【作者】 李峰；

【导师】 胡三元；

【作者基本信息】 山东大学 ， 外科学， 2010， 博士

【摘要】 研究背景随着生活水平的提高,糖尿病成为严重危害人类健康的慢性疾病之一,其中2型糖尿病占90%以上,而且患病率不断上升,发病年龄趋于年轻化。目前糖尿病患者全球逾1.7亿,到2030年患者数将达到3.66亿,新增患者主要集中在中国、印度等发展中国家。糖尿病引起的神经、心血管并发症造成人体严重损害,社会医疗负担巨大。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、加强运动、口服降糖药和使用胰岛素等,由于需要长期实施甚至终生维持,治疗费用高昂,不良反应等原因,患者的生活质量下降,以致患者的依从性差,很难将血糖控制在理想水平并获得长期疗效,因而最终不能避免糖尿病各种并发症的出现及进一步加重。胰腺移植和胰岛细胞移植的方法因移植排异、免疫抑制剂的副作用及手术难度大、并发症多等诸多因素制约了其临床应用及效果。胚胎干细胞和胰腺干细胞,又分别受到伦理学争论及取材不便的影响。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。上世纪80年代初,人们偶然发现胃旁路术(GBP)、胆胰转流术(BPD)治疗肥胖症患者术后,其中合并有2型糖尿病的患者不仅体质量显著下降,而且血糖得到良好控制,血浆胰岛素、糖化血红蛋白及胰岛素敏感性恢复正常,甚至可以完全脱离糖尿病药物。而且,这些患者血糖恢复到正常范围始于术后早期(<10天),远远早于体质量明显下降时间,表明外科手术控制血糖的作用是直接、独立的。此后,对袖状胃切除术、回肠转位术等其它术式进行了回顾性分析及前瞻性研究,同样发现这些外科手术对糖尿病有良好的治疗效果,2型糖尿病患者得到显著改善甚至治愈,而且治疗效果持久稳定。但有相当数量的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?如果手术治疗有效,其机理如何?国内外学者进行了相关研究,并提出了多种理论假说,但大部分的研究侧重于糖尿病的手术效果及治疗机制的初步研究,没有形成完整的、系统的理论体系,而且在关键因素的探讨上,没有形成定论。目前,对于何种术式治疗效果更佳及手术作用机理的明确阐述,尚未见相关的文献报道。为了进一步研究胃肠道重组对非肥胖2型糖尿病的近远期治疗效果,本研究选择非肥胖的2型糖尿病动物模型进行手术干预,研究不同的胃肠道手术方式对2型糖尿病的治疗效果,阐明其可能的作用机制,并对不同胃肠道重组术式进行分析比较,评估不同术式的治疗效价,以期寻找安全有效的最佳术式,为手术治疗2型糖尿病提供理论和实践依据。第一部分非肥胖2型糖尿病大鼠胃肠道重组模型的建立目的本研究对随机分组的手术组及假手术组2型糖尿病大鼠进行手术干预,建立胃肠道重组的2型糖尿病动物模型。观察胃肠道重组术式对糖尿病大鼠肝功、肾功、血脂等机体生理功能的影响。方法(1)2型糖尿病动物的选择术前检测8-10周龄GK雄性大鼠血糖、血清总蛋白(TP)、白蛋白(ALB)、肌酐(Cr)、尿素氮(BUN)、血清胆固醇(CH)、甘油三酯(TG)、游离脂肪酸(FFA)等指标,评估2型糖尿病动物模型的稳定性及是否符合实验要求。将达到2型糖尿病标准的GK大鼠以区组随机化分组法随机分组,每组GK大鼠20只,分别行三种胃肠道重组手术及相应假手术。(2)手术分组及模型的建立各手术组如下：A组：袖状胃切除术(sleeve gastrectomy, SG)组。手术方式：游离胃大弯侧至胃底,自幽门稍上方切除胃大弯侧胃体至胃底,将胃底完整切除,切除胃容积占全部胃容量的70%—80%,胃残端间断缝合。B组：Roux-en-Y胃旁路术(Roux-en-Y gastric bypass, RYGBP)组。手术方式：切除远端胃后,距Treitz韧带30cm切断空肠,远端空肠与残胃端端吻合,近端空肠距胃肠吻合口远端50cm处行肠肠侧侧吻合。C组：毕(Billroth)Ⅱ式胃空场吻合术(BⅡ)组。手术方式：切除远端胃后,缝闭十二指肠残端,距Treitz韧带20cm处上提空肠,于结肠前行胃空肠吻合。完成吻合后,排列肠道,以大网膜覆盖。袖状胃切除术、Roux-en-Y胃旁路术、毕(Billroth)Ⅱ式胃空场吻合术的假手术组分别为：Sham-A、Sham-B及Sham-C组,其手术方式为：于各手术组相应的解剖位点做原位胃肠切开及吻合术。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。记录各组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,评估2型糖尿病动物模型对各术式的耐受性。结果(1)A组(袖状胃切除术组)手术完成20例,手术成功率100%,平均手术时间47.6±7.5min,平均术后恢复时间22.4±4.8h,术后无动物死亡(2)B组手术完成20例,手术成功率100%,平均手术时间77.8±8.7min,平均术后恢复时间49.9±4.9h,术后死亡率15%,死亡原因为肠梗阻及代谢并发症。(3)C组手术完成20例,手术成功率100%,平均手术时间53.0±5.1min,平均术后恢复时间29.3±4.2h,术后死亡率5%,死亡原因为吻合口漏。(4)总计60例假手术组大鼠手术均获成功,存活率100%。(5)A、B、C三组大鼠术后肝功、肾功降低,术后第5天逐渐恢复,但尚未完全恢复至术前水平,术后2周各手术组大鼠肝功和肾功基本恢复至术前水平。此后上述指标进一步改善并维持至36周观察期结束。(6)A组手术时间、术后恢复时间、术后并发症发生率明显少于B、C组,差异有统计学意义(P<0.05)；B组手术时间、术后恢复时间、术后并发症发生率及死亡率明显高于A、C组(P<0.01)。结论(1)非肥胖2型糖尿病GK大鼠胃肠道重组模型稳定可靠。(2)不同术式对糖尿病大鼠机体的影响不同,差异有统计学意义。第二部分胃肠道重组对非肥胖2型糖尿病大鼠的治疗效果目的对胃肠道重组2型糖尿病动物模型及其相应假手术组动物进行糖尿病治疗效果的监测；分析比较不同手术方式的治疗效果。方法(1)测定术前及术后多个时间点各手术组及假手术组大鼠体质量、空腹血糖水平、胰岛素水平及游离脂肪酸等指标,并进行糖耐量试验(OGTT)和胰岛素敏感性试验(ITT),比较不同手术方式对2型糖尿病的治疗效果。(2)总观察期(术后36周)结束后,处死大鼠并取重要脏器进行病理组织学检测,以探讨手术对糖尿病所致重要脏器损伤的远期保护效果。结果(1)A、B、C三组大鼠术后体质量较术前及假手术组明显降低(P<0.05),体质量均值的大小顺序为B组<C组<A组。B组大鼠体质量显著低于A、C两组,差异有统计学意义(P<0.05)；A、C两组大鼠间体质量差异无统计学意义(P>0.05)。(2)A、B、C三组大鼠术后空腹血糖较术前及假手术组得到控制和改善(P<0.05)。血糖均值的大小顺序为B组<A组<C组,但三组间差异无统计学意义(P>0.05)。(3)A、B、C三组大鼠术后糖耐量和胰岛素敏感性均较术前及假手术组得到控制和改善(P<0.05)。A组大鼠糖耐量改善程度明显高于B、C组大鼠,差异有统计学意义(P<0.05)；B组大鼠胰岛素敏感性改善程度高于A、C两组,但三组间差异无统计学意义(P>0.05)。(4)A、B、C三组大鼠术后胰岛素水平较术前及假手术组增加(P<0.01),但三组间比较差异无统计学意义(P>0.05)(5)观察期结束后病理组织学检测显示：A、B、C三组大鼠的心脏、肝脏、肾脏和大动脉(胸主动脉)均未见明显糖尿病相关的病理学异常表现。结论(1)胃肠道重组手术对非肥胖2型糖尿病有治疗效果,能够显著改善糖代谢,获得稳定持久的血糖控制。(2)术后血糖获得持久稳定的改善,而体重量短期内即恢复并超过术前,因此,手术对2型糖尿病的治疗作用是直接的、独立的,不依赖于手术后的体质量减轻。(3)不同术式对2型糖尿病的治疗效果不同。第三部分胃肠道重组对非肥胖2型糖尿病治疗机制的研究目的检测术前、术后各组大鼠血清中与2型糖尿病发病主要相关的胃肠道激素、细胞因子水平的变化,研究手术对2型糖尿病可能的治疗机制；评价各术式疗效并选择2型糖尿病的最佳手术方式,为手术治疗2型糖尿病应用于临床提供实验支持并奠定理论基础。方法(1)检测指标：肠-胰岛轴相关激素,脂肪-胰岛轴相关因子,生长激素释放肽(Ghrelin)等,进而研究手术对2型糖尿病治疗的可能机制。(2)检测各组大鼠术后胃和小肠代偿增生情况。(3)统计方法：以上三部分数据以均数±标准差(x±s)表示,均数间采用t检验。葡萄糖曲线下面积(AUC)以梯形积分法计算。以SPSS17.0软件进行统计学分析,重复测量数据以重复测量数据的方差分析法检验,P<0.05认为差异有统计学意义。结果(1)A、B、C三组大鼠GLP-1的分泌显著高于术前及假手术组,差异有统计学意义(P<0.01)。其中B组及C组大鼠GLP-1水平高于A组,差异有统计学意义(P<0.01)(2)A、B、C三组大鼠GIP水平显著低于术前及假手术组,差异有统计学意义(P<0.05)。A组大鼠生长激素释放肽(Ghrelin)显著低于术前及假手术组(P<0.01),而B、C两组无明显改变(P>0.05)。(3)A、B、C三组大鼠游离脂肪酸(FFA)及瘦素(Leptin)显著低于术前及假手术组,差异有统计学意义(P<0.05)；脂联素(Adiponectin)显著高于术前及假手术组,差异有统计学意义(P<0.01)。(4)术后A组大鼠残胃代偿性增大；B、C组大鼠小肠粘膜重量增加。结论(1)胃肠道重组手术通过肠-胰岛轴、脂肪-胰岛轴等多种途径参与对2型糖尿病的调控。其中,多种胃肠道激素和细胞因子协同作用实现对血糖的控制。(2)胃肠道激素GLP-1及GIP通过调节肠-胰岛轴改善糖代谢,控制血糖水平,并对血糖的稳定维持起重要作用。(3) Leptin、FFA水平的降低与Adiponectin水平的升高通过脂肪-胰岛轴参与维持血糖正常和增加胰岛素敏感性,从而达到长期缓解2型糖尿病的作用。(4) Ghrelin水平的降低解除了对胰岛素分泌的抑制,并且提高了胰岛素敏感性,降低了胰岛素抵抗,改善了糖耐量,从而起到治疗糖尿病的作用。(5)2型糖尿病患者胃肠道重组术后早期血糖的快速降低及胰岛素抵抗的快速减退与肠-胰岛轴分泌激素的改变有关,而糖尿病的长期改善则与脂肪-胰岛轴中脂肪细胞因子水平的变化相关。(6)在血糖水平和胰岛素敏感性改善方面,RYGBP术式优于SG和毕Ⅱ两种术式,在手术时间、术后恢复时间、手术并发症发生率及糖耐量改善方面,SG术式优于其它两种术式。更多还原

【Abstract】 BackgroundType 2 diabetes mellitus presently afflicts more than 170 million people worldwide and is expected to affect about 366 million persons by 2030. The complications of diabetes have rendered the disease a major cause of morbidity and mortality that strains public health care funding. Diet contrrol, exercise, behavior modification, oral hypoglycemic agents and insulin therapy have little efficacy with respect to the return of euglycemia. Because of graft rejection, the side effect of immunosuppression, higher complication rate and other factors, it has restricted the clinical application of the pancreatic gland transplant and the islet cells transplant. Therefore, seeking for a safe, satisfying compliance and long-term effective method of improving the diabetes is a key problem at present which is worth discussing intensively.Early in 1980s, a wide array of investigations has shown bariatric surgery to be an effective means of not only resolving obesity and its co-morbidities, but also well improving the blood sugar, insulin, glycosylated hemoglobin and insulin sensibility, even people can be completely far away from any drug. Because euglycemia often occurs long before significant weight loss after bariatric surgery, the control of diabetes likely stems from direct effects of the operations rather than amelioration of obesity. Moreover, a great amount of retrospective analysis and prospective study were made on the efficacy of other bariatric surgeries, such as gastric bypass (GBP), biliopancreatic diversion (BPD), sleeve gastrectomy (SG), and ileal transposition (IT), it also showed better control of diabetes what most patients in the study were cured or well improved and no recurrence after 10 years. It is put forward if it is possible that the bariatric surgery is effective for non-morbidly obese individuals? And then what are the mechanisms? Researchers have conducted related research and proposed kinds of theory hypotheses, but no comprehensive or systematic theories formed.It supposed that the alteration of gastrointestinal hormone and some endocrine factors plays an important role in the progress of type 2 diabetes. Hormones such as GLP-1 and GIP are related to enteroinsular axis, leptin, resistin and adiponectin are related to adipoinsular axis, insulin-like growth factor-1 (IGF-1) and inflammation mediators also changed and have a synergistic effect.In order to study the effect and mechanism involved in the associations between gastrointestinal intervention and type 2 diabete, and provide the most effective procedure for the treatment of type 2 diabetes, nonobese type 2 diabetic animals were chosen and with respect to the purpose:Firstly, compare and evaluate the efficacy of different kinds of operations to further confirm the resolution of gastrointestinal surgery. Secondly, provide the optimal choice for controlling type 2 diabetes. Finally, study certain factors related to the pathogenesis of diabetes, explore and bring forward the possible mechanisms of gastrointestinal intervention for type 2 diabetes for the purpose of providing supports for the transition of gastrointestinal interventios to clinical application.PartⅠConstruction of animal models of nonobese type 2 diabetesObjectivGK rats, the nonobese spontaneously diabetes rats were randomly divided into the operation groups and the sham operation groups, and all rats underwent different kinds of gastrointestinal operations. The live function and renal function of the GK rats were detected after operations.Methods(1) Selection of type 2 diabetes animalFirst, the 8-10 weeks old GK rats were selected and fasting glycemia, plasma lipid (cholesterol, triglycercide and free fatty acid), liver function (serum total protein and albumin), and renal function (creatinine and blood urea nitrogen) were measured before the intervention in order to evaluated the stability of type 2 diabetes animal models. Then,120 male GK rats were randomly divided into three gastrointestinal operation groups and the corresponding sham operation groups (n=20 in each) that all rats underwent different kinds of gastrointestinal operations. (2) Surgical groups of gastrointestinal operationsGroup A:Sleeve gastrectomy (SG) surgery was conducted in this group. The SG operation was performed as described by de Bona Castelan J, et al. First, define the the line of incision for the longitudinal sleeve gastrectomy which covered the entire gastric fundus and much of the lumen. Then 70% to 80% of total stomach was dissected and removed.Group B:Roux-en-Y gastric bypass (RYGBP) surgery was performed in this group. The RYGBP operation involved (1) a midline abdominal incision was made, (2) distal gastrectomy, (3) the jejunal was divided 30 cm distal to the ligament of Treitz and the distal limb connected to the stomach, (4) small bowel continuity was maintained by an entero-enterostomy between the previously divided proximal jejunum and the distal limb which was so-called Roux limb and at least 50 cm long.Group C:BillrothⅡsurgery was taken in this group. The BillrothⅡoperation was performed as follows:(1) the duodenum was separated from the stomach, (2) distal gastrectomy, (3) an gastrojejunostomy was made with vestige stomach and proximal jejunum.Sham-A、Sham-B and Sham-C group:Sham surgeries involved the same abdominal incisions, transections and re-anastomosis of the gastrointestinal tract at corresponding sites where performed in the SG, RYGBP and BillrothⅡgroups. If necessary, sham operations were prolonged to achieve similar operative times and anesthetic effect as those observed for SG, RYGBP and BillrothⅡ.The surgery time of different groups was strictly recorded, the first defaecation time, serving as an indication of postoperative recovery time, and the occurrence of postoperative complications were observed and recorded carefully.Results(1) Group A:Rate of operative success was 100% of 20 cases and no death occurred in sleeve gastrectomy group. Mean operative time was 47.6±7.5 min, and mean postoperative recovery time (defined to begin at the end of the operation and to end with the first defecation) was 22.4±4.8h.(2) Group B:Rate of operative success was 100% of 20 cases. Mean operative time was 77.8±8.7 min, and mean postoperative recovery time was 49.9±4.9h. Two rats died of intestinal obstruction and one metabolic complication at postoperative 5 day,34 day and 92 day respectively. The postoperative mortality was 15%.(3) Group C:Rate of operative success was 100% of 20 cases. Mean operative time was 53.0±5.1 min, and mean postoperative recovery time was 29.3±4.2h. One rats died of anastomotic leakage on the fourth day. The postoperative mortality was 5%.(4) All the 60 rats of sham surgery groups were performed successfully with no death, and the postoperative survival rate was 100%.(5) Liver function and renal function were gradually recovered during the first week postoperatively and returned to the preoperative level two weeks later. Afterwards, liver function and renal function resumed to normal level and maintained until the end of the 36-week observation period.(6) The operative time, postoperative recovery time and postoperative morbidity were much shorter or lower in group A and there were statistically significant differences between group A and group B or C (P<0.05). The indexes above-mentioned were much longer and higher in group B and there were statistically significant differences between group B and group A or C (P<0.01)Conclusions(1) GK rats exhibit stable as the type 2 diabetes animal models and can meet the requirement for gastrointestinal interventions.(2) Different surgical operations resulted in different effects on the GK rats, and statistically significant differences were observed between groups.PartⅡThe treatment effect of gastrointestinal intervention on nonobese type 2 diabetesObjectivOn the one hand, different kinds of indexes related to type 2 diabetes were measured before and after operation in order to evaluate the efficacy of gastrointestinal surgery, on the other hand, comparisons of the therapeutic effect of different gastrointestinal interventions were made.Methods(1) Weight, fasting glycemia and serum insulin were measured and well documented. In addtion, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were also performed before and at several time points after operation.(2) After the full 36-week observation period ended, all the animals were sacrificed and specific organs were studied by means of pathologic examinations in order to clear the protection of gastrointestinal operation for type 2 diabetes.Results(1) There were statistically significant differences in weight loss of GK rats underwent A, B, and C gastrointestinal surgeries, compared with sham surgery groups and preoperation (P<0.05)(2) It resulted in statistically significant improvement in fasting glucose level, insulin sensibility and serum insulin in GK rats underwent A, B, and C gastrointestinal surgery, compared with sham surgery groups and preoperation (P<0.05). However, there were no statistically significant differences among the three groups (P>0.05).(3) It leaded to significant amelioration on oral glucose tolerance test (OGTT) in GK rats underwent A, B, and C gastrointestinal surgery, compared with sham surgery groups and preoperation (P<0.05).The improvement of OGTT in group A was much better than that in other two groups (P<0.05)(4) The gastrointestinal surgical intervention significantly prevented the aggravation of diabetes in heart, aorta, liver and kidney compared with sham groups by means of pathological examination.Conclusions(1) Remarkably therapeutic effect was observed on type 2 diabetes and sustainable control on glucose metabolism was obtained as a result of gastrointestinal intervention.(2) The surgical procedure played an important and direct role in the improvement of type 2 diabetes which was independent on the weight loss.(3) Different surgical procedure leads to dissimilar effect.PartⅢThe mechanism of gastrointestinal intervention as a treatment for type 2 diabetesObjectivTo further explore the therapeutic mechanism of gastrointestinal intervention as a treatment for type 2 diabetes, gastrointestinal hormones, endocrine factors and inflammation mediators which related to the pathogenesis of type 2 diabetes are selected and measured periodically before and postoperation. Apart from the key goal mentioned above, an optimal surgical procedure should be described and confirmed not only to elucidate the correlation between surgical intervention and type 2 diabetes but to establish and enforce the theoretical basis for the transition to clinical application.Methods(1) Hormones or cytokines related to enteroinsular axis and adipoinsular axis and plasma Ghrelin (growth hormone releasing peptide) were put into study and further to clarify the therapeutic mechanism.(2) The stomach and small intestine were studied in order to explore the compensatory hyperplasia in response to surgical intervention.(3) Data are expressed as mean±SD and trapezoidal integration was applied to calculating areas under curves for OGTT and ITT. Statistical analysis was performed using repeated-measures ANOVA for repeated measures and the Student’s t-test as appropriate. All statistical procedures were performed using SPSS version 17.0, P values<0.05 were considered to be statistically significant.Results(1) The result displayed that gastrointestinal intervention significantly increased insulin, GLP-1 and adiponectin (P<0.01), while dramaticlly reduced GIP, FFA and leptin (P＜0.05)(2) Ghrelin levels in group A decreased dramatically after surgery and there were statistically significant differences compared with other groups (P<0.01). GLP-1 levels for group B and group C were statistically significant higher compared with other groups (P＜0.01).(3) The stomach was hyperplastic and hypertrophic after the intervention, and the weight of mucosal in group B and group C was significant higher compared with the sham groups.Conclusions(1) Gastrointestinal intervention is an effective method in the treatment for type 2 diabetes that result in significant and durable glycemic control, and lead to improvement or resolution of diabetes related co-morbidities.(2) The enteroinsular axis performs an important function in the mechanism of diabetes pathogenesis which is related to the short-term effect of gastrointestinal intervention.(3) GLP-1 and GIP play an important role in the improvement of glycometabolism and treatment of diabetes after gastrointestinal intervention via the regulation of enteroinsular axis.(4) Leptin、FFA and adiponectin contribute to the management of diabetes by means of adipoinsular axis which is responsible for the long-term resolution of type 2 diabetes.(5) Due to the decrease of plasma Ghrelin, the inhibition of insulin secretion is released, as a result, the glucose levels, insulin sensitivity and insulin resistance were improved. Therefore the lower level of Ghrelin is considered playing a therapeutic role in type 2 diabetes.(6) SG surgery is the optimal procedure with advantages of acceptably low morbidity and mortality, reliable and durable therapeutic effect on type 2 diabetes.更多还原