【摘要】：第一部分手術(shù)模型的建立與術(shù)式效果的評(píng)價(jià)一、胃大彎折疊聯(lián)合十二指腸-空腸旁路手術(shù)模型的建立【目的】建立肥胖型2型糖尿病大鼠模型,探索新型代謝手術(shù)GCP-DJB治療T2DM的可行性�！痉椒ā侩S機(jī)選取經(jīng)高脂高糖飼料喂養(yǎng)及小劑量STZ(30mg/kg)腹腔注射成功的T2DM大鼠模型22只,平衡組間體重差異后,分為:GCP-DJB組(n=12)、假手術(shù)Sham組(n=10)。測(cè)量術(shù)前、術(shù)后1周、2周、4周時(shí)的體重,檢測(cè)空腹血糖、空腹血清胰島素(INS),計(jì)算胰島素抵抗指數(shù)(IRI)�！窘Y(jié)果】術(shù)前2組大鼠的各項(xiàng)指標(biāo)之間無(wú)差異(P0.05);術(shù)后1周時(shí),與Sham組相比,GCP-DJB組的大鼠空腹血糖明顯下降(P0.05),空腹胰島素水平升高(P0.05),IRI開始下降(P0.05);術(shù)后2周至4周時(shí),與Sham組相比,GCP-DJB組的大鼠體重明顯下降(P0.05),空腹血糖明顯下降(P0.05),空腹胰島素水平顯著升高(P0.05),胰島素抵抗指數(shù)IRI明顯降低(P0.05)�！窘Y(jié)論】成功建立T2DM大鼠GCP-DJB手術(shù)模型,可為研究代謝手術(shù)治療糖尿病機(jī)制提供穩(wěn)定的動(dòng)物模型。二、GCP-DJB與SG對(duì)T2DM大鼠降糖效果相關(guān)代謝指標(biāo)的影響【目的】研究比較GCP-DJB與常用的SG對(duì)T2DM大鼠的治療效果及代謝指標(biāo)差異�！痉椒ā繉⒔３晒Φ腡2DM大鼠,平衡體重差異后,隨機(jī)分成3組:Sham組(n=6)、SG組(n=6)、GCP-DJB組(n=6)。檢測(cè)術(shù)前及術(shù)后2、4、6、8、10、12W體重及日均攝食量;并于術(shù)后12W行OGTT檢測(cè)糖耐量、胰島素釋放實(shí)驗(yàn),計(jì)算IRI;檢測(cè)餐后血清GLP-1、GIP、PYY、膽汁酸釋放曲線,計(jì)算AUC�！窘Y(jié)果】術(shù)前,3組大鼠體重、日均攝食量無(wú)顯著差異(P0.05)。術(shù)后12周時(shí),SG組和GCP-DJB組體重、攝食量較Sham組更低(P0.05),且GCP-DJB組較SG組更低(P0.05);SG組及GCP-DJB組在糖耐量、餐后血清INS、GLP-1、PYY、膽汁酸方面較Sham組顯著提高(P0.05),且除血清胰島素外,GCP-DJB組的糖代謝相關(guān)指標(biāo)較SG組更高(P0.05);SG組及GCP-DJB組IRI及GIP分泌較Sham組顯著下降(P0.05),且GCP-DJB組GIP分泌量較SG組更低(P0.05)�！窘Y(jié)論】GCP-DJB及SG均可作為治療T2DM的選擇術(shù)式之一,其緩解機(jī)制可能與體重及攝食量的下降,INS、GLP-1、PYY、膽汁酸的升高,GIP及IRI的下降有關(guān)。從多種與T2DM降糖效果相關(guān)的代謝指標(biāo)來(lái)看,GCP-DJB術(shù)式的治療效果優(yōu)于SG。第二部分減少LPS誘導(dǎo)的炎癥因子釋放是GCP-DJB緩解T2DM的內(nèi)在機(jī)制之一一、高糖和LPS對(duì)SD大鼠胰腺β細(xì)胞胰島素分泌的影響【目的】探索T2DM環(huán)境下高糖(HG)及LPS對(duì)SD大鼠胰腺β細(xì)胞胰島素分泌的影響�！痉椒ā矿w外原代培養(yǎng)8周齡大鼠胰腺組織,根據(jù)析因分析設(shè)計(jì)為4組: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)。培養(yǎng)48h后,利用實(shí)時(shí)熒光定量-聚合酶鏈反應(yīng)(Q-PCR)檢測(cè)這4組培養(yǎng)基內(nèi)胰島素m RNA表達(dá),免疫熒光觀察比較HG和LPS對(duì)大鼠胰腺內(nèi)胰島素合成情況的差異�！窘Y(jié)果】HG和LPS均能顯著降低大鼠胰腺胰島素的表達(dá)量(P0.05)。HG能使胰腺中胰島素m RNA的表達(dá)下降39.58%(P0.05),高LPS能使得使胰腺中胰島素m RNA的表達(dá)下降40.50%(P0.05),兩種因素之間存在交互作用(P0.05)�！窘Y(jié)論】HG和LPS均能對(duì)胰腺β細(xì)胞造成損傷,顯著減少胰島素的合成與釋放。LPS對(duì)胰島β細(xì)胞的損傷作用在T2DM的發(fā)生發(fā)展過程中起著重要作用。二、GCP-DJB對(duì)T2DM大鼠血清LPS及炎癥因子IL-1β、IL-6、TNF-α的影響【目的】從LPS所誘導(dǎo)的炎癥級(jí)聯(lián)反應(yīng)角度,探索GCP-DJB緩解T2DM的內(nèi)在機(jī)制。研究GCP-DJB術(shù)后T2DM大鼠血清LPS及炎癥因子IL-1β、IL-6、TNF-α的變化。【方法】將20只T2DM大鼠隨機(jī)分為Control組(n=10)和GCP-DJB組(n=10)。術(shù)前及術(shù)后12周時(shí),ELISA檢測(cè)2組大鼠血清LPS、血清IL-1β、IL-6、TNF-α等炎癥相關(guān)指標(biāo),HE光鏡及透射電鏡觀察胰腺內(nèi)外分泌部以及超微結(jié)構(gòu)變化,Q-PCR檢測(cè)胰腺胰島素m RNA表達(dá)水平,免疫組化及WB檢測(cè)胰島素蛋白表達(dá)量。組間數(shù)據(jù)比較采用t檢驗(yàn)或Wilcoxon秩和檢驗(yàn)�！窘Y(jié)果】術(shù)前2組大鼠LPS、IL-1β、IL-6、TNF-α等炎癥相關(guān)指標(biāo)差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。術(shù)后12周時(shí),Control組大鼠胰腺外分泌部的腺細(xì)胞線粒體脫嵴腫脹,粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張,內(nèi)分泌部β細(xì)胞分泌顆粒數(shù)量減少,粗面內(nèi)質(zhì)網(wǎng)脫粒,線粒體減少;GCP-DJB組較Control組明顯改善。與Control組相比,GCP-DJB能顯著降低T2DM大鼠血清LPS(P0.05)及炎癥因子IL-1β、IL-6、TNF-α(P0.05),提高胰腺胰島素m RNA及蛋白表達(dá)量(P0.05)。【結(jié)論】GCP-DJB能顯著改善T2DM大鼠胰腺胰島素分泌功能,其內(nèi)在機(jī)制可能與降低LPS所誘導(dǎo)的炎癥級(jí)聯(lián)反應(yīng)有關(guān)。第三部分探索GCP-DJB降低T2DM大鼠血清LPS的原因一、GCP-DJB對(duì)T2DM大鼠腸道菌群結(jié)構(gòu)的影響【目的】GCP-DJB能夠降低LPS所誘導(dǎo)的炎癥級(jí)聯(lián)反應(yīng),從而提高胰島素儲(chǔ)備功能。因此,從LPS來(lái)源角度,探索GCP-DJB術(shù)后腸道菌群結(jié)構(gòu)的變化。【方法】將10只同一批次2型糖尿病大鼠隨機(jī)分成2組:GCP-DJB組(n=5)、Control組(n=5)。于術(shù)后12周時(shí),處死大鼠采集盲腸內(nèi)容物樣本,進(jìn)行16S r DNA測(cè)序(可變區(qū)V3+V4)。測(cè)序數(shù)據(jù)經(jīng)過統(tǒng)計(jì)、優(yōu)化及相關(guān)多變量統(tǒng)計(jì)分析,對(duì)比2組大鼠細(xì)菌分類及豐度差異。【結(jié)果】從多樣性指數(shù)來(lái)看,與Control組相比,GCP-DJB降低了總的細(xì)菌豐度,提高了某些特異菌群的比例。在門水平上,與Control組相比,GCP-DJB組厚壁菌門、軟壁菌門菌群比例顯著降低(P0.05);擬桿菌門、黏膠球形菌門、疣微菌門、脫鐵桿菌門顯著增高(P0.05);GCP-DJB組變形菌門雖較Control組高,但2組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。從科水平上來(lái)看,與Control組相比,GCP-DJB組氨基酸球菌科、脫鐵桿菌科、Gastranaerophilales_norank、螺桿菌科、普雷沃氏菌科、紅螺菌科、黏膠球形菌科的群落豐度顯著增加(P0.05);芽胞桿菌科、梭菌科、腸球菌科、柔膜細(xì)菌科、支原體科、消化鏈球菌科、瘤胃菌科的群落豐度顯著減少(P0.05)�！窘Y(jié)論】GCP-DJB術(shù)后厚壁菌門、軟壁菌門菌群比例顯著降低,擬桿菌門、黏膠球形菌門、疣微菌門、脫鐵桿菌門顯著增高,這些菌群結(jié)構(gòu)的變化可能與LPS的減少及T2DM的改善有關(guān)。二、GCP-DJB對(duì)T2DM大鼠腸道黏膜屏障功能的影響【目的】GCP-DJB能夠降低LPS所誘導(dǎo)的炎癥級(jí)聯(lián)反應(yīng),從而提高胰島素儲(chǔ)備功能。因此,從LPS入血途徑角度,探索GCP-DJB術(shù)后腸道黏膜屏障功能的變化�！痉椒ā繉⑼慌�10只2型糖尿病大鼠隨機(jī)分成2組:GCP-DJB組(n=5)、Control組(n=5)。術(shù)后12周時(shí),處死大鼠采集回腸組織標(biāo)本,HE、電鏡觀察腸道粘膜形態(tài)及腸道上皮緊密連接結(jié)構(gòu),采用免疫組化、免疫熒光、WB檢測(cè)腸道粘膜上皮緊密連接結(jié)構(gòu)的閉合蛋白claudin-1、咬合蛋白o(hù)ccludin、和閉合小環(huán)ZO-1蛋白的表達(dá)量�！窘Y(jié)果】與Control組相比,GCP-DJB組遠(yuǎn)端回腸黏膜形態(tài)明顯改善,表面絨毛細(xì)胞間隙致密,微絨毛下端連接復(fù)合體更為緊密。腸黏膜上皮細(xì)胞緊密連接蛋白Occuldin、Claudin-1、ZO-1表達(dá)量顯著升高(P0.05)�！窘Y(jié)論】GCP-DJB可通過提高緊密連接蛋白Occuldin、Claudin-1、ZO-1表達(dá),改善緊密連接結(jié)構(gòu),從而增強(qiáng)腸黏膜屏障功能,減少血清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..
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R656.6

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