【作者】 高娟；

【导师】 朱国庆；

【作者基本信息】 南京医科大学 ， 生理学， 2010， 硕士

【摘要】 背景慢性心力衰竭(CHF)是严重危害人类健康的常见病。过度激活的交感神经活动是CHF的重要特征,在CHF的病程进展中起重要作用。下丘脑室旁核(paraventricular nucleus,PVN)是心血管活动整合的重要中枢之一,在交感神经活动调控中起重要作用。本实验室既往研究表明PVN中血管紧张素Ⅱ(Ang II)与CHF大鼠过度增强的交感神经活动密切相关,PVN内NAD(P)H氧化酶来源的活性氧,尤其是超氧阴离子和过氧化氢都参与了Ang II对交感神经活动的调节作用。铜锌过氧化物歧化酶(CuZnSOD),是一种胞浆内的抗氧化同工酶,能够清除超氧阴离子。CHF大鼠长期口服SOD拟似剂tempol可降低交感神经活动,但是否因为长期清除PVN中的超氧阴离子引起的CHF大鼠的交感神经活动降低和心功能改善尚不清楚。由于CuZnSOD本身存在半衰期短、代谢快的特点,给长期PVN中清除超氧阴离子制造了瓶颈。随着分子生物学的发展,基因治疗作为传统药物治疗外的新治疗手段显示出广阔的前景,重组腺病毒载体携带目的基因的蛋白过表达技术日益成熟。PVN中腺病毒转染CuZnSOD基因,能够长期降低PVN中超氧阴离子水平,为在中枢水平探讨CHF的基因治疗奠定理论和实验基础,开辟新途径。目的1.观察PVN内微量注射携带人类CuZnSOD基因的腺病毒载体(Ad-SOD),能否降低PVN中超氧阴离子浓度。2.探讨降低PVN中超氧阴离子浓度能否使心肌梗死导致的CHF大鼠过度增强的交感神经活动恢复正常。3.探讨降低PVN中超氧阴离子浓度能否改善CHF大鼠心肌重构和心功能。方法和结果SD大鼠随机分为冠状动脉结扎(CAL)致心肌梗死(MI)组和假手术(Sham)组,各组再随机分为双侧PVN内微量注射携带CuZnSOD基因的腺病毒载体组(Ad-SOD)组和空载体组(Ad-null)组。冠状动脉结扎术或假手术后,将Ad-SOD和Ad-null立即微量注射入PVN。术后8周,通过Western Blotting检测PVN内CuZnSOD的表达,紫外分光光度法测定CuZnSOD的活性,化学发光法测定超氧阴离子水平;测定基础交感神经活动(RSNA),用高效液相色谱法(HPLC)测定血浆去甲肾上腺素(NE)的浓度,以心室表面应用辣椒素引起的肾交感神经活动变化评价心交感传入反射(CSAR);以心肌组织切片的苏木素-伊红(hematoxylin-eosin,HE)染色、Masson’s三重染色、TUNEL染色评价心肌重构;用超声心动图和左心室血流动力学方法测定心功能。结果如下:1.CuZnSOD蛋白表达水平、CuZnSOD活性和超氧阴离子水平MI-null组大鼠PVN内CuZnSOD蛋白表达水平和活性均显著低于Sham-null组,而超氧阴离子显著升高。CuZnSOD过表达组大鼠PVN内CuZnSOD蛋白表达和活性水平显著提高,并且降低超氧阴离子水平。2.基础交感神经活动和血浆NE水平MI组大鼠的基础RSNA和血浆NE水平均比Sham组大鼠高,PVN中CuZnSOD过表达可以抑制基础RSNA的升高,并使血浆NE浓度降至正常水平。3.CSAR和Sham组大鼠相比,MI组大鼠引起CSAR显著增强。CuZnSOD过表达可以减弱MI大鼠显著增强的CSAR。4.组织重量和疤痕MI组大鼠心脏/体重比值和湿肺/体重比值升高,心室前壁有较大的心肌梗死疤痕。PVN内CuZnSOD过表达可以降低心脏/体重比值和湿肺/体重比值,但并未减小疤痕面积。5.心肌切片HE染色、Masson’s三重染色、TUNEL染色在HE染色的心肌切片上观察到MI大鼠心肌细胞肥大,而CuZnSOD过表达明显减小了梗死周边区和非梗死区心肌细胞直径;Masson’s三重染色的心肌切片上观察到MI大鼠有明显的胶原沉积,而CuZnSOD过表达明显改善MI后梗死周边区的胶原沉积;TUNEL染色的心肌切片上中观察到MI大鼠心肌细胞凋亡增多,而CuZnSOD过表达显著降低梗死周边区的TUNEL阳性细胞比例,但是并未改善非梗死区的心肌细胞凋亡。6.超声心动图和Sham-null组大鼠相比,MI组大鼠左室舒张末期容积(LVEDV)和左室收缩末期容积(LVESV)增大,短轴缩短率(FS)和射血分数(EF)的降低。PVN的CuZnSOD过表达显著改善LVEDV、LVESV增大和FS、EF降低。7.左心室血流动力学MI-null组大鼠左室舒张末期压力(LVEDP)显著升高而左心室内压最大上升速率(LVdP/dtmax)显著降低。CuZnSOD过表达显著改善MI引起的LVEDP升高和LVdP/dtmax降低。结论1.PVN内微量注射Ad-CuZnSOD能够成功在体转染并持续表达和维持活性,降低MI大鼠PVN中超氧阴离子水平。2.PVN内过表达CuZnSOD使MI大鼠过度增强的交感神经活动、CSAR和血浆NE恢复正常。3.PVN中CuZnSOD过表达减轻了心肌梗死引起的梗死周边区心肌细胞肥大、胶原沉积和细胞凋亡,并且改善了心功能。更多还原

【Abstract】 BackgroundChronic heart failure has become one of the most important cardiovascular diseases around the world. One of its characteristics is the overenhanced sympathetic nerve activity, which play an important role in the progress of CHF. Paraventricular nucleus is one of the important cardiovascular integrative centers. Our previous studies showed that in acute experiments the reactive oxygen species in PVN generated from NAD(P)H oxidase mediated the effects of Ang II on sympathetic nerve activity, especially the superoxide anions and hydrogen peroxide.Cooper-zinc superoxide dismutase is a kind of antioxide isoenzyme in cytoplasm, which catalyzes the conversion of superoxide radicals into hydrogen peroxides. Long term administration of tempol, a SOD mimic, normalizes the enhanced baseline sympathetic outflow of CHF rats. However, it is unknown whether scavenging the superoxide anions in the PVN in long term has beneficial effects on the sympathetic nerve activity and dysfunction of CHF rats. And CuZnSOD has short half life and high metabolic rate, which prevent the research from long term scanvenging the superoxide anions in PVN. With the development of molecular biology, gene therapy, as new treatment methods, show large prospects, in addition to traditional medication. Protein overexpression technology with the recombinational adenovirus vector carrying the target gene has become more sophisticated. CuZnSOD gene transfer into PVN can facilitate the research on the effects of the central superoxide anions on sympathetic nerve activity, which lay the theoretical and experimental foundation for further exploration of the CHF gene therapy.Objective1. To determine whether recombinant adenoviral vectors harboring human CuZnSOD can be transferred in PVN and continue to express in long term.2. To determine whether the enhanced sympathetic nerve activity after myocardial infarction can be normalized by CuZnSOD overexpression in PVN of CHF rats.3. To determine whether the CuZnSOD overexpression in PVN can improve the myocardial remodeling and cardiac dysfunction in CHF rats. Methods and ResultsThe rats were randomly divided into MI and Sham groups which were respectively subjected to the coronary ligation or sham operation. The Ad-SOD or Ad-null (1×1012 particles·ml-1) was respectively microinjected into the PVN immediately after the coronary ligation or sham operation. The final experiment was carried out at the end of the eighth week of the surgery. CuZnSOD expression in the PVN was determined with Western blotting. The activity of CuZnSOD in the PVN was measured according to its scavenging ability of the homogenates to superoxide anions using Ultraviolet Spectrophotometry. Determine baseline RSNA. Plasma norepinephrine level was determined by high-performance liquid chromatography. The cardiac sympathetic afferent reflex was evaluated by application of capsaicin to the epicardial surface of anterior wall of the left ventricle. Heart slices were stained with hematoxylin-eosin staining for morphologic examination or Masson’s trichrome staining for assessment of interstitial fibrosis or TUNEL assay for apoptosis. Echocardiography and hemodynamic measurement were performed to envaluate the ventricular function.Results:1. CuZnSOD expression and activity, superoxide anionsCompared with sham rats, MI rats show lower CuZnSOD expression and activity, but higher superoxide anions level. Compared with null rats, Ad-CuZnSOD treated rats showed higher expression and activity of CuZnSOD and lower superoxide anions level.2. Baseline sympathetic nerve activity and plasma norepinephrine concentrationMI rats show higher baseline sympathetic nerve activity and plasma norepinephrine concentration than sham rats. Overexpression of CuZnSOD in PVN attenuated the increased baseline sympathetic nerve activity and plasma norepinephrine concentration in MI rats.3. CSARMI could induce enhanced CSAR. CuZnSOD gene transfer improved the enhanced CSAR after maycardial infarction.4. Weight and ScarMI rats showed significant increase in heart to body weight ratio and wet lung to body weight ratio compared with Sham rats. A dense scar was found in the anterior ventricular wall in each MI rat, but not in any Sham rat. Overexpression of CuZnSOD in PVN decreased the heart weight, but there was no significant difference in the infarct area between MI-SOD rats and MI-null rats.5. Heart slices for HE staining, Masson’s trichrome staining and TUNEL assayCardiomyocyte size was considerably increased in MI rats in both border and remote zones of the infarction, which was attenuated by Ad-SOD. Ad-SOD inhibited the collagen deposition caused by MI. The collagen volume fraction significantly increased in MI rats, which was attenuated by Ad-SOD. In the border zone, the percentage of TUNEL-positive cardiomyocytes was markedly reduced in the MI-SOD group relative to MI-null group. In the remote zone, PVN CuZnSOD overexpression did not show significant effect on the increased percentage of TUNEL-positive cardiomyocytes in MI rats.6. EchocardiographyCompared with Sham-null rats, the LVEDD, LVESD, LVEDV and LVESV increased, while the IVSd, IVSs, SI, FS and EF decreased in MI-null rats. These changes caused by the MI were significantly attenuated by Ad-SOD.7. HemodynamicsThe LVEDP increased and LVdP/dtmax decreased in MI-null rats. The left ventricular dysfunction was significantly improved by treatment with Ad-SOD.Conclusions1. CuZnSOD can be successfully transferred into the PVN, and keep persistent expression and stable activity.2. Overexpression of CuZnSOD in the PVN normalized the enhanced baseline sympathetic nerve activity, and attenuated the enhanced CSAR in MI rats.3. Overexpression of CuZnSOD in the PVN attenuated the cardiac hypertrophy, collagen deposition and apoptosis in the border zone after myocardial infarction, and improved the ventricular dysfunction in MI rats.更多还原