【作者】 赵永辉；

【导师】 姜勇；

【作者基本信息】 山东大学 ， 外科学， 2013， 硕士

【摘要】 研究目的从全球各个国家来看,蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)都是致死和致残的重要的疾病之一。在过去,蛛网膜下腔出血的研究主要集中在蛛网膜下腔出血后的脑血管痉挛。但至目前为止,有效治疗蛛网膜下腔出血后脑损伤的方法尚未发现。新的研究资料认为蛛网膜下腔出血后的早期脑损伤(Early brain injury, EBI)是蛛网膜下腔出血患者死亡的一个重要原因。蛛网膜下腔出血后,发生神经组织广泛的细胞凋亡,特别是海马(Hippocampus,HIP)神经元的凋亡,这些组织细胞的凋亡参与了EBI发生发展的病理过程。因此,减轻SAH后EBI可以有效地降低蛛网膜下腔出血患者的死亡率,从而改善SAH患者的神经功能。近期的研究表明,在48小时内孕酮对脑损伤具有明显的神经保护作用,可以减轻组织损害,恢复和改善创伤性脑损伤,稳定血脑屏障和减少脑水肿,抑制脂质过氧化,减少谷氨酸盐的释放,尤其是可以减轻神经细胞的凋亡。由于目前孕酮对SAH后EBI的研究较少,孕酮对蛛网膜下腔出血后早期脑损伤的作用仍不十分清楚。我们假设：孕酮可能通过抑制细胞凋亡减轻蛛网膜下腔出血后的早期脑损伤。因此,本研究通过制备大鼠蛛网膜下腔出血模型,探讨孕酮对大鼠蛛网膜下腔出血后早期脑损伤的保护作用及其可能机制。研究方法1.实验对象选择标准：48只SD(Sprague Dawley,SD)雄性大鼠,体重在250g至300g之间,随机分配到三个实验组,假手术对照组(Sham control group,sham组)、蛛网膜下腔出血组(Subarachnoid hemorrhage group,SAH组)、和孕酮治疗组(Progesterone treatment group,PG组)。2.蛛网膜下腔出血模型的建立：利用自体血二次注入枕大池法建立大鼠蛛网膜下腔出血模型,48h后取脑。3.给药：孕酮治疗组分别于24h给予16mg/kg的孕酮腹腔注射,假手术对照组和蛛网膜下腔出血组则给予相同剂量的生理盐水。4.凋亡的检测：海马神经元的组织形态学变化应用苏木素-伊红(HE)染色观察；海马神经元凋亡应用原位末端标记法(TUNEL)检测；大鼠海马中Bcl-2、Bax的表达运用免疫组织化学法检测。5.统计学分析：应用SPSS18.0统计学软件对所得资料进行统计分析,数据以x±s表示。应用单因素方差分析,P<0.05为差异有统计学意义。结果1.孕酮对细胞凋亡的影响：Sham对照组无明显TUNELP日性细胞发现,而SAH组阳性细胞数明显增多(P<0.05)；应用孕酮治疗组凋亡数目明显少于SAH组,且具有统计学意义(P<0.05),PG组与Sham组相比凋亡细胞增多(P<0.05)。2.免疫组化法检测凋亡相关基因发现,与蛛网膜下腔出血组相比,孕酮组促凋亡蛋白Bax表达下降(P<0.05),抗凋亡蛋白Bcl-2表达升高(P<0.05),并且海马凋亡细胞数显著减少。因此,孕酮可能通过促进抗凋亡蛋白Bcl-2的表达,抑制促凋亡蛋白Bax的水平,升高Bcl-2与Bax之间的比值,进而抑制海马神经元的凋亡,对SAH后EBI发挥脑保护作用。结论孕酮可能通过抑制大鼠蛛网膜下腔出血后早期脑损伤神经元的凋亡,减轻大鼠蛛网膜下腔出血后早期脑损伤。更多还原

【Abstract】 ObjectiveSubarachnoid hemorrhage (Subarachnoid hemorrhage,SAH) is an important cause of death and disability worldwide. In the past, researches have concentrated primarily on cerebral vasospasm after SAH.To date, there is still not a definitive treatment to absolutely prevent brain injury after SAH. Early brain injury (Early brain injury,EBI) has been pointed to be the primary cause of mortality in SAH patients.Apoptosis occurred in neuronal tissues,particularly in the hippocampus (Hippocampus,HIP)after SAH, is involved in the pathological process of early brain injury.Therefore, reducing early brain injury may effectively reduce the mortality and improve the neurological outcome in subarachnoid hemorrhage patients. The protective effect of progesterone in EBI after trauma has been supported by numerous studies.Progesterone is effective in decreasing cell death within48h after injury, reducing tissue damage and improving behavioral recovery following traumatic brain injury,stabilizing the BBB and reducing brain edema, and reducing lipid peroxidation, glutamate release,especially in neuronal apoptosis. However, due to the less research for the effect of progesterone on EBI after SAH,so the effect and mechanism of progesterone for the EBI remains not clear.we hypothesized that progesterone might reduce EBI after SAH and improve neurological outcomes by inhibiting cell apoptosis.Therefore,we explored the effect of progesterone on cell apoptosis in rats subjected to subarachnoid hemorrhage-induced EBI in this study. To investigate the effect of progesterone on early brain injury(EBI) after subarachnoid hemorrhage(SAH) and its possible mechanism.Methods1. The selection standards of the experiment objects:Forty and eight Sprague-Dawley male rats weighing between250and300g were randomly assigned to three groups:sham control group and experimental SAH group、progesterone treatment group.2. Rat SAH model:The SAH model was induced by injecting autologous blood into the cisterna magna,the rat brain was obtained after48h.3. Drug administration:The initial injection of progesterone (16mg/kg) was given intraperitoneallylh after SAH for progesterone group, and subsequent injections were given subcutaneously6h and24h after SAH. The sham control group and the SAH group rats were injected identically with same volume of water.4. Assay of apoptosis:HE staining, morphology of hippocampal neural cells of rats, neuronal apoptosis in hippocampus and the expression of Bcl-2and Bax were assayed with immunohistochemistry after48h of SAH.5. Statistical analysis:All the data was performed using SPSS18.0statistical software. Results were expressed as mean±standard deviation(SD). The simple correlation analysis statistical methods were used to analysis data. The level of significance was set to P<0.05.Results1. Effect of progesterone on cell apoptosis:In sham group, no TUNEL-positive cells were detected,TUNEL-positive cells were observed in the rat brain after48SAH(P<0.05). Progesterone reduced the number of TUNEL-positive cells in the rat brain compared with the SAH group (P<0.05). Compared with the Sham group, PG group have more apoptosis cells (P<0.05).2. Compared with SAH group, PG group markedly reduce the apoptosis of neurons in hippocampus(P<0.05).The expression of anti-apoptotic protein Bcl-2was rising(P<0.05).The expression of pro-apoptotic protein Bax was down-regulating(P <0.05). The mechanism may though promotes expression of the anti-apoptotic protein Bcl-2, reduces expression of the pro-apoptotic protein Bax,and increases the Bcl-2/Bax ratio, thus inhibiting the apoptosis of hippocampus neuronal cells and exerting a protective effect on the EBI after SAH.ConclusionProgesterone may reduce EBI after SAH by inhibiting cell apoptosis.更多还原