PBNA-413對心肌缺血再灌注損傷保護作用機制初探(碩士)

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PBNA-413對心肌缺血再灌注損傷保護作用機制初探(碩士)(論文23000字)
摘要
目的:
研究拳參正丁醇提取物(PBNA-413)對大鼠心肌缺血再灌注(I/R)損傷心電圖的影響及對其保護作用的最佳劑量,并從自噬、心肌細胞凋亡及縫隙連接蛋白Cx-43的角度,初步探討拳參有效成分PBNA-413對心肌缺血/再灌注損傷的保護機制。
方法:
將40只雄性大鼠隨機分為5組:假手術組、MI/R模型組,PBNA-43高、中、低(1.0 mg/kg、0.3 mg/kg、0.1 mg/kg)劑量組。通過對大鼠心臟左冠狀動脈前降支進行接渣,至心肌缺血45 min后,恢復血流并持續1 h,復制MI/R損傷模型,并記錄心電圖。各組分別予以不同濃度藥物處理。取部分結扎線以下左心室組織心肌組織,采用實時熒光定量PCR(Real-time PCR)和蛋白免疫印跡法(Western blot)檢測各組大鼠心肌組織Cx-43和p-Cx-43、凋亡相關蛋白B細胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相關X蛋白(Bax),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),Caspase-9及自噬相關蛋白Beclin-1、LC3Ⅱ的mRNA及蛋白表達。
結果:
結扎大鼠冠狀動脈左前降支后,模型組大鼠心電圖T波及ST段與假手術組比明顯抬高,PBNA-43處理后心肌ST段降低,且呈劑量依賴性,以高劑量的效果最好。與假手術組相比,模型組心肌組織Cx-43 mRNA、p-Cx-43和Cx-43、Beclin-1、LC3Ⅱ、Bcl-2蛋白水平均降低;心肌組織凋亡相關蛋白Bax,Caspase-3及Caspase-9蛋白表達升高。與模型組相比,PBNA-413高劑量組Cx-43 mRNA、p-Cx-43和Cx-43蛋白、Beclin-1、LC3Ⅱ、Bcl-2表達均升高。Bax及Caspase-3和Caspase-9蛋白表達降低,除Caspase-9外,Bax,Bcl-2和Caspase-3的基因水平出現與蛋白類似變化,LC3Ⅱ、Beclin-1的基因水平變化不明顯。
結論:
PBNA-43可能通過提高縫隙連接通道相關蛋白Cx43的表達及活化,適度促進細胞自噬及抑制細胞凋亡改善缺血/再灌注損傷性心律失常,進而對缺血/再灌注損傷心肌起保護作用。
關鍵詞:拳參正丁醇提取物-413;心肌缺血/再灌注;縫隙連接蛋白43,細胞自噬,細胞凋亡

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

目錄
常用縮寫詞中英文對照表...............................................3
前言.................................................................4
1材料與方法.........................................................5
  1.1主要藥物、儀器和試劑............................................6
1.2主要實驗試劑配制............................................
1.3動物及分組..............................................
  1.4動物模型制備及方法步驟..................................
1.5心肌缺血/再灌注(MI/R)模型制備..............................
1.6PBNA藥物配制及給藥.....................................
  1.7 Western blot法檢測心肌組織自噬相關蛋白、凋亡相關蛋白及p-cx43/
       Cx43的表達.................................................
   1.7.1取材......................................................
   1.7.2組織蛋白提取...............................................
   1.7.3 BCA法測蛋白濃度...........................................
   1.7.4制備上樣蛋白...............................................
   1.7.5SDS-PAGE凝膠制備...........................................
   1.7.6轉模.......................................................
   1.7.7孵育一抗、二抗、發光.......................................
   1.8 q-PCR法檢測心肌組織自噬相關基因、凋亡相關基因及縫隙連接蛋白
       Cx43的mRNA表達...........................................
   1.8.1組織總RNA提取.............................................
   1.8.2反轉錄、實時定量PCR的擴增.................................
   1.9 數據處理與統計分析..........................................
2 實驗結果.................................................
2.1 PBNA-43對MI/R大鼠心電圖影響.................................
   2.2 PBNA-43 對MI/R大鼠心肌組織p-Cx43/Cx43表達的影響...........
   2.3 PBNA-43對MI/R大鼠心肌組織自噬相關蛋白表達的影響..........
   2.4 PBNA-43對MI/R大鼠心肌組織凋亡相關蛋白的影響..............
   2.5 PBNA-43對MI/R大鼠心肌組織Cx43mRNA表達的影響..............
   2.6 PBNA-43對MI/R大鼠心肌組織自噬相關........................
   2.7 PBNA-43對MI/R大鼠心肌組織凋亡相關基因表達的影響..........
3 討論...................................................
4 結論.....................................................
5 參考文獻...............................................
6 綜述....................................................
7 參考文獻................................................
8 個人簡介................................................
常用縮寫詞中英文對照表
英文縮寫    英文名稱    中文名稱
MI/R    Myocardial ischemia/reperfusion    心肌缺血再灌注
LAD    Anterior descending coronary artery    左冠狀動脈前降支
I/R    Ischemia/reperfusion                  缺血再灌注
PBNA    Polygonum bistorlaL n-Butyl alcohol extract    拳參正丁醇提取物
WB    Western Blot    蛋白免疫印跡
SDS    Sodium dodecyl sulfate    十二烷基磺酸鈉
PVDF    polyvinylidene fluoride    聚偏二氟乙烯膜
TBS    Tris buffered solution    Tris緩沖液
APS    Ammonium persulfate    過硫酸銨
DEPC    Diethyl pyrocarbonate    焦碳酸二乙脂
GJ    gapjunction    縫隙連接通道
NS     Normal saline             生理鹽水
SPF    Specific pathogen Free    無特定病原體
SD       Sprague Dawley    SD大鼠
EP      Eppendorf    微量離心管
BCA    Bicinchonininc acid 2    2-聯喹啉-4,4- 二甲酸
RT-PCR    Real-time Polymerase Chain Reaction    實時熒光定量
Cx43    Connexin43    連接蛋白43

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