【摘要】 目的分析肺动脉高压时肺动脉平滑肌Ryanodine受体(ryanodine receptor,RyR)的结合功能改变及基因序列的变化。方法梯度离心法提取肺动脉平滑肌肌浆网膜蛋白组分,放射配基结合方法测定平衡解离常数(Kd)、最大结合容量(Bmax)的变化;体外提取肺动脉平滑肌细胞总RNA,反转录合成RyR2cDNA,以cDNA为模板使用特异性引物对目的片段进行PCR扩增,切胶回收后Sanger双脱氧末端终止法测定序列。结果野百合碱(monocrotaline,MCT)所致肺动脉高压组RyR最大结合容量(Bmax)为(197±21.4)nmol.g-1protein,较对照组(168±13.7)nmol·g-1protein明显增加(P<0.01);平衡解离常数(Kd)为(0.276±0.026)nmol·L-1与对照组(0.282±0.031)nmol·L-1相比差异无显著性(P>0.05);野百合组RyR2C-末端基因序列与对照组无差异。结论MCT所致肺动脉高压大鼠RyR最大结合容量(Bmax)增加,提示受体的数量发生了上调;Kd无明显变化,说明与受体的亲和力无改变。MCT所致肺动脉高压大鼠2型RyR(RyR2)C-末端基因序列无变化。更多还原

【Abstract】 Aim To investigate the changes in binding features and C-terminal gene sequence of ryanodine receptors in the pulmonary artery smooth muscle of pulmonary arterial hypertension (PAH) rats induced by monocrotaline (MCT). Methods Sarcoplasmic reticulum (SR) were fractionated with density gradient centrifugation and the characteristic of ryanodine receptor was assayed with a method of radioreceptor binding assay; The total RNA was obtained from the PAMSC of rat. The purity of total RNA was detected by agarose electrophoresis. The C-terminal fragment of RyR2 cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR), amplified by nest PCR, and then purified and retrieved by agarose electrophoresis separation. The sequence was analyzed using Sanger method. Results The Bmax of ryanodine receptors in the SR of pulmonary artery smooth muscle in MCT group was(197±21.4) nmol·g-1 protein, and significantly higher than that in the control group (168±13.7) nmol·g-1 protein, P<0.01. The Kd of ryanodine binding to SR remained unchanged (P>0.05). Sequence analysis showed that the C-terminal fragment sequence of RyR2 cDNA was the same in MCT group as in the control group. Conclusion These results demonstrated that up-regulation of ryanodine receptors in the pulmonary artery smooth muscle cells (PASMC) occurred during PAH, which may be one of the possible mechanisms of PAH.更多还原