【作者】 刘英辉；

【导师】 王正荣；

【作者基本信息】 四川大学 ， 生物医学工程， 2005， 博士

【摘要】 目的 研究原癌基因c-fos在正常小鼠大脑颞叶皮质、海马和体外培养小鼠NIH3T3成纤维细胞中的生物节律变化,并阐述其与吗啡成瘾的关系。 方法 饲养正常小鼠,按时间点(间隔4小时)灌注固定小鼠,取全脑作冰冻切片,用原位杂交法测定小鼠颞叶皮质和海马c-fos mRNA的变化。 体外培养小鼠NIH3T3成纤维细胞,血清休克诱导基因节律表达,按时间点提取细胞总RNA,用RT-PCR方法半定量检测c-fos mRNA和per1 mRNA的节律变化。 制备吗啡成瘾小鼠模型,一组按时间点(间隔4小时)灌注固定小鼠,取全脑作冰冻切片,用原位杂交法测定小鼠颞叶皮质和海马c-fos mRNA的变化;另一组按时间点注射盐酸纳络酮催瘾,连续观察30min戒断体征指标(跳跃、湿狗样抖动、爪颤、齿颤)出现次数。更多还原

【Abstract】 OBJECTIVETo study the rhythm of c-fos mRNA in the mouse temporal lobe cortex and hippocampus and in NIH3T3 cells in vitro. Furthermore, to study the possible relationship between the rhythm of c-fos mRNA and chronic morphine dependence.METHODSNormal mice were killed every 4 h on the day of the experiment and the brains were fixed by perfusion. The level of c-fos mRNA was assayed by in situ hybridization.NIH3T3 cells were cultured and RNA was isolated according to the time points. C-fos mRNA and perl mRNA were assayed by RT-PCR.Morphine addiction mice were modeled. On the day of the experiment, one group of the mice were killed every 4 h and the brains were fixed by perfusion. The level of c-fos mRNA was assayed by in situ hybridization. The other group were injectedwith naxolone at different time points. Withdrawal signs were precipitated by the injection of naloxone-HCl. Mice were placed individually in a clear area and the number of withdrawal signs such as jumping, wet-dog shaking, paw tremors, and teeth chattering were counted for 30 min after the naloxone challenge. The recombined plasmid pcDNA 3. 1-per1 RZ DNA was injected into the ventricles of morphine addition mice to transcript the corresponding riboenzyme which cleaves perl mRNA particularly. And then, the brains of mice were fixed by perfusion. The level of c-fos mRNA was assayed by in situ hybridization and its protein was detected by immunohistochemical staining.RESULTSOur results from in situ hybridization with c-fos specific probes revealed that c-fos expressed in the neuron cytoplasm of temporal lobe cortex and hippocampus of normal mice. Studies showed the circadian rhythm of the level of c-fos mRNA in the normal mice temporal lobe cortex and hippocampus.In NIH3T3 cells, perl mRNA showed the circadian rhythm, but c-fos mRNA didn’ t show the change.NS and Mor mice both showed the circadian rhythm in the temporal lobe cortex and hioppocampus. Under NS, c~fos hybridization signal in mice hippocampus continuously declined from a high day-time value at 1600 to a nighttime level at 0400 and rised at 0400, significant at 1200 vs. 0400 (P<0.05).Different from NS, under Mor, c-fos mRNA level declined from a high nighttime level at 2000 to a low nighttime level at 2400 and then increased continuously to its highest level at 2000. As compared with NS, the expression levels of c-fos in Mor at per time point all increased (P<0. 05). The number of occurrences of the indicated withdrawal behaviors (jump, wet-dog shake, paw tremors, teeth chattering) was recorded over a 30-min period following administration of naloxone (1mg/kg i.p.) to mice chronically treated with morphine (10mg/kg). The severity of morphine withdrawal symptoms was least pronounced at the naloxone injection time points of 2400 and most at 1200.The levels of c-fos mRNA and its protein decreased induced by the riboenzyme which specially cleaves perl mRNA.CONCLUSIONThe activities of the temporal lobe cortex and hippocampus neurons in normal mice are maintained the circadian rhythm.Chronic morphine treatment leads to the change of activities of the temporal lobe cortex and hippocampus neurons. Though the circadian rhythm is maintained in morphine addiction group, the mesor, amptitude and acrophase are changed. They maybe play important roles in morphine addiction.The riboenzyme specially cleaving perl mRNA has potential function in inhibiting the transcription and expression of c-fos and blocking the procedure of morphine addition.更多还原