【作者】 王基野；

【导师】 徐玫； 陈景元； 郑刚；

【作者基本信息】 第四军医大学 ， 运动人体科学， 2010， 硕士

【摘要】 研究背景寒冷地区的冬季,人们在户外活动时会暴露于低温环境中。从事低温环境作业的人员和参加特殊环境军事活动的官兵也经常暴露于低温环境中。低温环境对机体的影响和如何预防低温暴露相关疾病一直是人们研究的热点,具有重要意义。低温环境对人体造成损伤的机制以其特殊的军事地位和作用,很少有相关内容的公开报道。目前,人们对于低温环境对机体损伤机制的研究多限于长期的慢性暴露模型,而急性的低温暴露模型尚未见到相关的报道。在北方高寒地区冬季,部队执行野外巡逻和站岗任务的士兵需要暴露在寒冷环境中,如果防护措施不当则很容易造成急性冻伤。目前,对于急性寒冷损伤的研究多集中在体表皮肤及肌肉组织的冻伤,而体内脏器在急性低温暴露时受到的影响及其发挥的作用尚未见到相关报道。人体暴露于低温环境后,需要动员产热以维持体温。虽然在此时肌肉的寒颤产热占主导作用,但肝脏作为体内最大的糖原储存器官和静息状态的主要产热器官,也可能参与到急性低温暴露下机体的应激过程中。在大鼠和人的正常生长过程中,肝脏可以形成一定数量的双核细胞,双核细胞的形成可能与肝脏损伤和增殖能力相关。在能量缺乏或其他损伤效应作用下,肝细胞有丝分裂失败而形成双核细胞。PI3K-Akt-mTOR通路是重要的生存信号通路,参与细胞的能量代谢,增殖分化,生长繁殖等重要过程。其在低温暴露过程中起到的作用已在慢性模型中进行相关研究,但在急性低温暴露模型中及在肝脏中的相关作用尚未阐明。本研究试图通过建立急性低温暴露模型,探讨急性低温暴露是否可以引起肝脏病理生理学改变,并探讨PI3K-Akt-mTOR通路是否参与了急性低温暴露过程中的应答过程及其调控机制,为探索更多的低温暴露效应标志物及预防靶点提供理论依据。研究目的1.在大鼠急性低温暴露模型中,研究急性低温暴露引起大鼠肝脏的病理生理学改变,观察肝脏有丝分裂细胞及双核细胞在低温暴露过程中数量的变化及细胞能量水平的变化。同时观察PI3K-Akt-mTOR通路的变化情况及其与肝脏病理生理改变的关系。2.通过使用通路抑制剂,观察PI3K-Akt-mTOR通路与肝细胞能量水平变化的关系及其是否参与了肝细胞有丝分裂增加及双核细胞形成的过程。研究方法1.利用低温实验舱,建立大鼠急性低温暴露模型:暴露温度为﹣15℃,暴露时间为0.5 h、1 h、2 h和4 h。2.利用组化方法,通过HE染色观察急性低温暴露后大鼠肝脏的损伤情况及双核细胞的形成。3.利用电镜,观察急性低温暴露对大鼠肝脏超微结构的影响。4.利用western blot方法,观察PI3K-Akt-mTOR通路在急性低温暴露中的变化情况。研究结果1.从HE染色结果可以发现,随暴露时间延长,大鼠肝脏的损伤程度逐渐加深。在低温暴露0.5 h即发生轻度肝细胞水肿,在低温暴露4 h后,可发现细胞出现变性、水肿,肝细胞有丝分裂增加并且出现大量双核细胞和少数凋亡细胞。从电镜结果可以发现,低温暴露0.5 h可导致肝细胞糖原减少,线粒体轻度肿胀;低温暴露4 h可致肝细胞内糖原耗竭,线粒体明显肿胀,内质网被压缩,包绕于线粒体周围。2.通过对肝细胞ATP含量的测定,发现在低温暴露0.5 h,肝细胞ATP含量明显增加,并随暴露时间延长而逐渐降低(p<0.05)。3. western blot的检测结果发现,PI3K-Akt-mTOR通路相关蛋白出现先升高后降低的一过性激活, Akt蛋白的Ser-473位点、Thr-308位点;mTOR蛋白的Ser-2448位点、Ser-2481位点和p70 S6K蛋白的Thr-389位点在低温暴露0.5 h的磷酸化程度明显升高,随暴露时间延长而逐渐降低。在对线粒体蛋白的测定发现,Akt蛋白和mTOR蛋白在线粒体内的表达与其在胞质中的表达趋势一致。另外,与能量相关的AMPK蛋白的Thr-172位点随暴露时间延长而磷酸化程度逐渐增加。4.为进一步证实急性低温暴露可以诱导大鼠肝脏有丝分裂增加及双核细胞形成增加,利用免疫组织化学方法对大鼠肝脏双核细胞进行计数,结果发现在低温暴露4 h后,大鼠肝脏有丝分裂期细胞数量明显增加,并且双核细胞数量明显增加(p<0.05)。5.通过腹腔注射抑制剂渥曼青霉素(wortmannin),可以发现低温暴露4 h大鼠的肝脏细胞ATP水平与非抑制剂处理组相比明显降低,与室温对照组相比明显升高(p<0.05)。大鼠肝脏有丝分裂期细胞所占比例无明显改变,双核细胞的数量明显增加(p<0.05)。研究结论1.急性低温暴露4 h可以引起大鼠肝脏损伤,引起肝细胞ATP水平的异常增加,诱导静息期肝细胞进入有丝分裂期并伴随大量双核细胞形成。2. PI3K-Akt-mTOR通路参与了急性低温暴露对大鼠肝脏的损伤作用,参与了低温暴露过程中ATP的生成调节并促进肝细胞有丝分裂及双核细胞的形成。更多还原

【Abstract】 Background:People usually expose to cold environment in out-door activities in the cold winter. And workers who engage in the low temperature environment work and soldiers who execute special environment military missions will commonly expose to harsh weather. The mechanism of healthy effects caused by cold exposure and the method of preventing cold exposure relative diseases is of great interest to scientist and is of great importance.Being at the important military status, the mechanism of cold relative injuries is hardly to be seen in the open press. Up to now, the experimental models used by the study on mechanisms of cold exposure induced healthy effects are mostly the long lasting chronic ones, while the acute cold exposure models are rarely adopted. Soldiers who execute border patrolling or sentry duty will expose to the low tempareture environment in cold winter, and when they were insufficiently prevented, they will easily get acute cold injuries. The studies on acute cold injury are mainly focusing on the frostbite of skin and superficial structures, while the cold effects on the internal organs and the potential functions of the internal organs during cold exposure is rarely studied or reported. When exposed to the cold environment, adult mammals will sponsor the thermogenic activities to maintain body temperature. Although liver is less efficient on calorification than muscle, as one of the largest clycogen storage organ and the dominant thermogenic organ in the resting status, liver may participate in the response to acute cold stressing. Human and rat liver can form a number of binuclear hepatocytes during development. Under some pathological situations, the formation of binuclear hepatocyte may be relative with the liver injury and generative ability changing. The hepatocyte mitosis will be failure under energy stressing or other injurious status and then the binuclear hepatocyte formed. The PI3K-Akt-mTOR signaling is an important pathway that participates in the regulative progression of metabolism, differentiation, development and survival. The potential functions of the pathway in cold exposure have been studied in the chronic cold exposure model, while its role in the acute cold exposure circumstances is still to be illustrating.As expected, using the acute cold exposure animal model, we attempt to explain whether liver could get any physiological or pathological changes during acute cold exposure, and to explain whether and what role the PI3K-Akt-mTOR pathway participates in the responsive reactivities. This will be of great importance to find more effective biomarkers and targets of prevention in acute cold exposure situations.Aims:1. To observe the physiological and pathological changes of liver during acute cold exposure: The changes of number of mitotic hepatocytes and binuclear hepatocytes and the changes of energy level in the hepatocytes during cold exposure as well as the activity changing of the PI3K-Akt-mTOR pathway and its relative potential functions.2. By the utility of inhibitor, explores whether the PI3K-Akt-mTOR pathway participates in the regulation of liver ATP level during acute cold exposure. And whether this change can affect the process of promoting hepatocyte enter into the mitosis phase and the formation of binuclear hepatocyte.Methods:1. Adult male rats weighing 220-230 g are exposed to the cold environment at﹣15℃for 0.5, 1, 2 and 4 hours.2. To observe the pathological changes of liver during acute cold exposure by HE staining and immuno-histochemistry detecting.3. To observe the ultramicrostructure changes of the hepatocytes during acute cold exposure.4. To observe the changes of activities of the PI3K-Akt-mTOR pathway by western blot method.Results:1. Acute cold exposure could injure the liver by a time dependent manner. After 4 hours exposure, the rat hepatocytes were vacuolate degeneration and there were an increasing number of mitosis hepatocytes and binuclear hepatocytes, as well as some apoptosis hepatocytes. 4 hours exposure also caused the microstructure injuries such as the swollen of mitochondria and the depletion of clycogen.2. The ATP level elevated rapidly and peaked at the 0.5 hours during cold exposure and then declined as the exposure time prolonged.3. The activity of the PI3K-Akt-mTOR pathway paralleled with the changes of ATP level. And the AMPK activated in a time dependent manner.4. After injecting of inhibitor wortmannin intraperitoneally, the ATP level declined magnificently and the binuclear hepatocyte increased. Conclution:1. 4 hours acute cold exposure causes rat liver injury and an abnormally transiently increasing in the ATP level, and promotes the liver entering into the mitosis phase as well as forming an increasing number of binuclear hepatocytes.2. PI3K-Akt-mTOR pathway participates in the progression of liver injury during acute cold exposure that regulates the ATP genesis and promotes the hepatocyte getting into the M phase and forming the binuclear hepatocyte.更多还原