本文關鍵詞： 妊娠 TRB3 AKT CHOP 高脂高糖　出處：《鄭州大學》2014年碩士論文　論文類型：學位論文

【摘要】：背景與目的 妊娠期糖尿病(gestational diabetes mellitus,GDM)在全球范圍內比例逐年上升，帶來嚴重的經濟及公共衛(wèi)生健康問題。妊娠期糖尿病女性罹患各種高危妊娠并發(fā)癥風險增加，如妊娠期高血壓、難產及剖宮產比例升高、巨大兒等，尤其增加了日后發(fā)展為糖尿病及心血管疾病的風險。多種因子介導的胰島素信號傳導通路缺陷、內質網應激誘導的β細胞凋亡不同程度的參與了妊娠期糖尿病的發(fā)生發(fā)展。除此之外，，氧化應激、妊娠期肥胖、高齡等危險因素也在GDM的發(fā)病機制中發(fā)揮一定作用。最新研究發(fā)現：Tribbles同源蛋白3（TRB3）通過TRB3/AKT信號通路、CHOP/TRB3信號通路廣泛參與糖尿病的發(fā)病機制。但在妊娠期間TRB3與GDM發(fā)病機制的關聯研究較少，且妊娠期間飲食干預能否有效減輕胰島素抵抗、降低β細胞凋亡程度的分子機制需要我們進一步證實研究。 本實驗通過建立高脂高糖飲食妊娠大鼠模型，并觀察大鼠模型肝臟組織TRB3和AKT基因的表達水平，觀察胰腺組織TRB3及CHOP蛋白表達水平，且對比飲食干預前后的變化，初步探討TRB3參與妊娠期糖耐量異常發(fā)病機制的研究。 材料與方法 選取105只雌性和30只雄性健康42日齡SD大鼠（體重約200g）。采用普通飼料適應性喂養(yǎng)1周后雌鼠隨機分為5組:普通飲食非妊娠組（CV組15只）、普通飲食妊娠組（CG25只）、高脂高糖非妊娠組（FV15只）、高脂高糖妊娠組（FG25只）及高脂高糖妊娠后飲食干預組（DI組25只）（干預組，妊娠前給予高脂高糖飼料，妊娠后給予普通飲食干預）。雄性大鼠均用普通飼料飼養(yǎng)。分別喂養(yǎng)6周后將雄性大鼠與雌性大鼠合籠備孕，擬妊娠雌鼠與雄性大鼠以2∶1比例合籠，次晨鏡檢雌鼠陰道，以鏡下可見精子為標準，標記為1d，并隔離妊娠大鼠。合籠1周后未孕大鼠棄之不用。 妊娠第20天進行口服葡萄糖耐量試驗（OGTT）檢查，分別在0min,60min,120min尾靜脈取血檢測血糖水平。 妊娠第22天時心內取血離心，檢測TC、TG、FFA等指標，檢測空腹血糖及胰島素，得出HOMA-IR。應用逆轉錄聚合酶鏈反應(RT-PCR)方法測定各組大鼠肝臟組織中TRB3mRNA和AKT mRNA的表達。應用Western Blot方法檢測胰腺組織中TRB3和CHOP蛋白的表達水平。 妊娠第22天時CG、FG及DI3組各10只大鼠行剖宮產術取胎鼠，稱取胎鼠個體及整體重量，稱取胎盤重量，取血液檢測胎鼠的FBG、胰島素、血脂水平。 采用SPSS19.0對數據進行統(tǒng)計分析。 結果 (1)妊娠、高脂高糖飲食均可致大鼠HOMA-IR升高，F妊娠=2318.491，F飲食=2888.237，F交互=993.094，P均＜0.001； (2)相較于其他4組，妊娠、高脂高糖飲食均可致肝組織中TRB3mRNA的表達明顯升高，（F妊娠=256.887，F飲食=1749.406，F交互=2.579，P均＜0.001）；AKT mRNA的表達顯著降低（F妊娠=221.091，F飲食=1416.984，F交互=28.918，P均＜0.001）。FG組、DI組、CG組三組TRB3mRNA、AKT mRNA的表達水平差異有統(tǒng)計學意義（F=445.986和390.334，P均＜0.001）； (3)相較于其他4組，妊娠、高脂高糖飲食均可致大鼠胰腺組織中TRB3蛋白的表達水平明顯升高，（F妊娠=475.219，F飲食=2580.320，F交互=206.142，P均＜0.001）；CHOP蛋白的表達也顯著升高（F妊娠=1275.615，F飲食=4308.857，F交互=245.461，P均＜0.001）。FG組、DI組、CG組三組TRB3及CHOP蛋白的表達水平差異有統(tǒng)計學意義（F=764.890和1462.573，P均＜0.001）； (4)FG組體質量增加值、OGTT各時段數據檢測值、血糖、胰島素、TG、FFA、TC等均高于其他4組，差異具有統(tǒng)計學意義； (5)剖宮產取出的單個胎鼠重量在FG、CG、DI三組間無明顯差異，但是總胎體重三組之間有差異，具有統(tǒng)計學意義（P0.05）。三組胎鼠的FBG、Insulin和胎盤重量無明顯差異。 結論 1.可通過長期高脂高糖飲食誘導妊娠期糖耐量異常大鼠模型，高脂高糖及妊娠因素相互疊加作用于妊娠期糖耐量異常甚或妊娠期糖尿病的發(fā)病機制； 2. TRB3可通過TRB3/AKT、CHOP/TRB3等信號通路廣泛參與高脂高糖喂養(yǎng)大鼠糖耐量異常的發(fā)病機制，作用機制有一定相互交叉作用； 3.對妊娠期糖尿病進行飲食干預，可有效改善妊娠期糖尿病病情。
[Abstract]:Background and purpose
Gestational diabetes mellitus (gestational diabetes, mellitus, GDM) increased year by year in the global scope of the proportion of serious economic and public health problems. Pregnant women suffering from high-risk pregnancy increases the risk of complications, such as gestational hypertension, dystocia and cesarean section increased the proportion of macrosomia increased, especially after the development of Japan the risk of diabetes and cardiovascular disease. Insulin signal transduction defects mediated by multiple factors, endoplasmic reticulum stress induced beta cell apoptosis in different degree in the occurrence and development of gestational diabetes. In addition, oxidative stress, pregnancy, obesity, age and other risk factors also play a role in the pathogenesis of GDM. The latest research found: Tribbles homolog 3 (TRB3) through the TRB3/AKT signaling pathway, CHOP/TRB3 signaling pathway is widely involved in the pathogenesis of diabetes in pregnancy but. There is less study on the relationship between TRB3 and GDM pathogenesis, and whether the dietary intervention during pregnancy can effectively reduce insulin resistance and reduce the molecular mechanism of the degree of apoptosis of beta cells, we need further confirmation.
The high fat diet rat model of pregnancy, and to observe the expression of TRB3 and AKT gene in liver tissue of rats, to observe the expression of TRB3 and CHOP protein levels in pancreatic tissue, and compared the changes before and after the dietary intervention, a preliminary study of TRB3 participated in the study of gestational impaired glucose tolerance and pathogenesis.
Materials and methods
A total of 105 female and 30 male healthy 42 day old SD rats (weighing about 200g). The normal diet after 1 weeks of feeding female rats were randomly divided into 5 groups: normal diet and non pregnancy group (CV Group 15), normal diet pregnancy group (CG25), high fat and high glucose non pregnancy group (FV15), high fat and high glucose pregnancy group (FG25) and high fat and high glucose diet after pregnancy intervention group (25 rats in group DI (intervention group), given the high fat and high glucose diet before pregnancy, pregnancy after given normal diet intervention). Male rats were fed with normal diet. After 6 weeks of feeding respectively. Male rats and female rats mated to prepare pregnant, intends to 2 to 1 the proportion of mated female rats and male rats during pregnancy, the next morning microscopic examination of female rats with vaginal microscopically sperm as the standard, labeled 1D, and isolated pregnant rat cage. After 1 weeks of non pregnant rats abandoned.
The oral glucose tolerance test (OGTT) was performed on the twentieth day of pregnancy, and blood glucose levels were measured in 0min, 60min, 120min caudal veins respectively.
The twenty-second day of pregnancy when the heart blood centrifugation, detection of TC, TG, FFA and other indicators, detection of fasting blood glucose and insulin, HOMA-IR. obtained by reverse transcriptase polymerase chain reaction (RT-PCR) method for determination of the expression of TRB3mRNA and AKT mRNA in liver of rats in each group. The expression level of TRB3 and CHOP protein in pancreatic tissue was detected by Western Blot method.
On the twenty-second day of pregnancy, 10 rats in CG, FG and DI3 groups were taken cesarean section to get the fetuses, and the weight and weight of placenta were weighed. The placental weight was weighed, and the levels of FBG, insulin and blood lipid were detected by blood.
SPSS19.0 is used to analyze the data.
Result
(1) pregnancy, high fat and high sugar diet can increase HOMA-IR in rats, F pregnancy =2318.491, F diet =2888.237, F interaction =993.094, P < 0.001;
(2) compared to the other 4 groups of pregnancy, the expression of high fat diet can be induced by TRB3mRNA in liver tissue was significantly elevated (F =256.887 F =1749.406 of pregnancy, diet, F interaction =2.579, P < 0.001); the expression of AKT mRNA decreased significantly (F =221.091 F =1416.984 of pregnancy, diet, F interaction =28.918 P < 0.001) in.FG group, DI group, CG group and three TRB3mRNA group, there was significant difference in the expression level of AKT mRNA (F=445.986 and 390.334, P < 0.001);
(3) compared to the other 4 groups of pregnancy, the expression level of high fat diet can be induced by TRB3 protein in rat pancreatic tissue was significantly elevated (F =475.219 F =2580.320 of pregnancy, diet, F interaction =206.142, P < 0.001); the expression of CHOP protein was also significantly increased (F =1275.615 F =4308.857 pregnancy diet. F, interactive =245.461, P < 0.001) in.FG group, DI group, there was significant difference in the expression level of CG group three groups of TRB3 and CHOP proteins (F=764.890 and 1462.573, P < 0.001);
(4) the value of body mass increase in FG group, data detection value at each time of OGTT, blood glucose, insulin, TG, FFA, TC were all higher than those of the other 4 groups, and the difference was statistically significant.
(5) there was no significant difference in the weight of single fetuses taken from cesarean section among FG, CG and DI groups, but the total fetal weight was different between the three groups (P0.05). There was no significant difference between the three groups of FBG, Insulin and placental weight in the three groups of fetuses.
conclusion
1., we can induce a gestational impaired glucose tolerance rat model through long-term high-fat and high carbohydrate diet. The effects of high fat, high sugar and pregnancy factors on the pathogenesis of gestational impaired glucose tolerance or gestational diabetes mellitus were also observed.
2. TRB3 can be widely involved in the pathogenesis of impaired glucose tolerance in rats fed by high fat and high glucose through TRB3/AKT, CHOP/TRB3 and other signal pathways, and the mechanisms of action are interacted.
3. the diet intervention of gestational diabetes can effectively improve the condition of gestational diabetes.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714.256

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