PDGF-CCK转基因小鼠的建立及CCK对摄食行为影响的初步研究

发布时间：2018-01-24 06:22

本文关键词： 转基因胆囊收缩素饮食　出处：《中国协和医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 胆囊收缩素(Cholecystokinin，CCK)是一种广泛存在于胃肠道和中枢神经系统的脑肠肽，具有多种生理学功能。肠道中的CCK除了促进胆囊收缩以外，还可以调节胰酶的分泌和胰腺的生长、胃肠蠕动，抑制胃酸分泌，参与饱腹信号的传导。作为在脑内含量最丰富的脑肠肽之一，CCK在神经系统内参与了多种生理过程并发挥着重要的功能，如抑制进食、抗焦虑、镇痛、调节血压和增强记忆等。但是，迄今为止人们对CCK在脑中发挥作用的具体机制还不清楚；对CCK在脑中的许多功能还存在着很大的争论，如CCK是否能通过中枢神经系统对动物的饮食实现长期的调节?CCK是否具有神经保护作用并能增强记忆?CCK与某些神经精神疾病(如Parkinson’s disease、精神分裂症)的关系也是研究人员力在解决的问题。目前，研究人员对CCK多肽及其受体进行了多方面研究，仍没有建立CCK转基因的小鼠。为了更好的研究CCK在中枢神经系统的功能，本实验采用神经特异性启动子PDGF，建立了在神经系统特异性表达CCK的转基因小鼠并对CCK对小鼠摄食行为和体重的影响进行了初步探讨。实验分为3部分：转基因表达载体的构建；表达载体在神经母细胞瘤细胞中的表达；转基因小鼠的鉴定及CCK对饮食调节的初步探讨。扩增CCK cDNA编码区，克隆到含有神经特异性启动子PDGF的载体里。用表达质粒瞬时转染人神经母细胞瘤SHSY-5Y细胞，Western Blot显示构建质粒在体外表达。酶切含有PDGF启动子、CCK目的片断、PolyA的片断进行显微注射。筛选F_0代转基因阳性鼠，13只小鼠中3只为阳性。Western Blot显示CCK在转基因鼠脑、小肠组织特异性表达。比较3个首建鼠后代与野生鼠脑组织CCK表达量的差异，3个Founder鼠脑中外源CCK都有表达。其中，在两只(8号、12号)脑中，表达量增加约1倍，在一只(20号)脑内表达量增加约2倍。将动物分为转基因组和阴性对照组，并给与正常饮食和高脂饮食。初步动物实验结果经t检验显示：正常饮食情况下，转基因雄鼠体重低于阴性对照鼠8.02％(27.61±1.67g vs 30.02±1.04g，，P＜0.05)，转基因雌鼠低于对照鼠2.36％(21.48±0.41g vs 22.00±1.02g，P=0.364)；高脂饮食情况下，转基因雌鼠体重低于阴性对照鼠6.55％(21.83±0.54g vs 23.36±1.09g，P＜0.05)，转基因雄鼠低于对照鼠5.39％(27.56±2.04g vs 29.13±1.47g，P=0.144)。对各组小鼠进食进水量监测显示：给与正常饮食时，CCK降低了雄鼠摄食量，增加雌鼠摄食量；高脂饮食时，CCK增加了小鼠摄食量；对摄水量无显著影响。分析认为，CCK可能通过调节小鼠摄食量和物质代谢而降低其体重。实验结果初步显示，CCK对小鼠摄食行为有影响，是调节小鼠摄食和体重的一个重要神经肽。
[Abstract]:Cholecystokinin (CCK) is a kind of brain intestinal peptide widely distributed in the gastrointestinal tract and central nervous system. In addition to promoting gallbladder contraction, CCK can also regulate the secretion of trypsin and pancreatic growth, gastrointestinal peristalsis, and inhibit the secretion of gastric acid. As one of the most abundant brain intestinal peptides in the brain CCK participates in a variety of physiological processes and plays an important role in the nervous system such as inhibition of eating anti-anxiety analgesia. Regulation of blood pressure and enhancement of memory, etc., however, so far, the specific mechanism of the role of CCK in the brain is still unclear. There is much debate about the many functions of CCK in the brain, such as whether CCK can regulate the diet of animals for a long time through the central nervous system. Does CCK have neuroprotective effects and enhance memory? The relationship between CCK and certain neuropsychiatric disorders, such as Parkinson's disease, schizophrenia, is also a problem that researchers are addressing. CCK peptides and their receptors have been studied in many ways, but no CCK transgenic mice have been established. In order to better study the function of CCK in the central nervous system (CNS), the neuro-specific promoter PDGF was used in this study. Transgenic mice expressing CCK specifically in nervous system were established and the effects of CCK on feeding behavior and body weight of mice were preliminarily discussed. The experiment was divided into three parts: construction of transgenic expression vector; Expression of expression vector in neuroblastoma cells; Identification of transgenic mice and preliminary study of dietary regulation by CCK. The coding region of CCK cDNA was amplified and cloned into the vector containing neuro-specific promoter PDGF. Human neuroblastoma SHSY-5Y cells were transiently transfected with the expression plasmid. Western Blot showed that the constructed plasmid was expressed in vitro. The target fragment of PDGF promoter was detected by restriction endonuclease digestion. The PolyA fragments were microinjected. Three of the 13 mice in the F _ (0) generation of transgenic mice were selected. Western Blot showed that CCK was in the transgenic brain. The difference of CCK expression between the three progenies of the first born rat and the wild brain was compared. The expression of CCK was found in the brain of the three Founder mice, two of them (8). (12) in the brain, the expression increased by about 1 fold, and in one brain (20), the expression increased by about 2 times. Animals were divided into transgenic group and negative control group, and were given normal diet and high-fat diet. The body weight of transgenic male mice was lower than that of negative control mice (27.61 卤1.67g vs 30.02 卤1.04g, P < 0.05). The transgenic female mice were lower than the control mice (21.48 卤0.41g vs 22.00 卤1.02g / kg) and 0.364g / kg respectively. Under the condition of high fat diet, the body weight of transgenic female mice was lower than that of negative control mice (21.83 卤0.54g vs 23.36 卤1.09g / g vs 6.55g 卤0.54g vs 23.36 卤1.09g / g, P < 0.05). The transgenic male mice were lower than the control mice 5.39% 27.56 卤2.04g vs 29.13 卤1.47g. The results showed that CCK decreased the intake of male mice and increased the intake of female mice. CCK increased the food intake of mice during high fat diet. It is believed that CCK may decrease the body weight of mice by regulating their food intake and substance metabolism. The preliminary results showed that CCK had an effect on the feeding behavior of mice. It is an important neuropeptide that regulates food intake and body weight in mice.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332

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