钙结合蛋白S100A16基因敲除小鼠的构建以及S100A16对高脂饮食诱导下肥胖小鼠糖脂代谢的影响及其机制研究

发布时间：2017-12-31 09:20

本文关键词：钙结合蛋白S100A16基因敲除小鼠的构建以及S100A16对高脂饮食诱导下肥胖小鼠糖脂代谢的影响及其机制研究　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:构建钙结合蛋白(Calcium-binding protein)S100A16基因全身敲除小鼠模型,高脂喂养构建饮食诱导肥胖小鼠模型(Diet induced obesity model,DIO),同时利用HepG2细胞系构建胰岛素抵抗模型,进一步探讨S100A16的生物学功能及其在肥胖和胰岛素抵抗中的作用及其机制。方法:第一步:利用基因表达调控系统(即Cre/loxP系统)构建S100A16全身敲除小鼠。采用聚合酶链式反应(PCR)、实时荧光定量PCR(RT-PCR)、蛋白质印迹(Western blot,WB)方法验证其转录及翻译水平的表达。第二步:选用5周龄的S100A16全身敲除小鼠(杂合子小鼠,S100A16Ko+/-)与正常C57BL/6(B6,即野生型Wild type,WT)雄性小鼠为研究对象,进行高脂喂养构建饮食诱导肥胖小鼠模型。将2种不同基因型的小鼠随机分成正常饮食组(No high fat,NFD)和高脂饮食组(High fat,HFD),共4组,①正常对照组(NFDWT),n=10、②B6高脂组(HFDWT),n=10、③S100A16 Ko+/-普脂组(NFD+/-),n=10、④S100A16Ko+/-高脂组(HFD+/-),n=10。不同饮食喂养13周,每周称量并记录小鼠体重、随机血糖、于5周龄和17周龄时进行腹腔糖耐量试验(Introperitoneal glucose tolerance test,IPGTT)和于6周龄和18周龄时进行胰岛素耐量试验(Insulin tolerance test,ITT)。18周时取材,取血测定血清甘油三脂(Triglyceride,TG)、总胆固醇(Cholesterol,TC)等糖脂代谢相关生化指标;记录附睾、肾周等内脏脂肪重量,并利用病理染色方法观察病理学改变,同时应用RT-PCR、WB等实验手段检测S100A16及相关基因在小鼠脂肪及肝脏组织中的表达,初步探索其相关作用机制。构建动物模型的同时细胞模型。采用10-7μmol/L胰岛素诱导人HepG2肝癌细胞系产生胰岛素抵抗,分析胰岛素抵抗与S100A16的关系。结果:(1)S100A16基因全身敲除杂合子(S100A16 Ko+-)小鼠可成功饲养繁殖,子代雌雄性别出生率无差异;目前仍未生出纯合子小鼠(KO-/-)。S100A16Ko+/-小鼠脂肪、骨骼肌、肝脏、心肌、肺组织中S100A16转录水平与蛋白表达量显著下降。(2)DIO模型中,普脂S100A16 Ko+/-组与正常对照组相比:体重、脂肪重量、TG、TC、LDL无明显差异。给予高脂饮食后,高脂组与普脂组相比:体重、脂肪重量、TG、TC、LDL明显增加,S100A16、Wnt和β-catenin上调。高脂S100A16 Ko+/-组与高脂WT组相比:体重、脂肪重量、TG明显下降,S100A16、Wnt和 β-catenin下调,HDL上调。(3)胰岛素诱导人HepG2细胞系发生胰岛素抵抗,结果发现IRS-2下调,S100A16表达上调。结论:(1)成功建立了 S100A16全身敲除小鼠模型,为进一步研究S100A16的生物学功能及分子机制提供了动物模型。(2)S100A16与体重的增加和脂肪的聚集密切相关,S100A16可能通过影响Wnt/β-catenin通路从而对肥胖进行调节。(3)S100A16与胰岛素抵抗相关。S100A16在机体广泛表达,具有多种生物学功能。未生出纯合子小鼠,可能提示我们S100A16与生殖或胚胎致畸相关。后期,我们可以在细胞水平利用Wnt/β-catenin通路的特异性阻断剂ICG-001阻断这一通路,观察S100A16的变化。同时可以利用高通量技术(基因芯片、质谱分析等)筛选相关作用底物。S100A16与肥胖和胰岛素抵抗相关,但其具体的分子机制还需进一步深入研究。
[Abstract]:Objective: to construct a calcium binding protein (Calcium-binding protein) S100A16 gene knockout mouse model of systemic, construction of high fat diet induced obese mouse model (Diet induced obesity model, food DIO), at the same time using HepG2 cell line construction of the model of insulin resistance, to further explore the biological function of S100A16 in obesity and insulin resistance in rats and its mechanism. Methods: the first step: using gene expression system (Cre/loxP system) to construct S100A16 systemic knockout mice. Using polymerase chain reaction (PCR), real time fluorescence quantitative PCR (RT-PCR), Western blotting (Western blot WB) method to verify the expression of the transcription and translation level. The second step: a total of 5 weeks of age the body of S100A16 knockout mice (heterozygous mice, S100A16Ko+/-) and normal C57BL/6 (B6, the wild type Wild type, WT) male mice as the research object, construct the high-fat feeding diet Induced obese mice model. 2 different genotypes of mice were randomly divided into normal diet group (No high, fat, NFD) and high-fat diet group (High, fat, HFD), a total of 4 groups: normal control group (NFDWT), n=10, B6 in the high fat group (HFDWT), n=10, S100A16 Ko+/- P (NFD+/-), n=10 fat group, the S100A16Ko+/- high fat group (HFD+/-), n=10. of different diet for 13 weeks, body weight, blood glucose and record the mice were weighed every week, at 5 and 17 weeks of intraperitoneal glucose tolerance test (Introperitoneal glucose tolerance test, IPGTT) and at 6 weeks of age and 18 weeks of age were insulin tolerance test (Insulin tolerance test, ITT) at.18 weeks, blood serum glycerin three greases (Triglyceride, TG), total cholesterol (Cholesterol, TC) and other lipid metabolism related biochemical indexes; records of epididymis, perirenal fat weight, visceral, and by pathological staining observe the pathologic features and methods. The application of RT-PCR, the expression of WB experimental methods to detect S100A16 and related genes in the liver and adipose tissue in mice, and explore the related mechanism. At the same time cell model animal model. Using 10-7 mol/L human hepatoma cell line HepG2 induced by insulin induced insulin resistance, analysis of the relationship between insulin resistance and S100A16. Results: (1 General) S100A16 gene knockout heterozygous mice (S100A16 Ko+-) can be successfully breeding, progeny sex birth rate had no difference; not yet born mice homozygous (KO-/-).S100A16Ko+/- mice fat, skeletal muscle, liver, myocardium, the expression of the transcription level of S100A16 and protein in lung tissue was significantly decreased (2) DIO model. In general, lipid S100A16 Ko+/- group compared with the normal control group, body weight, fat weight, TG, TC, LDL have no significant difference. Given the high fat diet, high fat group and P group compared fat: body weight, fat weight, TG, TC, LDL increased significantly, S100A16, Wnt and beta -catenin up-regulated. High fat S100A16 Ko+/- group compared with WT group: high fat weight, fat weight, decreased TG, S100A16, Wnt and beta -catenin down regulated by HDL. (3) insulin induced HepG2 cells insulin resistance, the results showed that the down-regulation of IRS-2, the expression level of S100A16 increased. Conclusion: (1) S100A16 has been successfully established systemic knockout mice model and provides an animal model for further study on the biological function and molecular mechanism of S100A16. (2) increased S100A16 and body weight and fat accumulation is closely related to S100A16 may influence the Wnt/ beta -catenin pathway to regulate obesity. (3).S100A16 S100A16 is widely expressed in the body associated with insulin resistance, with a variety of biological functions. Which would suggest that the S100A16 homozygous mice, and reproductive or teratogeny. Later, we can at the cellular level Using specific Wnt/ beta -catenin pathway blocker ICG-001 blocked this pathway, to observe the change of S100A16. At the same time can use high-throughput technologies (microarray, mass spectrometry) screen substrate of.S100A16 with obesity and insulin resistance, but the molecular mechanism needs further study.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R58;R-332

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