【作者】 赵占正；

【导师】 刘章锁；

【作者基本信息】 郑州大学 ， 内科学， 2010， 博士

【摘要】 背景和目的终末期肾脏病(end stage renal disease, ESRD)已成为21世纪全人类面临的主要公共健康问题之一。腹膜透析(peritoneal dialysis, PD)是终末期肾脏病有效的治疗方法,具有保护残余肾功能、花费相对血液透析低等优势。但由于腹膜透析的并发症,如腹膜炎、营养不良、腹膜超滤衰竭,尤其透龄4-5年的患者,腹膜超滤衰竭发生率高达36%,已经成为腹膜透析患者被迫退出腹透的主要原因之一,因此探讨并干预腹膜透析超滤衰竭的发生与发展非常重要。目前认为超滤衰竭的发病机制主要有三方面的因素：1、腹膜血管表面积的不断增加：约50%到75%的超滤衰竭是由于腹膜血管新生导致腹膜血管表面积的不断增加,使得小分子溶质转运加快致腹腔渗透压递度迅速消失,使腹膜超滤功能丧失；2、因为水通道蛋白失功能；3、腹膜间质/淋巴系统吸收增加。腹膜血管新生可能是超滤衰竭发生的主要机制,调节血管新生的诱导剂有血管内皮生长因子(vascular endothelial growth factor,VEGF)、成纤维细胞生长因子(fibroblast growth factor,)等其它可溶性因子；抑制剂包括血管新生抑制素、内皮细胞抑制素(endostain, ES)等。目前认为血管生成时受到诱导因子和抑制因子的双重调节,正常情况下处于平衡状态,一旦诱导剂占优势,则激活血管长出新生血管,反之则使血管硬化。长期行腹膜透析的病人在其腹膜透析液中发现一些血管诱导剂增加,如VEGF、bFGF等,而且目前公认为血管内皮生长因子在腹透失超滤的发病机制中,是促进血管新生的主要因子,在血管新生中居于主导地位,并发现它在腹膜毛细血管内皮细胞和腹膜间皮细胞(peritoneal mesothelial cells, PMc)有阳性表达,但这些导致血管新生的因子表达增加的具体机制目前仍不清楚。综上所述我们有必要探讨腹膜血管新生在腹透失超滤发生机制所起的作用并进行干预研究,以便为延缓腹透失超滤的发生提供理论根据和实验依据,从而为多环节防治腹膜透析超滤衰竭的发生发展提供有效的治疗手段。因此本研究共分三部分：第一部分腹膜血管新生引起腹膜超滤衰竭的动物实验研究构建5/6肾切除尿毒症大鼠模型,进而构建尿毒症腹膜透析大鼠模型。检测大鼠腹膜组织VEGF、bFGF及ES基因和蛋白质水平的表达变化,以及检测腹膜组织毛细血管密度(MVD),探讨腹膜新生血管引起大鼠腹膜透析超滤衰竭的机制,为了下一步干预实验打好理论基础。第二部分腹膜血管新生引起腹膜超滤衰竭的临床研究留取正常对照者(取自非尿毒症的腹部择期手术患者,排除任何可累及腹膜的疾病)、尿毒症非透析患者(尿毒症患者首次腹透置管时)以及腹透超滤衰竭患者(尿毒症患者拔出腹透管时)的腹膜活检标本。检测血管内皮生长因子、碱性成纤维生长因子和内皮抑素基因和蛋白质水平的表达,检测腹膜组织毛细血管密度。为下一步干预腹膜透析超滤衰竭的研究奠定理论基础。第三部分重组人内皮抑素(恩度)干预腹膜血管新生的动物实验研究应用重组人内皮抑素(恩度),干预大鼠腹膜透析腹膜血管新生,从而起到抑制腹膜血管新生,干预腹膜透析超滤衰竭的作用,为下一步治疗临床腹膜透析患者超滤衰竭提供实验根据。第一部分腹膜血管新生引起腹膜超滤衰竭的动物实验研究一.资料与方法SD雄性大鼠42只,体重180-200g。构建5/6肾切除尿毒症大鼠模型,进而构建尿毒症腹膜透析大鼠模型。实验设立正常组(n=8只),手术组三组：尿毒症组(n=8只)、1.5%腹膜透析组(n=8只)、4.25%腹膜透析组(n=8只)。对各透析组大鼠进行规律腹透,每日每只20ml腹透液从大鼠颈部腹透管注入,腹腔保留2h后放出液体,规律透析28天后,取各组大鼠新鲜腹膜组织分别做逆转录聚合酶链反应(RT-PCR)及组织免疫组化,检测VEGF、bFGF及ES基因和蛋白质水平的表达变化,以CD34染色检测腹膜组织毛细血管密度(MVD),并进行统计学比较和基因水平的相关性分析,选取a=0.05作为差异具有统计学意义的检验水准。二.结果1. VEGF、bFGF及ES在正常大鼠腹膜组织中呈阳性表达。2.与正常组相比较,尿毒症组、1.5%腹膜透析组、4.25%腹膜透析组,VEGF、bFGF在基因及蛋白的表达水平均升高,差异具有统计学意义(p<0.05)。3.正常组、尿毒症组、1.5%腹膜透析组及4.25%腹膜透析组大鼠腹膜组织中均有ES mRNA表达。其中正常组为0.47±0.05；尿毒症组为0.45±0.04；1.5%腹膜透析组为0.46±0.04；4.25%腹膜透析组为0.47±0.03；各组间表达差异无统计学意义(P>0.05)。4.正常组大鼠腹膜组织中ES蛋白表达积分0分；尿毒症组大鼠腹膜组织中积分2分；1.5%腹膜透析组大鼠腹膜组织中积分4分；4.25%腹膜透析组大鼠腹膜组织中呈高表达,积分9分。5.与正常组相比较,尿毒症组、1.5%腹膜透析组、4.25%腹膜透析组大鼠腹膜组织中MVD的表达均增高,组间差异均有统计学意义(p<0.05)。第二部分腹膜血管新生引起腹膜超滤衰竭的临床研究一.资料与方法留取正常对照者、尿毒症非透析患者以及腹透超滤衰竭患者的腹膜活检标本。将所留取的腹膜标本分别行逆转录聚合酶链反应(RT-PCR)和组织免疫组化染色,检测血管内皮生长因子、碱性成纤维生长因子和内皮抑素基因和蛋白质水平的表达。行CD34染色,计数腹膜组织毛细血管密度。二.结果1. VEGF、bFGF及ES的mRNA在各组人腹膜组织均有表达。与正常相比较,尿毒症非透析组、PD组腹膜组织VEGF、bFGF mRNA表达上调,差异具有统计学意义(P<0.05)；与尿毒症非透析组相比较,PD组腹膜组织VEGF、bFGF mRNA表达上调,差异具有统计学意义(P<0.05)。ES mRNA各组表达无差异。2.各组腹膜组织均表达VEGF、bFGF及ES。与正常相比较,尿毒症非透析组、PD组腹膜组织VEGF、bFGF、ES蛋白表达上调,差异具有统计学意义(P<0.05)；与尿毒症非透析组相比较,PD组腹膜组织VEGF、bFGF、ES蛋白表达上调,差异具有统计学意义(P<0.05)。3.MVD计数结果在正常组可见新生毛细血管较少或无,与正常组相比较,尿毒症非透析组、腹膜透析超滤衰竭组人腹膜组织MVD计数增加(P<0.05),差异具有统计学意义。第三部分重组人内皮抑素(恩度)干预腹膜血管新生的动物实验研究一.资料与方法清洁级雄性SD大鼠43只,体质量为180-200g。构建5/6肾切除尿毒症大鼠模型,进一步构建尿毒症腹膜透析大鼠模型。实验分为A：对照组(正常组：n=8只),B：尿毒症非透析组(5/6肾切除组：n=8只)、C：尿毒症腹膜透析组(生理盐水对照组：n=8只)、D1：重组人内皮抑素治疗1组(10mg/kg治疗组：n=8只),D2：重组人内皮抑素治疗2组(40mg/kg治疗组：n=8只)。对C、D1、D2组大鼠进行规律腹透,每日4.25%的腹透液从大鼠颈部腹透管注入,腹腔保留2h后放出液体,规律透析28天。D1、D2组(重组人内皮抑素治疗1、2组)在规律腹透期间,隔天皮下注射重组人内皮抑素,连续给药14次,直至腹透第28天结束,留取各组大鼠新鲜腹膜组织,采用免疫组织化学及逆转录聚合酶链反应(RT-PCR)的方法检测腹膜组织VEGF基因及蛋白水平的表达,用CD34染色观察各组大鼠腹膜血管数并计算腹膜组织毛细血管密度(Microvessel Density, MVD),所得结果进行统计学分析,选取α=0.05作为差异具有统计学意义的检验水准。二.结果1.与正常组相比较,尿毒症组、尿毒症腹膜透析组,VEGF基因及蛋白水平的表达均升高(p<0.05),差异具有统计学意义。与尿毒症腹膜透析组相比较,重组人内皮抑素治疗1组、重组人内皮抑素治疗2组,VEGF基因及蛋白水平的表达均降低(p<0.05),差异具有统计学意义。与重组人内皮抑素治疗1组相比较,重组人内皮抑素治疗2组,VEGF-A基因及蛋白水平的表达均降低(p<0.05),差异具有统计学意义。2.与正常组相比较,尿毒症组、尿毒症腹膜透析组,MVD的值均增加(p<0.05),差异具有统计学意义。与尿毒症腹膜透析组相比较,重组人内皮抑素治疗1组、重组人内皮抑素治疗2组,MVD的值均降低(p<0.05),差异具有统计学意义。与重组人内皮抑素治疗1组相比较,重组人内皮抑素治疗2组,MVD的值降低(p<0.05),差异具有统计学意义。3. VEGF基因水平的表达与MVD值呈现正相关,VEGF和尿毒症长期腹透时腹膜血管新生相关。结论1.大鼠和人的腹膜组织均表达VEGF、bFGF及ES,体内尿毒症环境和非生理性腹透液的刺激可以上调腹膜组织VEGF、bFGF基因和蛋白的表达。2.尿毒症腹膜透析大鼠和人腹膜组织VEGF、bFGF基因及蛋白水平表达升高,可能在长期透析所致腹膜组织新生毛细血管形成过程中发挥一定的促血管新生作用,是腹透失超滤发生的重要因素之一。3.体内尿毒症毒素和非生理性腹透液刺激可使大鼠和人的腹膜组织ES蛋白表达升高,在长期透析所致腹膜组织毛细血管生成增多中可能发挥一定的抑制血管生成作用。4.重组人内皮抑素(恩度)能抑制尿毒症腹膜透析大鼠腹膜新生血管的形成,并且其抑制作用的强弱与药物的剂量有关。5.重组人内皮抑素(恩度)可能是通过下调尿毒症腹膜透析大鼠腹膜组织中VEGF的表达而发挥抑制腹膜新生血管形成的作用。更多还原

【Abstract】 Backgroud and ObjectiveEnd-stage renal disease (ESRD) has become one of the major public health problems which mankind must face up in the 21st century. Peritoneal dialysis (PD) is an effective way to treat end-stage renal disease. Compared with hemodialysis, PD has its own advantages, such as protecting residual renal function and lower cost. However, some complications often occur during peritoneal dialysis procedure, for example infections, malnutrition, ultrafiltration failure and so on. In particular, the rate of ultrafiltration failure was as high as 36% in these patients who have maitained peritoneal dialysis for 4-5 years. Ultrafiltration failure has become the main reason for peritoneal dialysis patients to withdraw from peritoneal dialysis. So it is greatly important to explore the reason of ultrafiltration failure and furthermore to intervent the occurrence of ultrafiltration failure of peritoneal dialysis.The pathogenesis of ultrafiltration failure was currently considered to relate with three main factors. The first was the increased peritoneal vascular surface area:about 50% to 75% ultrafiltration failure was due to peritoneal angiogenesis that lead to the increased peritoneal vascular surface area, which would make the transport of small solutes speeded up and then induced abdominal osmotic pressure gradient rapidly disappearing, then the peritoneal ultrafiltration capacity lost.The second was the loss of function of aquqporins(AQPs) and the last was the increasing of peritoneal interstitial/lymph fluid reabsorbing rate.Peritoneal neoangiogenesis was the main mechanism that dued to ultrafiltration failure. Regulating angiogenesis inducers included vascular endothelial growth factor (VEGF), fibroblast growth factor (aFGF and bFGF) and other soluble factors. Inhibitors included angiogenesis inhibin, Endostatin (ES) and so on. At present angiogenesis was regulated by inducing agents and inhibitors. Under normal circumstances they were in equilibrium, once the inducer was dominant, it would activate the blood vessels grow and shape the new blood vessels. Otherwise angiosclerosis would occur. A number of angiogenesis inducers increased in the peritoneal dialysis effluents of a long time peritoneal dialysis patients, such as VEGF, bFGF etc. Currently VEGF was recognized as the key factor in the pathogenesis of ultrafiltration failure and the promotion of angiogenesis. VEGF was in the dominant position during neoangiogenesis. The positive expression of VEGF was found in the peritoneal microvascular endothelial cells and peritoneal mesothelial cells (PMc), which was a hot topic in the field of international medical research. But the specific mechanism of angiogenesis factors was still not clear. In conclusion, it was necessary to study the role and the mechanism of peritoneal angiogenesis in peritoneal dialysis patients and to explore the ways to delay the occurrence of peritoneal dialysis ultrafiltration failure and provide a theoretical basis and experimental evidence. So it would provide an effective treatment for multiple control the occurrence of peritoneal dialysis ultrafiltration failure.Therefore, the study was further divided into three parts.Part I:Build 5/6 nephrectomy uremic rat model and then build uremic peritoneal dialysis rats. Detect the gene and protein expression of VEGF, bFGF and the ES, as well as detecting the microvessel desity of peritoneal tissue. Explore the mechanism of peritoneal angiogenesis. Set up the theoretical foundation for the intervention study.Part II:Select peritoneal biopsies which were obtained from normal subjects, uremic predialysis patients and PD patients. And then the gene and protein expressions of VEGF、bFGF and ES were detected by human’s peritoneum. Microvessel Density (MVD) was counted in each group. Set up a theoretical foundation for the further intervention study.PartⅢ:Use recombinant human endostatin (Endostar) to inhibit peritoneal neoangiogenesis of urimic peritoneal dialysis rats. The aim is to provide clinical experimental basis for treating the ultrafiltration failure of peritoneal dialysis.PartⅠAnimal experimentent Study of Neoangiogensis in Peritoneal Dialysis Ultrafiltration failureMaterials and MethodsThe study was done in male SD rats of clean grade weighing 180-200g.We randomly selected 8 rats as normal group and established the uremia rats model of 5/6 nephrectomy and then from these uremia rats, we randomly established the peritoneal dialysis (PD) rats model. At last, there were 4 groups in this study:normal rats (n=8), uremia rats (n=8), dialysized rats exposed to PD 1.5% solution (n=8) and 4.25%(n=8) respectively. The catheters implanted in dialysis rats were tunneled subcutaneously to the neck and solutions 20ml per rat were kept in the abdominal cavity for 2 hours per day without anesthesia. After regularly peritoneal dialysis for 28 days, tissue immunohistochemistry methods and reverse transcript polymerase chain reaction (RT-PCR) were applied to detect the mRNA and protein expressions of VEGF、bFGF and ES in rats’peritoneal tissues of each group. Statistical analysis was performed to compare expressions and co relationship in each group. Significance was defined as a=0.05.Results1. VEGF、bFGF and ES were positively expressed in peritoneal tissues of normal rat.2. Compared with normal group, the protein expression of VEGF、bFGF and ES were up-regulated in uremia rats and dialysized rats. 3. Compared with normal group, the mRNA expression of VEGF and bFGF were up-regulated in uremia rats and dialysized rats (p<0.05), but there was no significant difference (p>0.05).4. In uremia rats (r=0.990, P=0.000),1.5% dialysized rats (r=0.925, P=0.000), 4.25% dialysized rats (r=0.968, P=0.000), there was a positive correlation between VEGF and bFGF in mRNA level.5. MVD was up-regulated in uremia rats and dialysized rats compared with normal group (P<0.05).PartⅡClinical research of the Mechanism of Neoangiogensis inPeritoneal dialysis Ultrafiltration failureMaterials and MethodsPeritoneal biopsies were obtained from normal controls (from non-uremic patients with abdominal surgery, excluding any abdominal membrane disease), uremic predialysis patients at catheter insertion and PD patients at the time of catheter remove, reinsertion or renal transplantation. RT-PCR techniques and Immunohistochemical staining were used to investigate VEGF, bFGF and ES expression in peritoneal tissue. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody.Results1. The mRNA expression of VEGF and bFGF were found in all peritoneal samples in each group. Base on normal group, the mRNA expression of VEGF、bFGF in uremic predialysis and PD group were significantly up-regulated (P<0.05) Compared with uremic predialysis group, the mRNA expression of VEGF, bFGF in PD group were significantly up-regulated (P<0.05). The mRNA expression of ES was no significantly difference in each group.2. The protein expression of VEGF and bFGF were found in all peritoneal samples in each group. Compared with normal group, the protein expression of VEGF、 bFGF and ES in uremic predialysis and PD group were significantly up-regulated (P<0.05). Compared with uremic predialysis group, the protein expression of VEGF, bFGF and ES in PD group were significantly up-regulated (P<0.05)3. The new micro vascular vessels in normal group shows little or none. MVD was up-regulated in uremic predialysis and PD group compared with normal group (P<0.05).PartⅢIntervention Study of Preventing the occurrence of Peritoneal Dialysis Ultrafiltration FailureMaterials and Methods43 male SD rats of clean grade weighing 180-200g were used in the study. The uremia rats model was established by 5/6 nephrectomy and then from these uremia rats, we randomly established the peritoneal dialysis (PD) rats model. At last, there were 5 groups in this study:A:normal group (control group:n=8), B:uremia group (5/6 nephrectomy group:n=8), C:uremia peritoneal dialysis group (4.25% dialysized group:n=8), D1:the first treatment group with Endostar (10mg/kg treatment group: n=8), D2:the second treatment group with Endostar (40mg/kg treatment group:n=8). The rats from C, D1, D2 groups were given regular peritoneal dialysis. The catheters implanted in dialysis rats were tunneled subcutaneously to the neck and exchanged PD solutions per rat were kept in the abdominal cavity for 2 hours per day without anesthesia. The rats from D1, D2 groups accepted subcutaneous injection of Endostar during peritoneal dialysis period every other days, total administration 14 times. After regularly peritoneal dialysis for 28 days, tissue immunohistochemistry methods and reverse transcript polymerase chain reaction (RT-PCR) were applied to detect the mRNA and protein expressions of VEGF and in rats’peritoneal tissues of each group. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody. Statistical analysis was performed to compare with their expressions and co relationship in each group. Significance was defined as a=0.05.Results1. Base on normal group, the mRNA and protein expression of VEGF were significantly up-regulated in uremia group and uremia peritoneal dialysis group (P<0.05). Compared with uremia peritoneal dialysis group, the mRNA and protein expression of VEGF were significantly down-regulated in the first and second treatment groups with Endostar (P<0.05). Compared with the first treatment group with Endostar, the mRNA and protein expression of VEGF were significantly down-regulated in the second treatment group with Endostar (P<0.05)2. Base on the normal group, MVD was up-regulated in uremia group and uremia peritoneal dialysis group (P<0.05). Compared with uremia peritoneal dialysis group, MVD was significantly down-regulated in the first and second treatment groups with Endostar (P<0.05). Compared with the first treatment group with Endostar, MVD was significantly down-regulated in the second treatment group with Endostar (P<0.05)3. There was a positive correlation between VEGF and MVD in protein level (r=0.987, P=0.000)Conclusions1. The expression of VEGF、bFGF and ES was positive in peritoneal tissues both normal rats and human; the expression of VEGF、bFGF and ES was up-regulated by uremia circumstance and non-physiological compatibility peritoneal dialysis liquid.2. The upregulation of VEGF and bFGF in peritoneal dialysis rat and patient peritoneum might participate in the increasement of the peritoneum neoangiogensis and then cause the incidence of ultrafiltration.3. The protein level expression of ES was up-regulated by uremia circumstance and non-physiological compatibility peritoneal dialysis liquid. ES might play an important role in inhibitting peritoneum neoangiogensis4. Recombinant human endostatin (Endostar) can effectively inhibit rat peritoneum neoangiogensis inducing by peritoneal dialysis, and the role of inhibit strength is related with the dose of the drug.5. Recombinant human endostatin (Endostar) can inhibit rat peritoneum neoangiogensis by down-regulating the expression of VEGF in peritoneum.更多还原