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
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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(P<0.05)。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(P>0.05), 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(P<0.05),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(P>0.05)。
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(P<0.01)、平均速度(P<0.01)、活动时间(P<0.05)、活动次数(P<0.01)、在中央区域的活动次数(P<0.05)均明显低于KLH对照组,休息时间(P<0.05)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(P>0.05)。在第5天的探索阶段,两组的表现也无明显统计学差异(P>0.05)。
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(P<0.05),而嗅球及小脑的脑糖利用率无明显差别(P>0.05)。
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。
Introduction
Gastric Iinhibitory Peptide (GIP), also known as Glucose-dependent Insulinotropic Polypeptide, is a 42 amino acid-long peptide hormone synthesized in and released from duodenal and jejunal K-cells in response to the ingestion of nutrients, especially dietary lipids in which it facilitates the disposal of both glucose and fat. Of the incretins, GIP has received much interest in the role of regulation of glucose and lipid metabolism.By binding to a seven-transmembrane domain G-protein-coupled receptor expressed in adipose, GIP achieves effects such as regulation of adipocyte differentiation, increase in adipocyte glucose uptake, modulation of adipocyte lipolysis and reesterification and promotion of the storage of triglycerides by increasing the activity of lipoprotein lipase. Moreover, it has been shown that GIP is a key molecule linking overnutrition to obesity. Previous reports about GIP receptor knockout mice resistant to obesity suggest that inhibition of GIP signaling might be a novel target for anti-obesity drug development. The administration of the GIP-specific receptor antagonist and targeted ablation of GIP-producing cells in diet-induced obese mice have also been confirmed to improve metabolic activity. Collectively, inhibition of GIP action may offer exciting possibilities for the development of future therapies available for patients with obesity-related problems.
Conventionally, vaccines are usually used to induce specific immune responses against foreign, pathogen-derived structures. In marked contrast, vaccination against self-molecules is an unusual non-traditional method. Using this novel approach of vaccinating immune bodies against their own endogenous GIP may prove a promising design for combating obesity and related comorbidities. In this respect, the data presented by Fulurija A, Irwin N and colleagues support the neutralization of biologically active GIP by immunopharmacotherapy. The potential advantages of vaccines in the treatment of non-communicable and chronic diseases were thought be the beneficial long-term effects of a relatively short-term treatment, which required less compliance at the same time. However, the treatment may become more complicated with a GIP-targeted vaccine. It has been shown that GIP receptors are widely distributed in the brain and that they may modulate neural progenitor cell proliferation and protect neurons from toxic effects. To a certain extent, GIP may regulate brain function and behavior. Recently, it has been demonstrated that the high circulating GIP concentration brings about significant changes in behaviors. One example is that GIP-overexpressing transgenic mice have better sensorimotor coordination and memory recognition. Therefore the question remains that if immunization methods are used to change the circulating physiological GIP concentration, will there be a resultant change in brain function and behavior.
Here, we constructed GIP vaccine and first observed that the influence of active vaccination of mature rats with GIP immunoconjugates on body weight as well as on brain function and behavior.
Objective
To construct GIP vaccine. Virus-like particles (VLPs) formation by modified Hepatitis B virus core (HBc) or keyhole limpet hemocyanin (KLH), acting as a kind of immune-enhancing vaccine carrier, was fused with the specified epitope of gastric inhibitory peptide(GIP) to construct GIP vaccine (HBc-GIP and GIP-KLH) and the immune effects were investigated. To investigate the effects of active immunisation against GIP on weight gain, behavior and brain function after active immunization against GIP in high-fat-diet SD rats, which may provide theoretical and experimental basis of immune therapy of obesity.
Methods
GIP (1-12a.a.) cDNA was synthesized and fused to HBc VLPs (1-144a.a.) cDNA. Through the bacterial expression and purification processes, GIP vaccine attached to the VLPs carrier derived from HBc was constructed. Hapten GIP peptide (GPRYAEGTFISDC), added a cysteine in the N-terminal region of GIP-(1-12) amides segment, were synthesized by using solid phase peptide synthesis. And Synthetic hapten peptide was purified by HPLC, and coupled to the carrier keyhole limpet hemocyanin (KLH), its reaction with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and yielding immunoconjugates GIP-KLH. The immunogenicity of HBc-GIP and GIP-KLH vaccine in rats was studied.By monitoring of body weight, open field test (OFT), Morris water maze (MWM) test, 18F-fluorodeoxyglucose (FDG)-Positron emission tomography/computed tomography (PET/CT) examination, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) or proliferating cell nuclear antigen (PCNA) method, the differences in weight gain, behavior and brain function between the GIP-KLH-treated group and the KLH-treated group were investigated.
Results:
1. GIP vaccine attached to the VLPs carrier derived from HBc was successfully achieved. Vaccination with DNA Encoding HBc-GIP fusion protein (pVAX1-HBc-GIP) was also gotten. Both the immunization with DNA vaccine (HBc-GIP) or DNA vaccine (pVAXl-HBc-GIP) alone and the combination of DNA and adjuvanted protein vaccination resulted in high, peptide-specific IgG antibody. HBc-GIP and pVAX1-HBc-GIP vaccine displayed a significant strong immunogenicity, whereas control animals failed to exhibit this change. Immunization strategy of prime-boost immunization approach using a combination of DNA and protein vaccine (pV-HBc-GIP*2+HBc-GIP*2) resulted more higher antibody than using DNA or ptotein vaccine alone.After the third immunization, immunized animals induced higher titers of anti-GIP antibodies. Following the vaccination protocol, experimental rats had further increases in antibody levels. Increased immune responses in high-fat diet-induced SD rats by DNA vaccination combined with protein vaccine showed good results.
2. GIP vaccine attached to the KLH carrier was successfully achieved. On day 7 after the second immunization, GIP-KLH-immunized animals induced higher titers of anti-GIP antibodies compared to control rats (KLH-immunized animals). Following the vaccination protocol, experimental rats had further increases in antibody levels, whereas control animals failed to exhibit this change. Increased immune responses in high-fat diet-induced SD rats by GIP-KLH vaccination showed good results.
3. Although no significant differences were found in food intake (p>0.05), differences in increased body weight emerged among groups over the study period. GIP-KLH-vaccinated rats displayed significantly reduced body weight gain compared to KLH-treated rats. Even though there was no difference in the initial body weight, GIP-KLH-vaccinated rats began to gain weight more slowly after a 63-day high-fat feeding compared to other vaccinated rats. At the end of the experiment (98 days after the first vaccination), these experimental rats had gained 48g less weight than control animals (17% reduction in weight gain from the beginning). Although GIP is a key incretin in metabolism, fasting plasma glucose levels and serum insulin levels were unaffected after vaccination in our study (p>0.05).
4. In OFT, Compared with control rats, movement distance (P<0.01), movement speed (P <0.01), active times (P<0.05), the number of activities (P<0.01) the time in the center squares(P< 0.05) decreased and rest time increased (P<0.05) significantly in GIP immunised rats. Thus, the GIP-KLH-immunized rats'automated locomotor ability and the activities of the center squares were significantly decreased compared to the KLH-treated group. Therefore, it is likely that GIP vaccine could lead to behavioral side effects. In Morris water maze (MWM), all rats learned to locate the hidden platform as evidenced by a significant decrease in escape latency and swim distance (F=6.5; P=0.001 and F=5.3; P=0.002, respectively) and a significant increase in swim speed (F=3.3; P=0.024) over the 4-day trial period. However, no statistical difference was shown between GIP-KLH-immunized rats and KLH-treated rats with regard to the previously mentioned data (P>0.05). In the retention test (probe trial) 4-days later, neither GIP-KLH-immunized nor KLH-treated rats exhibited a better retention over the other or had more crossings over the previous location of the platform (P>0.05).
5. In the vivo, the 98th day after application of vaccine, brain glucose utilization assessed by 18F-FDG PET was reduced in hippocampus and cerebral cortices in the GIP-KLH-treated group compared to the KLH-treated group (P<0.05). However, there was no significant difference in olfactory bulbs, or cerebella (P>0.05).
6. Granule cells were marked by the TUNEL or PCNA method in the areas of hippocampal dentate gyrus of GIP-KLH-immunized rats and KLH-treated rats. TUNEL-positive (brown nuclear staining) apoptotic cells and PCNA-positive cells (brown nuclear staining) were found in both groups. But, Comparing the number and intensity of positive cells between two groups, we noticed a significantly larger number of marked cells by TUNEL or PCNA method in the hippocampus of the animals treated with GIP-KLH vaccine.
Conclusion:
HBc(1-144a.a.) or KLH acts as a kind of immune-enhancing vaccine carrier for GIP successfully. Both HBc-GIP and GIP-KLH vaccine are able to overcome GIP-specific B cell unresponsiveness. Active immunisation against GIP results in high, GIP-specific antibody titers. The results encourage further work towards the development of GIP vaccine against obesity. At the same time, active vaccination of mature rats with GIP immunoconjugates may be associated with significant changes in not only body weight, but also brain function and behavior.
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(P<0.05)。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(P>0.05), 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(P<0.05),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(P>0.05)。
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(P<0.01)、平均速度(P<0.01)、活动时间(P<0.05)、活动次数(P<0.01)、在中央区域的活动次数(P<0.05)均明显低于KLH对照组,休息时间(P<0.05)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(P>0.05)。在第5天的探索阶段,两组的表现也无明显统计学差异(P>0.05)。
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(P<0.05),而嗅球及小脑的脑糖利用率无明显差别(P>0.05)。
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。
Introduction
Gastric Iinhibitory Peptide (GIP), also known as Glucose-dependent Insulinotropic Polypeptide, is a 42 amino acid-long peptide hormone synthesized in and released from duodenal and jejunal K-cells in response to the ingestion of nutrients, especially dietary lipids in which it facilitates the disposal of both glucose and fat. Of the incretins, GIP has received much interest in the role of regulation of glucose and lipid metabolism.By binding to a seven-transmembrane domain G-protein-coupled receptor expressed in adipose, GIP achieves effects such as regulation of adipocyte differentiation, increase in adipocyte glucose uptake, modulation of adipocyte lipolysis and reesterification and promotion of the storage of triglycerides by increasing the activity of lipoprotein lipase. Moreover, it has been shown that GIP is a key molecule linking overnutrition to obesity. Previous reports about GIP receptor knockout mice resistant to obesity suggest that inhibition of GIP signaling might be a novel target for anti-obesity drug development. The administration of the GIP-specific receptor antagonist and targeted ablation of GIP-producing cells in diet-induced obese mice have also been confirmed to improve metabolic activity. Collectively, inhibition of GIP action may offer exciting possibilities for the development of future therapies available for patients with obesity-related problems.
Conventionally, vaccines are usually used to induce specific immune responses against foreign, pathogen-derived structures. In marked contrast, vaccination against self-molecules is an unusual non-traditional method. Using this novel approach of vaccinating immune bodies against their own endogenous GIP may prove a promising design for combating obesity and related comorbidities. In this respect, the data presented by Fulurija A, Irwin N and colleagues support the neutralization of biologically active GIP by immunopharmacotherapy. The potential advantages of vaccines in the treatment of non-communicable and chronic diseases were thought be the beneficial long-term effects of a relatively short-term treatment, which required less compliance at the same time. However, the treatment may become more complicated with a GIP-targeted vaccine. It has been shown that GIP receptors are widely distributed in the brain and that they may modulate neural progenitor cell proliferation and protect neurons from toxic effects. To a certain extent, GIP may regulate brain function and behavior. Recently, it has been demonstrated that the high circulating GIP concentration brings about significant changes in behaviors. One example is that GIP-overexpressing transgenic mice have better sensorimotor coordination and memory recognition. Therefore the question remains that if immunization methods are used to change the circulating physiological GIP concentration, will there be a resultant change in brain function and behavior.
Here, we constructed GIP vaccine and first observed that the influence of active vaccination of mature rats with GIP immunoconjugates on body weight as well as on brain function and behavior.
Objective
To construct GIP vaccine. Virus-like particles (VLPs) formation by modified Hepatitis B virus core (HBc) or keyhole limpet hemocyanin (KLH), acting as a kind of immune-enhancing vaccine carrier, was fused with the specified epitope of gastric inhibitory peptide(GIP) to construct GIP vaccine (HBc-GIP and GIP-KLH) and the immune effects were investigated. To investigate the effects of active immunisation against GIP on weight gain, behavior and brain function after active immunization against GIP in high-fat-diet SD rats, which may provide theoretical and experimental basis of immune therapy of obesity.
Methods
GIP (1-12a.a.) cDNA was synthesized and fused to HBc VLPs (1-144a.a.) cDNA. Through the bacterial expression and purification processes, GIP vaccine attached to the VLPs carrier derived from HBc was constructed. Hapten GIP peptide (GPRYAEGTFISDC), added a cysteine in the N-terminal region of GIP-(1-12) amides segment, were synthesized by using solid phase peptide synthesis. And Synthetic hapten peptide was purified by HPLC, and coupled to the carrier keyhole limpet hemocyanin (KLH), its reaction with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and yielding immunoconjugates GIP-KLH. The immunogenicity of HBc-GIP and GIP-KLH vaccine in rats was studied.By monitoring of body weight, open field test (OFT), Morris water maze (MWM) test, 18F-fluorodeoxyglucose (FDG)-Positron emission tomography/computed tomography (PET/CT) examination, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) or proliferating cell nuclear antigen (PCNA) method, the differences in weight gain, behavior and brain function between the GIP-KLH-treated group and the KLH-treated group were investigated.
Results:
1. GIP vaccine attached to the VLPs carrier derived from HBc was successfully achieved. Vaccination with DNA Encoding HBc-GIP fusion protein (pVAX1-HBc-GIP) was also gotten. Both the immunization with DNA vaccine (HBc-GIP) or DNA vaccine (pVAXl-HBc-GIP) alone and the combination of DNA and adjuvanted protein vaccination resulted in high, peptide-specific IgG antibody. HBc-GIP and pVAX1-HBc-GIP vaccine displayed a significant strong immunogenicity, whereas control animals failed to exhibit this change. Immunization strategy of prime-boost immunization approach using a combination of DNA and protein vaccine (pV-HBc-GIP*2+HBc-GIP*2) resulted more higher antibody than using DNA or ptotein vaccine alone.After the third immunization, immunized animals induced higher titers of anti-GIP antibodies. Following the vaccination protocol, experimental rats had further increases in antibody levels. Increased immune responses in high-fat diet-induced SD rats by DNA vaccination combined with protein vaccine showed good results.
2. GIP vaccine attached to the KLH carrier was successfully achieved. On day 7 after the second immunization, GIP-KLH-immunized animals induced higher titers of anti-GIP antibodies compared to control rats (KLH-immunized animals). Following the vaccination protocol, experimental rats had further increases in antibody levels, whereas control animals failed to exhibit this change. Increased immune responses in high-fat diet-induced SD rats by GIP-KLH vaccination showed good results.
3. Although no significant differences were found in food intake (p>0.05), differences in increased body weight emerged among groups over the study period. GIP-KLH-vaccinated rats displayed significantly reduced body weight gain compared to KLH-treated rats. Even though there was no difference in the initial body weight, GIP-KLH-vaccinated rats began to gain weight more slowly after a 63-day high-fat feeding compared to other vaccinated rats. At the end of the experiment (98 days after the first vaccination), these experimental rats had gained 48g less weight than control animals (17% reduction in weight gain from the beginning). Although GIP is a key incretin in metabolism, fasting plasma glucose levels and serum insulin levels were unaffected after vaccination in our study (p>0.05).
4. In OFT, Compared with control rats, movement distance (P<0.01), movement speed (P <0.01), active times (P<0.05), the number of activities (P<0.01) the time in the center squares(P< 0.05) decreased and rest time increased (P<0.05) significantly in GIP immunised rats. Thus, the GIP-KLH-immunized rats'automated locomotor ability and the activities of the center squares were significantly decreased compared to the KLH-treated group. Therefore, it is likely that GIP vaccine could lead to behavioral side effects. In Morris water maze (MWM), all rats learned to locate the hidden platform as evidenced by a significant decrease in escape latency and swim distance (F=6.5; P=0.001 and F=5.3; P=0.002, respectively) and a significant increase in swim speed (F=3.3; P=0.024) over the 4-day trial period. However, no statistical difference was shown between GIP-KLH-immunized rats and KLH-treated rats with regard to the previously mentioned data (P>0.05). In the retention test (probe trial) 4-days later, neither GIP-KLH-immunized nor KLH-treated rats exhibited a better retention over the other or had more crossings over the previous location of the platform (P>0.05).
5. In the vivo, the 98th day after application of vaccine, brain glucose utilization assessed by 18F-FDG PET was reduced in hippocampus and cerebral cortices in the GIP-KLH-treated group compared to the KLH-treated group (P<0.05). However, there was no significant difference in olfactory bulbs, or cerebella (P>0.05).
6. Granule cells were marked by the TUNEL or PCNA method in the areas of hippocampal dentate gyrus of GIP-KLH-immunized rats and KLH-treated rats. TUNEL-positive (brown nuclear staining) apoptotic cells and PCNA-positive cells (brown nuclear staining) were found in both groups. But, Comparing the number and intensity of positive cells between two groups, we noticed a significantly larger number of marked cells by TUNEL or PCNA method in the hippocampus of the animals treated with GIP-KLH vaccine.
Conclusion:
HBc(1-144a.a.) or KLH acts as a kind of immune-enhancing vaccine carrier for GIP successfully. Both HBc-GIP and GIP-KLH vaccine are able to overcome GIP-specific B cell unresponsiveness. Active immunisation against GIP results in high, GIP-specific antibody titers. The results encourage further work towards the development of GIP vaccine against obesity. At the same time, active vaccination of mature rats with GIP immunoconjugates may be associated with significant changes in not only body weight, but also brain function and behavior.
引文
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