【作者】 王甜甜；

【导师】 胡三元；

【作者基本信息】 山东大学 ， 外科学， 2009， 博士

【摘要】 研究背景:糖尿病是危害人类健康的重要疾病,目前糖尿病患者全球逾1.7亿,到2030年患病人数将翻一番,新增患者主要集中在中国、印度等发展中国家。糖尿病患者致残和早亡的重要原因为其血糖控制不佳所致的各种并发症,而目前用于控制血糖的主要措施包括控制饮食、运动、口服降糖药和使用胰岛素等,由于它们自身的缺陷、不良反应、费用及需要终生维持等原因,以致患者的依从性差,很难将血糖控制在正常水平。因此,寻找长效、安全、依从性高的糖尿病治疗新方法并验证其临床可行性,是目前值得深入探讨的问题。1995年,针对肥胖症治疗手术(bariatric surgery)效果的大宗回顾性病例分析研究发现,肥胖症患者接受胃旁路术(gastric bypass,GBP)后,不仅体重显著下降,而且其伴发的2型糖尿病也意想不到的得到了治愈或改善。随后的大量临床研究进一步证实,GBP治疗患有肥胖症的2型糖尿病患者有着良好的疗效,且其作用似乎是直接、独立的。但更多的2型糖尿病患者是非肥胖的或不伴肥胖症的,肥胖症治疗手术对此类患者是否有效?如果有效,其治疗机理如何?对于这些问题,已有学者开始着手进行研究,主要研究的术式有:胃旁路术,回肠转位术,胆胰转流术,胃成形术等,并对各种术式对糖尿病的治疗机理提出了多种假说;但大部分的研究是非常初步的,其假说也没有形成完整的、系统的理论,而且在治疗关键因素的探讨上,多年来的研究更是众说纷纭。对于何种术式更佳的问题,尚不能检索到相关文献。为了进一步研究胃肠道重组对糖尿病的治疗效果,明确其作用机理,并对不同术式进行比较,以期找到一个最佳的治疗方案,我们选择对非肥胖型的糖尿病动物模型进行手术干预,从而研究不同的手术方式对糖尿病各有怎样的影响及其可能机制,进一步证实外科手术对糖尿病的治疗作用;同时在不同术式间进行对比分析,为糖尿病外科治疗的临床化提供实验依据;最后系统检测多项治疗相关因子,探索手术治疗糖尿病的可能机制。目的:1.对随机分组的手术组及假手术组糖尿病大鼠行手术干预,建立胃肠道重组的糖尿病动物模型,并观察大鼠模型的稳定性及是否符合实验要求,以确定动物模型是否适宜可靠。2.观察手术前后各组动物糖尿病相关指标的变化,检测各组动物糖尿病并发症所致的器官损伤的情况,以评估各术式对糖尿病的治疗作用,以期寻找最佳治疗方案。3.检测各手术组及假手术组大鼠多项与糖尿病发病和治疗效果相关的体液因子在血清中的水平变化,以探讨手术对糖尿病可能的治疗机制。总之,通过以上研究,为胃肠道重组治疗糖尿病应用于临床提供动物实验支持并奠定理论基础。方法:第一部分:非肥胖2型糖尿病大鼠胃肠道重组模型的建立及其评价。将GK大鼠以区组随机化分组法随机分组,每组雄性GK大鼠20只,分别行三种胃肠道重组手术及相应假手术。各实验组命名如下:A组:胃旁路术(gastric bypass,GBP)组。GBP术式:自幽门处断十二指肠,远端封闭,近端备用;距屈式韧带远端8cm断空肠,远端空肠与幽门吻合,近端空肠与距吻合口12cm处空肠行端侧吻合。B组:胆胰转流术(biliopancreatic diversion,BPD)组。BPD术式:幽门处断十二指肠,远端封闭,近端备用;然后,自距屈式韧带远端约8cm处切断空肠,远端空肠与幽门吻合,近端空肠与距回盲瓣10cm的回肠作端侧吻合。C组:回肠转位术(ilealtransposition,IT)组。IT术式:自距回盲部5-15cm处取长8cm的部分回肠,保留肠系膜,转位至距Treitz韧带5-10cm的空肠与之吻合。Sham-A、Sham-B及Sham-C组:胃旁路术、胆胰转流术及回肠转位术的假手术(Sham-operation)组。其术式为:于手术组相应的解剖位点做原位肠切开及肠吻合术。必要时,延长手术时间,以产生与手术组同等程度的麻醉及手术影响。检测术前术后各组肝功能、血清总蛋白及白蛋白水平、肾功能等指标,判断大鼠模型的稳定性及是否符合实验要求以确定动物模型是否适宜可靠。记录手术时间及手术成功率,观察术后恢复情况及术后并发症情况,判断动物模型对各术式的耐受性。第二部分:对构建成功的胃肠道重组糖尿病动物模型及其相应对照组进行糖尿病治疗效果监测并对各手术组间不同术式的手术效果进行比较。总观察期为术后24周。测定术前及术后多个时间点各手术组及假手术组大鼠体重、空腹血糖水平、胰岛素水平、血脂水平、肝肾功能,并进行糖耐量试验和胰岛素敏感性试验。观察期结束后,处死大鼠并取各脏器行病理学检测以探讨手术对糖尿病各脏器损伤程度的远期影响。记录各组手术时间和术后恢复时间,以及术后并发症的发生率,以比较不同手术方式对糖尿病的治疗效果。第三部分:对胃肠道重组动物模型进行手术干预从而研究手术治疗糖尿病的可能机制。针对糖尿病发病机制设计检测指标如下:血胰岛素水平,肠-胰岛轴相关激素,脂肪-胰岛轴相关因子,炎症介质,血胰岛素样生长因子-1水平以及手术组动物小肠消化液定量检测。通过对各种糖尿病发病相关因子的检测来探讨手术对糖尿病的影响,进而探讨手术对2型糖尿病治疗效果的可能机制。以上三部分数据以(?)±s表示,以SPSS13.0软件进行统计学分析。均数间采用t检验,曲线下面积以梯形积分法计算,重复测量数据以重复测量数据的方差分析检验,以P＜0.05为检验水准。结果:第一部分:非肥胖2型糖尿病大鼠胃肠道重组模型的建立及其评价。A组手术完成20例,手术成功率100%,平均手术时间79.8±10.71min,平均术后恢复时间45.25±11.4h,术后死亡率10%,死因均为肠梗阻;B组手术完成20例,手术成功率90%,平均手术时间83.44±15.26min,平均术后恢复时间51.25±14.7h,术后死亡率15%,死因为吻合口漏及代谢并发症;C组手术完成20例,手术成功率100%,平均手术时间50.82±8.49 min,平均术后恢复时间19.29±7.26h,术后无动物死亡。总计60例假手术组大鼠手术均获成功,存活率100%。术后1周各手术组肝功、肾功均恢复至术前水平,术后2周各手术组大鼠总蛋白及白蛋白恢复至术前水平,随后各项指标持续好转至正常水平并维持至24周观察期结束。C组较A、B组的手术时间、术后恢复时间、术后并发症发生率之间差异有显著性。第二部分:对构建成功的胃肠道重组糖尿病动物模型及其相应对照组进行糖尿病治疗效果监测,并对不同术式对糖尿病的治疗效果进行比较。对糖尿病动物模型的体重、血糖、糖耐量、胰岛素敏感性等反映近期直接治疗效果的指标进行观察,同时通过对各重要脏器的生化检测及组织学检测来评价手术对糖尿病所致远期器官损伤的保护效果。A、B、C组大鼠术后体重、空腹血糖、糖耐量及胰岛素敏感性较术前及假手术组均有好转,手术对大鼠胰岛素水平影响无统计学意义;三种手术术后肝肾功能及血脂较术前及假手术组均有好转。三手术组之间比较:最低体重均值的大小顺序为B组＜A组＜C组,但差异无显著性;A组、B组及C组大鼠最低血糖均值的大小顺序为B组＜C组＜A组,但差异无显著性;C组大鼠糖耐量改善程度低于A组及B组大鼠(AUC分别超过其它两组11%及12.5%);C组大鼠表现出较A、B组更好的胰岛素敏感性(AUC较A组减少14%,较B组减少10.1%)。观察期结束后病理学检测显示,A、B、C三组大鼠的心脏、肝脏及肾脏均未见明显糖尿病相关的病理学异常表现。第三部分:以胃肠道重组动物模型对手术的糖尿病治疗机制进行研究。24周观察期中持续监测多项指标,包括胰岛素,肠-胰岛轴相关激素,脂肪-胰岛轴相关因子,炎症介质,胰岛素样生长因子-1(Insulin-like growth factor-Ⅰ,IGF-1)以及实验组动物小肠消化液定量检测。结果显示,A、B、C组大鼠术后胰岛素、TNF-α和皮质醇分泌较术前及假手术组的变化均无统计学意义;GLP-1,GIP,IGF-1的分泌均显著高于术前及假手术组水平,且其差别有统计学意义。而FFA,瘦素,脂连素,抵抗素,则显著低于术前及假手术组水平,其差别亦有统计学意义。在各手术组之间的比较中,A组及B组大鼠GLP-1水平均低于C组大鼠,差异有统计学意义。三手术组大鼠的小肠粘膜重量均较假手术组增加,差异有显著性。C组大体标本观察可见,转位的回肠增生肥大。结论:(1)胃肠道重组的非肥胖型2型糖尿病大鼠模型是可靠稳定的,可用于长期观察及检测研究,有助于临床评估相关手术的远期效果。(2)胃肠道重组对2型糖尿病治疗有效,可获得稳定而持久的血糖控制。而且手术对糖尿病的治疗效果是直接独立的,并不依赖于手术后的体重减轻。(3)三种术式均可稳定持久的降低动物模型的体重。(4) IT术式对糖尿病动物模型的治疗效果稳定,手术创伤小、并发症少,有望成为可应用于临床的非肥胖及轻度肥胖2型糖尿病患者的替代治疗途径。(5)未完全消化的食物提前进入回肠而产生的一系列肠道及肠道外激素应答性反应是胃肠道重组手术治疗糖尿病的关键因素之一。(6)行胃肠道重组后,胃肠道激素GLP-1及GIP通过调节肠-胰岛轴起到改善血糖水平的作用。(7)胃肠道重组术后,血清低游离脂肪酸水平通过脂肪-胰岛轴对术后长期的血糖改善起到一定的作用。(8)胃肠道重组术后,IGF-1升高对血糖的长期稳定维持起一定的作用。(9)胃肠道重组手术通过多种血糖调节机制控制血糖,其中肠-胰岛轴的作用占据关键地位。更多还原

【Abstract】 BackgroundDiabetes mellitus is a severe disease which is very harmful to human being, the population of people who suffer diabetes is over 170 millions, but the prediction showed that this number will be bouble by 2030, and most of these patients will come from developing countries, such as India, China and so on. The main determinant to the risk of disability and even death from diabetes mellitus is the complications due to out of control to blood glucose levels during total lifetime. The most popular ways at present to restore permanently are diet control, physical exercises, give hypoglycemic agents, intensive insulin therapy and so on. Unfortunately, all these treatments mentioned above do not achieve normal levels of blood glucose, and is also difficult to implement for many patients, because of the defect and untoward reaction of their own, high cost, long-time treatment and other adverse factors. So, how to find safe, satisfying complience and long-term effective methods of achieving and maintaining normoglycemia is a problem which is worth to be studied deeply.In 1995, The information, that Type-2 diabetes can apparently be "cured" by surgery was first published. The researchers documented that, besides of the remarkable loss of weight, all signs of Type 2 diabetes the regressed in large amount of patient with daibetes, who had undergone gastric bypass (GBP) for morbid obesity. Moreover, lots of researches show that the normoglycemia after GBP is reliable, and a direct and independent effect of the operation rather than a secondary outcome of the amelioration of obesity-related abnormalities. This makes us ask is it possible that the Bariatric Surgery is effective not only to patients of Type-2 diabetes with morbid obesity, but also to non-morbidly obese individuals? And if it is possible, what kinds of mechanisms are operating? There are some investors try to answer these questions through search and also raise several kinds of hypothesis about mechanism of the treatment, the most popular ones of which are GBP, biliopancreatic diversion (BPD), gastroplasty and so on. But most of these researches are just at the beginning, which means the theories are not comprehensive and systematic. Especially we can not find the related article about the comparison between different procedures.In order to study the effect and mechanism of gastrointestinal surgery as a treatment for diabetes and find the best protocol that can be used in clinical research, we chose animal model of non-obese type 2 diabetes to give gastrointestinal operation as the treatment. The purpose of which is: (1) Find the effect and possible mechanism of different kinds of operations, and confirm resolution of all signs of Type 2 diabetes after gastrointestinal operation is reliable. (2) Compare different kinds of procedures to establish the foundation and give evidence to surgical therapy for type 2 diabetes. (3) Study several factors related to surgical treatment of diabetes and find the mechanism why gastrointestinal operations can cure or release type-2 diabetes.ObjectiveIn the first step of experiments, rats randomly underwent different kinds of gastrointestinal operations and sham gastrointestinal operations to establish the animal models of type-2 diabetes which were given gastrointestinal operations as interventions. We observed the stability of these kinds of animal models in order to confirm reliability and suitability to the research of gastrointestinal surgery as a treatment for diabetes. Secondly, for the purpose of evaluating various kinds of procedures and finding the best treatment protocol, different kinds of indexes related to type-2 diabetes before and after operation were measured and disfunction in different organs due to complications of type 2-diabetes were observed. The last aim of our work was to investigate the several kinds of hormone levels that control glycemic status after gastrointestinal operations in an animal model of non-obese type-2 diabetes mellitus, with the purpose of exploring the mechanism to surgical therapy for type 2 diabetes. In a word, we want to establish the foundation of theory and give evidence through experimental research of animal to clinical application of gastrointestinal operation as a treatment for type-2 diabetes.MethodsPart oneFirst of all, we chose Goto-Kakizaki rat (G-K rat) as the animal model of non-morbidly type 2 diabetes, and then the 120 male G-K rats were randomly divided into six groups (n=20 in each) that were given three kinds of gastrointestinal operations or the corresponding sham gastrointestinal operations as follow:Group A: Gastric bypass (GBP) surgery was given in this group. The GBP surgery was performed as described by Rubino F, et al. The duodenum was separated from the stomach, and bowel continuity was interrupted at the level of the distal jejunum (8 cm from the ligament of Treitz ligament). The distal of the 2 limbs was directly connected to the stomach (gastrojejunal anastomosis) and the proximal limb carrying the biliopancreatic juices was reconnected downward to the alimentary limb at a distance of 12 cm from the gastrojejunal anastomosis (Roux-en-Y reconstruction).Group B: The rats in this group were given biliopancreatic diversion (BPD) surgery. First of all, the duodenum was separated from the stomach and closed the distal limb, and then bowel continuity was interrupted at the level of the distal jejunum (8 cm from the ligament of Treitz ligament). The distal of the 2 limbs was directly connected to the stomach (gastrojejunal anastomosis) and the proximal limb carrying the biliopancreatic juices was end-to-side anastomosed to the distal ileum 10 cm above the ileocecal valve to form the common limb.Group C: Ileal transposition (IT) surgery was given in this group. IT surgery was performed as described by Strader AD, et al. The cecum was located and removed from the abdomen and placed on saline-soaked sterile gauze. An 8-cm segment of ileum located 5-15 cm proximal to the ileocecal valve was isolated and transected. An anastomosis was then made with the two open ends of the ileum. The remaining small intestine was then transected 5-10 cm distal to the ligament of Treitz. The isolated ileal segment with full neural innervation and intact vascular supply to the transposed segment was then inserted in the original peristaltic direction by making two additional end-to-end anastomoses.Group Sham-A, Sham-B and Sham-C: Sham surgeries were performed by making transections and re-anastomosis of the gastrointestinal tract at multiple sites (corresponding to where enterotomies were performed for the GBP, BPD and IT). After transection, the intestines were immediately attached by anastomosis. When needed, surgery time was prolonged to produce a similar degree of anesthesiological stress as those rats which underwent GBP, BPD or IT.Vital signs, liver function, ALB, TP and renal function were measured in preoperative period in order to evaluate the stability and suitability of the animal models we setup for this research.Part twoCompare treatment effectiveness of the animal model of non-morbidly type-2 diabetes to give gastrointestinal operation as the treatment among different kinds of procedures. The total time of follow up is 24 weeks after the operation. We measured weight, fasting glycemia, insulin, carbohydrate tolerance, insulin sensibility, liver function, renal function and blood fat at different time of perioperative period. After 24 weeks of follow-up period for each set, all the animals were sacrificed, and then several kinds of organs were achieved to do study in pathology. We also recorded of operative time and the status of postoperative recovery. With all these documents we got, treatment effectiveness of different kinds of gastrointestinal operation as the treatment for type-2 diabetes in G-K rats can be evaluated and compared. We also observed the operative time, achievement ratio, postoperative recovery and complication ratio to evaluate the animal’s tolerance of different kinds of operation.Part threeThis part is about the study to explore the mechanism of gastrointestinal surgery as a treatment for type-2 diabetes in G-K rats. On the basis of the theory to pathogenesis in type-2 diabetes, insulin, hormones related to enteroinsular axis and adipoinsular axis, mediators of inflammation and IGF-1 were chosen and measured. The weight of enteric digestive juice was realized also. The main objective of this part was to explore the mechanism which means the reason why gastrointestinal operations can cure or release type-2 diabetes.Data are expressed as mean±SD and analysised by SPSS13.0. Areas under curves were calculated by trapezoidal integration. Statistical analysis was performed using repeated-measures ANOVA and the Student’s t-test as appropriate. P values<0.05 were considered to be statistically significant.ResultsPart oneGroup A: The achievement ratio of group A was 100% of 20 cases, the mean operative time was 79.8±10.71min, and the mean postoperative recovery time was 45.25±11.4h.There are two rats that died of intestinal obstruction, which means the postoperative mortality was 10%.Group B : The achievement ratio of group A was 90% of 20 cases, the mean operative time was 83.44±15.26min, and the mean postoperative recovery time was 51.25±14.7h.There are two rats that died of stomal leak and complication about metabolism, which means the postoperative mortality was 15%.Group C: The achievement ratio of group A was 100% of 20 cases, the mean operative time was 50.82±8.49 min, and the mean postoperative recovery time was 19.29±7.26h.There wasn’t any death of rat. All the 60 cases of sham operation group underwent successful without any death, which means the survival rate was 100%. All the animals’ vital sign, liver function and renal function recovered to preoperative normal level one week after the operation, and it cost two weeks for total protein and albumin in serum recovered to normal level. All the index keep going well and stayed in the normal limits until the end of observation after 24 weeks. There were statistically significant differences of operative time, postoperative recovery time and complication ratio between group C and group A, B (P < 0.05).Part twoThe treatment effectiveness to the animal models of non-morbidly type-2 diabetes which were given gastrointestinal operation as the treatment among different kinds of procedures was compared. On one hand, we measured weight, fasting glycemia, glycosylated hemoglobin, insulin, carbohydrate tolerance, insulin sensibility, liver function, renal function and blood fat, with the purpose of evaluating the direct effectiveness in the near future. On the other hand, in order to study whether gastrointestinal operation can protect the vital organ or not, histological and biochemical examination of the organs closely related to complications due to type-2 diabetes were also made. All these three kinds of intervention in G-K rats resulted in a statistically significant decreases in body weight, fasting glycemia, carbohydrate tolerance and insulin sensibility, compared with condition of group sham operation and preoperative status. However, the differences of insulin in serum among these groups are not statistically significant. The liver function, renal function and blood fat of group A, B and C were all improved compared to sham-operated animals and preoperative status. Among the three intervention group, weight remained lower in the BPD rats compared to the IT rats, and meanwhile, the IT group was lower than GBP group. But there were not statistically significant differences among these animals (P>0.05). Furthermore, the mean number of fasting glycemia in group B was lower than group C, the group C’s was lower than group A’s simultaneously. The carbohydrate tolerance was better improved in group A than in group B and C. But the rats in group C showed better glucose tolerance than the ones in group A and C. The gastrointestinal surgical intervention significantly prevented the aggravation in heart, vessel, liver and kidney as compared to their respective groups, with methods of pathological examination.Part threeWe explored the mechanism of gastrointestinal surgery as a treatment for type-2 diabetes in G-K rats in this part. Several kinds of hormones related to type-2 diabetes were measured during 24 weeks’ follow up, which included insulin, hormones related to enteroinsular axis and adipoinsular axis, mediators of inflammation and reactive oxygen species (ROS). The weight of enteric digestive juice was realized also. Theresult showed that, the treatment with gastrointestinal operation significantly and dose-dependently increased GLP-1, GEP, IGF-1 and reduced FFA, leptin, adiponectin, resistin in serum. However, the level of insulin, TNF-a and cortisol in serum were not significantly altered in of group A, B and C after the intervention compared to preoperative status and sham operation group. Among all the operation groups, the increase of GLP-1 in group C was statistically significant different as compared with group A and B. The mucosal weight of group A, B and C was significantly higher compared with that of sham operation group (P<0.05). Meanwhile, the ileums of transposition were notablely hyperplastic and hypertrophic after the intervention.Conclusions(1) The animal model of non-obese type 2 diabetes to be given gastrointestinal operation as the treatment is stable and reliable, which means can be used in long-term observation of foundation research and evaluation the effectiveness of clinical work.(2) Gastrointestinal surgery is an effective method to cure type-2 diabetes, which can control the blood glucose levels in G-K rats stably and persistently. And most of these effects were independent and have direct relation to gastrointestinal operation itself, not only related to weight loss and/or dietary restriction brought about by the surgery.(3) All of the three kinds of operations resulted in a stable and persistant decreases in body weight of G-K rats.(4) With advantage of reliable effect, less surgical trauma and lower rate of complication, the IT has a promising future that can be applied in clinical work as a treatment of non-obese or mild-obese type-2 diabetes to replace insulin therapy.(5) One of the most important and fundamental mechanisms that why gastrointestinal operation can release type-2 diabetes is the response of series hormones were secreted by the endocrine cell in and out of gastrointestinal tract due to exposure to meal contents soon after food intake. All these effects were independent and direct.(6) Another factor contributing to the homeostasis was a significant decrease of FAA in serum after the intervention through the adipoinsular axis way (7). GLP-1 and GIP, which were secreted by gastrointestinal endocrine cell, play very important role in the improvement of hyperglycemia in G-K rats following gastrointestinal operation through the regulation of enteroinsular axis.(8).IGF-1 also resulted in the hemeostasisi of blood glucose for long run after the gastrointestinal surgical intervention.(9) Although the glycemia level in animal model of non-obese type 2 diabetes to be given gastrointestinal operation as the treatment was regulated by varies mediators, the most important one is the mechanism of enteroinsular axis更多还原