PBNA-413对心肌缺血再灌注损伤保护作用机制初探(硕士)

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. [来源：http://www.doc163.com]
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.............................................

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   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....................................................

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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

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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