【作者】 韩张；

【导师】 倪道凤； 张德昌；

【作者基本信息】 中国协和医科大学 ， 耳鼻咽喉科学， 2002， 博士

【摘要】 高胆红素血症是造成新生儿听力损伤的高危因素之一。部分高胆红素患儿的听性脑干反应结果提示听觉脑干中枢存在病变，遗传性高胆红素血症动物模型Gunn大鼠的听觉脑干中枢及斜方体有明显病变，高胆红素血症也是听神经病的最重要诱因之一。因此，有必要研究胆红素对神经系统的损伤机理。 细胞凋亡是细胞对周围环境变化所做出的反应之一。诱导细胞凋亡的因素很多，包括氧化应激，氮化应激，高渗应激，损伤基因组DNA的化合物，紫外线照射，热休克，低氧，生长因子剥夺，某些细胞因子如肿瘤坏死因子，转化生长因子β等。介导凋亡反应的信号通路很多，如蛋白激酶A，蛋白激酶C，MAPK等，其中属于MAPK超家族的P38MAPK和JNK家族在凋亡信号的转导中发挥重要的作用。 本文以胆红素作用于体外培养的人神经母细胞瘤细胞系SH-SY5Y细胞，探讨胆红素损伤神经细胞的分子机制。运用分子生物学和细胞生物学技术，观察了胆红素诱导SH-SY5Y细胞凋亡，及对其线粒体膜电势的影响；应用细胞转染技术，建立了bcl-2过表达的细胞株，观察了bcl-2对胆红素诱导SH-SY5Y细胞凋亡的影响；应用P38MAPK特异性抑制剂SB203580研究了P38在胆红素诱导SH-SY5Y细胞凋亡的信号转导中的作用，然后，应用反义基因表达技术，建立了P38MAPK低表达的细胞株，进一步证实P38的作用；最后利用过氧化氢短暂激活JNK，探讨JNK通路在胆红素诱导SH-SY5Y细胞凋亡中扮演的角色。 1．胆红素诱导SH-SY5Y细胞凋亡 显微镜下见SH-SY5Y细胞经0.02g／L胆红素作用2h后即开始皱缩，变圆；Hochest33258染色显示在胆红素作用6h后，SH-SY5Y细胞出现染色质凝集，片段化等典型的凋亡改变；western blot检测在胆红素作用2h后多聚核糖腺苷二磷酸聚合酶(PARP)蛋白出现明显的断裂，提示caspase3被激活；流式细胞仪的定量分析显示SH-SY5Y细胞的凋亡率随胆红素作用时间的延长而增多，3h为19.4％，4h达到76.4％，6h后达到79％。 2．胆红素诱导SH-SY5Y细胞线粒体膜电势的崩解 经Rodanmin 123染色后，流式细胞仪结果显示SH-SY5Y细胞经0.02g／L胆红素作用1h后即出现线粒体膜电势的崩解，由16.6下降到7.11。而此时更多还原

【Abstract】 Neonatal hyperbilirubinemia is one of the risk factors leading to hearing loss of neonates. Auditory brainstem responses of neonates with severe hyperbilirubinemia might indicate that there were pathological changes in the auditory central nucleus; Research showed morphological changes in the cochlear and nucleus of trapezoid body in Gunn rat pups which is the genetic animal model of hyperbilirubinemia; Hyperbilirubinemia also is the most frequent pathogeny of auditory neuropathy. Consequently, It is very significant to study the mechanism that bilirubin damage central nerve system.Apoptosis is a response of cell to the dramatic changes during the surrounding environment. Diverse extracellular stimuli including irradiation, ultraviolet light, heat shock, high osmotic, proinflammatory cytikines such as interleukin-1 and tumor necrosis factor, oxidative stress, nitric oxide, hypoxia, transforming growth factor- 3 can trigger apoptosis. Such stimuli are sensed by receptors at the cell surface, and transmitted to cytosolic and nuclear targets by the activation of intracellular signal transduction pathways. Diverse signal pathways including protein kinase A, protein kinase C, mitogen-activated protein kinase(MAPK) mediate the apoptotic response, in particular, the family of P38MAPK and c-jun N-terminal kinase both belonging to the MAPK superfamily play a important roles.We use the bilirubin directly acting on human neuroblastoma cell line SH-SY5Y cells to study the mechanism by which bilirubin damage neurons, especially the signaling transduction pathway. Utilizing the techniques of molecular biology and cell biology we observe that bilirubin induce apoptosis in SH-SY5Y cells, and its effect on the mitochondrial membrane potential; we establish a cell line SH-SY5Ybcl-2 with overexpression of bcl-2 protein, and observe the effect of bcl-2 on bilirubin-induced apoptosis and disruption of mitochondrail membrane potential of SH-SY5Y cells. Applying the P38MAPK specific inhibitor SB203580 we observe the role of P38 in the signal transduction of biliribin-induced apoptosis in SH-SY5Y cells. To further prove this we set up a cell line SH-SY5Yantip38 with low expression of P38MAPK更多还原