间歇性冷暴露提高机体抗寒能力的研究

发布时间：2018-02-20 00:06

本文关键词： 中心体温冷适应冷暴露 Akt ERK 线粒体融合线粒体分裂细胞凋亡　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景寒冷是常见的环境因素，当机体暴露于寒冷环境时，若防护措施不当或暴露时间过长便会对机体造成损伤，影响健康，甚至威胁生命。寒冷可以显著降低人的劳动能力。对于军队而言，寒冷环境的暴露可能严重削弱部队的战斗力。因此，研究寒冷对人体的损伤机制和防护措施，无论对保护人民的生产作业能力，还是提高部队寒冷环境下的战斗力都至关重要。我们实验室以往研究发现PI3K-Akt调控通路参与急性冷暴露时肝脏能量代谢和损伤的发生，但其机制还尚不清楚。本实验通过对急性冷暴露环境下肝脏损伤的机理加以探讨，同时通过模拟现实生活规律建立冷适应动物模型，探讨冷适应对机体提高寒冷耐受能力和肝脏耐受急性冷暴露损伤的机制，为以后采取提高机体的防护措施提供依据。研究目的研究间歇性冷暴露提高机体抗寒能力及其可能机制。研究方法 1.建立急性冷暴露引起肝损伤的动物模型，通过模拟现实作业规律进行间歇性冷暴露建立冷适应动物模型。 2.通过TUENL染色的方法研究大鼠肝细胞凋亡情况，利用线粒体提取试剂盒提取线粒体，根据JC-1发光原理检测线粒体膜电位。 3.采用透射电镜方法观察冷暴露对大鼠肝脏超微结构的影响 4.利用Western blot方法研究冷暴露对线粒体融合相关蛋白、Caspase3的表达水平及Akt和ERK磷酸化水平的影响研究结果 1.在急性冷暴露过程中，随着暴露时间的延长，大鼠中心体温逐渐降低；在暴露4h时，中心体温显著降低，血清谷丙转氨酶和谷草转氨酶升高，肝功能受损。 2.通过对大鼠肝脏细胞凋亡检测发现急性冷暴露4h后大鼠肝细胞的凋亡水平显著增加，具有统计学意义（p0.05）；随着冷暴露时间的增加，大鼠肝脏内Caspase3活性逐渐增加。 3.对线粒体膜电位检测发现，急性冷暴露4h时，大鼠肝脏线粒体膜电位较对照组降低；透射电镜结果显示急性冷暴露4h后大鼠肝脏线粒体透光度增加，肿胀明显，小线粒体较多；Wentern blot检测结果显示大鼠肝细胞线粒体融合蛋白mfn1/2在在冷暴露1h较对照组升高，但随着暴露时间延长而显著下降；ERK磷酸化水平随着冷暴露时间的增加逐渐降低；Akt的磷酸化水平在冷暴露1h时较对照有一过性增加，之后逐渐降低，在4h时显著低于对照组。 4.冷适应大鼠接受急性冷暴露时，中心体温下降程度较单纯急性冷暴露组显著降低（p0.01）；其肝脏细胞凋亡数量也较急性冷暴露组减少（p0.05），ERK和Akt磷酸化水平以及线粒体融合蛋白mfn1/2水平均较单纯急性冷露组高。研究结论 1.急性冷暴露4h后，大鼠中心体温显著降低，肝细胞凋亡发生增加，，线粒体分裂增加，能量代谢受限，这些病理变化可能是导致肝脏结构、功能受损的机制。 2.冷适应大鼠对寒冷的耐受能力显著提高，可能是通过提高线粒体能量代谢功能，减少细胞凋亡等途径实现的。在此过程中，Akt和ERK通路可能发挥着一定作用。
[Abstract]:Research background
Cold is a common environmental factors, when the body is exposed to a cold environment, if improper protective measures or long time exposure will cause damage to the body, affect the health, even life-threatening. Cold can significantly reduce the labor ability. For the army, the cold exposure may seriously weaken the combat effectiveness. Therefore, study on cold injury mechanism and protective measures of the human body, regardless of the production capacity to protect people, or improve the cold environment of combat are crucial.
Our previous study found that PI3K-Akt pathway involved in energy metabolism and liver injury induced by acute cold exposure, but the mechanism is still unclear. This experiment explored the mechanism of liver injury in acute cold exposure environment, at the same time through the simulation of real life law of cold adaptation animal model, to enhance the body to adapt to cold cold tolerance and liver tolerance to acute cold exposure injury mechanism, for the future to provide a basis for improving the body's protective measures.
research objective
The study of intermittent cold exposure to improve the body's cold resistance and its possible mechanism.
research method
1. animal models of liver injury caused by acute cold exposure were established, and cold adapted animal models were established by intermittent cold exposure through simulated realistic operation.
2., the apoptosis of rat hepatocytes was studied by TUENL staining. Mitochondria were extracted from mitochondria and extracted from mitochondria. The mitochondrial membrane potential was detected according to the principle of JC-1 luminescence.
3. the effect of cold exposure on the ultrastructure of rat liver by transmission electron microscopy
4. Western blot method was used to study the effects of cold exposure on mitochondrial fusion related protein, Caspase3 expression level and the level of Akt and ERK phosphorylation
Research results
1. in acute cold exposure process, with the extension of exposure time, the core body temperature of rats decreased gradually; after the exposure of 4h, the central body temperature decreased significantly, serum alanine aminotransferase and aspartate aminotransferase, impaired liver function.
2., by detecting the apoptosis of rat liver cells, we found that the apoptosis level of hepatocytes in rats after acute cold exposure increased significantly, with statistical significance (P0.05). With the increase of cold exposure time, the activity of Caspase3 in rat liver increased gradually. (4h)
To detect 3. of the mitochondrial membrane potential, acute cold exposure 4h, rat liver mitochondrial membrane potential were lower than the control group; transmission electron microscopy results showed that acute cold exposure after 4H of rat liver mitochondria light increased, mitochondrial swelling significantly, little more; Wentern blot showed that the hepatic mitochondrial fusion protein mfn1/2 in rats cold exposure 1H increased compared with the control group, but with the extension of exposure time decreased significantly; the phosphorylation level of ERK decreased gradually with the increase of time of exposure to cold; the level of Akt phosphorylation in 1H when compared with the control of cold exposure had increased, then gradually decreased, significantly lower than the control group at 4H.
4. cold adapted rats received acute cold exposure, the central body temperature decreased acute cold exposure group decreased significantly (P0.01); the number of cell apoptosis in the liver with acute cold exposure group decreased (P0.05), ERK and Akt phosphorylation and mitochondrial fusion protein mfn1/2 levels were compared with acute cold exposed groups.
research conclusion
1. after acute cold exposure 4h, the central body temperature of rats decreased significantly, the apoptosis of hepatocytes increased, the mitochondrial division increased, and the energy metabolism was limited. These pathological changes may be the mechanism of liver structure and function damage.
2. cold adaptation to the cold tolerance of rats is significantly improved. It may be achieved by increasing mitochondrial energy metabolism function and reducing cell apoptosis. During this process, Akt and ERK pathway may play a role.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R82

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