【作者】 王志青；

【导师】 胡三元；

【作者基本信息】 山东大学 ， 外科学（专业学位）， 2014， 博士

【摘要】 第一部分2型糖尿病肾病大鼠模型的建立背景在世界范围内,糖尿病发病率呈逐渐上升趋势。其中90%以上是2型糖尿病。糖尿病肾病(Diabetes Nephropathy, DN)是糖尿病的严重并发症,是导致终末期肾衰竭的主要原因之一。在西方人群中己成为终末期肾脏疾病(end stage renal disease, ESRD)最常见的病因。美国肾脏病数据系统(USRDS)2012年最新数据显示：在2010年,新增透析患者中,糖尿病肾病患者超过了50%。在我国,糖尿病肾病患病率仅次于慢性肾小球肾炎,排第二位。研究显示糖尿病病程达10-20年者50%合并糖尿病肾病,并且已成为糖尿病的主要死亡原因。针对糖尿病及其并发症的基础研究大多通过建立动物模型来进行,可靠的糖尿病肾病动物模型是研究糖尿病肾病发生机制及干预措施的重要手段。理想的动物模型应该具有在任何实验室均可复制,又可以模拟人类糖尿病肾病的发展病程及特点,同时,兼具价格便宜,方便普及的优点。糖尿病肾病模型同样分为1型和2型糖尿病肾病模型。建立2型糖尿病动物模型的方法较多,主要包括自发性糖尿病模型及化学药物诱导模型。链脲佐菌素(streptozotocin, STZ)是最常用药物。小剂量STZ+高脂饮食是诱导2型糖尿病最常用的方法。大鼠高脂饮食饲养1个月左右形成胰岛素抵抗,然后腹腔注射小剂量STZ损伤部分胰岛功能。此方法能模拟人2型糖尿病过程,形成非胰岛素依赖性糖尿病。另有报道,单肾切除+高脂饮食+小剂量STZ用于糖尿病肾病模型建立。但尚无应用于手术治疗糖尿病肾病的实验研究的报道。为检验适合用于糖尿病手术研究的糖尿病肾病动物模型,我们采用上述两种方法建立糖尿病大鼠模型,然后根据其病程自然发展为糖尿病肾病模型大鼠。筛选成模大鼠进行糖尿病手术干预,初步了解术后大鼠死亡率及肾功能改变情况。方法将30只SD大鼠随机分为3组,即对照组、两联组及肾切除两联组。其中对照组大鼠采用高脂饲料喂养4周,然后喂养普通饲料。两联组大鼠进行高脂饲料喂养4周以诱导胰岛素抵抗,然后按35mg/kg剂量进行一次性腹腔STZ注射。然后普通饲料喂养。三联组先行右肾切除,一周后处理同两联组。大鼠于STZ注射后3天尾静脉测量随机血糖,血糖>16.7mmol/L视为2型糖尿病诱导成功。大鼠继续普通饲料喂养至12周。然后分别对两联组及三联组大鼠实施十二指肠空肠旁路术干预。术后继续喂养观察至16周期间检测大鼠体重、进食量、尿量等一般指标,并进一步检测检测大鼠血糖变化以及血脂、肌酐变化。检测大鼠尿蛋白排泄率,肾小球滤过率等肾功能指标。结果1.两联组及三联组大鼠总体死亡率分别为25%和66.67%,其中实施十二指肠空肠旁路术后死亡率分别为18.18%及55.56%。2.两联组及三联组大鼠STZ诱导DM后均出现明显体重增长变缓、进食量增加、尿量增加血糖升高等糖尿病表现。DJB术后再次出现一过性体重下降,而后缓慢上升。3.血肌酐第8、12、16周,三联组相比对照组可见明显升高,差异有显著性(p<0.05)。三联组大鼠血肌酐呈上升趋势,在第8、12、16周相比实验初始有显著性升高(p<0.05)。两联组及三联组大鼠尿肌酐排泄在第12及16周出现下降,与对照组相比差异有显著性(p<0.05)。而两联组与三联组相比无显著性差异。4.检测血总胆固醇及甘油三酯,可见两联组及三联组大鼠TG以及TC水平均在第8及12周出现显著性升高。高于对照组(p<0.01)。5.检测尿微量白蛋白排泄率,三联组在8周开始出现上升,与对照组相比有显著性差异(p<0.05)。而两联组则至第12周出现明显上升,差异有显著性(P<0.05)。在第8、12周以及16周(DJB手术干预后),三联组尿微量白蛋白排泄均高于两联组(P<0.05)。6.肾小球滤过率检测发现两联组及三联组大鼠GFR在8周开始出现上升,与对照组相比差异有显著性(P<0.05)。至第12、16周上升明显。手术干预后可见曲线上升变缓,两联组及三联组变化趋势一致。结论1.利用高脂饮食辅助小剂量STZ腹腔注射诱导2型糖尿病大鼠模型技术成熟,成功率高。利用该方法所建立的2型糖尿病大鼠持续喂养8周可出现明显的蛋白尿及肾小球率过滤升高。2.单肾切除配合高脂饮食辅助小剂量STZ腹腔注射后所建立糖尿病模型大鼠在STZ注射后4周即出现糖尿病肾病表现,出现较高的尿蛋白排泄率及肾小球滤过率。该模型比非肾切除者出现糖尿病肾病症状早,缩短建模时间。3.两者均实施DJB手术干预后其血糖水平均可得到良好控制。尿蛋白排泄率及肾小球滤过率变化趋势基本一致。4.三联组大鼠实施DJB手术后死亡率及总体死亡率较高,如应用于糖尿病手术研究尚待进一步完善。第二部分十二指肠空肠旁路术对2型糖尿病肾病大鼠肾功能的影响前言糖尿病肾病是糖尿病病人疾病进展所出现的临床综合征之一,蛋白尿为其典型的临床表现。在国外,大约30%的新增2型糖尿病患者尿蛋白水平异常,其中约75%的患者患有微量白蛋白尿,25%的患者存在较明显的糖尿病肾病表现。国内研究显示糖尿病病程达10-20年者50%合并糖尿病肾病,并且已成为糖尿病的主要死亡原因。足细胞(podocyte),是附着与肾小球基底膜(GBM)外侧的肾小囊脏层上皮细胞,足细胞损伤往往表现为明显的蛋白尿。足细胞的丢失往往是糖尿病肾病早期的病理改变,并预示着临床糖尿病肾病的发生和发展有研究表明,通过药物和饮食锻炼等严格的血糖控制能够延缓微量白蛋白尿的发展,稳定或减少尿蛋白的排泄,并且减慢慢性肾衰竭的进展。但治疗效果有限,并不能完全阻止糖尿病肾病的进展。针对减肥手术的实验及临床研究发现,减肥手术在治疗肥胖的同时,其合并的糖尿病亦得到缓解。减肥手术抑或糖尿病手术在国外临床上已得到广泛应用。其治疗糖尿病的效果已得到肯定。然而,肥胖是慢性肾功能障碍的一个独立因素,减肥手术治疗糖尿病的同时带来体重的下降,而体重下降本身可使肾功能得到改善。所以,减肥手术本身带来的肾功能改善原因是多方面的,包括体重、血糖及其他方面的因素。故并不能够代表糖尿病手术本身对肾功能的影响。十二指肠空肠旁路术(Duodenal-Jejunal Bypass, DJB)假设糖尿病的改善不依赖于手术的减肥作用,而是手术本身所致。那么直接将十二指肠及空肠上段旷置,不改变胃容积,同样可以起到改善糖尿病作用。实验结果证实,旷置十二指肠及空肠上段可直接起到控制糖尿病的目的。在本实验中,我们采用第一部分两联组大鼠的建模方式建立糖尿病肾病大鼠模型,然后实施DJB手术,并与假手术及对照组进行比较,从而进一步对糖尿病手术后大鼠肾功能的影响进行研究。方法将40只SD大鼠分为两组,对照组(CON)8只,采用常规饲料喂养。其余32只作为实验组给予高脂饲料喂养。4周后,将该组大鼠禁食过夜(12h),然后行腹腔注射STZ(按35mg/kg)以诱导高血糖。3天后取尾静脉血测随机血糖。随机血糖≥16.7mmol/L者纳入下一步实验组,未达标者再给予10mg/kg一次STZ注射。将实验组大鼠随机分为三组如下：DJB手术组(DJB组),假手术组(S-DJB组),糖尿病组(DM组)。8周后分别对DJB和S-DJB组实施手术干预,DM和对照组继续喂养观察,不进行任何干预。所有大鼠继续喂养观察至术后8周。期间检测大鼠体重、进食量、尿量等一般指标,并进一步检测检测大鼠血糖变化以及血脂、肌酐、胱抑素-C变化。检测大鼠尿蛋白排泄率,肾小球滤过率等肾功能指标。实验结束,将大鼠处死,左‘肾称重,右肾甲醛固定,进一步行病理分析,检测大鼠肾小球系膜扩张率等。然后进行免疫组化,检测大鼠肾小球足细胞突触极蛋白表达率,明确DJB手术对大鼠肾功能及病理变化的影响。结果1. DJB、S-DJB及DM组大鼠体重基本保持一致,只有在手术干预后2周内,DJB及S-DJB组大鼠出现明显的体重下降p<0.05)。在整个实验过程中,各个时间点DJB及S-DJB组大鼠进食量相比均无显著性差异。只有在实施手术干预后最初2周内DJB及S-DJB组与DM组相比出现进食量下降,差异有显著性(p<0.05)。2. S-DJB和DM组大鼠拥有较高的肾重及肾重／体重比值,与对照组及DJB组相比有显著性差异p<0.05)。但S-DJB和DM组相比以及对照组和DJB组相比差异均无显著性。3.DJB组大鼠在2周内出现血糖水平下降,接近正常水平,其后血糖水平一直保持稳定。与S-DJB和DM组相比,差异有显著性(p<0.01)。4.实验结束测DJB组大鼠总胆固醇及甘油三脂水平均低于对照组、S-DJB和DM组,差异有显著性(P<0.05)。血清胱抑素-C水平检测发现,实验结束时DJB、S-DJB及DM组大鼠血清胱抑素-C水平相比对照组均有明显下降(P<0.05)。但DJB组下降幅度要明显低于S-DJB及DM组大鼠(P<0.05)。四组大鼠血肌酐水平检测未发现显著差异。5.各组大鼠在术后检测尿24小时微量白蛋白排泄率呈逐渐上升趋势。DJB组大鼠较S-DJB及DM组上升较慢。在术后2、4、6及8周DJB组大鼠尿微量白蛋白排泄率均明显低于S-DJB及DM组,差异有显著性(P<0.05)。6. DJB、S-DJB及DM组大鼠术前比较GFR并无显著性差异,但均高于对照组。术后均呈先上升后下降趋势。DJB组大鼠GFR曲线低且平缓,第2、4、6、8周DJB组大鼠GFR水平均明显低于S-DJB及DM组,有显著性差异(p<0.01)。但S-DJB与DM组之间比较并无显著性差异发现。7.S-DJB及DM组大鼠平均肾小球面积较对照组及DJB组有明显增大,差异有显著性(P<0.05)。但DJB组及对照组相比,肾小球面积无显著性差异发现。而DJB、S-DJB及DM组大鼠肾小球系膜扩张率均较对照组有明显上升,差异有显著性(p<0.05)。8.肾小球突触极蛋白在对照组大鼠肾小球内有较高表达,而在S-DJB及DM组大鼠中均有较大程度的表达降低。DJB组大鼠突触极蛋白表达则处于对照组和S-DJB及DM组之间。低于对照组但高于S-DJB及DM组,差异均有显著性(P<0.05)。结论1.DJB手术可使糖尿病肾病模型大鼠24小时尿微量白蛋白排泄率、肾小球滤过率及血清胱抑素-C水平得到改善。2.DJB手术可改善糖尿病肾病大鼠血清总胆固醇及甘油三酯水平。3.免疫组化结果显示,DJB手术可抑制糖尿病肾病大鼠肾小球足细胞的损伤及丢失。4.糖尿病肾病大鼠肾小球系膜扩张率在DJB术后8周时仍未得到明显改善。5.DJB手术有可能通过抑制糖尿病肾病大鼠肾小球足细胞的丢失及损伤从而改善术后肾小球滤过率及24小时尿微量白蛋白排泄率。更多还原

【Abstract】 PART I THE ESTABLISHMENT OF RAT MODELS OF TYPE2DIABETIC NEPHROPATHYBackgroundDiabetic nephropathy is a leading cause of end-stage renal disease, and its incidence is increasing worldwide. More than ninety percent of them are type2diabets mellitus.Once diabetic nephropathy becomes overt, there is no curative therapy, and most patients eventually progress to end-stage renal disease.Animal models of diabetic nephropathy is wildly used to investigate diabetes and the complications. Although numerous animal models have been established in rodents and these diabetic animals develop kidney disease that resembles human disease, no single animal model develops renal changes identical to those seen in humans. Therefore, to fully investigate the disease pathogenesis, appropriate animal models are essential.MethodsThirty SD rats were randomly divided into three groups:the control group(CON), model group(MOD) and the singal-nephrectomy group(NEP). A high fat diet(HFD) and standard diet (STD) were provide. In the NEP group, all rats were treated with singal-nephrectomy. One week later, all rats were fed on HFD for4weeks,and then rats of CON received STD. Rats of MOD and NEP were treated with STZ to induce hyperglycemia. After three days, random blood glucose was measured in duplicate from tail vein blood. Rats with random blood glucose>16.7mmol/L were considered to have diabetes established. At week12, the MOD and NEP group were treated with surgery, while the control group did not undergo any experimental manipulations. All rats were then observed for4weeks post operatively. Body weight, food intake and random blood glucose were measured every week preoperatively and postoperatively. Blood samples were collected from the tail vein and the serum creatinine, Cys_C levels, fasting TC and TG levels were measured. Individual rats were placed in metabolic cages to obtain urine collections. Then the urine volume (Vu), urine creatinine (Cu), and UAER were measured and the GFR was calculated by equation.ResultsThe total mortality of MOD and NEP group were25%and66.67%and the operative mortality were18.18%and55.56%。The features of DM were observed either in MOD group or in NEP group. The serum creatinine levels in NEP group were significantly higher than in CON group at week8, week12and week16. The TC and TG levels in MOD and NEP groups were significantly higher than in CON group at week8and week12. The urine creatinine levels in MOD and NEP groups were significantly lower than in CON group at week12and16. No significant was found within MOD and NEP groups. The urine albumin excretion rate was ascending at week8in NEP group and week12in MOD group.The UAER level in NEP group was significantly higher than in MOD group at week8,12and16.The GFR was ascending at week8in MOD and NEP groups and was significantly higher in NEP group than in MOD group at week8,12and16.ConclusionThe rat model of DN through high fat diet and low dose of STZ injection was representing high UAER and GFR8weeks after STZ injection. While the NEP model represented at4weeks after injection. The NEP method uses less time to establish a DN model. The two models represented similar tendency in blood glucose, UAER and GFR levels even after surgery was performed. While further and long-term research is still needed for the NEP model of DN before its using on diabetes surgical procedures. PART II THE EFFECTIVENESS OF DUODENAL-JEJUNAL BYPASS ON RENAL FUNCTION IN A TYPE2DIABETIC NEPHROPATHY RAT MODELBackgroundDiabetic nephropathy(DN) is one of the most serious complications of type2diabetes mellitus (T2DM), usually accompanied by a progressive rise in proteinuria followed by decline in renal function. In the past two decades, there has been a continual increase in the incidence of ESRD among patients with diabetes, predominantly those with T2DM. Up to30%of people with newly diagnosed T2DM have abnormally high urine albumin levels, and about75%of these people have microalbuminuria and about25%overt DN.Diabetes surgery is growing in popularity and has been shown to have marked effects on diabetes. The improvement of diabetes through RYGB and several other surgical procedures is always accompanied with obvious weight loss, although such loss occurs several weeks after glucose regulation. While several studies have shown it also induces some renal injury. However, the interaction of diabetes surgery on renal function is less clear. In this study, we examined renal function and histological changes in rats with diabetic nephropathy through a duodeno-jejunal bypass (DJB) operation.MethodsRats had diabetes induced through injection of streptozotocin (STZ) and were randomly assigned to the DJB group (DJB), Sham-DJB group (S-DJB) and diabetes group (DM). Six age-matched normal rats were assigned as the control group. DJB and sham surgery were performed. Body weight, kidney weight, food intake and random blood glucose were measured every week preoperatively and postoperatively. The serum creatinine (creatinine in plasma, Cp), Cys_C levels, fasting TC and TG levels were measured using an automatic biochemistry analyzer at the laboratory of the Fourth Hospital of Jinan. The urine volume, urine creatinine, UAER and GFR were measured. Kidney sections were stained with periodic acid-Schiff (PAS), and then the glomerular mesangial area, glomerular area and the ratio of mesangial area/glomerular area which showed the index of mesangial expansion was determined. The immunohistochemical studies of synaptopodin for podocytes in renal sections were also performed.Results1. After surgical intervention In DJB and S-DJB groups, there was a significant decrease in body weight compared with the DM group within2weeks (P<0.05), while no significant differences were found between DJB and S-DJB groups. At the end of study, no significant differences were found between DJB, S-DJB and DM groups in body weight. No significant difference in food intake between DJB and S-DJB groups were found at any stage of the study.2. The KW and KW/BW ratio was higher in S-DJB and DM groups than in DJB and control groups (P<0.05. No significant differences were found in KW and KW/BW ratio between DJB and control groups or S-DJB and DM groups.3. The blood glucose was significantly dropped in the DJB group within2weeks after DJB surgery.4. DJB surgery demonstrated a significant reduction in TC and TG levels compared with S-DJB, DM, and control groups(P<0.05).5. The increase of24h urinary albumin excretion was blocked in the DJB group after surgery (P<0.05).6. DJB group showed less GFR increase than that of S-DJB and DM groups after surgery.7. DJB surgery significantly ameliorated the increase of the mesangial matrix but not the IME compared with that of DM and S-DJB groups.8. In the DJB group, the positive area of synaptopodin staining was higher than that of DM and S-DJB groups (P<0.05). It showed an amelioration of synaptopodin expression area and a return toward the values of normal rats. Conclusion1.DJB surgery ameliorated renal function by inducing an improvement of UAER, GFR and Cys-C levels4and8weeks after surgery.2. It also improved lipid metabolism by decreasing fasting total serum cholesterol (TC) and triglyceride (TG) levels.3. Immuno-staining of synaptopodin showed podocyte injury was also improved in DJB glomeruli compared with sham and DM groups.4.Histological analysis showed that the mesangial expansion was not significantly prevented8weeks after DJB surgery.5.DJB ameliorated renal function in UAER and GFR but not mesangial expansion in a DN rat model. The improvement of renal function may be attributed to reversing the injury or loss of podocytes after DJB surgery.更多还原