抑胃多肽疫苗的构建及主动免疫后对大鼠行为学和脑功能的影响

发布时间：2018-06-16 19:05

本文选题：抑胃多肽 + 葡萄糖依赖性的促胰岛素分泌多肽　；参考：《第二军医大学》2010年博士论文

【摘要】： [背景]抑胃多肽(gastric inhibitory peptide;又称葡萄糖依赖性促胰岛素分泌多肽,glucose-dependent insulinotropic polypeptide; GIP)是由分布于十二指肠和空肠的K细胞分泌的一种含42个氨基酸的直链多肽。血液中GIP的浓度与饮食成份密切相关,进餐的质与量决定了餐后GIP分泌的高低。葡萄糖、脂肪或氨基酸的摄入,均可促使GIP释放,其中高脂饮食会明显增加GIP的分泌；而低脂饮食,则GIP的分泌量偏低。GIP是一种较为重要的调控糖脂代谢的肠促胰岛素(incretin)。GIP通过与其七次跨膜的G蛋白偶联受体(GIPR)结合,发挥着调节脂肪细胞的分化成熟；调控脂肪细胞的脂解及再酯化；增加脂蛋白酯酶(LPL)的合成、分泌及活性,从而促进甘油三酯(TG)的贮存；以及促进脂肪细胞对葡萄糖(G)的摄取等作用。现有的研究结果表明,GIP可能是过剩营养导致肥胖一个关键激素。在动物试验中,采用GIP受体基因敲除鼠、GIP受体拮抗剂、减少小肠K细胞GIP的分泌等方法,均能有效抑制脂肪合成、控制肥胖、减少体重增加等。因此,阻断或削弱GIP生物活性的方法可能是今后肥胖药物治疗的又一新靶点,干扰GIP/GIPR信号通路在抗肥胖治疗方面具有潜在研究前景。基因工程疫苗领域的快速发展为抗肥胖领域研究提供了另一种可能的药物治疗策略。采用疫苗免疫获得抗体,中和自身内源性的GIP的方法,为战胜肥胖及其相关并发症带来了新希望。目前已有研究者以噬菌体-病毒样颗粒(Qβ-VLP)或卵清蛋白(ovalbumin)为载体,分别与GIP1-15位多肽序列或GIP1-11位多肽序列,采用化学耦联方式结合,构建蛋白疫苗,在小鼠模型上进行了抗肥胖相关研究,两种疫苗形式均能使小鼠打破免疫耐受,产生针对GIP的特异性抗体,从而获得了抗肥胖或改善血糖的作用。疫苗治疗的主要优势在于其仅需短期治疗,却可获得长期收益,从而使患者依从性问题得到很好的解决。但这一点也使得疫苗的免疫治疗存在一定潜在风险,免疫治疗产生的抗体后会在较长时间内持续发挥效应,如一旦产生不良作用,短期之内则难以祛除,因此GIP靶向的免疫治疗在应用前尚需在更多动物模型上进行研究比较,并给与相对综合及长期地观察和评价。目前认为GIP/GIPR信号通路除调控能量代谢外,尚维系着机体的其他方面的很多重要功能。有研究发现GIPR在脑组织中广泛表达,同时GIP具有调节中枢神经系统神经祖细胞的增殖,减少有害物质神经元损伤的作用。因此,从一定程度上推断,GIP可能具有调控机体行为和脑功能的作用。已有实验证实GIP过表达转基因小鼠的感觉运动协调能力、认知记忆能力明显提升,说明高水平的GIP会引起行为学的改变。那么,通过免疫的方法改变血循环中的GIP水平,是否会对行为学及脑功能带来影响,目前尚无关于这方面的相关研究和报道,因此,本实验采用构建GIP疫苗,主动免疫大鼠的方法,观察免疫治疗影响GIP/GIPR信号通路后对大鼠体重、行为学及脑功能发面的影响。 [目的]以乙肝核心抗原-病毒样颗粒(HBc-VLP)或钥孔戚血蓝蛋白(KLH)为载体分别构建携带GIP序列的蛋白疫苗HBc-GIP及GIP-KLH,主动免疫高脂饮食大鼠,观察疫苗免疫效果；高脂饮食大鼠主动免疫GLP-KLH蛋白疫苗后,观察其在体重、行为学以及脑功能方面的变化。初步探讨以GIP为靶向的免疫干预治疗的可行性及安全性问题,为肥胖的免疫治疗提供理论和实验依据。 [方法]克隆GIP N端12肽的编码cDNA序列,并与HBc VLP(1-144a.a.)cDNA序列进行融合,通过原核表达及纯化,制备携带GIP序列的HBc VLP疫苗(HBc-GIP);采用固相合成方法合成GIP成熟体多肽N端1-12序列,并将其C端与KLH耦联构建GIP蛋白疫苗(GIP-KLH);免疫高脂饮食大鼠并进行免疫学指标评价。通过对高脂饮食大鼠的体重称量、行为学试验(开放场测试及Morris水迷宫测试)、正电子发射计算机断层显像仪(PET/CT)检测脑糖利用率、TUNEL及PCNA的方法检测海马齿状回细胞的增殖和凋亡情况来评估GIP-KLH疫苗免疫组与对照组(如期皮下多点注射KLH)在体重、行为学及脑功能方面存在的差异。 [结果] 1、成功制备了携带GIP抗原序列的HBc-GIP蛋白疫苗,并同时制备了编码该融合蛋白序列的核酸疫苗pVAXl-HBc-GIP。两种疫苗分别免疫或联合免疫高脂饮食大鼠后,均可以产生GIP特异性抗体,而做为对照的HBc免疫组和PBS免疫组则没有GIP特异性抗体的产生。说明我们制备的GIP核酸疫苗及蛋白疫苗均能够打破宿主对自身GIP的免疫耐受而产生抗体。在免疫策略组合上,我们发现两次免疫GIP核酸疫苗后再两次免疫HBc-GIP蛋白疫苗的策略(pV-HBc-GIP*2+HBc-GIP*2)可以有效地诱导高滴度GIP抗体,其抗体滴度在第3轮免疫后开始显著提高,并在第4轮加强免疫后继续上升,最终持续产生高滴度的GIP特异性抗体,抗体最终滴度优于4轮免疫HBc-GIP蛋白疫苗或GIP核酸疫苗的水平(P0.05)。因此,经过两种疫苗的联合免疫,可以获得高滴度的GIP特异性抗体IgG,显示了良好的免疫效果。 2、成功制备了携带GIP抗原序列的GIP-KLH蛋白疫苗,免疫高脂饮食大鼠后,GIP-KLH疫苗组第二次免疫接种后7天,即产生了高滴度GIP特异性抗体,如期继续免疫接种,抗体水平进一步增高,而KLH对照组则(如期皮下多点注射KLH)没有此变化。结果说明,经过多次免疫GIP-KLH蛋白疫苗,可以获得高滴度的GIP特异性抗体IgG,显示了的良好免疫效果。 3、各组动物在试验中,进食情况无明显统计学差异(P0.05), GIP-KLH疫苗免疫组与KLH对照组(如期皮下多点注射KLH)相比,体重增长减少。在初始体重无统计学差异的前提下,实验组与对照组于初次免疫后第63天,体重增长开始出现统计学差异(P0.05),在试验结束时(即初次免疫后第98天),GIP-KLH疫苗组与KLH对照组相比,体重少增长了48g(17%)。实验中GIP-KLH疫苗组空腹血糖及胰岛素与KLH对照组相比无明显统计学差异(P0.05)。 4、在行为学实验中,开放场试验(OFT)的测试结果显示,GIP-KLH疫苗组与KLH对照组相比较,总路程(P0.01)、平均速度(P0.01)、活动时间(P0.05)、活动次数(P0.01)、在中央区域的活动次数(P0.05)均明显低于KLH对照组,休息时间(P0.05)明显高于对照组,具有统计学差异。GIP-KLH疫苗组与KLH对照组相比,自发活动性及在中央区域的活动次数减低,因此GIP-KLH疫苗的主动免疫影响到了大鼠的行为活动及精神状态。在Morris水迷宫测试(MWM)中,GIP-KLH疫苗组与KLH对照组动物在4天的训练阶段中均表现为逃避潜伏期和游泳距离的缩短(分别为F=6.5；P=0.001 and F=5.3；P=0.002,),以及游泳速度的明显增快(F=3.3；P=0.024),结果表明两组动物都在努力学习定位隐藏的站台,但是两组间无明显统计学差异(P0.05)。在第5天的探索阶段,两组的表现也无明显统计学差异(P0.05)。 5、在高脂饮食大鼠初次免疫后第98天,以18氟-脱氧葡萄糖(18F-FDG)为示踪剂,通过PET/CT检测大鼠脑糖利用率。GIP-KLH疫苗组与KLH对照组相比,海马区、大脑皮层的脑糖利用率减低(P0.05),而嗅球及小脑的脑糖利用率无明显差别(P0.05)。 6、通过末端脱氧核苷酰基转移酶介导性dUTP切口末端标记(TUNEL)和增殖细胞核抗原(PCNA)的方法,观察海马齿状回颗粒细胞的增殖及凋亡情况。在GIP-KLH疫苗组与KLH对照组的病理切片中均可观察到TUNEL阳性细胞(细胞核棕色着色)及PCNA阳性细胞(细胞核棕色着色),但GIP-KLH疫苗组TUNEL阳性细胞数及PCNA阳性细胞数较KLH对照组明显增多,说明GIP-KLH疫苗组海马齿状回颗粒细胞的增殖及凋亡均增加。 [结论]以HBc(1-144a.a.)或KLH为载体,制备GIP疫苗,可以有效地诱导靶向大鼠GIP的特异性体液免疫反应,打破大鼠自身抗原耐受,诱生GIP特异性抗体。该疫苗可以做为一种新的控制肥胖的免疫干预药物来进一步深入研究。GIP-KLH主动免疫干预治疗高脂饮食大鼠,具有抗肥胖作用,但同时在一定程度上也影响到大鼠的行为学及脑功能活动。
[Abstract]:[background] gastric polypeptide (gastric inhibitory peptide; also called Glucose dependent insulin secretion polypeptide, glucose-dependent insulinotropic polypeptide; GIP) is a 42 amino acid direct chain polypeptide secreted by K cells distributed in the duodenum and jejunum. The concentration of GIP in the blood is closely related to the dietary ingredients, and meals. Quality and quantity determine the level of GIP secretion after meal. The intake of glucose, fat or amino acids can promote the release of GIP, in which the high fat diet can significantly increase the secretion of GIP; while low fat diet, the low secretion of GIP by.GIP is a more important regulation of glycolipid metabolism of insulin (incretin).GIP through its seven transmembrane G Protein coupled receptor (GIPR) binding, which regulates the differentiation and maturation of adipocytes, regulates adipocyte lysis and re esterification, increases the synthesis, secretion and activity of lipoprotein esterase (LPL), thus promotes the storage of triglycerides (TG), and promotes the uptake of glucose (G) by adipocytes. The existing research results show that GIP In animal trials, the use of GIP receptor knockout mice, GIP receptor antagonists, and reducing the secretion of GIP in small intestinal K cells, can effectively inhibit fat synthesis, control obesity, and reduce weight gain in animal tests. Therefore, the method of blocking or weakening the biological activity of GIP may be a future obesity drug. Another new target of drug therapy is the interference of GIP/GIPR signaling pathway, which has potential research potential in anti obesity therapy.
The rapid development in the field of genetic engineering vaccines provides another possible therapeutic strategy for the field of anti obesity research. The use of vaccine immunization to obtain antibodies and neutralize endogenous GIP has brought new hope to defeat obesity and its related complications. Currently, researchers have phage virus like particles (Q beta -VLP) or egg white. Protein (ovalbumin) was used as a carrier, combined with GIP1-15 bit polypeptide sequence or GIP1-11 bit polypeptide sequence, combined with chemical coupling method to construct a protein vaccine. The anti obesity related research was carried out on the mouse model. The two vaccine forms can make mice break immune tolerance and produce specific antibodies against GIP, thus gaining anti obesity or modification. The main advantage of good blood sugar is that it is the main advantage of the vaccine treatment, but it only needs short-term treatment, but it can obtain long-term benefits, so that the patient compliance problem is well solved. But this also makes the vaccine immunotherapy have a certain potential risk, immune treatment produced antibodies will continue to play a long time after the effect, such as once. It is difficult to remove the adverse effects in the short term, so GIP targeted immunotherapy needs to be compared on more animal models before application, and gives a relatively comprehensive and long-term observation and evaluation. At present, the GIP/GIPR signaling pathway still maintains a lot of other important functions in other aspects of the body besides regulating energy metabolism. It has been found that GIPR is widely expressed in brain tissue, and GIP can regulate the proliferation of neural progenitor cells in the central nervous system and reduce the damage of harmful substance neurons. Therefore, to a certain extent, GIP may play a role in regulating the body's behavior and brain function. It has been proved that GIP overexpressed the sensory transport of transgenic mice. The ability of dynamic coordination and cognitive memory is obviously improved, indicating that high level of GIP can cause behavioral changes. Then, whether the GIP level in the blood circulation can be changed by immune method will affect the behavior and brain function. There is no related research and report on this. Therefore, this experiment uses the construction of GIP vaccine. To observe the effect of immune therapy on body weight, behavior and brain function of rats after immunization with GIP/GIPR signaling pathway.
[Objective] to construct the protein vaccine HBc-GIP and GIP-KLH carrying GIP sequence with hepatitis B core antigen virus like particle (HBc-VLP) or key keyhole hemocyanin (KLH) as the carrier, and actively immunization high fat diet rats and observe the immune effect of the vaccine. The weight, behavior and behavior of the high fat diet rats after the active immunization of the GLP-KLH protein vaccine are observed. The changes in brain function. The feasibility and safety problems of GIP targeted immunotherapy are preliminarily discussed in order to provide theoretical and experimental basis for the immunotherapy of obesity.
[method] the encoding cDNA sequence of GIP N terminal 12 peptide was cloned and the HBc VLP (1-144a.a.) cDNA sequence was fused. The HBc VLP vaccine (HBc-GIP) carrying GIP sequence was prepared by prokaryotic expression and purification. The solid-phase synthesis method was used to synthesize the 1-12 sequence of the GIP mature polypeptide. Lipid diet rats were evaluated by immunological indexes. By weighing the body weight of high fat diet rats, behavioral test (open field test and Morris water maze test), positron emission computed tomography (PET/CT) test of brain sugar utilization, TUNEL and PCNA methods were used to detect the proliferation and apoptosis of hippocampal gyrus, and to evaluate GIP There were differences in body weight, behavior and brain function between -KLH vaccine group and control group (subcutaneous multiple injection KLH).
[results]
1, the HBc-GIP protein vaccine carrying the GIP antigen sequence was successfully prepared, and the nucleic acid vaccine pVAXl-HBc-GIP. of the DNA vaccine encoding the fusion protein was also prepared and two vaccines were immunized or combined with high fat diet rats respectively, all of which could produce GIP specific antibodies, while the HBc immunization group and the PBS immune group did not have the specific resistance to GIP. The GIP nucleic acid vaccine and protein vaccine we prepared can break the host's immune tolerance to the GIP and produce antibodies. In the combination of immunization strategies, we found that the strategy of immunization of HBc-GIP protein (pV-HBc-GIP*2 +HBc-GIP*2) after two immunization of GIP nucleic acid vaccine (pV-HBc-GIP*2 +HBc-GIP*2) can effectively induce high titer GIP resistance. The antibody titer began to increase significantly after third rounds of immunization, and continued to rise after the fourth round of immunization, eventually producing a high titer GIP specific antibody. The final titer of the antibody was superior to the level of the 4 round immune HBc-GIP vaccine or the GIP nucleic acid vaccine (P0.05). Therefore, a high titer could be obtained by combined immunization of two vaccines. The GIP specific antibody IgG showed a good immunological effect.
2, the GIP-KLH protein vaccine carrying the GIP antigen sequence was successfully prepared. After immunization of the high fat diet rats, the GIP-KLH vaccine group produced a high titer GIP specific antibody after 7 days of immunization. The antibody level was further increased, while the KLH control group had no change in the subcutaneous multipoint injection of KLH. Ming, after repeated immunization with GIP-KLH protein vaccine, can obtain high titer of GIP specific antibody IgG, showing good immune effect.
3, there was no significant difference in food intake between the animals in each group (P0.05). The weight increase of the GIP-KLH vaccine group and the KLH control group (KLH as the subcutaneous multipoint injection) decreased, and the weight gain began to appear in the experimental group and the control group on the premise of no statistical difference (P 0.05) at the end of the experiment (the first ninety-eighth days after the first immunization), the GIP-KLH vaccine group increased the weight less 48g (17%) compared with the KLH control group. There was no significant difference between the fasting blood glucose and the insulin in the GIP-KLH vaccine group compared with the KLH control group (P0.05).
4, in the behavioral experiment, the open field test (OFT) test showed that the GIP-KLH vaccine group was compared with the KLH control group, the total distance (P0.01), the average speed (P0.01), the activity time (P0.05), the activity times (P0.01), the activity times in the central region (P0.05) were obviously lower than the KLH control group, and the rest time (P0.05) was obviously higher than that of the control group, and the rest time (P0.05) was significantly higher than that of the control group. Compared with the KLH control group, the spontaneous activity and the number of activities in the central region decreased in the.GIP-KLH vaccine group, so the active immunity of the GIP-KLH vaccine affected the behavior and the mental state of the rats. In the Morris water maze test (MWM), the GIP-KLH and KLH control groups were all fleeing in the 4 day training stage. The shortening of the incubation period and swimming distance (F=6.5; P=0.001 and F=5.3; P=0.002) and the rapid increase of swimming speed (F=3.3; P=0.024) showed that the two groups of animals were trying to learn to locate the hidden platform, but there was no significant difference between the two groups (P0.05). The performance of the two groups was not obvious at the fifth day of the exploration stage. Statistical difference (P0.05).
5, ninety-eighth days after the first immunization of high fat diet rats, 18 fluoro deoxyglucose (18F-FDG) was used as tracer. Compared with the KLH control group, the brain sugar utilization rate of the rat brain was compared with the control group of the KLH control group. The brain sugar utilization rate of the hippocampus and the cerebral cortex was decreased (P0.05), but the brain sugar utilization rate of the olfactory bulb and cerebellum was not significantly different (P0.05).
6, the proliferation and apoptosis of hippocampal dentate gyrus were observed by terminal deoxy nucleoside acyl transferase mediated dUTP incision terminal labeling (TUNEL) and proliferating cell nuclear antigen (PCNA). The TUNEL positive cells (brown color of nuclei) and PCNA positive cells were observed in the pathological sections of the GIP-KLH vaccine group and the KLH control group. The number of TUNEL positive cells and the number of PCNA positive cells in the GIP-KLH vaccine group were significantly higher than those in the KLH control group, indicating that the proliferation and apoptosis of the dentate gyrus cells in the GIP-KLH vaccine group were all increased.
[Conclusion] the preparation of GIP vaccine with HBc (1-144a.a.) or KLH as a carrier can effectively induce the specific humoral immune response of the target rat GIP, break the rat's self antigen tolerance and induce the GIP specific antibody. This vaccine can be used as a new immune intervention agent for controlling obesity to further study the.GIP-KLH active immune intervention treatment. The rats fed high-fat diet had anti obesity effects, but at the same time, they also affected the behavior and brain function of rats.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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