心内科，黑龙江 哈尔滨 150086
Author(s):
LUAN Ying;  CAO Wei;  YIN XinHua
Department of Cardiovascular Disease, Second Affiliated Hospital, Harbin Medical University, Harbin 150086, Heilongjiang, China
关键词:
荧光定量PCR; 钙调磷酸酶; 血管紧张素II; 基因表达
Keywords:
realtime PCR ;  calcineurin ;  angiotensin II;  gene expression
分类号:
Q344
DOI:
-
文献标识码:
A
摘要:
目的 检测不同血管紧张素II（Ang II）的浓度对大鼠心肌细胞内钙调磷酸酶（CaN）Aβ基因表达水平的影响。方法 设计并合成大鼠CaN Aβ和β肌动蛋白(βactin)基因的特异性引物和TaqMan探针，将PCR 扩增的CaN Aβ和βactin基因片段分别克隆入pMD18T载体,用于标准曲线的绘制和样品检测。用ROX 标记的TaqMan 探针对CaN Aβ基因的表达进行定量；用FAM 标记的TaqMan 探针对βactin 看家基因的表达进行定量。通过看家基因βactin表达水平的定量结果对CaN Aβ表达水平的定量结果进行校正。改变培养的大鼠心肌细胞中Ang II的浓度并定量检测其CaN Aβ基因的表达水平。结果 应用重组质粒绘制的定量曲线循环阈值与模板的浓度具有良好的线性关系；与定量对照曲线比较计算出正常大鼠心肌细胞内CaN Aβ是βactin含量的90％。随着Ang II浓度的增高，大鼠心肌细胞内CaN mRNA的表达水平亦增加。结论 成功地建立了利用TaqMan探针检测CaN Aβ和βactin基因的多重荧光定量PCR方法并绘制标准曲线,从定量角度进一步证实Ang II能刺激CaN基因表达的水平。
Abstract:
AIM To detect the effect of angiotensin II (Ang II) at different concentrations on the expression of Calcineurin (CaN ) Aβ mRNA in rat myocardiocytea. METHODS To design and synthesis specific primer and TaqMan probe were designed and synthesized respectively. CaN Aβ and βactin cDNA from primary culture cardiocytes were amplified by RTPCR and were cloned into pMD18T vector for standard curve. The level of CaN Aβ mRNA was detected by multiplex PCR, altering the concentrations of Ang II in vitro myocardiocytes, and the result was corrected by the expression of housekeeping gene βactin. RESULTS The concentration of CaN Aβ of rat cardiocyte was about 90% of that of βactin. The expression of CaN Aβ mRNA increased with the increase of the concentration of Ang II. CONCLUSION We have successfully established a method to measure CaN Aβ and βactin mRNA expression with TaqMan probe. Ang II can increase the level of CaN Aβ mRNA in rat myocardiobcytes.

参考文献/References

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备注/Memo

备注/Memo:
收稿日期：2007-03-16.基金项目: 黑龙江省自然科学基金项目资助(No.D01 64)通讯作者:尹新华,主任医师, 主要从事心血管病学研究Email:xinhua_yin@163.com作者简介:栾颖, 主治医师,博士Email:qiuyang1021@yahoo.com.cn
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