【作者】 孟立锋；

【导师】 谢桂权；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的本课题以中医“痰瘀互结”是腹膜纤维化发生的病机关键为理论依据,采用临床与动物实验相结合的方法,观察并探讨化痰祛瘀法防治腹膜透析患者腹膜纤维化的临床效果。并重点从TGF-β/Smads信号通路,进一步探讨化痰祛瘀法防治腹膜纤维化大鼠模型的作用和机制。综合评价化痰祛瘀法防治腹膜纤维化的临床疗效,为防治腹膜透析患者腹膜纤维化提供新的中医药理论依据与方法。方法通过收集我院2010年1月至2011年1月符合纳入标准的腹膜透析患者,观察化痰祛瘀中药对腹膜透析患者的临床症状及实验室指标如血肌酐、尿素氮、白蛋白、血脂、血分析、肝纤四项及腹透液中TGF-β1、VEGF因子表达等的影响,客观评价化痰祛瘀中药防治腹膜纤维化的临床疗效。通过建立大鼠腹膜纤维化模型,观察经用化痰祛瘀中药干预后,腹膜纤维化大鼠模型腹膜结构的组织学变化,阐明化痰祛瘀中药拮抗腹膜纤维化大鼠模型的有效性。并应用间接免疫荧光法测定各组大鼠壁层腹膜TGF-β1、p-Smad2／3和α-SMA、E-cadherin及p-Smad7蛋白的表达,探讨化痰祛瘀中药拮抗腹膜纤维化与调控TGF-β/Smads信号通路的关系,初步揭示化痰祛瘀法防治腹膜纤维化的作用机制。结果(一)化痰祛瘀法防治腹膜纤维化的临床研究1、化痰祛瘀中药对中医症候积分的影响治疗组经用化痰祛瘀中药治疗后,面色晦暗,肌肤甲错,肢体麻木或刺痛,身重困倦,脘腹胀满,口中粘腻,口干不欲饮,食少纳呆,大便粘滞不爽的改善作用明显优于对照组(P<0.05),表明化痰祛瘀中药在改善中医痰瘀互阻症候方面,治疗组优于对照组。2、化痰祛瘀中药对腹膜纤维化相关指标的影响治疗组疗后腹透液中TGF-β1因子水平显著下降(130.56±14.54 pg/ml),与治疗前相比(164.84±32.38 pg/ml),有显著性差异(P<0.05)；对照组治疗前好比较无显著性差异(P>0.05),治疗后,两组组间比较,治疗组腹透液中TGF-β1因子下降水平明显高于对照组(P<0.01)。治疗组疗后腹透液中VEGF因子水平显著下降(63.62±12.95pg/ml),与治疗前相比(95.87±18.59pg/ml),有显著性差异(P<0.05)；对照组治疗前好比较无显著性差异(P>0.05),治疗后,两组组间比较,治疗组腹透液中VEGF因子下降水平明显高于对照组(P<0.01)。反映纤维化程度的指标：肝纤四项,治疗组与对照组疗后与疗前比较,均有所降低,但仅治疗组差异有统计学意义(P<0.05),对照组差异无统计学意义(P>0.05)；治疗后,两组组间比较,统计学有显著性差异(P<0.01)；表明腹膜透析患者服用化痰祛瘀中药对肝纤四项指标有一定的改善作用。3、化痰祛瘀中药对实验室指标的影响治疗组经用化痰祛瘀中药治疗后,治疗组的肌酐、尿素氮、总胆固醇、甘油三酯、低密度脂蛋白、纤维蛋白原等指标的改善作用优于对照组(P<0.05),表明化痰祛瘀中药在改善患者肾功能、血脂、凝血功能方面,有良好疗效。4、化痰祛瘀中药药物安全性指标的观察治疗组患者服用化痰祛瘀中药期间,未出现各种不良反应症状,反映心脏功能、肝功能的指标肌酸激酶-MB同工酶(CK-MB).谷草转氨酶(AST)、谷丙转氨酶(ALT)、谷氨酰转移酶(GGT),治疗前后,差异无统计学意义(P>0.05),表明腹膜透析患者服用化痰祛瘀中药,对心脏及肝功能无不良影响,服用安全。(二)化痰祛瘀法防治腹膜纤维化的实验研究1、腹膜间皮细胞内存在TGF-β1、Smad2／3、Smad7、SMA和E-cadherin蛋白的表达,并证实高浓度葡萄糖透析液和LPS有协同作用,能促进大鼠腹膜组织TGF-G1/Smads信号通路的活化,产生更加明显的腹膜纤维化。2.TGF-β1/Smads信号通路是腹膜纤维化的共同作用途径,化痰祛瘀中药是通过对TGF-β/Smads信号通路的影响来拮抗腹膜纤维化的,中药高剂量组有更好的防治腹膜纤维化的作用。3、化痰祛瘀中药能上调TGF-β抑制性信号蛋白Smad7的表达,这可能是通过抑制TGF-β受体调控信号蛋白(Smad2/3)的活化,还显示化痰祛瘀中药能减轻E—cadhefin蛋白水平下调,及α-SMA蛋白水平上调的程度,进而说明化痰祛瘀中药可以减轻腹膜间皮细胞转分化的程度,防止腹膜纤维化的发展,保护腹膜功能。结论1、通过对2010年1月至2011年1月符合纳入标准的30例行标准连续性非卧床腹膜透析(CAPD)治疗者临床研究提示：化痰祛瘀中药可以改善腹膜透析患者临床症状,提高腹膜透析治疗效果。2、化痰祛瘀中药复方能明显降低反映腹膜纤维化的相关指标(肝纤四项、腹透液中TGF-β1、VEGF因子),可以明显减轻腹膜纤维化大鼠模型腹膜组织的炎症反应病理改变程度,保护腹膜组织形态结构的完整,减少腹膜纤维化大鼠模型腹膜的致密层厚度,从而起到拮抗腹膜纤维化的作用,这一作用呈量效关系,以中药高剂量组效果最佳。初步显示化痰祛瘀中药有较好的防治腹膜纤维化作用。3、通过对5/6肾切除的大鼠应用4.25%的高糖腹膜透析液和脂多糖(LPS)造成腹膜纤维化模型的研究发现：化痰祛瘀中药拮抗腹膜纤维化的作用机制可能是通过上调TGF-β抑制性信号蛋白Smad7的表达,进而抑制TGF-β受体调控信号蛋白(Smad2／3)的活化,阻止腹膜纤维化动物模型腹膜间皮细胞转分化的发生,从而阻止或减轻腹膜纤维化的发生和进展,保护腹膜功能。更多还原

【Abstract】 ObjectiveThe topics to Chinese medicine, "the phlegm stasis ties mutually" is the pathogenesis key which the peritoneum fibrosis occurs as the theoretical basis, Uses the clinical method which unifies with the animal experimentation, observes and discusses the phlegm reduction to remove extravasted blood the law prevention in peritoneal dialysis patients the clinical effect. And key from TGF-β/Smads signal passage, further discusses the phlegm reduction to remove extravasted blood the law prevention peritoneum fibrosis big mouse model the function and the mechanism. Comprehensive evaluation of the use of phlegm and blood stasis and the prevention and treatment of peritoneal fibrosis in clinical efficacy for the prevention of peritoneal fibrosis in peritoneal dialysis patients to provide a new theoretical basis and methods of Chinese medicine.MethodsThrough the collection of our hospital from January 2010 to January 2011 met the inclusion criteria of the peritoneal dialysis patients Phlegm and blood stasis observed in patients on peritoneal dialysis clinical symptoms and laboratory parameters such as serum creatinine, urea nitrogen, albumin, blood lipids, blood analysis, liver filament four item and peritoneal fluid TGF-β1, VEGF expression factor such as the impact of phlegm and blood stasis objective evaluation of the clinical efficacy of prevention and treatment of peritoneal fibrosis. Through the establishment of big mouse peritoneum fibrosis model, after the observation durable phlegm reduction removes extravasted blood the traditional Chinese medicine intervention, the peritoneum fibrosis big mouse model peritoneum structure’s histology change, expounded that the phlegm reduction removes extravasted blood the traditional Chinese medicine to oppress the anti-big mouse model peritoneum fibrosis the validityAnd applies the indirect immunity fluorescence method to determine each group of big mouse somatopleura peritoneum TGF-β1, p-Smad2/3 andα-SMA, E-cadherin and the p-Smad7 protein expression, the discussion phlegm reduction removes extravasted blood the traditional Chinese medicine to oppress the anti-peritoneum fibrosis and the regulative TGF-β/Smad signal passage’s relations, promulgates the phlegm reduction to remove extravasted blood the law prevention peritoneum fibrosis initially the function mechanism.Result(一) The phlegm reduction removes extravasted blood the law prevention peritoneum fibrosis the clinical research1、Phlegm and blood stasis syndrome influnce score of Chinese medicine After the treatment group durable phlegm reduction removes extravasted blood the traditional Chinese medicine treatment, the facial color is gloomy, the flesh armor is wrong, the body numb or the stabbing pain, the feeling of heaviness in the limbs is sleepy, wan abdomen puffiness, in the mouth sticks greasily, dry mouth do not want to drink, the food few losses of appetite, stool viscosity the improvement function surpasses the control group obviously (P<0.05), whichshow phlegm reduction removes extravasted blood the traditional Chinese medicine to improve the symptoms, the treatment group better than the control group.2、Phlegm and blood stasis related indicators of the impact of peritoneal fibrosisEffluent treatment group after treatment factors in the TGF-β1 levels decreased significantly (130.56±14.54 pg/ml), compared with before treatment (164.84±32.38 pg/ml), there was significant difference (P<0.05); the control group Good pre-treatment showed no significant difference (P> 0.05), after treatment, comparison between the two groups, treatment group, dialysate factor in the decreased levels of TGF-β1 was significantly higher (P<0.01). Effluent treatment group after treatment factors in the VEGF levels were significantly decreased (63.62±12.95pg/ml), compared with before treatment (95.87±18.59pg/ml), there was significant difference (P<0.05); the control group before treatment well there was no significant difference (P>0.05), after treatment, comparison between the two groups, the treatment group factor of decline in peritoneal dialysis fluid VEGF levels were significantly higher (P<0.01).Indicators reflect the degree of fibrosis:hepatic-fibrosis four, treatment and control groups before treatment after treatment compared were decreased, but only statistically significant difference between the treatment group (P<0.05), no statistical difference between the control group significance (P>0.05); after treatment between the two groups was statistically significant difference (P<0.01); showed that patients treated with peritoneal dialysis on liver blood stasis Phlegm fiber four indicators have some improvement.3、Phlegm and blood stasis effect on laboratory parametersTreatment group after treatment with phlegm and blood stasis, the i mprovement of creatinine, urea nitrogen, total cholesterol, triglycerides, low density lipoprotein, fibrinogen better than the control group (P<0. 05), indicating that the phlegm and blood stasis in improving renal funct ion, blood lipid, have a good effect.4、Phlegm and blood stasis observed indicators of drug safetyTreatment group, reflecting the heart and liver function indicators of CK-MB, AST, ALT, GGT, before and after treatment, the difference was not statistically significant (P>0.05), showed that patients treated with peritoneal dialysis phlegm and blood stasis, on cardiac function and liver no functional effect, taking safety.(二) Experimental study of phlegm and blood stasis to Prevent and Treat the peritoneal fibrosis1、Peritoneal mesothelial cells exist in TGF-β1, Smad2/3, Smad7, SMA, and E-cadherin protein expression, and confirmed that high glucose dialysate and LPS have synergistic effects, can signal in rat peritoneal tissue TGF-β1/Smads pathway activation, resulting in more significant peritoneal fibrosis.2、TGF-β1/Smads signaling pathway is a common pathway of peritoneal fibrosis, phlegm and blood stasis TGF-β/Smad signaling pathway through the influence of antagonistic to peritoneal fibrosis, the Chinese high-dose group had a better prevention and treatment of peritoneal fibrosis.3、phlegm and blood stasis can increase TGF-βinhibitory Smad7 expression of signaling proteins, which may inhibit the regulation of TGF-βreceptor signaling protein (Smad2/3) activation, also showed that blood stasis can reduce phlegm E-cadhefin lower protein levels, and increased levels ofα-SMA protein level, then explain the phlegm and blood stasis can reduce the transdifferentiation of peritoneal mesothelial cells in the extent and prevent the development of peritoneal fibrosis, peritoneal membrane function protectionConclusion1、Through January 2010 to January 2011 30 patients met the inclusion criteria of the standard continuous ambulatory peritoneal dialysis (CAPD) treatment of clinical studies suggest that:phlegm and blood stasis can improve clinical symptoms of peritoneal dialysis patients, increased peritoneal dialysis treatment2、Blood stasis and phlegm reflect the:compound can significantly reduce the relevant indicator of peritoneal fibrosis (liver fiber four, peritoneal fluid TGF-β1, VEGF factor), can protect the structural integrity of peritoneal morphology and reduce the rat model of peritoneal fibrosis, peritoneal thickness of the dense layer, play the role of peritoneal fibrosis antagonist. Phlegm and blood stasis initially showed better prevention and treatment of peritoneal fibrosis.3, Through 5/6 nephrectomy rats application of 4.25% of the high-glucose peritoneal dialysis fluid and lipopolysaccharide (LPS) caused by retroperitoneal fibrosis were found:phlegm and blood stasis on the impact of TGF-β/Smad signaling pathway possibly by increased TGF-βsignaling inhibition protein expression of Smad7 and inhibit TGF-βreceptor signaling protein regulated (Smad2/3) activation, prevents peritoneal fibrosis in animal models of peritoneal mesothelial cell transdifferentiation occurs, thereby preventing or reducing the incidence and progression of peritoneal fibrosis, Protection of peritoneal function.更多还原