【作者】 马香芹；

【导师】 付润芳； 翁世艾；

【作者基本信息】 郑州大学 ， 药理学， 2003， 硕士

【摘要】 凋亡和坏死是急性缺血再灌注过程中心肌细胞死亡的两种不同形式。凋亡不同于坏死，它是一种能动性的生理性细胞死亡过程，具有特征形态和生化改变，这些改变是一系列基因活动引起的级联反应的结果。因此，深入研究急性心肌缺血再灌注过程中的细胞凋亡现象对心肌缺血再灌注损伤的防治具有重要的意义。前列腺素E₁（Prostaglandin E₁，PGE₁）是前列腺素的一种，具有抑制血小板聚集、扩张血管、改善微循环，保护缺血性心肌，缩小心肌梗死面积的作用。是目前用于治疗心绞痛、心肌梗死、血栓栓塞性疾病的药物之一。以往的研究证实PGE₁对冠状动脉结扎法所致的心肌缺血再灌注损伤具有明显的保护作用，但PGE₁能否抑制心肌细胞的凋亡，目前尚未见报道。本课题采用离体纯化培养的乳鼠心肌细胞建立缺氧复氧损伤模型，观察在该损伤过程中心肌细胞的凋亡现象及PGE₁对缺氧复氧乳鼠心肌细胞凋亡的影响，并探讨其作用机制。 方法 实验采用新生1-3d的Wistar大鼠乳鼠，无菌条件下开胸取出心脏，PBS缓冲液冲洗后用0.125％胰蛋白酶反复消化，收集心肌细胞悬液，差速贴壁法纯化后制成浓度为5×10⁵·ml^-1的细胞悬液，接种到25ml培养瓶中培养，实验采用培养72h后的原代心肌细胞。用缺氧溶液置换正常培养液密闭培养30min造成缺氧，用复氧溶液置换缺氧溶液培养40min后造成再复氧损伤。实验分五组：①正常细胞培养组，②缺氧复氧损伤（H／R）模型组，③缺氧复氧损伤+PGE₁小剂量(5μg·L^-1)组，④缺氧复氧损伤+PGE₁中剂量(15μg·L^-1)组，⑤缺氧复氧损伤+PGE₁大剂量(45μg·L^-1)组。分别从形态学、生化学、分子生物学等方面观察缺氧复氧损伤乳鼠心肌细胞的凋亡现象及PGE₁对凋亡的影响。郑州大学2003届硕士研究生论文PGE，对缺氧复氧乳鼠心肌细胞凋亡的影响 结果 1.心肌细胞形态学改变倒置显微镜下观察，正常心肌细胞培养3天后，细胞成簇生长，伸出伪足，折光性强，且搏动明显。细胞形态呈现多样化，如圆形、梭形及椎形。缺氧复氧损伤后心肌细胞伪足缩短或消失，折光性下降，搏动减弱或消失。PGEI各给药组心肌细胞形态得到明显改善。 2.心肌细胞电镜超微结构心肌细胞电镜超微结构显示，正常细胞培养组细胞基本结构完整、清晰，核染色质均匀分布，未见核固缩现象。缺氧复氧损伤组的多数细胞胞浆减少，核深染，固缩，染色质不均匀有边集现象，部分染色质呈典型半月体样改变。PGE:各给药组多数细胞结构接近正常，少数有染色质轻度边集，核深染等形态学变化。 3.DNA琼脂搪凝胶电泳检测心肌细胞DNA梯状条带正常心肌细胞培养组DNA电泳呈一条大分子DNA片段，为正常基因组DNA带形，紧靠加样孔。缺氧复氧损伤组DNA呈明显的非随机降解状态，在400bP、600bp、800bp、1 ooobp左右出现了3一4条区带，在电泳中呈特征分布，即凋亡独有的“梯状图谱”。PGE:各给药组随剂量的增加，电泳中DNA的梯状条带逐渐减弱。PGE;（45陀.L’’）组接近正常基因组DNA带形。 4.T1J’NEL法检测心肌细胞凋亡率正常心肌细胞培养组偶见阳性核染色标记。缺氧复氧损伤组心肌细胞有较多的阳性标记，细胞核呈紫蓝色颗粒，有的聚集成团，有的染色质边缘化。PGEI（45雌·L一‘）组只有散在的阳性标记，其凋亡率（6 .80士1.99）%与缺氧复氧损伤组（n.40士2.32）%相比差异有统计学意义（尸<0.01）。 5.原位杂交法检测心肌细胞内bel一2、bax mRNA含量结果用阳性细胞的积分光密度值表示阳性细胞的含量，PGE;（5此一’，15雌·u‘，45陀·U，）三个剂量组bcl一2mRNA的积分光密度值分别为3.40肚0.405、4.505士0.687、8.55矢0.602与缺氧复氧损伤组1.98妊0.522比较差异均有统计学意义（尸<0.01）;PGE;（5陀·L一，、15林gL一，、45林g·L一，）三个剂量组baxm胭A的积分光密度值分别为6.418士0.792、4.353士0.767、3.171士0.728与缺氧复氧损伤组8.547士0.852比较差异均有统计学意义（尸<0.01）;bax mRNA的积分光密度值与bel一2 mRNA的积分光密度值呈负相关（:二一0.868） （尸<0.01）。提示PGEI能促进抗凋亡基因bel一2 mRNA的表达，抑制促凋亡基因郑州大学2003届硕士研究生论文pGEI对缺氧复氧乳鼠心肌细胞调亡的影响bax mRNA的表达。 6．免疫组化法检测心肌细胞内bclZ、bax蛋白的含量 结果用阳性细胞的积分光密度值表示阳性细胞的含量，缺氧复氧损伤十PGE；6·L－’、15ng工“‘、45pg·L－’)各剂量组可分别使心肌细胞的bC12 蛋白的积分光密度值从缺氧复氧损伤组1．slbeo．457上升至 2．20po0．3399、3石67t 0．6611、5．30610．529（P＜0．of）；bax蛋白的积分光密度值从缺氧复氧损伤组7．19lfo．631下降至5石Zbeo．543、4．195f0．622、2．32310．358(P＜0刀1入 b。蛋白的积分光密度值与比上蛋白的积分光密度值呈负相关（r—O．912）（P＜O．01）。结果与原位杂交相一致。 结论 1．离体培养的乳鼠心肌细胞建立的缺氧复氧损伤模型可诱导心肌细胞的凋亡，进一步证实了心肌细胞凋亡是心肌缺血再灌注过程更多还原

【Abstract】 Apoptosis and necrosis are two different forms of cell death in myocardial ischemia/reperfusion injury(IRI). Apoptosis is a genetically determined, active process with typical morphological and biological changes, these changes are regulated by the actions of a series of genes. So there is important significance to study apoptosis in myocardial ischemia /reperfusion injury. Prostaglandin EI has the effects of dilating vessel, ameliorating microcirculation, inhibiting platelet aggregation, and lessening myocardial infarct area. Previous study has demonstrated that PGEi has evidently protective effects on myocardial ischemia/reperfusion injury induced by ligation of coronary artery, but there are no reports so far about whether PGEi can inhibit cardiomyocyte apoptosis. The purpose of this study is to investigate the effects of PGEi on hypoxia/reoxygenation apoptosis in cultured neonatal rat cardiomyocytes.MethodsThe hearts were isolated from l~3-day-old Wistar rats and the ventricles were minced in phosphate buffer solution. The myocardial cells were dissociated with 0.125% pancreatin. After dispersed, the cells were collected, purified and incubated in 25ml culture bottles(the cells density was adjusted to 5x105cells ml-1 with Dulbecco’s modified eagle culture medium consisting of 15% fetal calf serum). The models ofhypoxia/reoxygenation were made with the first generation of cultured cardiomyocytes. The normal cultured medium was replaced by hypoxia solution, the cells were cultured hermetically for 30min to induce hypoxia, then the hypoxia solution was replaced by reoxygenation solution, the cells were cultured for 40min to induce reperfusion. Cultured cardiomyocytes of neonatal rats were divided into five groups: control group(c); hypoxia/reoxygenation(H/R) group; hypoxia/reoxygenation±low dose of PGE1(5ug L-1) group; hypoxia/reoxygenation±medium dose of PGE1(15ug L-1) group; hypoxia/reoxygenation±high dose of PGE1(45ug L-1) group. The effects of PGE1 on hypoxia/reoxygenation apoptosis in cultured neonatal rat cardiomyocytes were studied at morphological, biochemical and molecular biological levels.ResultsBeing injured by hypoxia and reoxygenation, refracting power of the cells declined, pseudo-podium became shortened or disappeared, cell beat became weak or stopped. Most cells of H/R group by electron microscopy appeared as cytoplasmic concentration, nuclear chromatin condensation and margination. PGE^Sug-L’^lSug-L’^Sug-L"1) relieved the injury. Most cells approached to normal, only few cells showed above changes.The results of DNA electrophoresis in the H/R group showed the typical DNA ladder and the DNA ladder decreased gradually as the dose of PGE1 increased. The TUNEL staining showed that the sum of apoptotic cells in the H/R group was larger than that in PGE1(45u g L-1) group.The results of in situ hybridization showed that the integral light density of bcl-2 mRNA in PGE,(5ug L-1, 15ug L-1 ,45ug L-1) groups(3. 406 ±0.405, 4.505±0.687, 8.559 ±0.602) was significantly higher than that in H/R group(l. 989 ±0.522) (P < 0.01 ). The integral light density of bax mRNA in PGE1(5ug L-1, 15ug L-1, 45ug L-1) groups(6.418± 0.792, 4.353±0.767, 3.171 ±0.728) was significantly lower than that in H/R group(8.547 ±0.852) (P < 0.01) . The same results were also demonstrated by the method of immunohistochemical staining.ConclusionsThe results suggest that H/R injury can induce cardiomyocyte apoptosis, PGE1 has obviously anti-apoptotic effects on cardiomyocyte and the mechanisms are possibly by inhibiting the expression of bax and increasing the expression of bcl-2.更多还原