【作者】 张韶君；

【导师】 史轶蘩； 邓洁英； 金自孟； 龚凤英；

【作者基本信息】 中国协和医科大学 ， 内分泌代谢学， 2007， 博士

【摘要】 目的:肥胖症已成为危害全球人类健康的重要疾病,与2型糖尿病、高血压、心脑血管疾病、脂代谢异常、胰岛素抵抗等多种疾病的发生、发展密切相关,并可引起其他一些相关疾病如非酒精性肝病、哮喘以及关节炎等的发生。肥胖症形成的中心环节是脂肪细胞中脂质的过度积聚,多种因素参与了此过程,但是具体形成机制还不完全清楚,而关于该方面的研究,对肥胖症的防治有重要意义。锌α₂糖蛋白（zinc-α₂-glycoprotein,ZAG）是最近新发现的一个脂肪细胞因子,可以促进肿瘤恶液质患者的脂肪分解,增加脂肪利用。但是ZAG与肥胖症患者的脂肪堆积是否有关系,目前尚未有相关报道。本研究拟用基因表达技术,观察ZAG基因过表达对肥胖小鼠体重、内脏脂肪含量、糖脂代谢等的影响以及在脂肪代谢中起关键作用的脂肪酸合成酶（Fatty acid synthase,FAS）、激素敏感性脂肪酶（Hormone sensitive lipase,HSL）mRNA表达的变化,同时观察正常人和肥胖患者减肥前后血清中ZAG蛋白水平的变化。通过以上这些观察探讨ZAG与肥胖症的关系。方法:1小鼠ZAG蛋白表达质粒pcDNA3.1（-）-mZAG的构建:1.1 mZAG cDNA插入片段的扩增:从小鼠肝脏中提取总RNA,应用RT-PCR技术,获得小鼠ZAG cDNA全序列的大量扩增产物,并利用引物在其两端引入酶切位点。1.2载体片段的获得:将本室保存的人ZAG蛋白表达质粒pcDNA3.1（-）-hZAG大量拷贝,经EcoRⅤ、HindⅢ双酶切后,获得大量载体片段。1.3应用T₄DNA连接酶,将经EcoRⅤ、HindⅢ双酶切的小鼠ZAG cDNA全序列扩增产物与载体片段进行连接,获得小鼠ZAG表达质粒pcDNA3.1（-）-mZAG。2 pcDNA3.1（-）-mZAG表达质粒的体外鉴定:应用阳离子脂质体转染方法将pcDNA3.1（-）-mZAG质粒转染小鼠3T3-L1前脂肪细胞,40h后收集培养液,应用western-blotting方法观察ZAG蛋白的表达情况。3采用高脂饲料诱导的肥胖小鼠模型,观察ZAG过表达对小鼠体重、内脏脂肪重量及糖脂代谢的影响:选择6周龄的雄性昆明小鼠45只,分为5组:普通饲料组、高脂饲料组、ZAG过表达组、空白质粒组、曲美组,每组9只。除普通饲料组外,其余四组均用高脂饲料喂养。5周后肥胖小鼠模型建立,然后进行2周干预试验。ZAG过表达组用pcDNA3.1（-）-mZAG质粒25μg与lipofectamine2000脂质体40ul混合后给予小鼠尾静脉注射,隔日1次,共注射2周,空白质粒组以pcDNA3.1（+）质粒进行注射,曲美组每天以曲美10mg/kg体重/d灌胃。干预两周后处死小鼠,观察小鼠摄食量、体重、附睾周围脂肪重量、血糖、血脂等指标。4小鼠附睾周围脂肪组织FAS、HSL mRNA的表达:从动物实验中获得的小鼠附睾周围脂肪组织中提取总RNA,应用RT-PCR技术,检测FAS、HSLmRNA的表达。5正常人和肥胖患者减肥前后血清中ZAG蛋白表达水平的比较:正常人19人（男11人,女8人）,平均BMI为21.6。肥胖患者31例（男14人,女17人）,平均BMI为32.6,13例肥胖患者减肥后,BMI平均为29.6,减重5-10%,平均减重7.6kg,较减肥前显著降低（P＜0.05）。应用western-blotting技术,利用抗人ZAG的单克隆抗体,对血清中的ZAG的量进行比较,并以同组标本的血清β-actin的量作内参照,观察ZAG在不同BMl人群中的表达情况。结果:1成功克隆小鼠ZAG cDNA全序列,并将其与pcDNA3.1（-）载体片段连接,构建成pcDNA3.1（-）-mZAG表达质粒。2在体外培养的小鼠3T3-L1前脂肪细胞系中,证实ZAG表达质粒pcDNA3.1（-）-mZAG可以在脂肪细胞中良好表达。3高脂饲料组、空白质粒组、曲美组小鼠的血清ZAG水平分别为0.51±0.10、0.54±0.08、0.55±0.05,没有显著差别（P均＞0.05）,但与普通饲料组（0.75±0.07）相比,均明显降低（P均＜0.01）。ZAG过表达组小鼠的血清ZAG水平为1.01±0.16,显著高于普通饲料组及高脂饲料组（P均＜0.001）,提示pcDNA3.1（-）-mZAG表达质粒确实能在小鼠体内表达ZAG蛋白。4在2周的干预试验中,高脂饲料组、ZAG过表达组每只小鼠的摄食量平均分别为115.4±13.5g、115.8±7.5g,两组间无差异（P=0.82）,均明显高于普通饲料组（90.1±9.8g）（P均＜0.001）。高脂饲料组每只小鼠体重增加3.7±0.9g,明显高于普通饲料组（2.7±0.7g）（P＜0.01）。ZAG过表达组小鼠的总体重增加3.0±0.5g,明显低于高脂饲料组（P＜0.001）,与普通饲料组相比无差别（P=0.23）。高脂饲料组小鼠的内脏脂肪占体重比例为2.22±0.48%,显著高于普通饲料组（P＜0.05）。ZAG过表达组小鼠的内脏脂肪占体重比例平均1.16±0.10%,明显低于高脂饲料组小鼠（P＜0.001）及普通饲料组（P＜0.05）。高脂饲料组小鼠的空腹血糖为10.87±1.77 mmol/L,是普通饲料组的1.24倍（P＜0.05）。ZAG过表达组小鼠的空腹血糖水平平均9.13±1.00 mmol/L,为高脂饲料组小鼠的84%（P＜0.05）,与普通饲料组无差异（P=0.55）。高脂饲料组小鼠的低密度脂蛋白为1.10±0.24 mmol/L,明显高于普通饲料组（0.59±0.44 mmol/L）,为普通饲料组的1.86倍（P＜0.001）。ZAG过表达组小鼠的血清低密度脂蛋白水平平均0.78±0.17 mmol/L,为高脂饲料组的71%（P＜0.05）,与普通饲料组相比,无明显差异（P=0.49）。5高脂饲料组小鼠内脏脂肪组织FAS mRNA为4.10±0.64,明显高于普通饲料组（2.67±0.46）,为后者的1.54倍（P＜0.001）。ZAG过表达组小鼠脂肪组织FAS mRNA（1.54±0.39）表达显著降低,为普通饲料组的58%,为高脂饲料组的38%（P均＜0.001）。高脂饲料组小鼠内脏脂肪组织HSL mRNA表达为1.18±0.19,明显低于普通饲料组（1.98±0.25）,为后者的60%（P＜0.01）。ZAG过表达组小鼠脂肪组织HSL mRNA为4.39±0.95,较普通饲料组和高脂饲料组均明显增加,分别为普通饲料组和高脂饲料组的2.22倍和3.72倍（P均＜0.001）。6正常人血清ZAG蛋白水平平均为0.74±0.10,肥胖患者血清ZAG蛋白水平显著低于正常人（P＜0.05）。减肥后,ZAG蛋白水平较减肥前有所增加（P＜0.05）,但仍明显低于正常人水平（P＜0.05）。正常人、肥胖患者及肥胖患者减肥后,血清ZAG蛋白水平与BMI均不具有相关关系（r分别为0.16、0.34、0.25,P均＞0.05）。结论:1成功构建了pcDNA3.1（-）-mZAG表达质粒,在体内、体外均能良好表达鼠源ZAG蛋白,是研究ZAG作用的一种有效工具。2 ZAG可使高脂诱导的肥胖小鼠,体重增加减少,内脏脂肪含量减少,血清低密度脂蛋白降低,表明ZAG具有减轻体重,减少脂肪积聚的作用,有一定的减肥效果。3 ZAG可能通过作用于小鼠脂肪组织FAS和HSL两个靶点,抑制脂肪合成,促进脂肪分解,从而减轻体重。4 ZAG在肥胖患者中表达减少,减肥后表达有所增加,可能与肥胖症有一定关系。更多还原

【Abstract】 Object:Obesity has emerged as a worldwide health issue, and has close relationship with the occurrence of many disease, including type 2 diabetes, hypertension, coronary heart disease, stroke, dysregulation of lipid metabolism and insulin resistance, etc. It also causes non-alcoholic liver disease, asthma and arthritis. The much mor fat accumulation in adipocytes is the core factor in development of obesity. ZAG （ zinc-α₂-glycoprotein） is a new adipokine which has been shown to induce lypolysis in tumor patients with cachexia and increase the ultilization of fat. But as far as we know, there isn’t any reports concerning the relationship between ZAG and fat accumulation in obesity patients. In this study, we constructed pcDNA3.1（-）-mZAG vector containing murine ZAG full coding sequence. We observed the influence of ZAG overexpression on the weight, epididymal fat, glucose and lipid metabolism in obesity mices . We also observed the influence of ZAG overexpression on the expression of FAS mRNA and HSL mRNA in the adipose tissues. Furthermore we compared the serum ZAG level in normal people, obesity patients and obesity patients after weight loss.Methods:1 The construction of pcDNA3.1（-）-mZAG plasmid containing murine ZAG full coding sequence: Total RNA from BALB/c mice were extracted from murine liver, transcribed reversly into cDNA, then amplified and gained high copies of murine ZAG cDNA full sequences. The pcDNA3.1（-）-hZAG plasmid containing human ZAG full coding sequence were performed to high copies. EcoRⅤ、HindⅢwere used to cut the plasmid to have the vector fragments. Then we ligated the murine ZAG cDNA sequences and the vector fragments by T4 lignase and constructed the pcDNA3.1（-）-mZAG plasmid containing murine ZAG coding sequence successfully.2 The identification of pcDNA3.1（-）-mZAG plasmid containing murine ZAG coding sequence in vitro: The plasmid was transfected into murine 3T3-L1 cells by liposome transfection method and gathered the solution after 40 hours , then observed ZAG protein expression of culture solution by western-blotting method.3 The influcence of ZAG overexpression on the weight, epididymal fat, glucose and fat metabolism of obesity mices: The KunMing mices were distinguished to 5 groups: nomal food group, high-fat food group, ZAG overexpression group, pcDNA3.1（+） plasmid control group and QUMEI group. The mices were bred with high-fat food except nomal food group for 4 weeks to make them obesity. Then ZAG overexpression group were administered with the blending of 25ug pcDNA3.1（-）-mZAG plasmid and 40μl liposome by tail venus injection qod for 2 weeks. The influcence of pcDNA3.1（-）-mZAG plasmid on the weight, epididymal fat, glucose and fat metabolism was observed later.4 The expression of FAS、HSL mRNA in murine epididymal adipose tissues: Total RNA was reverse transcribed and then amplified by PCR. The PCR products were compared, murineβ-actin was used as a house-keeping gene.5 Compared the serum ZAG level among the normal people, obesity patients and obesity patients after weight loss: we used human monoclonal ZAG antibody to compare the serum ZAG level among the different BMI people, after weight loss, murineβ-actin was used as a house-keeping protein. The BMI of nomal people were 21.6, 32.6 of obesity patients, and 29.6 of obesity patients after weight loss.Results:1 Murine ZAG coding sequence was amplified successfully and ligated with vector fragments by T4 ligase. The construction of pcDNA3.1（-）-mZAG plasmid containing murine ZAG coding sequence was succeed.2 pcDNA3.1（-）-mZAG plasmid was transfected into murine 3T3-L1 cells by liposome transfection method. It was confirmed that pcDNA3.1（-）-mZAG plasmid could well expressed in murine 3T3-L1 adipocytes..3 The serum ZAG levels of high-fat food group （0.51±0.10）, pcDNA3.1（+） plasmid control group （0.54±0.08）and QUMEI group （0.55±0.05） didn’t have significant difference. And the serum ZAG levels of three groups all have significant decreased comparing to normal food group （P<0.01） . The serum ZAG level of ZAG-overexpression group（ 1.01±0.16） has significant increased comparing to normal food group and high-fat food group （P<0.001） . This suggested that pcDNA3.1（-）-mZAG plasmid could presented ZAG protein in murine.4 During the two-weeks intervention test, the ingested volume of high-fat food group and ZAG-overexpression group are respectively 115.4±13.5g and 115.8±7.5g, all have significant increased comparied with normal food group. It didn’t have significant difference between the high-fat food group and ZAG-overexpression group.The weight gain of high-fat food group is 3.7±0.9g , it has significant increased comparing to normal food group（2.7±0.7g）. The weight gain of ZAG-overexpression group is 3.0±0.5g, is significant decreased comparied with high-fat food group（P<0.001） and didn’t has significant difference with normal food group（P=0.23）.The ratio of visceral fat and body weight of high-fat food group is 2.22±0.48% , it has significant increased comparing to normal food group（P<0.05）. The ratio of visceral fat and body weight of ZAG-overexpression group is 1.16±0.10%, is significant decreased comparied with high-fat food group and normal food group （P<0.05）.The fasting blood glucose level of high-fat food group is 10.87±1.77 mmol/L, it has significant increased comparing to normal food group（P<0.05）. The fasting blood glucose level of ZAG-overexpression group is 9.13±1.00 mmol/L, is significant decreased comparied with high-fat food group（P<0.05） and didn’t has significant difference with normal food group（P=0.55）. The serum low density lipoprotein level of high-fat food group is 1.10±0.24mmpl/L , it has significant increased comparing to normal food group（0.59±0.44mmpl/L）. The serum low density lipoprotein level of ZAG-overexpression group is 0.78±0.17mmpl/L, is significant decreased comparied with high-fat food group（P<0.05） and didn’t has significant difference with normal food group（P=0.49）.5 The FAS mRNA expression in murine visceral adipose tissue of high-fat food group is 4.10±0.64, it has significant increased comparing to normal food group（2.67±0.46）（P<0.001）. The FAS mRNA expression in murine visceral adipose tissue of ZAG-overexpression group is 1.54±0.39, is significant decreased comparied with high-fat food group（P<0.001） and normal food group（P<0.001）.6 The HSL mRNA expression in murine visceral adipose tissue of high-fat food group is 1.18±0.19, it has significant decreased comparing to normal food group（P<0.01）. The HSL mRNA expression in murine visceral adipose tissue of ZAG-overexpression group is 4.39±0.95, is significant increased comparied with high-fat food group（P<0.001） and normal food group（P<0.001）.7 The serum ZAG level of normal people is 0.74±0.10, the serum ZAG level of obesity patients is significant decreased comparing to normal people（P<0.05）. After the obesity patients lost weights, the serum ZAG level is raised but still has significant difference with normal people（P<0.05）. The serum ZAG level and BMI in normal people, obesity people and the obesity patients who lost weights didn’t have correlativity（r=0.16, 0.34, 0.25, P>0.05）.Conclusion:1 mZAG expression plasmid pcDNA3.1（-）-mZAG was constructed successfully and could express murine ZAG protein in vitro murine preadipocytes 3T3-L3 cells and in vivo mices. This is a convenient tool for ZAG study.2 ZAG could decrease the weight increasing, visceral fat and serum LDL levels, but didn’t influence the fasting blood glucose, CHO, TG and HDL in obesity mices. This indicates that ZAG maybe reduce fat accumulation.3 ZAG can inhibit fat synthesis, promote lipolysis through murine adipose FAS and HSL.4 The expression of ZAG maybe has relationship with othe obesity.更多还原