抑制PTEN入核在新生鼠缺氧缺血性脑损伤治疗中的作用

发布时间：2018-02-14 11:39

本文关键词： HIBD动物模型抑癌蛋白PTEN 时间依赖性多肽Tat-K13剂量 PTEN泛素化行为学变化学习记忆　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：第一部分HIBD动物模型的建立和PTEN入核水平的变化目的：为了建立新生鼠缺氧缺血性脑损伤（HIBD）实验动物模型，采用7日龄SD新生鼠，观察抑癌蛋白PTEN是否在新生鼠缺氧缺血后入核增加。方法：新生7日龄SD大鼠，随机分成HIBD组、假手术组。用异氟烷进行麻醉，仰卧固定于手术板上，75%酒精消毒颈部皮肤，偏左剪开，轻轻分离皮下脂肪组织，找到左侧颈总动脉后用4-0丝线结扎两端并剪短血管，检查断流成功后，缝合切口。假手术组分离左侧颈总动脉后不结扎，缝合切口。术后放回母鼠身边休息两小时，HIBD组放入密闭缺氧箱，持续充以8%氧和92%氮混合气，持续2.5小时，取出放回母鼠身边饲养。假手术组不做缺氧。造模后分别于缺氧3h、6h、12h、24h、3d、7d，冰上分离左右侧海马和皮层，提取左侧海马核蛋白，用western blot检测核蛋白中PTEN水平的变化。结果：新生鼠缺氧缺血性脑损伤后3h、6h、12h， PTEN入核水平较假手术组高，24h后几乎恢复到假手术水平。结论：新生鼠缺氧缺血时，PTEN入核水平先增加后恢复正常，两者之间存在时效关系，，找到这种关系有利于我们找到最佳治疗时间窗。第二部分：多肽Tat-K13治疗HIBD后的动物行为学检测目的：在第部分实验结果的基础上，检测多肽Tat-K13对抑制PTEN入核的剂效关系。应用多种动物行为学检测方法，观察Tat-K13多肽对HIBD的治疗作用。方法：首先在完成模型的新生鼠中随机分组，腹腔注射不同剂量的Tat-K13多肽（0、2.5、5、10、20mg/kg）或Tat-K13R对照多肽，于缺氧前0h和缺氧3h各给次药，在缺氧12h时取脑组织，提左侧海马组织核蛋白，western blot检测PTEN入核水平的变化。剂效关系稳定后，在动物模型上给药，分别于缺氧0h、3h、24h、48h各腹腔注射次多肽。4-6周后观察行为学变化。分别做了体重测量、高架十字迷宫、转棒测试、抓力测试、水迷宫测试和避暗逃避实验。结果：根据剂效关系图，确定用药剂量为10mg/kg。体重在四个实验组中没有统计学差异；高架十字迷宫没有统计学差异，说明HIBD不造成新生鼠的情感记忆障碍；转棒实验，HIBD组较Sham组在棒上的时间长，给予多肽K13治疗能部分逆转。抓力实验，HIBD组右侧前肢肌力较左侧强，多肽K13能部分逆转，基本达Sham组水平；水迷宫实验，检测其空间记忆能力，HIBD组较Sham组找到平台的时间长，多肽K13组较HIBD组找到平台的时间长。避暗逃避实验主要也检测其空间记忆力，HIBD组较Sham组进入电击箱的潜伏期短，K13组较HIBD组潜伏期几乎达Sham组水平。说明HIBD对空间记忆能力有损害作用，多肽K13能部分逆转这种损害。结论：抑制PTEN入核对新生鼠缺氧缺血性脑损伤有定的治疗作用。
[Abstract]:Part I Establishment of HIBD animal model and changes of PTEN entry level. Aim: to establish an experimental model of hypoxic-ischemic brain injury (HIBD) in neonatal rats, 7-day-old SD neonatal rats were used to observe whether the tumor suppressor protein PTEN increased after hypoxic ischemia in neonatal rats. Methods: SD rats of 7 days old were randomly divided into HIBD group and sham operation group. Anesthetized with isoflurane, 75% alcohol was fixed on the surgical plate to sterilize the neck skin. The skin was cut to the left and subcutaneous adipose tissue was gently separated. After finding the left common carotid artery, the left common carotid artery was ligated with 4-0 silk thread and the blood vessels were cut off. After the disconnection was successful, the incision was sutured. The sham operation group did not ligate the left common carotid artery after separating the left common carotid artery. Suture incision. After the operation, the rats were put back to the mother for two hours to rest. In the HIBD group, the rats were placed in a closed hypoxia chamber, filled with 8% oxygen and 92% nitrogen mixture for 2.5 hours. The sham-operation group did not do anoxia. After 3 h of hypoxia, the left and right hippocampus and cortex were isolated from the left and right hippocampus and cortex for 7 days. The changes of PTEN level in the left hippocampus were detected by western blot. Results: the level of PTEN entry in neonatal rats was higher than that in sham-operated group for 24 h and almost recovered to the level of sham-operation after 3 hours and 12 hours after hypoxic-ischemic brain injury. Conclusion: the nuclear entry level of PTEN in neonatal rats increased first and then returned to normal, and there was a time-dependent relationship between the two. Finding this relationship is helpful for us to find the best therapeutic time window. The second part: the detection of animal behavior after polypeptide Tat-K13 treatment of HIBD. Aim: based on the results of the first part of the experiment, the effects of polypeptide Tat-K13 on inhibiting the entry of PTEN into the nucleus were detected. The therapeutic effect of Tat-K13 polypeptide on HIBD was observed by various animal behavioral methods. Methods: first of all, the neonate rats were randomly divided into two groups. The rats were injected intraperitoneally with different doses of Tat-K13 polypeptide (0 2. 5%) or Tat-K13R control peptide (10 mg / kg). The brain tissues were taken at 12 h after hypoxia, 0 h before hypoxia and 3 h after hypoxia. Western blot was extracted from the left hippocampal tissue to detect the changes of PTEN entry level. After stable dose-effect relationship, the rats were given the drug on the animal model, and the behavioral changes were observed after 48h intraperitoneal injection of polypeptide at 3h, 24h or 48h, respectively, and the body weight was measured. Elevated cross maze, rotating bar test, grip test, water maze test and escape test. Results: according to the dose-effect diagram, the dosage was determined to be 10 mg / kg. There was no significant difference in body weight among the four experimental groups, and there was no statistical difference in the elevated cross maze, which indicated that HIBD did not cause emotional memory disorder in newborn rats. HIBD group had a longer time on the stick than Sham group, and the treatment of peptide K13 could be partially reversed. The muscle strength of right forelimb was stronger than that of left side in the grip test group, and the peptide K13 could be partially reversed to the level of Sham group, and the water labyrinth test showed that the muscle strength of right forelimb in HIBD group was stronger than that in left side. The spatial memory ability of HIBD group was longer than that of Sham group. The time of finding platform in K13 group was longer than that in HIBD group, and the spatial memory in HIBD group was shorter than that in Sham group. The incubation period of K13 group was almost equal to that of Sham group compared with HIBD group. Memory is impaired, Peptide K 13 can partially reverse this damage. Conclusion: inhibition of PTEN entry nucleus has definite therapeutic effect on hypoxic ischemic brain injury in neonatal rats.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742

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