| It has been widely known that nicotine can induce the cancer, and that the exposure of pregnant women to nicotine has many harmful effects on the development of fetus. Tobacco use in several countries has been estimated to cause nearly 20% of all deaths, making it the largest single cause of premature death. But, the accumulation of evidence express that nicotine is the primary component of tobacco that motivates continued use despite harmful effects. The addictive mechanism for tobacco by humans is not clear. But now, some studies suggest that a - 7 nAChRs which locate in the mesocorticolimbic and nigrostriatal system play pivotal roles in drug reinforcement, motility and associative motor learning. The mesocorticolimbic system, with cell bodies in VTA, plays a key role in mediating the reinforcing effects of natural rewards as well as of drugs of abuse. The nigrostriatal system, with cell bodies in the SN, inervates the caudate nucleus and putamen of the corpus striatum. These receptors are also involved in mediating nicotine's effects on addiction.Although all combinations of subunits that form functional nicotinic receptors can bind nicotine, α - 7 containing receptors form the high affinity nicotine binding site in the brain and are therefore thought to be the primary nAChRs subtype affected by the relatively low nicotine concentration ( 100 - 500nM) found in the blood of smokers. Therefore, the current experiments focus on the regulation of nAChRs by nicotine.In a word, The α - 7 nAChRs gene and protein expression can be study on transcriptional and translational levels by Immunohistochemistry, In Situ Hybridization, Western -blot and Immunofluorescence in rats and PC 12 cells.Methods1. Establishment of rat animal model that were acutely exposed to nicotine. Male Wistar rats (180 -200g) were grouped at random. Nicotine (0.4mg/kg/day,30mg/kg/day s. c. ) was administrated in experiment groups and saline of the same volume was given in control groups.2. Nissle staining3. Immunohistochemistry (including immune - fluorescent method) Sections and PC 12 cells were examined by immunohistochemistry and immune - fluorescent to study the expression and distribution of a - 7 nAChRs, respectively, and the results were analyzed by MetaMorph/DP10/BX41 system and statistical methods.4. In situ hybridization methodsSections were examined by ISH in order to observe the mRNA of a - 7 nAChRs in VTA, SN and cerebral cortex, and the results were analyzed by MetaMorph/DP10/BX41 system and statistical methods.5. PC 12 cell cultureFor experiments,PC 12 cells were cultured, and then added NGF.6. Western-blotThe protein were extracted from PC 12 cells for detecting the expression of a -7 nAChRs through western -blot. Gray values are qualified with Band Scan image analysis software.Results1. Animal model acutely exposed nicotine was establishedThe rats were feed individually, there are no significant difference in weight gain between NIC and CON group.2. Results of Nissle stainingThe body of neuron were big and round, and the nucleuses were stained weakly. So the area of Cortex, VTA and SN can be different3. Results of Immunohistochemistrya. Results of Immunohistochemistry: The positive cells in Cortex in group NIC were significantly strong than in group CON. Otherwise, there was no changes in VTA and SN.b. Results of Immunofluorescence: It was obviouse that a -7 nAChRs expressed in PC 12 cells which effected by NGF or not.4. Results of In Situ HybridizationResults of ISH: The positive cells in VTA and SN in group NIC were significantly more than that in group Con. Otherwise, there was no changes in Cortex.5. Results of Western - blotIn PC 12 cells, the protein levels of a -7 nAChRs was increased by NIC. IOOjxM, 48h > 100jxM,24h > 10jxM,24h > CON. Comparison of gray values both a - 7 nAChRs and (3 - acting were done through Band Scan analysis soft, and the results were significant.Conclusions1. The number of a - 7 nAChRs mRNA seems to be regulated by a tran-scriptional mechanism that increased a - 7 nAChRs mRNA in VTA and SN, and not in Cortex. On the contrary, the changes of a - 7 nAChRs protein were different.2. The protein level was drasticly increased by nicotine in PCI2 cells. It is unambiguous that the changes of protein were regulated by nicotine through translation level.
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