胃大弯折叠联合十二指肠空肠旁路术对2型糖尿病大鼠治疗作用的探索

发布时间：2018-07-26 17:56

【摘要】：第一部分手术模型的建立与术式效果的评价一、胃大弯折叠联合十二指肠-空肠旁路手术模型的建立【目的】建立肥胖型2型糖尿病大鼠模型,探索新型代谢手术GCP-DJB治疗T2DM的可行性。【方法】随机选取经高脂高糖饲料喂养及小剂量STZ(30mg/kg)腹腔注射成功的T2DM大鼠模型22只,平衡组间体重差异后,分为:GCP-DJB组(n=12)、假手术Sham组(n=10)。测量术前、术后1周、2周、4周时的体重,检测空腹血糖、空腹血清胰岛素(INS),计算胰岛素抵抗指数(IRI)。【结果】术前2组大鼠的各项指标之间无差异(P0.05);术后1周时,与Sham组相比,GCP-DJB组的大鼠空腹血糖明显下降(P0.05),空腹胰岛素水平升高(P0.05),IRI开始下降(P0.05);术后2周至4周时,与Sham组相比,GCP-DJB组的大鼠体重明显下降(P0.05),空腹血糖明显下降(P0.05),空腹胰岛素水平显著升高(P0.05),胰岛素抵抗指数IRI明显降低(P0.05)。【结论】成功建立T2DM大鼠GCP-DJB手术模型,可为研究代谢手术治疗糖尿病机制提供稳定的动物模型。二、GCP-DJB与SG对T2DM大鼠降糖效果相关代谢指标的影响【目的】研究比较GCP-DJB与常用的SG对T2DM大鼠的治疗效果及代谢指标差异。【方法】将建模成功的T2DM大鼠,平衡体重差异后,随机分成3组:Sham组(n=6)、SG组(n=6)、GCP-DJB组(n=6)。检测术前及术后2、4、6、8、10、12W体重及日均摄食量;并于术后12W行OGTT检测糖耐量、胰岛素释放实验,计算IRI;检测餐后血清GLP-1、GIP、PYY、胆汁酸释放曲线,计算AUC。【结果】术前,3组大鼠体重、日均摄食量无显著差异(P0.05)。术后12周时,SG组和GCP-DJB组体重、摄食量较Sham组更低(P0.05),且GCP-DJB组较SG组更低(P0.05);SG组及GCP-DJB组在糖耐量、餐后血清INS、GLP-1、PYY、胆汁酸方面较Sham组显著提高(P0.05),且除血清胰岛素外,GCP-DJB组的糖代谢相关指标较SG组更高(P0.05);SG组及GCP-DJB组IRI及GIP分泌较Sham组显著下降(P0.05),且GCP-DJB组GIP分泌量较SG组更低(P0.05)。【结论】GCP-DJB及SG均可作为治疗T2DM的选择术式之一,其缓解机制可能与体重及摄食量的下降,INS、GLP-1、PYY、胆汁酸的升高,GIP及IRI的下降有关。从多种与T2DM降糖效果相关的代谢指标来看,GCP-DJB术式的治疗效果优于SG。第二部分减少LPS诱导的炎症因子释放是GCP-DJB缓解T2DM的内在机制之一一、高糖和LPS对SD大鼠胰腺β细胞胰岛素分泌的影响【目的】探索T2DM环境下高糖(HG)及LPS对SD大鼠胰腺β细胞胰岛素分泌的影响。【方法】体外原代培养8周龄大鼠胰腺组织,根据析因分析设计为4组:C+LPS40(Glucose 5mmol/ml+LPS 40pg/ml),C+LPS 200(Glucose 5mmol/L+LPS 200pg/ml),HG+LPS40(Glucose 25mmol/L+LPS 40pg/ml),HG+LPS 200(Glucose 25mmol/L+LPS200pg/ml)。培养48h后,利用实时荧光定量-聚合酶链反应(Q-PCR)检测这4组培养基内胰岛素m RNA表达,免疫荧光观察比较HG和LPS对大鼠胰腺内胰岛素合成情况的差异。【结果】HG和LPS均能显著降低大鼠胰腺胰岛素的表达量(P0.05)。HG能使胰腺中胰岛素m RNA的表达下降39.58%(P0.05),高LPS能使得使胰腺中胰岛素m RNA的表达下降40.50%(P0.05),两种因素之间存在交互作用(P0.05)。【结论】HG和LPS均能对胰腺β细胞造成损伤,显著减少胰岛素的合成与释放。LPS对胰岛β细胞的损伤作用在T2DM的发生发展过程中起着重要作用。二、GCP-DJB对T2DM大鼠血清LPS及炎症因子IL-1β、IL-6、TNF-α的影响【目的】从LPS所诱导的炎症级联反应角度,探索GCP-DJB缓解T2DM的内在机制。研究GCP-DJB术后T2DM大鼠血清LPS及炎症因子IL-1β、IL-6、TNF-α的变化。【方法】将20只T2DM大鼠随机分为Control组(n=10)和GCP-DJB组(n=10)。术前及术后12周时,ELISA检测2组大鼠血清LPS、血清IL-1β、IL-6、TNF-α等炎症相关指标,HE光镜及透射电镜观察胰腺内外分泌部以及超微结构变化,Q-PCR检测胰腺胰岛素m RNA表达水平,免疫组化及WB检测胰岛素蛋白表达量。组间数据比较采用t检验或Wilcoxon秩和检验。【结果】术前2组大鼠LPS、IL-1β、IL-6、TNF-α等炎症相关指标差异无统计学意义(P0.05)。术后12周时,Control组大鼠胰腺外分泌部的腺细胞线粒体脱嵴肿胀,粗面内质网扩张,内分泌部β细胞分泌颗粒数量减少,粗面内质网脱粒,线粒体减少;GCP-DJB组较Control组明显改善。与Control组相比,GCP-DJB能显著降低T2DM大鼠血清LPS(P0.05)及炎症因子IL-1β、IL-6、TNF-α(P0.05),提高胰腺胰岛素m RNA及蛋白表达量(P0.05)。【结论】GCP-DJB能显著改善T2DM大鼠胰腺胰岛素分泌功能,其内在机制可能与降低LPS所诱导的炎症级联反应有关。第三部分探索GCP-DJB降低T2DM大鼠血清LPS的原因一、GCP-DJB对T2DM大鼠肠道菌群结构的影响【目的】GCP-DJB能够降低LPS所诱导的炎症级联反应,从而提高胰岛素储备功能。因此,从LPS来源角度,探索GCP-DJB术后肠道菌群结构的变化。【方法】将10只同一批次2型糖尿病大鼠随机分成2组:GCP-DJB组(n=5)、Control组(n=5)。于术后12周时,处死大鼠采集盲肠内容物样本,进行16S r DNA测序(可变区V3+V4)。测序数据经过统计、优化及相关多变量统计分析,对比2组大鼠细菌分类及丰度差异。【结果】从多样性指数来看,与Control组相比,GCP-DJB降低了总的细菌丰度,提高了某些特异菌群的比例。在门水平上,与Control组相比,GCP-DJB组厚壁菌门、软壁菌门菌群比例显著降低(P0.05);拟杆菌门、黏胶球形菌门、疣微菌门、脱铁杆菌门显著增高(P0.05);GCP-DJB组变形菌门虽较Control组高,但2组之间差异无统计学意义(P0.05)。从科水平上来看,与Control组相比,GCP-DJB组氨基酸球菌科、脱铁杆菌科、Gastranaerophilales_norank、螺杆菌科、普雷沃氏菌科、红螺菌科、黏胶球形菌科的群落丰度显著增加(P0.05);芽胞杆菌科、梭菌科、肠球菌科、柔膜细菌科、支原体科、消化链球菌科、瘤胃菌科的群落丰度显著减少(P0.05)。【结论】GCP-DJB术后厚壁菌门、软壁菌门菌群比例显著降低,拟杆菌门、黏胶球形菌门、疣微菌门、脱铁杆菌门显著增高,这些菌群结构的变化可能与LPS的减少及T2DM的改善有关。二、GCP-DJB对T2DM大鼠肠道黏膜屏障功能的影响【目的】GCP-DJB能够降低LPS所诱导的炎症级联反应,从而提高胰岛素储备功能。因此,从LPS入血途径角度,探索GCP-DJB术后肠道黏膜屏障功能的变化。【方法】将同一批次10只2型糖尿病大鼠随机分成2组:GCP-DJB组(n=5)、Control组(n=5)。术后12周时,处死大鼠采集回肠组织标本,HE、电镜观察肠道粘膜形态及肠道上皮紧密连接结构,采用免疫组化、免疫荧光、WB检测肠道粘膜上皮紧密连接结构的闭合蛋白claudin-1、咬合蛋白occludin、和闭合小环ZO-1蛋白的表达量。【结果】与Control组相比,GCP-DJB组远端回肠黏膜形态明显改善,表面绒毛细胞间隙致密,微绒毛下端连接复合体更为紧密。肠黏膜上皮细胞紧密连接蛋白Occuldin、Claudin-1、ZO-1表达量显著升高(P0.05)。【结论】GCP-DJB可通过提高紧密连接蛋白Occuldin、Claudin-1、ZO-1表达,改善紧密连接结构,从而增强肠黏膜屏障功能,减少血清LPS。
[Abstract]:The establishment of the first part of the operation model and the evaluation of the effect of the operation. The establishment of a model of large bending of the stomach and duodenal jejunum bypass surgery. [Objective] to establish a model of obese type 2 diabetic rats and explore the feasibility of a new type of metabolic operation GCP-DJB for the treatment of T2DM. TZ (30mg/kg) intraperitoneal injection of a successful T2DM rat model of 22 rats, after the balance group weight difference, divided into: GCP-DJB group (n=12), the sham operation Sham group (n=10). Measure the weight before operation, 1 weeks, 2 weeks, 4 weeks, test the fasting blood glucose, fasting serum insulin (INS), and calculate the insulin resistance index (IRI). [results] the indexes of the 2 groups of rats before operation At 1 weeks after operation, the fasting blood glucose of rats in group GCP-DJB decreased significantly (P0.05), the level of fasting insulin increased (P0.05) and IRI began to decrease (P0.05) at 1 weeks after operation, and the body weight of rats in group GCP-DJB decreased significantly (P0.05) at 2 to 4 weeks after operation (P0.05), fasting blood glucose decreased significantly (P0.05), and fasting insulin level was significantly lower than that of group Sham. Significantly increased (P0.05), insulin resistance index IRI significantly decreased (P0.05). [Conclusion] the successful establishment of T2DM rat GCP-DJB operation model can provide a stable animal model for the study of the mechanism of metabolic surgery for the treatment of diabetes. Two, GCP-DJB and SG on the effects of metabolic indices related to the hypoglycemic effect of T2DM rats [Objective] to compare GCP-DJB and common use The effect of SG on the treatment of T2DM rats and the difference of metabolic index. [Methods] the successful T2DM rats were divided into 3 groups randomly: Sham group (n=6), SG group (n=6), GCP-DJB group (n=6). The 2,4,6,8,10,12W weight and daily food intake were measured before and after the operation. GLP-1, GIP, PYY, and bile acid release curves were measured after the meal, and AUC. [results] before operation, the body weight of 3 groups of rats was not significantly different (P0.05). At the 12 week after operation, the weight of SG group and GCP-DJB group was lower than that of the Sham group (P0.05), and the GCP-DJB group was lower than that of the SG group. GLP-1, PYY, and bile acids were significantly higher than those in the Sham group (P0.05), and the glucose metabolism related indexes in GCP-DJB group were higher than those in the SG group (P0.05), and the secretion of IRI and GIP secretion in SG and GCP-DJB groups was significantly lower than that in the Sham group. One of the alternative mechanisms may be associated with a decline in weight and intake of weight and intake of INS, GLP-1, PYY, bile acid, GIP and IRI. From a variety of metabolic indicators associated with T2DM hypoglycemic effect, the therapeutic effect of GCP-DJB is superior to SG. second in reducing LPS induced inflammatory factor release is the intrinsic machine for GCP-DJB to alleviate T2DM. The effect of high sugar and LPS on the insulin secretion of pancreatic beta cells in SD rats [Objective] to explore the effect of high glucose (HG) and LPS on pancreatic beta cell insulin secretion in SD rats. [Methods] the primary culture of pancreatic tissue in 8 weeks old rats in vitro was designed. According to the analysis of factorial analysis, the expression of C+LPS40 (Glucose 5mmol/ml+LPS 40pg/ml), C+ LPS 200 (Glucose 5mmol/L+LPS 200pg/ml), HG+LPS40 (Glucose 25mmol/L+LPS 40pg/ml), HG+LPS 200 (Glucose 25mmol/L+LPS200pg/ml). After cultivating 48h, the 4 groups of intracellular insulin expressions were detected by real-time fluorescence quantitative polymerase chain reaction. [results] [results] both HG and LPS can significantly reduce the expression of insulin in pancreas (P0.05).HG can decrease the expression of M RNA in pancreas by 39.58% (P0.05), high LPS can reduce the expression of M RNA in pancreas by 40.50% (P0.05), and there is a interaction between the two factors (P0.05). Beta cell damage, significantly reducing the insulin synthesis and release of.LPS damage to islet beta cells plays an important role in the development of T2DM. Two, the effect of GCP-DJB on the serum LPS and inflammatory factors IL-1 beta, IL-6, TNF- a in T2DM rats [Objective] to explore the T2DM of the inflammatory cascade induced by LPS, and to explore the T2DM of GCP-DJB. The changes in serum LPS and inflammatory factors IL-1 beta, IL-6, TNF- alpha in serum of T2DM rats after GCP-DJB were studied. [Methods] 20 rats were randomly divided into Control group (n=10) and GCP-DJB group (n=10). Before and 12 weeks after operation, ELISA detected the serum levels of 2 groups of rat serum, serum beta, serum, and transmission electron microscopy The changes in the secretory part and ultrastructure of the pancreas were observed. The expression of pancreatic insulin m RNA was detected by Q-PCR, and the expression of Insulin protein was detected by immunohistochemistry and WB. The data were compared with t test or Wilcoxon rank test. [results] there was no statistical difference between the 2 groups of rats (P0.0, LPS, IL-1 beta, IL-6, TNF- a). 5). At 12 weeks after operation, the mitochondria of the exocrine pancreas of group Control rats were swollen, the rough endoplasmic reticulum expanded, the number of secretory granules in the endocrine part decreased, the rough endoplasmic reticulum threshing, and the mitochondria decreased, and the group GCP-DJB was obviously better than the Control group. Compared with the group Control, GCP-DJB could significantly reduce the LPS (P0.05) and the serum LPS (P0.05) in the T2DM rats, as compared with the Control group. Inflammatory factors IL-1 beta, IL-6, TNF- alpha (P0.05), increase pancreatic insulin m RNA and protein expression (P0.05). [Conclusion] GCP-DJB can significantly improve the secretion of insulin in the pancreas of T2DM rats. The intrinsic mechanism may be related to the reduction of the cascade of inflammation induced by LPS. The third part explores the reason why GCP-DJB reduces the causes of the serum of T2DM rats. The effect of DJB on the intestinal microflora structure in T2DM rats [Objective] GCP-DJB can reduce the inflammatory cascade induced by LPS and improve the function of insulin reserve. Therefore, from the point of view of LPS, the changes in the intestinal microflora structure of GCP-DJB have been explored. [Methods] 10 rats of the same batch of type 2 diabetic rats were randomly divided into 2 groups: GCP-DJB group (n=5), Group Control (n=5). At 12 weeks after the operation, the rats were sacrificed to collect the cecum content samples and carry out 16S R DNA sequencing (variable region V3+V4). The sequencing data were statistically analyzed, optimized and correlated multivariate statistical analysis, and compared the classification and abundances of the 2 groups of rats. [results] from the diversity index, GCP-DJB reduced the total. Bacterial abundance increased the proportion of certain specific bacteria groups. Compared with the Control group, the proportion of the GCP-DJB group was significantly lower than that of the Control group (P0.05); the bacteriobacteria, the mucous glogate, the verruca Microbacterium and the deformiobacteria gate were significantly higher (P0.05), while the GCP-DJB group was higher than the Control group, but there was no difference between the 2 groups. Statistical significance (P0.05). Compared with group Control, the community abundances of group GCP-DJB, deonobacteriaceae, deonobacteriaceae, Gastranaerophilales_norank, helicobacteraceae, Poulet Was, snail, and viscose were significantly increased (P0.05); Bacillus sp., clostridiaceae, Enterococcus, soft membrane bacteria, Mycoplasma The community abundances of the family of Streptococcus, digestible Streptococcus and rumen bacteria decreased significantly (P0.05). [Conclusion] after GCP-DJB, the proportion of the bacilli was significantly reduced, the bacteriobacteriaceae, the mucous glomen, the verruca microbacteriae, and the deferate gate were significantly increased. The changes of these bacteria groups may be related to the decrease of LPS and the improvement of T2DM. Two, GCP-DJ The effect of B on the intestinal mucosal barrier function of T2DM rats [Objective] GCP-DJB can reduce the inflammatory cascade reaction induced by LPS and improve the function of insulin reserve. Therefore, from the angle of LPS blood pathway, the changes of intestinal mucosal barrier function after GCP-DJB operation are explored. [Methods] 10 rats of the same batch of type 2 diabetic rats were randomly divided into 2 groups. Group GCP-DJB (n=5) and group Control (n=5). At 12 weeks after the operation, the rats were sacrificed to collect the ileum tissue specimens. HE, the morphology of the intestinal mucosa and the close connection structure of the intestinal epithelium were observed by electron microscopy. Immunofluorescence, immunofluorescence, and WB were used to detect closed protein claudin-1, occlusal protein occludin, and closed small loop ZO-1 protein in intestinal mucosa closely connected structure. Compared with the Control group, the morphology of the distal ileum mucosa in the GCP-DJB group was obviously improved, the surface villi space was dense and the lower end of microvilli were more tightly connected. The expression of close connexin Occuldin, Claudin-1 and ZO-1 in the intestinal mucosa epithelial cells increased significantly (P0.05). [Conclusion] GCP-DJB can be tighten by increasing the density of GCP-DJB. The expression of connexin Occuldin, Claudin-1 and ZO-1 improves tight junction structure, thereby enhancing intestinal mucosal barrier function and reducing serum LPS..
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R656.6

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