核因子κB、TNF-a、IL-6在PCOS大鼠模型中的表达及二甲双胍的干预研究

发布时间：2018-04-01 06:46

本文选题：NF-κB　切入点：二甲双胍　出处：《福建医科大学》2014年硕士论文

【摘要】：[目的]1、建立可靠的PCOS大鼠模型，并设计二甲双胍的给药干预，研究NF-κB、IL-6、TNF-a在正常组、PCOS组、二甲双胍干预组大鼠的表达，及与终体质量、T、LH、HOMA-IR、E2表达的相关性，了解NF-κB及炎症因子在PCOS中的作用；2、研究二甲双胍对NF-κB、IL-6、TNF-a及终体质量、T、LH、HOMA-IR、E2表达的影响，为临床治疗提供实验依据；3、探讨二甲双胍对多囊卵巢大鼠的促排卵作用及可能的机制。 [方法]将32只雌性S/D大鼠随机分为三组，正常对照组（A组，n=10）：自由摄取食物和水31d；PCOS组(B组，n=12)：来曲唑1mg/kg灌胃21d后予以2.6mL/kg生理盐水灌胃10d；二甲双胍干预组（C组，n=10）：来曲唑1mg/kg灌胃21d后予以6%的二甲双胍2.6mL/kg灌胃10d。每日阴道涂片及称重，测定各组大鼠血清E2、T、LH、FPG、FINS水平，通过HE染色观察卵巢形态，通过免疫组化、荧光定量PCR方法监测各组大鼠卵巢NF-κB p65蛋白及mRNA的表达，通过Elisa测定各组大鼠循环中IL-6、TNF-a的表达，通过RT-PCR测定各组大鼠卵巢IL-6、TNF-a mRNA的相对表达。 [结果] 1.用来曲唑1mg/kg灌胃共21天，模型组大鼠于造模第9天均失去动情周期，卵巢病理多囊改变，并具有高雄激素血症和胰岛素抵抗的特点，建立近似于人类PCOS的动物模型； 2. PCOS组大鼠血清TNF-a显著高于正常对照组（P0.05），二甲双胍干预组血清TNF-a较PCOS组显著下降（P0.05）；血清IL-6水平在三组大鼠间无显著性差异（P0.05）；PCOS大鼠二甲双胍干预后血清INS、T、LH显著下降； 3.各指标之间进行Pearson相关性分析，很多各指标之间存在相关，形成恶性循环；在相关的基础上进行多元线性回归分析：TNF-a和体质量是引起胰岛素抵抗的独立影响因素,可能是胰岛素抵抗的机制之一；T和IL6对体质量具有显著性影响； IL-6是除HOMA-IR外，对T的独立影响因子； 4.在二甲双胍药物干预的第6天，二甲双胍组大鼠均出现动情周期，恢复排卵；而PCOS组持续处于动情间期，提示无排卵，直到第10天12只中仅1只恢复动情周期。二甲双胍干预组大鼠的卵巢形态均恢复正常，而PCOS组大鼠的卵巢形态仍为多囊改变； 5. PCOS大鼠卵巢局部TNF-a、IL-6mRNA的表达较正常对照组增高（P0.05），二甲双胍干预组大鼠卵巢局部炎症因子mRNA的表达较PCOS组显著减少（P0.05）； 6.卵巢组织免疫组化显示：NF-κB P65均一致性的表达在卵巢颗粒细胞的细胞核，但各组之间的表达无明显统计学差别（P0.05），荧光定量PCR检测卵巢组织NF-κB P65mRNA的表达，各组之间未发现显著性差别（P0.05）。 [结论] 1. S/D大鼠来曲唑建模可作为研究PCOS的理想动物模型； 2.本PCOS大鼠模型存在慢性低度炎症，可能由于临床异质性，，主要反映在TNF-a水平的升高，二甲双胍的干预显示有益的作用； 3. TNF-a是引起PCOS胰岛素抵抗的独立影响因素， IL-6是PCOS体质量的独立影响因素，可用于预测发生的风险性； 4.二甲双胍具有良好的促排卵作用，能恢复卵巢正常形态，卵巢局部炎症因子的改变可能直接参与发病机制； 5.炎症因子的变化并不是通过NF-κB介导，可能存在其他信号通路，或者与PCOS临床表现的异质性有关，具体机制有待进一步研究。
[Abstract]:Objective to establish the PCOS rat model of]1, reliable, and the design of metformin administration intervention, IL-6 of NF- kappa B, TNF-a, in the normal group, PCOS group, the expression of the intervention group rats and metformin, and the final body weight, T, LH, HOMA-IR, correlation between the expression of E2, to understand the role of B and inflammation factor NF- K in PCOS; 2, to study the effects of metformin on NF- kappa B, IL-6, TNF-a and final body mass, T, LH, HOMA-IR, E2 expression, and provide experimental basis for clinical treatment; 3, to investigate the effect of metformin on ovulation of polycystic ovary in rats and its possible mechanism.
[Methods] 32 female S/D rats were randomly divided into three groups, normal control group (group A, n=10): free access to food and water 31d; PCOS group (group B, n=12): letrozole intragastric administration of 1mg/kg 21d to 2.6mL/kg saline 10d; metformin intervention group (C group, n=10): letrozole intragastric Administration of 1mg/kg 21d to 6% 2.6mL/kg after intragastric administration of metformin 10d. daily vaginal smears and weighing, determination of serum E2, the rats of T, LH, FPG, FINS, observation of ovarian morphology by HE staining, immunohistochemistry, fluorescence quantitative PCR method for monitoring the expression of rats ovaries NF- p65 kappa B protein and mRNA, the rats were measured by Elisa TNF-a in the IL-6 cycle, the expression of ovarian IL-6 rats were measured by RT-PCR, the relative expression of TNF-a mRNA.
[results]
1., for 21 days, the rats in the model group were treated with trazole 1mg/kg for 21 days. The rats in the model group lost estrous cycle on the ninth day of modeling, ovarian pathology and polycystic changes, and had the characteristics of Kaohsiung hormone and insulin resistance. A animal model similar to human PCOS was established.
The serum TNF-a in 2. PCOS group was significantly higher than that in the normal control group (P0.05). The serum TNF-a in metformin intervention group was significantly lower than that in the PCOS group (P0.05), and there was no significant difference in serum IL-6 level between the three groups (P0.05). After PCOS metformin treatment, the serum INS, T and PCOS decreased significantly.
Pearson correlation analysis was performed between the 3. indexes, there are correlation between many indexes form a vicious spiral; multiple linear regression analysis in related on the basis of TNF-a and body mass is caused by the independent influencing factors of insulin resistance may be one of the mechanisms of insulin resistance; T and IL6 had significant effect on body weight; IL-6 is in addition to HOMA-IR, the independent impact factor of T;
4. in the metformin drug intervention for sixth days, the metformin group was observed in rat estrous cycle, ovulation; and PCOS Group continued in diestrus, suggesting no ovulation, until the tenth day of 12 in only 1 recovery of estrous cycle. Ovarian morphology in rats intervention group metformin were return to normal, and ovarian morphology in PCOS group the rat is polycystic change;
5., the expression of TNF-a and IL-6mRNA in the ovary of PCOS rats was higher than that in the normal control group (P0.05). The expression of mRNA in the ovary of the metformin treated group was significantly lower than that in the PCOS group (P0.05).
6. ovarian tissue immunohistochemistry showed that: NF- kappa B P65 were similar in the granulosa cell nuclei of expression, but the expression was no significant difference between the groups (P0.05), the expression of fluorescence quantitative PCR detection of ovarian tissue NF- kappa B P65mRNA, significant differences were found between the groups (P0.05).
[Conclusion]
1. S/D rat model of letrozole can be used as an ideal animal model for the study of PCOS.
2., there is chronic low grade inflammation in the PCOS rat model. It may be due to the clinical heterogeneity, which is mainly reflected in the increase of TNF-a level. Metformin intervention has a beneficial effect.
3. TNF-a is an independent factor causing PCOS insulin resistance, and IL-6 is an independent factor in the mass of PCOS body, which can be used to predict the risk of occurrence.
4. metformin has good ovulation stimulating effect and can restore the normal morphology of the ovary. The changes of local inflammatory factors of the ovary may be directly involved in the pathogenesis.
5., the change of inflammatory factors is not mediated by NF- kappa B. There may be other signaling pathways, or are related to the heterogeneity of PCOS clinical manifestations. The specific mechanism needs further study.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.75

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