【作者】 谢满江；

【导师】 余志斌；

【作者基本信息】 第四军医大学 ， 航空航天医学， 2002， 硕士

【摘要】 心力衰竭是一种以心功能进行性降低为特点的常见疾病。各种诱因引起的慢性压力／容积超负荷是心力衰竭的主要病因之一。其变化过程为慢性压力／容积超负荷首先引起代偿性心肌肥厚，继而发生失代偿，最后转向心力衰竭。近年的研究结果更新了对心力衰竭机制的理解，认识到心肌细胞凋亡在心衰发生与发展过程中可能起到重要作用。因此，探讨心肌细胞凋亡的发生机理，可能会为临床上预防与治疗心衰提供新的思路。但当前的研究仅仅是一个开端，有几个基本问题亟待解决：1)病理浓度的多种促心肌细胞肥大因子先后或联合作用是否会导致心肌细胞凋亡率增加?2)肥大的心肌细胞是否更容易发生凋亡?3)心肌细胞肥大刺激因子通过何种信号转导通路，诱发肥大心肌细胞向凋亡转化? 为了探讨以上问题，我们以原代培养的新生大鼠心肌细胞为实验模型，首先建立心肌细胞肥大与凋亡的检测方法：测量HE染色细胞的截面面积，并结合偶合FITC的鬼笔环肽荧光染色，观测心肌细胞的肥大特征，依此确定心肌细胞是否肥大。采用Hoechst 33258荧光直接进行细胞核染色、DNA降解片段的琼脂糖凝胶电泳与原位末端转移酶标记(TUNEL) 第口果巨大仆顷士纱让论文一三种不同方法进行细胞凋亡的检测。观测促心肌细胞肥大因子去甲肾上腺素（NE）、内皮素（ET－1）或前列腺素（PGFza）等单独或联合作用，对心肌细胞肥大与凋亡的影响。其主要结果如下： （一）建立了可靠的新生大鼠心肌细胞原代培养方法 新生大鼠心肌细胞在含10％胎牛血清的培养液中，可健康存活10 d以上：在含HS的无血请培养液中，亦可健康存活10 d以上。在整个培养过程中，细胞形态良好，心肌细胞生长与搏动正常。在培养的4～6d，其搏动频率基本保持在SO～100次／min。采用抗actin或心肌TnT的单克隆抗体进行免疫组化鉴定表明，心肌细胞所占比例大于 90％。 （二）正常培养的心肌细胞截面面积与基础凋亡百分率的范围 正常培养第 6 d的心肌细胞截面面积范围为 900～3000 p＼平均截面面积为1924士74 pm‘。培养6 d时，用 Hoechst 33258检测有血清培养心肌细胞的凋亡率约为 9．67土 0．47％；无血清培养心肌细胞的凋亡率为门刀5土 0厂0％，两者间无显著差别（HO刀5）。 （三）内皮素l可啊去甲肾上腺素诱导的心肌细胞凋亡 1卜mow的M作用心肌细胞48 h，心肌细胞凋亡率为 18．39士0．80％；10 pmoee的 NE作用 48 h，心肌细胞凋亡率升高到 30．27土 1．32％，与同步对照（11刀5士0．70％）相比较，两者的变化均具有显著性意义呼＜0刀1人IO或10O。OTh的ET－l作用48 h，。G’肌细胞凋亡率分别为11．88f0．46％与 12．06士 0．70％（P＞0．05）。当 10 ＂mow的 ET－l与 1卜mo皿的 NE联合作用时，心肌细胞凋亡率大幅度升高，达 35．20士 1．37％，与同步对照组相比具有显著性差异（P＜0刀1）；与NE作用组相比，亦有显著性差别（P＜0刀1）。当以＊l1受体＊T＾的秸抗剂＊Q123（1卜一W）作用时，icm＊t的ET－1与 lpmow 的 NE作用组凋亡率下降到 20．5土 1．9％（P＜0＊ IversuslonmoljL ET－l十1卜moooMi）。 ry 第口旱巴大学刃士学位论文一 （四）肥大心肌细胞的凋亡倾向性增加 1）ET－l或PGF。a所诱导的心肌细胞肥大呈明显剂量依赖性关系 同步对照培养第5天时心肌细胞截面面积为 1277．belll．2 pm＼ 10 urn。W＊l1作用24 h，使心肌细胞截面面积明显增至二150＊士13o．5卜m’（P＜O．01）。100 。了L＊＊－1作用24 h使心肌细胞截面面积呈进一步增加趋势，为刀50．8士18O二Vin’（P＞0刀5 versus 10 ＂mow＊＊ ）。虽然10 nmoMi的PGF。a作用 24 h仅引起心肌细胞截面面积小巾增加至 1636．5ti20．3 im’ （P＜0刀5），但是 100 11．－－－－－－。i的 PGFZCh作用 24 h却引起心肌细胞截面面积大幅增力至2637．8土200．9卜＜‘（P＜0刀1）。 2）ET－l与PGFZa联合作用诱导心肌细胞肥大，但仅有协同作用，缺乏叠加效应 10 nmoffe ET－l与 10 nmoCh的 PGF批联合作用 24 h，细胞截面面积增至230O．3士120＊卜m‘（P＜0＊1）；10 nmo凡＊l1与10O ＂mow的 PGF水联合作用 24 h，细胞截面面积进一步增大至2836．2if80．8 pm‘ （P＜0刀1）。而 100 nmoffe ET－l与 10 nmol／L的 PGF。a联合作用 24 h，细胞截面面积增力较J为 2500．41 130．2 pm‘（P＜0刀1）；100 nmoffe ET－l与100。。e 的 PGFZ。联合作用 24 h，细胞截面面积大幅增加至3825．5土＊0石卜m‘（P＜0刀1）。10 nmo凡＊＊1十IO nmoWP＊＊a组的细胞截面面积显著大于 10 nmol／L的 PGF。口组（P＜0刀1），但与 10 nxnoffe ET－l组差异不明显（Peq刀5）。10 nmol／L ET－l十 100 nmol／L PGF。a组的细胞截面面积大于更多还原

【Abstract】 Heart failure is a kind of important clinical diseases with progressive myocardial dysfunction. Recently accumulating evidences suggest that apoptosis contributes to heart failure as the loss of viable cardiac myocytes. In clinical, heart failure is mainly caused by chronic pressure or volume overload. Therefore, the importance of apoptosis in the transition form compensatory hypertrophy to decompensation of the heart has been recognized. To understand the molecular and cellular mechanisms involved in the regulation of cardiac myocyte hypertrophy and apoptosis may provide a new idea in the therapy to prevent and cure this kind of heart failure. However* Supported by Foundation for University Key Teacher by the Ministry of Education People’s Republic of Chinavmthis research is only in the beginning and there are three questions to be considered firstly: 1) Whether the action of various pro-hypertrophic factors may increase the apoptotic rate in combination or succession? 2) Whether hypertrophic cardiac myocytes are more susceptible to apoptosis than normal myocytes? 3) What are the critical signal transduction pathways during the transition from hypertrophy to apoptosis in cardiac myocytes?Based on these questions, Primary cardiac myocytes were cultured in serum-free media. First we established methods to detect hypertrophy and apoptosis in cardiac myocytes. To detect myocyte hypertrophy, we measured profile area of cardiac myocytes stained with HE and observed cardiac myocyte myofibril stained with FITC conjugated phalloidin under fluorescent microscope. To detect myocyte apoptosis, cardiac myocyte nuclei were stained with fluorescent DNA-binding dyes Hoechst 33258 and propidium iodide and viewed under fluorescent microscope, fragmentation of genomic DNA was isolated from the neonatal rat cardiac myocytes by electrophoresis on 2% agarose gel to observe DNA ladder and cardiac myocytes were stained with terminal deoxy-nucleotidyl transferase mediated UTP end labeling (TUNEL). We investigated and compared the effects of NE, ET-1 and PGF2a on hypertrophy and apoptosis in neonatal rat cardiac myocytes. The main results of present work are as follows:I) A stable method for the culture of primary neonatal rat cardiac myocytes: The cultured neonatal rat cardiac myocytes in 10% PCS media survived over 10 days and the cultured myocytes in serum-free media survived over 10 days too. The morphological features and beat rate of myocytes were in good condition during their growth. During 4-6 days, the beat rates areDC80-100 beats/min. The cardiac myocyte were identified by immunocytochemical staining with actin and cTnl monoclonal antibodies. The purity of cultured neonatal rat cardiac myocyte was 90%.II) Normal ranges of profile area of myocytes and the percentage of apoptotic rate in cultured cardiac myocytes: normal range of profile area of myocytes cultured for 6 days was 900~3000 urn2 and the mean profile area was 1924 + 74 um2. With Hoechst 33258 and PI, we know the percentage of apoptosis in myocytes cultured in serum-containing for 6 days was 9.67 + 0.47 % and the percentage of apoptosis in myocytes cultured in serum-free for 6 days was 11.05 ?.70%. There was no significant difference between serum-containing and serum-free conditions (.P>0.05) .III) Enhancement of NE-induced apoptosis in cardiac myocytes by ET-1: 1 umol/L of NE treatment caused an increase in the percentage of apoptotic cells (18.39 ?.80% versus 11.05 ?.70% in control cultures, PO.01); 10 umol/L of NE induced 30.27?.32 % apoptotic cells. ET-1 treatment did not increase the percentage of apoptotic cells (P>0.05 versus CON). The combination of 10 nmol/L of ET-1 and 1 umol/L of NE caused a marked increase in the percentage of apoptotic cells (35.20?.37% versus CON, PO.01). BQ-123 (the antagonist of ET-1 receptor A) inhibited this effect of ET-1 on enhancing NE-induced apoptosis in cardiac myocytes (20.5 ?1.9%, PO.01 versus 10 nmol/L of ET-1+1 umol/L of NE).IV) Increased susceptibility of hypertrophic更多还原