PBNA-413对心肌缺血再灌注损伤保护作用机制初探(硕士)

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PBNA-413对心肌缺血再灌注损伤保护作用机制初探(硕士)(论文23000字)
摘要
目的:
研究拳参正丁醇提取物(PBNA-413)对大鼠心肌缺血再灌注(I/R)损伤心电图的影响及对其保护作用的最佳剂量,并从自噬、心肌细胞凋亡及缝隙连接蛋白Cx-43的角度,初步探讨拳参有效成分PBNA-413对心肌缺血/再灌注损伤的保护机制。
方法:
将40只雄性大鼠随机分为5组:假手术组、MI/R模型组,PBNA-43高、中、低(1.0 mg/kg、0.3 mg/kg、0.1 mg/kg)剂量组。通过对大鼠心脏左冠状动脉前降支进行接渣,至心肌缺血45 min后,恢复血流并持续1 h,复制MI/R损伤模型,并记录心电图。各组分别予以不同浓度药物处理。取部分结扎线以下左心室组织心肌组织,采用实时荧光定量PCR(Real-time PCR)和蛋白免疫印迹法(Western blot)检测各组大鼠心肌组织Cx-43和p-Cx-43、凋亡相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),Caspase-9及自噬相关蛋白Beclin-1、LC3Ⅱ的mRNA及蛋白表达。
结果:
结扎大鼠冠状动脉左前降支后,模型组大鼠心电图T波及ST段与假手术组比明显抬高,PBNA-43处理后心肌ST段降低,且呈剂量依赖性,以高剂量的效果最好。与假手术组相比,模型组心肌组织Cx-43 mRNA、p-Cx-43和Cx-43、Beclin-1、LC3Ⅱ、Bcl-2蛋白水平均降低;心肌组织凋亡相关蛋白Bax,Caspase-3及Caspase-9蛋白表达升高。与模型组相比,PBNA-413高剂量组Cx-43 mRNA、p-Cx-43和Cx-43蛋白、Beclin-1、LC3Ⅱ、Bcl-2表达均升高。Bax及Caspase-3和Caspase-9蛋白表达降低,除Caspase-9外,Bax,Bcl-2和Caspase-3的基因水平出现与蛋白类似变化,LC3Ⅱ、Beclin-1的基因水平变化不明显。
结论:
PBNA-43可能通过提高缝隙连接通道相关蛋白Cx43的表达及活化,适度促进细胞自噬及抑制细胞凋亡改善缺血/再灌注损伤性心律失常,进而对缺血/再灌注损伤心肌起保护作用。
关键词:拳参正丁醇提取物-413;心肌缺血/再灌注;缝隙连接蛋白43,细胞自噬,细胞凋亡

A preliminary study on the protective mechanism of PBNA-413 on myocardial ischemia reperfusion injury
Abstract
Objectives:
The present study aims to investigate the effect of polygonum bistorta n-butyl alcohol extract (PBNA-413) on electrocardiogram (ECG) of myocardial ischemia reperfusion (I/R) injury and screen the best dosage of PBNA-413 for the protective
function to myocardial ischemia reperfusion injury. This study also preliminarily explores the protective mechanism of PBNA-413 on myocardial ischemia reperfusion injury by the study of myocardial cell apoptosis and autophagy as well asgap connexin Cx-43.
Methods:
40 male rats were randomly divided into 5 groups: the sham operation group, the MI/R model group, the groups with PBNA-43 high, medium and low-dose(1 mg/kg, 0.3 mg/kg, 0.1 mg/kg) treatment. The MI/R injury model was established by ligation of the left anterior descending branch of the coronary artery for 45min followed by reperfusion for 1 hour. The ECG was recorded. Each group was treated with different concentration of drugs. The mRNA or protein expression of Cx-43, p-Cx-43, Bcl-2, Bax, Caspase-3, Caspase-9, Beclin-1 and LC3 II in left ventricular myocardium tissue with I/R injury were detected by Real-time PCR and Western blotting.
Results:
The T wave and ST segment of the ECG in the MI/R model group were significantly higher than those in the sham operation group, but the ST segment decreased after PBNA-43 treatment in dose- dependent manner. Compared with the sham operation group, the mRNA expression levels of Cx-43 in MI/R model group decreased. So did the protein expression levels of p-Cx-43, Cx-43, Beclin-1, LC3Ⅱ and Bcl-2. But the protein expression levels of Bax, Caspase-3 and Caspase-9 in myocardial tissue with I/R injury increased. Compared with the model group, the mRNA expression level of Cx-43 in the group with PBNA-43 high-dose treatment increased. So did the protein expression level of p-Cx-43, Cx-43, Beclin-1, LC3Ⅱ and Bcl-2 in the group with PBNA-43 high-dose treatment. But the protein expression levels of Bax, Caspase-3 and Caspase-9 decreased. Except for Caspase-9, the similar changes were found in mRNA and protein expression level of Bax, Bcl-2 and Caspase-3. There were no significant differences in mRNA expression of LC3 II and Beclin-1 between them.
Conclusion:
PBNA-43 may promote the autophagy and inhibit cell apoptosis by improving the expression and activation of Cx43, and rectify the arrhythmias that originate from I/R injury, thus effectively protect myocardium from I/R injury
Keywords: PBNA -413; Myocardial ischemia / reperfusion; Connexin 43; Autophagy; Apoptosis

目录
常用缩写词中英文对照表...............................................3
前言.................................................................4
1材料与方法.........................................................5
  1.1主要药物、仪器和试剂............................................6
1.2主要实验试剂配制............................................
1.3动物及分组..............................................
  1.4动物模型制备及方法步骤..................................
1.5心肌缺血/再灌注(MI/R)模型制备..............................
1.6PBNA药物配制及给药.....................................
  1.7 Western blot法检测心肌组织自噬相关蛋白、凋亡相关蛋白及p-cx43/
       Cx43的表达.................................................
   1.7.1取材......................................................
   1.7.2组织蛋白提取...............................................
   1.7.3 BCA法测蛋白浓度...........................................
   1.7.4制备上样蛋白...............................................
   1.7.5SDS-PAGE凝胶制备...........................................
   1.7.6转模.......................................................
   1.7.7孵育一抗、二抗、发光.......................................
   1.8 q-PCR法检测心肌组织自噬相关基因、凋亡相关基因及缝隙连接蛋白
       Cx43的mRNA表达...........................................
   1.8.1组织总RNA提取.............................................
   1.8.2反转录、实时定量PCR的扩增.................................
   1.9 数据处理与统计分析..........................................
2 实验结果.................................................
2.1 PBNA-43对MI/R大鼠心电图影响.................................
   2.2 PBNA-43 对MI/R大鼠心肌组织p-Cx43/Cx43表达的影响...........
   2.3 PBNA-43对MI/R大鼠心肌组织自噬相关蛋白表达的影响..........
   2.4 PBNA-43对MI/R大鼠心肌组织凋亡相关蛋白的影响..............
   2.5 PBNA-43对MI/R大鼠心肌组织Cx43mRNA表达的影响..............
   2.6 PBNA-43对MI/R大鼠心肌组织自噬相关........................
   2.7 PBNA-43对MI/R大鼠心肌组织凋亡相关基因表达的影响..........
3 讨论...................................................
4 结论.....................................................
5 参考文献...............................................
6 综述....................................................
7 参考文献................................................
8 个人简介................................................
常用缩写词中英文对照表
英文缩写    英文名称    中文名称
MI/R    Myocardial ischemia/reperfusion    心肌缺血再灌注
LAD    Anterior descending coronary artery    左冠状动脉前降支
I/R    Ischemia/reperfusion                  缺血再灌注
PBNA    Polygonum bistorlaL n-Butyl alcohol extract    拳参正丁醇提取物
WB    Western Blot    蛋白免疫印迹
SDS    Sodium dodecyl sulfate    十二烷基磺酸钠
PVDF    polyvinylidene fluoride    聚偏二氟乙烯膜
TBS    Tris buffered solution    Tris缓冲液
APS    Ammonium persulfate    过硫酸铵
DEPC    Diethyl pyrocarbonate    焦碳酸二乙脂
GJ    gapjunction    缝隙连接通道
NS     Normal saline             生理盐水
SPF    Specific pathogen Free    无特定病原体
SD       Sprague Dawley    SD大鼠
EP      Eppendorf    微量离心管
BCA    Bicinchonininc acid 2    2-联喹啉-4,4- 二甲酸
RT-PCR    Real-time Polymerase Chain Reaction    实时荧光定量
Cx43    Connexin43    连接蛋白43