脂肪细胞型脂肪酸结合蛋白疫苗预防高脂喂养小鼠胰岛素抵抗及动脉粥样硬化的实验研究
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摘要
[背景]
随着社会的不断发展和人们生活方式的改变，大量脂肪摄入所引发的肥胖及其相关代谢性疾病尤其是2型糖尿病的发病率正逐年上升，严重危害着人民群众的身心健康。肥胖和糖尿病可能带来一系列生理功能的改变，其大血管并发症如动脉粥样硬化更是直接威胁生命，从而加重任何原因引致死亡的风险。传统治疗方法如控制饮食、加强运动、内科药物及外科手术等，均或多或少存在着难以坚持、依从性差、手术并发症和远期效果不明确等种种弊端。因此，对于上述疾病的治疗还需另辟蹊径。
目前已有大量研究证据表明，脂肪组织除了储存能量之外，还是一个重要的内分泌器官，能够释放大量生物活性物质入血，并通过局部和全身作用，协调能量代谢、胰岛素敏感性、炎症和血管反应。脂肪细胞型脂肪酸结合蛋白（A-FABP，又称FABP4，ap2）就是其中的一种，它属于脂肪酸结合蛋白大家族，主要存在于脂肪细胞和巨噬细胞，也可以分泌入血，其主要作用是参与游离脂肪酸及其它类型的脂质激素的转运和合成，从而调节全身胰岛素敏感性及能量代谢活动，并且在巨噬细胞中调节炎症细胞因子和胆固醇酯的聚集。大量的动物实验和临床研究已经证实，FABP4水平与肥胖和糖脂代谢紊乱相关的多种病理状态如高血糖、高胰岛素血症、高脂血症、代谢综合征、动脉粥样硬化等的发生和发展密切相关，FABP4基因敲除或口服其特异性抑制剂的小鼠，表现出几乎能够完全抵抗饮食性或遗传性肥胖诱导下的胰岛素抵抗、血脂紊乱、动脉粥样硬化、脂肪性肝病等。
因此，研究者们认为，从某种程度上抑制FABP4的表达和作用，能够有效改善外周的胰岛素抵抗，保护胰岛β细胞功能，而且对糖脂代谢和大血管病变产生有益的效应，它可能成为肥胖、糖尿病和动脉粥样硬化治疗的新靶点。然而，基因敲除技术难度高且可行性差，口服抑制剂同样存在难以坚持、依从性差等问题。
[目的]
基于上述理论和实践基础，本研究以与机体糖脂代谢密切相关的蛋白FABP4为靶点，拟构建能够诱导出针对FABP4特异性中和抗体的治疗性疫苗，主动免疫高脂喂养下小鼠，观察免疫效果及其安全耐受性，为高脂诱导下胰岛素抵抗及其大血管病变并发症的预防及治疗新途径提供理论和实验依据。
[方法]
1、采用生物合成的方法表达得到纯化的小鼠FABP4蛋白作为一种蛋白疫苗，为了增加其免疫原性，另选取两种不同的载体与之组成另两种蛋白疫苗，分别是：(1)、以乙肝病毒核心抗原病毒样颗粒（HBc-VLP）为载体的HBc-FABP4蛋白疫苗；(2)、以钥孔戚血蓝蛋白（KLH）为载体的KLH-FABP4蛋白疫苗。
2、分别以三种不同的蛋白疫苗（实验组）及其相应载体或佐剂（对照组）对小鼠进行免疫，观察小鼠安全耐受性，同时摸索最佳免疫剂量和免疫间隔，通过ELISA法检测各组FABP4抗体滴度以筛选出最佳免疫方案。
3、选取方法2中筛选出的最佳免疫方案，对高脂喂养下野生型小鼠进行免疫，并以其相应载体或佐剂为对照，测量小鼠体重、摄食量、血糖、胰岛素、脂联素等指标，并进行葡萄糖耐量实验；处死小鼠后，取肝脏组织进行油红O染色，比较肝脏脂肪沉积面积。
4、选取方法2中筛选出的最佳免疫方案，对高脂喂养下载脂蛋白E（ApoE）敲除的小鼠（ApoE-/-）进行免疫，并以其相应的载体或佐剂为对照，测量小鼠体重、摄食量、血糖、胰岛素、脂联素、总胆固醇（TC）、甘油三酯（TG）、游离脂肪酸（FFA）、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）等糖脂代谢相关指标，并进行葡萄糖耐量实验；处死小鼠后，分离小鼠主动脉进行油红O染色，比较动脉粥样斑块面积。
[结果]
1、采用生物合成的方法，成功表达了纯化的FABP4蛋白和对照组所需的免疫重组HBc蛋白，制备了携带FABP4抗原序列的HBc-FABP4蛋白疫苗，经western-blot验证确实为所需蛋白；采用化学耦联的方法，成功制备了KLH-FABP4蛋白疫苗。为进一步的小鼠免疫打下了良好基础。
2、三种候选蛋白疫苗分别免疫小鼠后，均可产生FABP4特异性抗体，而作为对照的HBc组、KLH组和佐剂组则没有FABP4特异性抗体的产生，说明这三种蛋白疫苗均能够打破宿主对自身对FABP4的免疫耐受。其中，FABP4组小鼠产生的抗体滴度最高，优于HBc-FABP4组和KLH-FABP4组(P＜0.05)。免疫间隔3周为有效的间隔时间，各组抗体滴度于第3轮加强免疫后达到平稳状态。免疫过程中，各组均有部分小鼠存在免疫接种局部轻度脱毛和皮下硬结现象，无其他显著不良事件及死亡发生。
3、选取最佳免疫方案（FABP4）对高脂喂养野生型雌性和雄性C57BL/6J小鼠分别进行免疫，产生了高滴度的FABP4特异性抗体。雌性：到首次免疫后第16w，疫苗组小鼠体重虽高于正常对照组，但明显低于同样高脂饮食的佐剂组（P＜0.05）；疫苗组小鼠日平均摄食量明显高于正常组（P＜0.05），但与佐剂组无差异（P＞0.05）；疫苗组空腹血糖、空腹胰岛素、HOMA-IR指数、腹腔葡萄糖耐量实验曲线下面积均明显低于佐剂组（P＜0.05），血清脂联素水平高于佐剂组（P＜0.05），与正常对无统计学差异（P＞0.05）。雄性：到首次免疫后第16w，疫苗组小鼠体重明显高于正常对照组（P＜0.05），与佐剂组未见明显差别（P＞0.05）；疫苗组日平均摄食量与其余两组未见明显区别（P＞0.05）；疫苗组空腹血糖、腹腔葡萄糖耐量实验曲线下面积低于佐剂组（P＜0.05），血清脂联素水平高于佐剂组（P＜0.05），与正常组无明显差异（P＞0.05）；疫苗组空腹胰岛素、HOMA-IR指数高于正常组但明显低于佐剂组（P＜0.05）。肝脏切片可见疫苗组脂肪阳性面积和积分光密度（IOD）较空白对照组高，但明显低于佐剂组（P<0.05）。
4、选取最佳免疫方案（FABP4）对高脂喂养雄性ApoE-/-小鼠进行免疫，产生了高滴度的FABP4特异性抗体。到首次免疫后第12w，疫苗组小鼠与对照组体重水平和日平均摄食量水平无明显差异（P＞0.05），疫苗组空腹血糖、空腹胰岛素、HOMA-IR指数、腹腔葡萄糖耐量实验曲线下面积、TC、HDL、LDL和TC/HDL比值均明显低于佐剂组（P＜0.05），血清脂联素水平高于佐剂组（P＜0.05），空腹TG、FFA、LDL/HDL比值与佐剂组无明显差异（P＞0.05）。主动脉切片可见疫苗组动脉斑块占管腔面积百分比明显低于佐剂组（P<0.05）。
[结论]
经过HBc-FABP4、KLH-FABP4和FABP4三种蛋白疫苗的免疫，均可获得高滴度的FABP4特异性抗体IgG，其中FABP4抗体滴度最高，显示了良好的免疫效果。以FABP4蛋白疫苗免疫高脂喂养野生型和ApoE-/-小鼠，能够有效降低血糖、胰岛素和血脂水平，减轻肝脏脂肪沉积和主动脉泡沫细胞聚集，改善糖脂代谢紊乱状态，为高脂诱导的胰岛素抵抗和动脉粥样硬化的治疗提供了新的途径和初步证据，可进行进一步的深入研究。
Background
With the development of society and changes in lifestyle, a large amount of fat intake cause the increasing incidence of obesity and its related metabolic disorders, especially type2diabetes mellitus year by year,which are seriously endangering people's physical and mental health.Obesity and diabetes may cause a series of changes in physiological functions,of which its macrovascular complications such as atherosclerosis is a direct threat to life, thereby increasing the risk of all-cause death.Traditional treatments including dietary control, physical exercises,medicine and surgery,are more or less in limit due to maintain difficulty,poor compliance,indefinition on surgical complications and long-term effect. Therefore, search of new therapies is absolutely necessary.
Lots of evidence has shown that adipose tissue is not only a energy store but also an important endocrine organ,which releases large amounts of bioactive substances into circulation.These substances then coordinate energy metabolism, insulin sensitivity, inflammation and vascular reaction through local and systemic action. Adipocyte fatty-acid-binding protein(A-FABP,FABP4,ap2),belonging to FABP family,is one of these substances,which expresses mainly in adipocytes and macrophages, and also can be secreted into circulation.It participates transport and synthesis of free fatty acid(FFA) and other kinds of lipid hormones,thus regulates systemic insulin sensitivity and energy metabolism, the aggregation of inflammatory cell factors and cholesterol esters in macrophage.
Studies in zoopery and clinic have proved the level of FABP4is closely related to the development of hyperglycemia, hyperinsulinism, hyperlipoidemia,metabolic syndrome and atherosclerosis,which are pathologic status of obesity and metabolic disturbance. FABP4-deficient mice and mice taking small-molecule inhibitor of FABP4have shown almost complete resistance to dietary or genetic obesity-induced insulin resistance, Dyslipidemia, atherosclerosis,fatty liver disease,and so on.
Therefore,researchers insist that inhibiting the function of this lipid chaperone can significantly improve several aspects of metabolic syndrome.FABP4may be a new target to the therapies of obesity,diabetes and atherosclerosis.However, gene knock-out technique is difficult and infeasible,and taking inhibitor is also of poor compliance.
Objective
Based on the theory and practice above,here we first constructed vaccines against FABP4with different carriers,and immunized wild-type and gene-deficient mice with high-fat diet respectively. Immune effects and security were observed in order to supply theoretical and experimental evidence in new prevention and treatments of high-fat-induced insulin resistance and its macroangiopathy complications.
Methods
1、First, biosynthesis method was used to obtain depurated mouse FABP4,as one of the vaccines.In order to enhance the immunogenicity,two types of carriers:Virus-like particles (VLPs) formation by modified Hepatitis B virus core (HBc) and keyhole limpet hemocyanin (KLH), acting as a kind of immune-enhancing vaccine carrier, were chosen and fused with FABP4.Thus we have constructed in all three types of vaccines,HBc-FABP4,KLH-FABP4,FABP4,respectively.
2、These three vaccines were used to immunized wild-type mice as experimental groups,with their corresponding carriers and adjuvant as control groups.The security and tolerance were observed,and the best immunizing dose and interval were researched.ELIS A was used to test the antibody titer of each group.
3、The best strategy of immunization was selected to immunize wild-type C57BL/6J mice with high-fat diet.Body weight,food-intake and fasting blood glucose were measured during the whole immune period.After16weeks of the first vaccination,IPGTT was performed on each mouse,and the levels of serum insulin and adiponectin were tested.Then the mice were executed and frozen sections of their livers were made.Oil red O staining and imag-pro plus were used to compare the areas of fat deposition.
4、The best strategy of immunization was also selected to immunize Apo E-/-mice with high-fat diet. Body weight,food-intake and fasting blood glucose were measured during the whole immune period. After12weeks of the first vaccination,IPGTT was performed on each mouse,and index related to glucose and lipid metabolism such as the levels of serum insulin,adiponectin,TC,TG,FFA,HDL,LDL were tested.Then the mice were executed and frozen sections of their aortas were made. Oil red O staining and imag-pro plus were used to compare the areas of atherosclerosis lesions.
Results:
1、Depurant FABP4and immune recombinant HBc protein were successfully expressed by the biosynthesis method.FABP4vaccines attached to the VLPs carrier derived from HBc and to the KLH respectively were successfully achieved.In conclusion,we successfully constructed3types of vaccines against FABP4, which established a good foundation for further immunization in mice.
2、After immunized by the3types of vaccinations respectively,mice in every immune group all response with high, peptide-specific IgG antibody titers,which were not observed in control groups injected with carriers or adjuvant.That proved the3types of vaccinations can break the immunologic tolerance to FABP4in the hosts. Among them,the titer induced by FABP4was higher than that induced by HBc-FABP4or KLH-FABP4(P<0.05),and was considered to be the best immune strategy.3-weeks interval was effective,and the antibody titer of each group was stable after the3rd booster.During the immune period,there are some mice both in immune groups and in control groups appeared depilation and subcutaneous induration among injection sites,and no other adverse events or death happened.
3、FABP4-immunized wild-type C57BL/6J mice with high-fat diet (treating group) were compared with adjuvant-injected mice with high-fat diet(control group) and mice with normal diet(normal group).In female mice:After16weeks of the first immunization,the body weight in treating group was higher than that in normal group but lower than that in control group (P<0.05). food-intake was higher than that in normal group (P<0.05),and had no difference with that in control group (P>0.05).The levels of fasting blood glucose,fasting insulin, HOMA-IR index,glucose AUC of IPGTT were all lower than those in control group(P<0.05),while adiponectin level was higher than that in control group (P<0.05),and had no differences with those in normal group (P>0.05). In male mice:After16weeks of the first immunization,the body weight in treating group was higher than that in normal group (P<0.05), and had no difference with that in control group (P>0.05). food-intake had no difference with that in either control group or normal group (P>0.05).The levels of fasting blood glucose, glucose AUC of IPGTT were lower than those in control group (P<0.05),while adiponectin level was higher than that in control group (P<0.05),and had no differences with those in normal group (P>0.05).The levels of fasting insulin and HOMA-IR index were higher than those in normal group but lower than those in control group (P<0.05).In both male and female mice,frozen sections of liver showed that the area and IOD of fat disposition in treating group were higher than those in normal group but much lower than those in control group (P<0.05)
4、FABP4-immunized male Apo E-/-mice with high-fat diet (treating group) were compared with adjuvant-injected male Apo E-/-mice with high-fat diet(control group). After12weeks of the first immunization,the body weight and food-intake in treating group had no difference with those in normal group (P>0.05).The levels of fasting blood glucose, fasting insulin,HOMA-IR index,glucose AUC of IPGTT,TC,HDL,LDL and TC/HDL were lower than those in control group (P<0.05),while adiponectin level was higher than that in control group (P<0.05) The levels of fasting TG,FFA,LDL/HDL had no difference with that in control group (P>0.05).Frozen sections of aortas showed that the percentage of plaque area to the whole lumens area in treating group was much lower than that in control group(P<0.05)
Conclusion
In conclusion,we successfully constructed3types of vaccines against FABP4,which could all induce specific humoral immunity and produce high titer of specific antibody in mice.FABP4vaccination effectively improved the levels of blood glucose,insulin,blood lipid of mice with high-fat diet,and also reduced fat disposition in liver and foam cell aggregation in aortas. Our study further proved the feasibility of treatment targeting to FABP4in high-fat induced insulin resistance and atherosclerosis,and offers theoretical and experimental basis for the brand-new immunologic intervention.
随着社会的不断发展和人们生活方式的改变，大量脂肪摄入所引发的肥胖及其相关代谢性疾病尤其是2型糖尿病的发病率正逐年上升，严重危害着人民群众的身心健康。肥胖和糖尿病可能带来一系列生理功能的改变，其大血管并发症如动脉粥样硬化更是直接威胁生命，从而加重任何原因引致死亡的风险。传统治疗方法如控制饮食、加强运动、内科药物及外科手术等，均或多或少存在着难以坚持、依从性差、手术并发症和远期效果不明确等种种弊端。因此，对于上述疾病的治疗还需另辟蹊径。
目前已有大量研究证据表明，脂肪组织除了储存能量之外，还是一个重要的内分泌器官，能够释放大量生物活性物质入血，并通过局部和全身作用，协调能量代谢、胰岛素敏感性、炎症和血管反应。脂肪细胞型脂肪酸结合蛋白（A-FABP，又称FABP4，ap2）就是其中的一种，它属于脂肪酸结合蛋白大家族，主要存在于脂肪细胞和巨噬细胞，也可以分泌入血，其主要作用是参与游离脂肪酸及其它类型的脂质激素的转运和合成，从而调节全身胰岛素敏感性及能量代谢活动，并且在巨噬细胞中调节炎症细胞因子和胆固醇酯的聚集。大量的动物实验和临床研究已经证实，FABP4水平与肥胖和糖脂代谢紊乱相关的多种病理状态如高血糖、高胰岛素血症、高脂血症、代谢综合征、动脉粥样硬化等的发生和发展密切相关，FABP4基因敲除或口服其特异性抑制剂的小鼠，表现出几乎能够完全抵抗饮食性或遗传性肥胖诱导下的胰岛素抵抗、血脂紊乱、动脉粥样硬化、脂肪性肝病等。
因此，研究者们认为，从某种程度上抑制FABP4的表达和作用，能够有效改善外周的胰岛素抵抗，保护胰岛β细胞功能，而且对糖脂代谢和大血管病变产生有益的效应，它可能成为肥胖、糖尿病和动脉粥样硬化治疗的新靶点。然而，基因敲除技术难度高且可行性差，口服抑制剂同样存在难以坚持、依从性差等问题。
[目的]
基于上述理论和实践基础，本研究以与机体糖脂代谢密切相关的蛋白FABP4为靶点，拟构建能够诱导出针对FABP4特异性中和抗体的治疗性疫苗，主动免疫高脂喂养下小鼠，观察免疫效果及其安全耐受性，为高脂诱导下胰岛素抵抗及其大血管病变并发症的预防及治疗新途径提供理论和实验依据。
[方法]
1、采用生物合成的方法表达得到纯化的小鼠FABP4蛋白作为一种蛋白疫苗，为了增加其免疫原性，另选取两种不同的载体与之组成另两种蛋白疫苗，分别是：(1)、以乙肝病毒核心抗原病毒样颗粒（HBc-VLP）为载体的HBc-FABP4蛋白疫苗；(2)、以钥孔戚血蓝蛋白（KLH）为载体的KLH-FABP4蛋白疫苗。
2、分别以三种不同的蛋白疫苗（实验组）及其相应载体或佐剂（对照组）对小鼠进行免疫，观察小鼠安全耐受性，同时摸索最佳免疫剂量和免疫间隔，通过ELISA法检测各组FABP4抗体滴度以筛选出最佳免疫方案。
3、选取方法2中筛选出的最佳免疫方案，对高脂喂养下野生型小鼠进行免疫，并以其相应载体或佐剂为对照，测量小鼠体重、摄食量、血糖、胰岛素、脂联素等指标，并进行葡萄糖耐量实验；处死小鼠后，取肝脏组织进行油红O染色，比较肝脏脂肪沉积面积。
4、选取方法2中筛选出的最佳免疫方案，对高脂喂养下载脂蛋白E（ApoE）敲除的小鼠（ApoE-/-）进行免疫，并以其相应的载体或佐剂为对照，测量小鼠体重、摄食量、血糖、胰岛素、脂联素、总胆固醇（TC）、甘油三酯（TG）、游离脂肪酸（FFA）、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）等糖脂代谢相关指标，并进行葡萄糖耐量实验；处死小鼠后，分离小鼠主动脉进行油红O染色，比较动脉粥样斑块面积。
[结果]
1、采用生物合成的方法，成功表达了纯化的FABP4蛋白和对照组所需的免疫重组HBc蛋白，制备了携带FABP4抗原序列的HBc-FABP4蛋白疫苗，经western-blot验证确实为所需蛋白；采用化学耦联的方法，成功制备了KLH-FABP4蛋白疫苗。为进一步的小鼠免疫打下了良好基础。
2、三种候选蛋白疫苗分别免疫小鼠后，均可产生FABP4特异性抗体，而作为对照的HBc组、KLH组和佐剂组则没有FABP4特异性抗体的产生，说明这三种蛋白疫苗均能够打破宿主对自身对FABP4的免疫耐受。其中，FABP4组小鼠产生的抗体滴度最高，优于HBc-FABP4组和KLH-FABP4组(P＜0.05)。免疫间隔3周为有效的间隔时间，各组抗体滴度于第3轮加强免疫后达到平稳状态。免疫过程中，各组均有部分小鼠存在免疫接种局部轻度脱毛和皮下硬结现象，无其他显著不良事件及死亡发生。
3、选取最佳免疫方案（FABP4）对高脂喂养野生型雌性和雄性C57BL/6J小鼠分别进行免疫，产生了高滴度的FABP4特异性抗体。雌性：到首次免疫后第16w，疫苗组小鼠体重虽高于正常对照组，但明显低于同样高脂饮食的佐剂组（P＜0.05）；疫苗组小鼠日平均摄食量明显高于正常组（P＜0.05），但与佐剂组无差异（P＞0.05）；疫苗组空腹血糖、空腹胰岛素、HOMA-IR指数、腹腔葡萄糖耐量实验曲线下面积均明显低于佐剂组（P＜0.05），血清脂联素水平高于佐剂组（P＜0.05），与正常对无统计学差异（P＞0.05）。雄性：到首次免疫后第16w，疫苗组小鼠体重明显高于正常对照组（P＜0.05），与佐剂组未见明显差别（P＞0.05）；疫苗组日平均摄食量与其余两组未见明显区别（P＞0.05）；疫苗组空腹血糖、腹腔葡萄糖耐量实验曲线下面积低于佐剂组（P＜0.05），血清脂联素水平高于佐剂组（P＜0.05），与正常组无明显差异（P＞0.05）；疫苗组空腹胰岛素、HOMA-IR指数高于正常组但明显低于佐剂组（P＜0.05）。肝脏切片可见疫苗组脂肪阳性面积和积分光密度（IOD）较空白对照组高，但明显低于佐剂组（P<0.05）。
4、选取最佳免疫方案（FABP4）对高脂喂养雄性ApoE-/-小鼠进行免疫，产生了高滴度的FABP4特异性抗体。到首次免疫后第12w，疫苗组小鼠与对照组体重水平和日平均摄食量水平无明显差异（P＞0.05），疫苗组空腹血糖、空腹胰岛素、HOMA-IR指数、腹腔葡萄糖耐量实验曲线下面积、TC、HDL、LDL和TC/HDL比值均明显低于佐剂组（P＜0.05），血清脂联素水平高于佐剂组（P＜0.05），空腹TG、FFA、LDL/HDL比值与佐剂组无明显差异（P＞0.05）。主动脉切片可见疫苗组动脉斑块占管腔面积百分比明显低于佐剂组（P<0.05）。
[结论]
经过HBc-FABP4、KLH-FABP4和FABP4三种蛋白疫苗的免疫，均可获得高滴度的FABP4特异性抗体IgG，其中FABP4抗体滴度最高，显示了良好的免疫效果。以FABP4蛋白疫苗免疫高脂喂养野生型和ApoE-/-小鼠，能够有效降低血糖、胰岛素和血脂水平，减轻肝脏脂肪沉积和主动脉泡沫细胞聚集，改善糖脂代谢紊乱状态，为高脂诱导的胰岛素抵抗和动脉粥样硬化的治疗提供了新的途径和初步证据，可进行进一步的深入研究。
Background
With the development of society and changes in lifestyle, a large amount of fat intake cause the increasing incidence of obesity and its related metabolic disorders, especially type2diabetes mellitus year by year,which are seriously endangering people's physical and mental health.Obesity and diabetes may cause a series of changes in physiological functions,of which its macrovascular complications such as atherosclerosis is a direct threat to life, thereby increasing the risk of all-cause death.Traditional treatments including dietary control, physical exercises,medicine and surgery,are more or less in limit due to maintain difficulty,poor compliance,indefinition on surgical complications and long-term effect. Therefore, search of new therapies is absolutely necessary.
Lots of evidence has shown that adipose tissue is not only a energy store but also an important endocrine organ,which releases large amounts of bioactive substances into circulation.These substances then coordinate energy metabolism, insulin sensitivity, inflammation and vascular reaction through local and systemic action. Adipocyte fatty-acid-binding protein(A-FABP,FABP4,ap2),belonging to FABP family,is one of these substances,which expresses mainly in adipocytes and macrophages, and also can be secreted into circulation.It participates transport and synthesis of free fatty acid(FFA) and other kinds of lipid hormones,thus regulates systemic insulin sensitivity and energy metabolism, the aggregation of inflammatory cell factors and cholesterol esters in macrophage.
Studies in zoopery and clinic have proved the level of FABP4is closely related to the development of hyperglycemia, hyperinsulinism, hyperlipoidemia,metabolic syndrome and atherosclerosis,which are pathologic status of obesity and metabolic disturbance. FABP4-deficient mice and mice taking small-molecule inhibitor of FABP4have shown almost complete resistance to dietary or genetic obesity-induced insulin resistance, Dyslipidemia, atherosclerosis,fatty liver disease,and so on.
Therefore,researchers insist that inhibiting the function of this lipid chaperone can significantly improve several aspects of metabolic syndrome.FABP4may be a new target to the therapies of obesity,diabetes and atherosclerosis.However, gene knock-out technique is difficult and infeasible,and taking inhibitor is also of poor compliance.
Objective
Based on the theory and practice above,here we first constructed vaccines against FABP4with different carriers,and immunized wild-type and gene-deficient mice with high-fat diet respectively. Immune effects and security were observed in order to supply theoretical and experimental evidence in new prevention and treatments of high-fat-induced insulin resistance and its macroangiopathy complications.
Methods
1、First, biosynthesis method was used to obtain depurated mouse FABP4,as one of the vaccines.In order to enhance the immunogenicity,two types of carriers:Virus-like particles (VLPs) formation by modified Hepatitis B virus core (HBc) and keyhole limpet hemocyanin (KLH), acting as a kind of immune-enhancing vaccine carrier, were chosen and fused with FABP4.Thus we have constructed in all three types of vaccines,HBc-FABP4,KLH-FABP4,FABP4,respectively.
2、These three vaccines were used to immunized wild-type mice as experimental groups,with their corresponding carriers and adjuvant as control groups.The security and tolerance were observed,and the best immunizing dose and interval were researched.ELIS A was used to test the antibody titer of each group.
3、The best strategy of immunization was selected to immunize wild-type C57BL/6J mice with high-fat diet.Body weight,food-intake and fasting blood glucose were measured during the whole immune period.After16weeks of the first vaccination,IPGTT was performed on each mouse,and the levels of serum insulin and adiponectin were tested.Then the mice were executed and frozen sections of their livers were made.Oil red O staining and imag-pro plus were used to compare the areas of fat deposition.
4、The best strategy of immunization was also selected to immunize Apo E-/-mice with high-fat diet. Body weight,food-intake and fasting blood glucose were measured during the whole immune period. After12weeks of the first vaccination,IPGTT was performed on each mouse,and index related to glucose and lipid metabolism such as the levels of serum insulin,adiponectin,TC,TG,FFA,HDL,LDL were tested.Then the mice were executed and frozen sections of their aortas were made. Oil red O staining and imag-pro plus were used to compare the areas of atherosclerosis lesions.
Results:
1、Depurant FABP4and immune recombinant HBc protein were successfully expressed by the biosynthesis method.FABP4vaccines attached to the VLPs carrier derived from HBc and to the KLH respectively were successfully achieved.In conclusion,we successfully constructed3types of vaccines against FABP4, which established a good foundation for further immunization in mice.
2、After immunized by the3types of vaccinations respectively,mice in every immune group all response with high, peptide-specific IgG antibody titers,which were not observed in control groups injected with carriers or adjuvant.That proved the3types of vaccinations can break the immunologic tolerance to FABP4in the hosts. Among them,the titer induced by FABP4was higher than that induced by HBc-FABP4or KLH-FABP4(P<0.05),and was considered to be the best immune strategy.3-weeks interval was effective,and the antibody titer of each group was stable after the3rd booster.During the immune period,there are some mice both in immune groups and in control groups appeared depilation and subcutaneous induration among injection sites,and no other adverse events or death happened.
3、FABP4-immunized wild-type C57BL/6J mice with high-fat diet (treating group) were compared with adjuvant-injected mice with high-fat diet(control group) and mice with normal diet(normal group).In female mice:After16weeks of the first immunization,the body weight in treating group was higher than that in normal group but lower than that in control group (P<0.05). food-intake was higher than that in normal group (P<0.05),and had no difference with that in control group (P>0.05).The levels of fasting blood glucose,fasting insulin, HOMA-IR index,glucose AUC of IPGTT were all lower than those in control group(P<0.05),while adiponectin level was higher than that in control group (P<0.05),and had no differences with those in normal group (P>0.05). In male mice:After16weeks of the first immunization,the body weight in treating group was higher than that in normal group (P<0.05), and had no difference with that in control group (P>0.05). food-intake had no difference with that in either control group or normal group (P>0.05).The levels of fasting blood glucose, glucose AUC of IPGTT were lower than those in control group (P<0.05),while adiponectin level was higher than that in control group (P<0.05),and had no differences with those in normal group (P>0.05).The levels of fasting insulin and HOMA-IR index were higher than those in normal group but lower than those in control group (P<0.05).In both male and female mice,frozen sections of liver showed that the area and IOD of fat disposition in treating group were higher than those in normal group but much lower than those in control group (P<0.05)
4、FABP4-immunized male Apo E-/-mice with high-fat diet (treating group) were compared with adjuvant-injected male Apo E-/-mice with high-fat diet(control group). After12weeks of the first immunization,the body weight and food-intake in treating group had no difference with those in normal group (P>0.05).The levels of fasting blood glucose, fasting insulin,HOMA-IR index,glucose AUC of IPGTT,TC,HDL,LDL and TC/HDL were lower than those in control group (P<0.05),while adiponectin level was higher than that in control group (P<0.05) The levels of fasting TG,FFA,LDL/HDL had no difference with that in control group (P>0.05).Frozen sections of aortas showed that the percentage of plaque area to the whole lumens area in treating group was much lower than that in control group(P<0.05)
Conclusion
In conclusion,we successfully constructed3types of vaccines against FABP4,which could all induce specific humoral immunity and produce high titer of specific antibody in mice.FABP4vaccination effectively improved the levels of blood glucose,insulin,blood lipid of mice with high-fat diet,and also reduced fat disposition in liver and foam cell aggregation in aortas. Our study further proved the feasibility of treatment targeting to FABP4in high-fat induced insulin resistance and atherosclerosis,and offers theoretical and experimental basis for the brand-new immunologic intervention.
引文
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