【作者】 王莉；

【导师】 姬秋和；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 多项临床研究证实促性腺激素释放激素(gonadotropin-releasing hormone;GnRH)与糖代谢关系密切,同时妊娠期母体内的GnRH发生明显变化,且有研究显示接受长效GnRH类似物治疗的体外受孕妇女中妊娠糖尿病的发病率显著增高。结合本课题组既往发现胰腺外分泌部和肠道存在GnRH及其受体,提示胰腺和肠道的GnRH可能在妊娠期生理性胰岛素抵抗或妊娠糖尿病中起重要作用。此研究拟从肠道激素的角度探讨GnRH在高脂妊娠状态时的变化,以期阐明胰肠系统的GnRH在妊娠糖尿病发生发展中的作用。目的:1.明确高血脂妊娠状态下,胰腺和不同肠段GnRH的表达水平。2.了解胰腺和不同肠段GnRH的变化与胰高血糖素、胰岛素及糖代谢变化之间的相关性。方法:1.采用免疫组织化学方法观察了高血脂妊娠大鼠胰腺GnRH及其受体蛋白的表达及分布特点。2.采用放免法测定高血脂妊娠大鼠及正常妊娠大鼠OGTT和进餐试验各点胰岛素水平;并对胰岛素敏感性进行评估。3.采用实时荧光定量PCR方法,观察高血脂妊娠大鼠及正常妊娠大鼠GnRH及其受体、胰高血糖素原和GLP-1受体的mRNA在胰腺和不同肠段表达的差异。4.采用ELISA法测定高血脂妊娠大鼠及正常妊娠大鼠GnRH在胰腺蛋白质水平的表达。5.采用Western Blot测定高血脂妊娠大鼠及正常妊娠大鼠胰高血糖素在胰腺蛋白质水平的表达。结果:1.胰腺GnRH及其受体、胰高血糖素、GLP-1受体表达及GnRH与高血糖素的相关性研究A. OGTT结果显示各组大鼠的血糖最高点均在服糖后30min,Control组胰岛素分泌最高点在15min ,而Hch组、Gestation组和Hch+Gestation组胰岛素分泌高峰为30min;说明高脂和妊娠两种状态均导致胰岛素高峰分泌延迟。Gestation组空腹胰岛素高于Control组(21.68±2.55 vs 14.35±0.86 mIU/L,P<0.05),而胰岛素AUC等与Control组无区别,说明妊娠中期胰岛素抵抗以基础胰岛素增高为主。Hch组胰岛素AUC高于Control组(230.25±13.19 vs 129.71±11.33 mIU/L, P<0.05);ISI低于Control组(2.84±0.52 vs 4.95±0.56, P<0.01),说明高脂血症时存在胰岛素抵抗。Hch+Gestation组的空腹胰岛素高于Control组(25.76±3.31 vs 14.35±0.86 mIU/L,P<0.01),而与Hch组的空腹胰岛素相比无差异,其血糖AUC低于Hch组(29.01±1.40 vs 35.49±2.06mmol/L,P<0.01);同时Hch+Gestation组的胰岛素AUC高于Control组( 204.60±27.95 vs129.71±11.33mIU/L,P<0.05),ISI低于Control组(2.21±0.32 vs 4.95±0.56, P<0.01)和Gestation组(2.21±0.32 vs 4.64±0.90, P<0.01);提示高脂妊娠中期已存在高胰岛素血症和胰岛素抵抗。B.胰腺GnRH组织化学染色显示Hch+Gestation组胰腺细胞GnRH阳性率高于Control组(P<0.01)。C. Hch+Gestation组胰腺GnRH mRNA相对表达量高于Control组1.76倍(P<0.01),高于Hch组1.4倍(P<0.05),高于Gestation组1.53倍(P<0.01)。Hch+Gestation组胰腺GnRH蛋白质表达高于Control组(7.06±0.28 vs 5.40±0.41 mg/L,P<0.05),Gestation组(7.06±0.28 vs 5.95±0.38mg/L,P<0.05)和Hch组(7.06±0.28 vs 5.97±0.30 mg/L,P<0.05)。D. Hch+Gestation组胰腺PG mRNA相对表达量高于Control组1.77倍(P<0.01),高于Hch组1.54倍(P=0.01),同时Gestation组PG mRNA相对表达量高于Control组1.46倍(P<0.05)。Hch+Gestation组胰高血糖素蛋白质表达量高于Control组3.44倍(P<0.01),高于Hch组2.9倍(P<0.01),与Gestation组表达量无差异;Gestation组胰高血糖素高于Control组2.57倍(P<0.01)。E.大鼠胰腺GnRH mRNA相对表达量与PGmRNA相对表达量呈正相关,相关系数r =0.521,P=0.01。2.肠道GnRH mRNA的表达及其与PG mRNA的相关性研究A.进餐试验提示进餐后各组大鼠血糖最高点和胰岛素分泌最高点均在60min;说明进餐试验时胰岛素高峰晚于OGTT。B. Hch+Gestation组空肠GnRH mRNA相对表达量高于Control组2.68倍(P<0.01),高于Hch组1.49倍(P<0.05),高于Gestation组1.45倍(P<0.05);同时Hch组高于Control组1.86倍(P<0.05)。而其PG mRNA相对表达量高于Control组1.78倍(P<0.05),高于Gestation组1.53倍(P<0.05),同时Hch组高于Control组1.52倍(P<0.05)。Gestation组空肠GnRHR mRNA相对表达量高于Control组1.85倍(P<0.05)。各组空肠GLP-1R mRNA相对表达量均未见差异。大鼠空肠GnRH mRNA相对表达量与PG mRNA相对表达量呈正相关,相关系数r =0.699,P<0.01。C. Hch+Gestation组回肠GnRH mRNA相对表达量高于Control组2.18倍(P<0.05)。各组间回肠PG mRNA相对表达量均未见差异。大鼠空肠GnRH mRNA相对表达量与PG mRNA相对表达量呈正相关,相关系数r =0.755 ,P<0.01。D.各组间结肠GnRH mRNA相对表达量均未见差异。Hch+Gestation组PG mRNA相对表达量高于Control组2.07倍(P<0.05),Gestation组高于Control组2.06倍(P<0.05);Hch组高于Control组2.11倍(P<0.05)。大鼠结肠GnRH mRNA相对表达量与PG mRNA相对表达量呈正相关,相关系数r =0.513,P<0.01。结论:高脂妊娠大鼠的胰腺GnRH增加,同时其胰岛素抵抗较高脂大鼠和正常妊娠大鼠加重,尽管这两种变化的关系和机制有待深入研究,但本研究中实验大鼠的胰腺GnRH与PG具有良好的正相关性,提示胰腺GnRH可能与胰腺胰高糖素样多肽的调节有关。另一方面,各组大鼠GnRH mRNA和PG mRNA在各肠段表达变化不同,但在各肠段GnRH mRNA和PGmRNA表达量均呈正相关性,说明肠道GnRH和PG表达水平存在相同的变化趋势,提示肠道GnRH也可能与相应部位胰高糖素样多肽的调节有关。更多还原

【Abstract】 Some clinical data confirm that the GnRH analogues are related to glucose metabolism. GnRH proceed a radical change during gestation, and studies showed treatment with long-acting triptorelin acetate made the prevalence of gestational diabetes mellitus (GDM) increased significantly in women who was undergoing vitro fertilization pregnancies. GnRH and its receptor showed widespread expression in digestive system, including exocrine pancreas, jejunum, ileum, colon and so on. Whether GnRH secreted by pancreas and intestine is changed during gestation? Whether the change is related to glucoregulation during gestation, and whether it plays a critical role in insulin resistance and GDM during gestation by acting on GnRH receptor in pancreas and intestine through effecting the secreting of islet hormones, intestinal absorption and motor function, or regulating secretion of other gastro-intestinal hormones, such as glucagon, glucagons like peptid-1, all these issues need to be investigated. The aim of this study is to explor the function of GnRH secreted by pancreas and intestine in the pathophysiology mechanism of GDM. OBJECTIVE:The purposes of this study were to identify the changes of the expression of GnRH, GnRH receptor, proglucagon and GLP-1 receptor in pancreas and intestine in gestational rat with diet-induced hyperlipidia, and to seek the relationships among the expression of GnRH, GnRH receptor, proglucagon and GLP-1 receptor in pancreas and intestine.METHOD:1. Immunohistochemical method was used to show the protein expression and the distribution of GnRH and its receptor in pancreas in gestational rat with diet-induced hyperlipidia at the time of 14 days after gestation.2. RIA was used to identify insulin during OGTT/meal test in gestational rat with diet-induced hyperlipidia at the time of 13 or 14 days after gestation.3. Real-time quantitative PCR was used to detect the changes of the mRNA expressions of GnRH, GnRH receptor, proglucagon and GLP-1 receptor in pancreas, jejunum, ileum and colon in gestational rat with diet-induced hyperlipidia at the time of 14 days after gestation.4. ELISA was used to detect the changes of the protein expression of GnRH in pancreas in gestational rat with diet-induced hyperlipidia at the time of 14 days after gestation.5. Western Blot was used to detect the changes of the protein expression of glucagon in pancreas in gestational rat with diet-induced hyperlipidia at the time of 14 days after gestation.RESULTS:1. The expression of GnRH in pancreas in hyperlipidemic gestational ratsA. OGTT showed insulin AUC in Hch+Gestation rats and Hch rats was higher than that in Control rats(204.60±79.06 vs 129.71±11.33mIU/L , P<0.05 ),(230.25±13.19 vs 129.71±11.33 mIU/L , P<0.05 ). ISI in Hch+Gestation rats was lower than that both in Control rats(2.21±0.32 vs 4.95±0.56, P<0.01)and Gestation rats(2.21±0.32 vs 4.64±0.90, P<0.01), while ISI in Hch rats was lower than Control rats (2.84±0.52 vs 4.95±0.56, P<0.01). Fasting insulin in Hch+Gestation rats was no difference with that in Hch rats. Fasting insulin in Gestation rats was higher than that in Control rats(21.68±2.55 vs 14.35±0.86,P<0.05).B. Immunohistochemical staining showed there are GnRH and GnRH receptor expressed in rat pancreas. The percentage of GnRH positive cell in Hch+Gestation rats was higher than in Control rats(P<0.05).C. In pancreas, GnRH relative mRNA level in Hch+Gestation rats was 1.76 fold higher than that in Control rats(P<0.01), 1.53 fold higher than that in Gestation rats (P<0.01)and 1.4 fold higher than that in Hch rats(P<0.05). GnRH protein in Hch+Gestation rats was higher than that in Control rats (7.06±0.28 vs 5.40±0.41 mg/L,P<0.05),higher than that in Gestation rats (7.06±0.28 vs 5.95±0.38 mg/L,P<0.05) and higher than that in Hch rats (7.06±0.28 vs 5.97±0.30 mg/L,P<0.05).D. PG relative mRNA level in Hch+Gestation was 1.77 fold higher than that in Control rats(P<0.01),1.54 fold higher than that in Hch rats(P=0.01),PG relative mRNA levels in Gestation rats was 1.46 fold higher than that in Control rats ( P<0.05 ) . The glucagon in Hch+Gestation rats was 3.44 fold higher than in that Control rats(P<0.01)and 2.9 fold higher than that in Hch rats (P<0.01), the glucagon in Gestation rats was 2.57 fold higher than in that Control rats(P<0.01). E. A significant positive correlation between the expression of GnRH mRNA and PG mRNA was observed in rat’s pancreas, r =0.521,P=0.01.2. The expression of GnRH in intestine in hyperlipidemic gestational ratsA. Meal test showed Hch+Gestation rats insulin AUC was higher than that in Control rat(s213.88±8.51 vs 155.87±12.39 mIU/L,P<0.01 )and that in Gestation rats (213.88±8.51 vs 169.27±6.55 mIU/L,P<0.01). Insulin AUC in Hch rats was higher than that in Control rats(188.52±16.70 vs 155.87±12.39 mIU/L,P<0.05 ); HOMA-IR in Hch+Gestation rats was higher than that in Control rats(7.66±0.93 vs 4.14±0.47,P<0.01), HOMA-IR in Hch rats was higher than that in Control rats(6.59±0.33 vs 4.14±0.47,P<0.01).B. In jejunum, GnRH relative mRNA level in Gestation rats was 2.68 fold higher than in Control rats (P<0.01), 1.45 fold higher than in Gestation rats(P<0.05)and 1.49 fold higher than in Hch rats(P<0.05). GnRH relative mRNA level in Hch rats was 1.86 fold higher than in Control rats(P<0.05). PG relative mRNA level in Hch+Gestation rats was 1.78 fold higher than in Control rats(P<0.05),1.53 fold higher than in Gestation rats(P<0.05). PG relative mRNA level in Hch rats was 1.52 fold higher than in Control rats (P<0.05), GnRHR relative mRNA level in Gestation rats was 1.85 fold higher than in Control rats(P<0.05). A significant positive correlation between the expression of GnRH mRNA and PG mRNA was observed in rat’s jejunum, r =0.699,P<0.01.C. In ileum, GnRH relative mRNA level in Hch+Gestation rats was 2.18 fold higher than in Control rats(P<0.05). PG relative mRNA level was no difference among rats. A significant positive correlation between the expression of GnRH mRNA and PG mRNA was observed in rat’s ileum, r =0.755,P<0.01.D. In Colon, GnRH relative mRNA level was no difference among rats. PG relative mRNA level in Hch+Gestation rats was 2.07 fold higher than in Control rat(sP<0.05),PG relative mRNA level in Gestation rats was 2.06 fold higher than in Control rats(P<0.05). PG relative mRNA level in Hch rats was 2.11 fold higher than in Control rats(P<0.05). A significant positive correlation between the expression of GnRH mRNA and PG mRNA was observed in rat’s colon, r =0.513,P<0.01.CONCLUSION:Pancreatic GnRH level in Hch+Gestation rats was higher than that in Control and Hch rats, while insulin resistance was more severe than Hch rats and Gestation rats. The mechanisms of these changes were unclear. But a significant positive correlation between the expression of GnRH mRNA and PG mRNA was observed in rat’s pancreas, which might suggest pancreatic GnRH might influence glucagon secretion in hyperlipidemia rat during gestation. Significant positive correlations between the expression of GnRH mRNA and PG mRNA were observed in rat’s jejunum, ileum and colon. These might also suggest intestinal GnRH may interact with intestinal PG, which influence glucose metabolism.更多还原