父代高血糖改变子代下丘脑摄食调节神经通路加速肥胖形成的分子机制研究
发布时间：2018-11-21 19:22
【摘要】：目的:研究表明,妊娠期合并糖尿病可引起子代代谢紊乱,增加子代患糖尿病和其他代谢性疾病的风险。而父代高血糖对子代的影响目前研究较少,且集中于流行病学调查和临床特征的观察,缺乏深度的机制研究。本研究旨在探讨父代高血糖对子代代谢特征的影响以及可能的中枢调控机制。方法:在重庆医科大学实验动物中心购买健康、年龄匹配(约6周龄)的雌性和雄性SD大鼠各15只,饲养于SPF房间。喂养2周后,按每只大鼠35 mg/kg的剂量,雄性SD大鼠腹腔注射链脲佐菌素(STZ)或者等体积柠檬酸盐缓冲液(CB),建立父代高血糖模型(血糖高于16.7mmol/L)和对照模型,两组雄性大鼠与正常雌性SD大鼠交配。两组子代(STZ-O和CB-O)离乳后以标准饲料喂养,连续每周监测体重;在子代6周龄时施以禁食-复进食试验;在24-26周龄期间监测连续3周的摄食量;成年期进行冷暴露(4℃)6h处理。HE染色观察脂肪组织脂质沉积情况;酶联免疫吸附法(ELISA)测定血清中瘦素(Leptin)和a-黑素细胞刺激素(a-MSH)水平;实时荧光定量PCR(real-time q-PCR)检测子代下丘脑中瘦素受体(Lepr)、神经肽Y(NPY)、阿片-促黑素细胞皮质素原(POMC)、细胞因子信号传导抑制蛋白-3(SOCS3)、前蛋白转化酶1(PC1)和2(PC2)、羧肽酶E(CPE)以及冷刺激下棕色脂肪组织中解偶联蛋白(UCP1)的m RNA表达水平;蛋白免疫印迹法(western blotting)检测下丘脑中瘦素信号通路相关基因磷酸化的信号转导和转录激活因子-3(p-STAT3)和SOCS3的蛋白表达水平;免疫组化石蜡切片法(IHC-P)检测两组子代棕色脂肪组织中UCP1的相对表达。结果:经多次检测STZ组大鼠的血糖水平显著高于CB组,并且持续高于16.7mmol/L,表明父代高血糖动物模型构建成功。对两组子代进行观察发现:与CB-O相比,STZ-O的体重和摄食量均明显增加;同时,STZ-O的脂肪组织重量增加,血清中TG水平和leptin水平明显升高,而a-MSH水平则显著降低;下丘脑中瘦素介导的JAK/STAT3通路中,p-STAT3的表达下降,Lepr的表达有下降的趋势,NPY的表达上调,POMC的表达则下调,PC1、PC2和CPE也相应下调;冷刺激下,STZ-O的棕色脂肪组织中UCP1的m RNA和蛋白表达均显著下降,产热活性受损。结论:父代高血糖可引起子代食欲旺盛、产热能力受损,导致体重增加,加速肥胖的形成;其机制可能与瘦素介导的JAK/STAT3信号通路受损引起下丘脑摄食调节肽的变化有关。
[Abstract]:Objective: studies have shown that gestational diabetes associated with diabetes may lead to metabolic disorders in offspring and increase the risk of diabetes and other metabolic diseases in offspring. However, the effects of hyperglycemia on offspring in the father generation are less studied at present, and focus on the epidemiological investigation and clinical characteristics observation, and lack of in-depth research on the mechanism. The aim of this study was to investigate the effects of paternal hyperglycemia on the metabolic characteristics of offspring and the possible central regulatory mechanisms. Methods: 15 healthy age-matched female and 15 male SD rats were collected from Experimental Animal Center of Chongqing Medical University and fed in SPF room. After feeding for 2 weeks, male SD rats were injected intraperitoneally with streptozotocin (STZ) or citrate buffer (CB),) at a dose of 35 mg/kg per rat. The paternal model of hyperglycemia (blood glucose was higher than 16.7mmol/L) and the control model were established. The two groups of male rats were mated with normal female SD rats. The two groups of offspring (STZ-O and CB-O) were fed with standard diet after weaning, their body weight was continuously monitored weekly, the force-refeeding test was performed at the age of 6 weeks, and the intake was monitored for 3 weeks during 24-26 weeks of age. The lipid deposition in adipose tissue was observed by HE staining, and the levels of leptin (Leptin) and a-melanocyte stimulating hormone (a-MSH) in serum were measured by enzyme linked immunosorbent assay (ELISA). Real-time fluorescence quantitative PCR (real-time q-PCR) was used to detect the expression of leptin receptor (Lepr), neuropeptide Y (NPY), opioid melanotrophil (POMC), signal suppressor protein 3 (SOCS3) in hypothalamus. The expression levels of m RNA of uncoupling protein (UCP1) in brown adipose tissue under cold stimulation, such as PC1 and 2 (PC2), carboxypeptidase E (CPE) and cold-stimulated brown adipose tissue, were detected. Western blot (western blotting) was used to detect the phosphorylation of leptin signaling pathway related genes and the expression of p-STAT3 and SOCS3 in hypothalamus. Immunohistochemical paraffin section method (IHC-P) was used to detect the relative expression of UCP1 in brown adipose tissue of the two groups. Results: the level of blood glucose in STZ group was significantly higher than that in CB group and was higher than 16.7 mmol / L, indicating that the animal model of paternal hyperglycemia was successfully constructed. Compared with CB-O, the body weight and food intake of STZ-O were significantly increased. At the same time, the weight of adipose tissue of STZ-O increased, the level of TG and leptin in serum increased significantly, while the level of a-MSH decreased significantly. In the JAK/STAT3 pathway mediated by leptin, the expression of p-STAT3 decreased, and the expression of Lepr decreased. The expression of NPY was up-regulated, the expression of POMC was down-regulated, and the expression of PC1,PC2 and CPE was down-regulated. Under cold stimulation, m RNA and protein expression of UCP1 in brown adipose tissue of STZ-O decreased significantly, and the heat production activity was impaired. Conclusion: the paternal hyperglycemia may lead to strong appetite, impaired heat production, weight gain and accelerated obesity in the offspring, and its mechanism may be related to the changes of hypothalamic feeding regulatory peptides caused by the damage of leptin mediated JAK/STAT3 signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
本文编号：2348045
[Abstract]:Objective: studies have shown that gestational diabetes associated with diabetes may lead to metabolic disorders in offspring and increase the risk of diabetes and other metabolic diseases in offspring. However, the effects of hyperglycemia on offspring in the father generation are less studied at present, and focus on the epidemiological investigation and clinical characteristics observation, and lack of in-depth research on the mechanism. The aim of this study was to investigate the effects of paternal hyperglycemia on the metabolic characteristics of offspring and the possible central regulatory mechanisms. Methods: 15 healthy age-matched female and 15 male SD rats were collected from Experimental Animal Center of Chongqing Medical University and fed in SPF room. After feeding for 2 weeks, male SD rats were injected intraperitoneally with streptozotocin (STZ) or citrate buffer (CB),) at a dose of 35 mg/kg per rat. The paternal model of hyperglycemia (blood glucose was higher than 16.7mmol/L) and the control model were established. The two groups of male rats were mated with normal female SD rats. The two groups of offspring (STZ-O and CB-O) were fed with standard diet after weaning, their body weight was continuously monitored weekly, the force-refeeding test was performed at the age of 6 weeks, and the intake was monitored for 3 weeks during 24-26 weeks of age. The lipid deposition in adipose tissue was observed by HE staining, and the levels of leptin (Leptin) and a-melanocyte stimulating hormone (a-MSH) in serum were measured by enzyme linked immunosorbent assay (ELISA). Real-time fluorescence quantitative PCR (real-time q-PCR) was used to detect the expression of leptin receptor (Lepr), neuropeptide Y (NPY), opioid melanotrophil (POMC), signal suppressor protein 3 (SOCS3) in hypothalamus. The expression levels of m RNA of uncoupling protein (UCP1) in brown adipose tissue under cold stimulation, such as PC1 and 2 (PC2), carboxypeptidase E (CPE) and cold-stimulated brown adipose tissue, were detected. Western blot (western blotting) was used to detect the phosphorylation of leptin signaling pathway related genes and the expression of p-STAT3 and SOCS3 in hypothalamus. Immunohistochemical paraffin section method (IHC-P) was used to detect the relative expression of UCP1 in brown adipose tissue of the two groups. Results: the level of blood glucose in STZ group was significantly higher than that in CB group and was higher than 16.7 mmol / L, indicating that the animal model of paternal hyperglycemia was successfully constructed. Compared with CB-O, the body weight and food intake of STZ-O were significantly increased. At the same time, the weight of adipose tissue of STZ-O increased, the level of TG and leptin in serum increased significantly, while the level of a-MSH decreased significantly. In the JAK/STAT3 pathway mediated by leptin, the expression of p-STAT3 decreased, and the expression of Lepr decreased. The expression of NPY was up-regulated, the expression of POMC was down-regulated, and the expression of PC1,PC2 and CPE was down-regulated. Under cold stimulation, m RNA and protein expression of UCP1 in brown adipose tissue of STZ-O decreased significantly, and the heat production activity was impaired. Conclusion: the paternal hyperglycemia may lead to strong appetite, impaired heat production, weight gain and accelerated obesity in the offspring, and its mechanism may be related to the changes of hypothalamic feeding regulatory peptides caused by the damage of leptin mediated JAK/STAT3 signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
【参考文献】
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1 Shikun HE;李晓华;;表观遗传学简介[J];中华实验眼科杂志;2011年07期
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