【作者】 杨国杰；

【导师】 曾秋棠；

【作者基本信息】 华中科技大学 ， 内科学， 2008， 博士

【摘要】 第一部分表达Livin的逆转录病毒载体的构建、病毒包装和产生目的：为了观察大鼠心肌livin基因的过表达对大鼠心肌细胞缺血再灌注后心肌细胞凋亡的影响，构建表达Livin的逆转录病毒载体，包装产生逆转录病毒颗粒。方法：（1）从鼠乳腺癌细胞MA782中提取RNA，RT-PCR扩增得到Livin cDNA．先克隆入T载体（pGEM-T），经XhoⅠ/HindⅢ双酶切和PCR扩增鉴定，得到重组子pGEM-T-Livin,进行DNA序列测定。XhoⅠ／HindⅢ双酶切下livin目的基因片段，重组入（插入）逆转录病毒载体pLNCX₂，XhoⅠ／HindⅢ双酶切和PCR扩增鉴定得到重组子pLNCX₂-Livin，将pLNCX₂-Livin转染入PT67细胞中进行病毒包装，纯化得到表达Livin的逆转录病毒颗粒。结果：RT-PCR扩增得到816bp的Livin cDNA目的基因片段：筛选得到重组子pGEM-T-Livin，DNA序列分析证实插入序列与GenBank中XM-914797 Livin基因序列完全一致；筛选得到重组子pLNCX₂-Livin；包装得到表达Livin的逆转录病毒颗粒，滴度为3．9×10⁷IU／ml。结论：成功构建出表达Livin的逆转录病毒表达载体pLNCX₂-Livin，并包装纯化得到逆转录病毒颗粒，为下游实验奠定实验基础。第二部分Livin对大鼠心肌缺血再灌注损伤的影响目的：近年来研究发现凋亡参与了心肌缺血再灌注损伤的发生和发展，天冬氨酸特异的半胱氨酸蛋白酶-3（Caspase-3）被认为是凋亡发生的关键酶和标志酶，Livin是近年来发现的凋亡抑制蛋白家族中的新成员，能够通过抑制Caspase-3而发挥抑制凋亡作用，但是正常心肌细胞内Livin表达较少。为此，本实验拟转染表达livin的逆转录病毒载体，并建立大鼠心肌缺血再灌注模型，观察大鼠心肌缺血再灌注过程中Livin、Caspase-3的表达变化和二者的相关性，以及Livin对心肌梗死范围和心肌细胞凋亡的影响。方法：健康SD大鼠90只，随机分为缺血再灌注组（IR组）、Livin组和假手术对照组（Control组）。缺血再灌注组结扎LAD缺血30min，再灌注120 min；Livin组为缺血再灌注前24h心肌内注射携带Livin的逆转录病毒载体；对照组只穿线而不结扎LAD。再灌注120 min后TTC染色测定梗死范围，硫磺素染色法测定无复流面积，SP免疫组化测定Caspase-3、Livin蛋白表达量，TUNEL法测定凋亡心肌细胞。结果：1．免疫组化法检测心肌细胞有Livin的表达，缺血再灌注增加Livin的表达量（3．10±0．52），但与Control组（2．35±0．47）比较无统计学差异。2．缺血再灌注过程中Caspase-3表达增多明显，IR组（103．39±8．24）较Control组（91．39±4．82）显著增加；注射Livin后则Caspase-3的表达减少，Livin组（97．43±11．15）与IR组比较差异显著。3．Livin组心肌梗死范围（12．80±1．19％）较IR组（20．82±2．82％）显著减少。4．Livin组无复流面积（19．39±6．25％）较IR组（25．93±6．45％）显著缩小；5．TUNEL法检测显示缺血再灌注过程中可见心肌细胞凋亡的发生，IR组（10．35±3．34）较Control组（1．10±0．42）显著增加；注射Livin后则细胞凋亡的发生明显减少，Livin组（1．70±1．57）与IR组比较差异显著，与Control组比较无显著差异。结论：1．正常大鼠心肌细胞有Livin的表达。2．Livin可以抑制缺血再灌注心肌细胞Caspase-3的表达。3．Livin可以减少缺血再灌注心肌梗死范围和无复流面积。4．Livin可以抑制缺血再灌注心肌细胞凋亡的发生。第三部分荧光定量PCR检测鼠心肌caspase-3，Livin mRNA目的：观察大鼠心肌livin mRNA表达水平以及转染表达Livin的逆转录病毒载体后Livin在心肌表达的变化情况、缺血再灌注过程中Livin，Caspase-3的mRNA表达水平，以及Livin对Caspase-3 mRNA表达水平的影响．方法：提取第二部分实验各组组织总RNA，逆转录为cDNA，用荧光定量PCR方法扩增检测Livin和Caspase-3 mRNA表达水平。结果：荧光定量PCR检测到大鼠心肌细胞有Livin表达，正常组Livin mRNA表达水平（8．41±1．39）×10^-3，空载体组为（7．45±2．51）×10^-3，IR组为（7．82±3．22）×10^-3；Livin组为（145±89）×10^-3)，显著高于其他三组（P<0．01）。正常组Caspase-3mRNA相对表达量（5．12±2．11）×10^-3；空载体组为（6．88±2．31）×10^-3；缺血再灌注过程中Caspase-3表达明显增加(（92．1±34．6）×10^-3，显著高于正常对照组和空载体组（P<0．01），说明IR可造成Caspase-3升高；Livin组Caspase-3 mRNA表达水平为（56．2±21．1）×10^-3，与IR组相比，显著降低（P<0．05）。说明Livin过表达可有效降低IR组细胞Caspase-3表达。结论：1．正常大鼠心肌细胞有Livin mRNA的表达；2．表达Livin的逆转录病毒载体可以成功转染大鼠心肌；3．转染Livin可以降低大鼠缺血再灌注心肌细胞Caspase-3 mRNA的表达水平，有助于IR中的抗凋亡。更多还原

【Abstract】 PartⅠConstruction and production of retroviral vectorexpressing LivinObjectives: Construction a retroviral vector carrying Livin cDNA. To explore the effect of Livin over-expression on cardiac ischemia reperfusion.Methods: Livin gene from mice MA782 cell was amplificated by reverse-transcription polymerase chain reaction （RT-PCR） and first cloned into pGEM-T vector. Then digested by XhoⅠ/HindⅢand identified by PCR.And then subcloned into the retroviral vector pLNCX₂. pLNCX₂-Livin was transferred into packaging cell PT67 after XhoⅠ/HindⅢdigestion and identified by PCR. PT67/pLNCX2-Livin cells were selected. A pure packaging cell line/pLNCX2-Livin cells expressing Livin was cultured. The virus titer was assayed.Results:816bp Livin cDNA was obtained by RT-PCR amplification. The recombinant vector pLNCX2-Livin containing Livin gene was confirmed. The virus titer of the pure packaging cell line/ PT 67/ pLLNCX2-Livin was 3.9×10⁷ IU/ml.Conclusion:A kind of recombinant retroviral vector pLNCX2-Livin as well as a pure packaging cell line producing high virus titer are constructed successfully. PART 2 Effects of Livin on Myocardial Ischemiareperfusion Injury in RatsObjectives: To observe the livin protein expression in rat myocardial after the transfection of retroviral vectors and its effects on ischemia-reperfusion （I/R） injury.Methods: （1）90 healthy SD rats were divided into Contrl group, I/R group and Livin group. Rats were subjected to 30 min of left coronary artery occlusion followed by 120 min of reperfusion with （Livin group） or without （IR group） treating the rats with retrovirus vector expressing Livin protein by intramyocardial injection 24h before left coronary artery occlusion. Rats in Control group underwent thoracotomy without coronary ligation and injection of vector.（2） No-reflow area was detected by thioflavin S and the myocardial infarction size was evaluated by TTC dyeing method.（3）Caspase-3 and Livin expression detected with S? immunohistochemical staining.（4）Cardiomyocytes apoptosis was determined with terminal deoxynucleotidyl transferase-mediated Dutp-fluorescein nick end labeling（ TUNEL） method .Results:1.The expression of Livin was detected in myocardial cells by immunohisto- chemistry. The level of Livin was increased by Ischemia-reperfusion injury. There is no statistical significance between IR group （3.10±0.52） and Control group （2.35±0.47）.2.The level of Caspase-3 increased significantly during ischemia-reperfusion; There was significant diference between IR group（103.39±8.24） and Control group （91.39±4.82）. The expression of Caspase-3 was decreased after livin injection, there was significant difference between Livin group （97.43±11.15） and IR group.3. The infarct size （12.80±1.19 vs 20.82±2.82） and the no-reflow area （19.39±6.25 vs 25.93±6.45） were also decreased in Livin groups compared with the IR hearts.4. Using TUNEL assays, we examined the presence of apoptosis during I/R, which was significantly higher in IR group compared with the control hearts （10.35±3.34 vs 1.1±0.42）. Apoptosis rate was significantly reduced after Livin rejection （1.7±1.57） versus IR, but had no difference with control group. Conclusion1.There is Livin expression in normal rat myocardial.2.Retrovirus vector expressing Livin protein could express successfully in rat myocardial.3.Livin could inhibit caspase-3 expression in myocardial during I/R.4.Livin inhibits apoptosis, reduces no-reflow area and infartion size in myocardial, and protects the heart against ischemia/reperfusion injury.PART 3 Caspase-3, Livin mRNA detection by real time polymerase chain reactionObjectives: To observe the livin and Caspase-3 mRNA expression in rat myocardial with and without retroviral vectors transfection and its effects on ischemia-reperfusion （I/R） injury.Methods: Both Caspase-3 and Livin mRNA expression were detected by real time polymerase chain reaction （PCR）.Results: The livin mRNA expression was positive in normal rats detected by real time quantification PCR. The livin mRNA expression was （8.41±1.39）×10^-3 in control group, （7. 45±2. 51）×10^-3 in vector without livin gene group, （7.82±3.22）×10^-3 in IR group, （145±89）×10^-3 in Livin group. Compared with the Control and IR groups, Livin vector transfection significantly increased Livin mRNA expression.The Caspase-3 mRNA expression was （5.12±2.11）×10^-3 in control group, （6.88±2.31）×10^-3 in vector without livin gene group. Compared with the control group, I/R significantly increased Caspase-3 mRNA expression [（92.1±34.6）×10^-3, I/R group , P<0.01], which indicated that reperfusion could increase Caspase-3 mRNA expression. The Caspase-3 mRNA expression was （56.2±21.1）×10^-3 in Livin group, which was significantly decreased compared to IR group （P<0. 05） . These suggest that Livin overexpression could depress Caspase-3 expression in myocardial during I/R. Conclusions:1.There is Livin expression in normal rat myocardial.2.Retroviral vector expressing Livin protein could express successfully in rat myocardial.3.Livin could depress caspase-3 expression in myocardial during I/R.更多还原