父代高血糖改變子代下丘腦攝食調(diào)節(jié)神經(jīng)通路加速肥胖形成的分子機制研究
發(fā)布時間：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謝紊亂,增加子代患糖尿病和其他代謝性疾病的風(fēng)險。而父代高血糖對子代的影響目前研究較少,且集中于流行病學(xué)調(diào)查和臨床特征的觀察,缺乏深度的機制研究。本研究旨在探討父代高血糖對子代代謝特征的影響以及可能的中樞調(diào)控機制。方法:在重慶醫(yī)科大學(xué)實驗動物中心購買健康、年齡匹配(約6周齡)的雌性和雄性SD大鼠各15只,飼養(yǎng)于SPF房間。喂養(yǎng)2周后,按每只大鼠35 mg/kg的劑量,雄性SD大鼠腹腔注射鏈脲佐菌素(STZ)或者等體積檸檬酸鹽緩沖液(CB),建立父代高血糖模型(血糖高于16.7mmol/L)和對照模型,兩組雄性大鼠與正常雌性SD大鼠交配。兩組子代(STZ-O和CB-O)離乳后以標(biāo)準(zhǔn)飼料喂養(yǎng),連續(xù)每周監(jiān)測體重;在子代6周齡時施以禁食-復(fù)進(jìn)食試驗;在24-26周齡期間監(jiān)測連續(xù)3周的攝食量;成年期進(jìn)行冷暴露(4℃)6h處理。HE染色觀察脂肪組織脂質(zhì)沉積情況;酶聯(lián)免疫吸附法(ELISA)測定血清中瘦素(Leptin)和a-黑素細(xì)胞刺激素(a-MSH)水平;實時熒光定量PCR(real-time q-PCR)檢測子代下丘腦中瘦素受體(Lepr)、神經(jīng)肽Y(NPY)、阿片-促黑素細(xì)胞皮質(zhì)素原(POMC)、細(xì)胞因子信號傳導(dǎo)抑制蛋白-3(SOCS3)、前蛋白轉(zhuǎn)化酶1(PC1)和2(PC2)、羧肽酶E(CPE)以及冷刺激下棕色脂肪組織中解偶聯(lián)蛋白(UCP1)的m RNA表達(dá)水平;蛋白免疫印跡法(western blotting)檢測下丘腦中瘦素信號通路相關(guān)基因磷酸化的信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子-3(p-STAT3)和SOCS3的蛋白表達(dá)水平;免疫組化石蠟切片法(IHC-P)檢測兩組子代棕色脂肪組織中UCP1的相對表達(dá)。結(jié)果:經(jīng)多次檢測STZ組大鼠的血糖水平顯著高于CB組,并且持續(xù)高于16.7mmol/L,表明父代高血糖動物模型構(gòu)建成功。對兩組子代進(jìn)行觀察發(fā)現(xiàn):與CB-O相比,STZ-O的體重和攝食量均明顯增加;同時,STZ-O的脂肪組織重量增加,血清中TG水平和leptin水平明顯升高,而a-MSH水平則顯著降低;下丘腦中瘦素介導(dǎo)的JAK/STAT3通路中,p-STAT3的表達(dá)下降,Lepr的表達(dá)有下降的趨勢,NPY的表達(dá)上調(diào),POMC的表達(dá)則下調(diào),PC1、PC2和CPE也相應(yīng)下調(diào);冷刺激下,STZ-O的棕色脂肪組織中UCP1的m RNA和蛋白表達(dá)均顯著下降,產(chǎn)熱活性受損。結(jié)論:父代高血糖可引起子代食欲旺盛、產(chǎn)熱能力受損,導(dǎo)致體重增加,加速肥胖的形成;其機制可能與瘦素介導(dǎo)的JAK/STAT3信號通路受損引起下丘腦攝食調(diào)節(jié)肽的變化有關(guān)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 Shikun HE;李曉華;;表觀遺傳學(xué)簡介[J];中華實驗眼科雜志;2011年07期
，本文編號：2348045
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