实验性糖尿病大鼠肾脏组织PEDF和MMP-2的表达及人工发酵虫草菌丝体干粉和罗格列酮对其表达的影响
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摘要
目的:糖尿病肾病(diabetic nephropathy, DN)是糖尿病(diabetes mellitus, DM)重要的慢性微血管并发症之一。长期高血糖及其引起的代谢紊乱,激活多种途径导致肾脏结构的不可逆改变,是终末期肾病的主要原因。主要病理特征为肾小球硬化、基底膜增厚及细胞外基质(extracellular matrix, ECM)沉积等。目前DN的发病机制尚未完全明确。色素上皮衍生因子(pigment epithelium derived factor, PEDF)是近年来发现的一种强有力的新生血管抑制因子,具有神经营养保护、调节血管通透性和高效抑制血管生成等作用。在DN的发生和发展过程中,常伴有ECM的合成和/或降解异常,而基质金属蛋白酶(matrix metalloproteinases, MMPs)在ECM代谢中起重要作用。按作用底物可将MMPs分为五类,MMP-2属于白明胶酶,在肾小球系膜细胞、内皮细胞、成纤维细胞等均有表达,主要降解Ⅳ型胶原。MMP-2的表达及活性改变可能参与了DN的发生发展。转化生长因子β1(transforming growth factor-β1, TGF-β1)在DN时过量表达,通过促进肾小球细胞增殖、肥大,导致ECM堆积,损伤滤过屏障,同时抑制MMPs的合成,减少ECM的降解,加速DN的形成。冬虫夏草Cordyceps sinensis(Berk.)Sacc是一种富含多糖、氨基酸、脂肪酸、甘露醇及多种微量元素的名贵中药。研究显示它可通过降低炎症反应、抑制肾小球系膜细胞增殖等多种途径起到肾脏保护作用。罗格列酮(rosiglitazone, RSG)作为噻唑烷二酮类胰岛素增敏剂,通过结合并激活过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor, PPARγ)发挥作用,具有降糖、抗炎、调节脂质代谢、抑制细胞生长因子及降低尿蛋白排泄等重要作用。
因此,本研究通过腹腔注射大剂量链脲佐菌素(streptozotocin, STZ)建立1型糖尿病大鼠模型,并应用人工发酵虫草菌丝体干粉和RSG进行干预治疗,运用生物化学、病理形态学等方法检测肾脏形态及功能的改变和PEDF、MMP-2及TGF-β1在肾脏表达情况,探讨PEDF和MMP-2在糖尿病肾损害中的作用和人工发酵虫草菌丝体干粉及RSG在其中发挥的保护作用。
方法:雄性8周龄SD大鼠60只,随机抽取15只作为正常对照组(A组),其余45只作为实验组。将实验组大鼠给予STZ 55mg/kg腹腔注射,72小时后测尾尖血糖大于16.7 mmol/L,持续3天,确认为1型糖尿病动物模型。把成模DM大鼠随机分为糖尿病对照组(B组)、人工发酵虫草菌丝体干粉2.4g/kg/d干预治疗组(C组)、RSG 4mg/kg/d干预治疗组(D组)。实验全程共持续12周,造模前及成模后0、4、8、12周末测量大鼠体重、血糖。第12周末实验结束前,代谢笼收集24小时尿液。股静脉放血处死动物,收集血液分离血清,测定血中总胆固醇(TC)、甘油三酯(TG)、尿素氮(BUN)、血肌酐(Scr)、谷丙转氨酶(ALT)、肾重指数(KWI)和24小时尿白蛋白排泄量(UAE)。肾组织制备光镜切片,运用HE、PAS染色方法观测肾脏形态学变化;用免疫组织化学方法检测PEDF、MMP-2和TGF-β1在肾组织的表达水平及部位;以逆转录PCR方法检测肾脏PEDF mRNA的表达情况。使用SPSS13.0统计软件处理实验数据,多组间比较采用方差分析,两组间比较采用LSD检验,等级资料采用秩和检验。
结果:
1造模72小时后,B组、C组和D组都较A组大鼠血糖明显升高,差异有显著性(P均<0.01),血糖均超过16.7 mmol/L,表明已成功建立1型糖尿病大鼠模型。4周、8周、12周末,B组、C组和D组血糖均高于A组,且差异有显著性(P均<0.01)。
2造模72小时后及4周、8周及12周末实验结束时,B组、C组和D组都较A组大鼠体重明显降低,差异有显著性(P均<0.01)。B组、C组和D组肾重指数均较A组升高,差异具有显著性(P均<0.01),C组和D组肾重指数均较B组有所改善。
3 12周末实验结束时,B、C、D组UAE较A组升高,差异具有显著性(P均<0.01)。C、D组UAE较B组有所降低,差异具有显著性(P均<0.01)。
4 B、C、D组血清TG、TC与A组相比均升高,且有显著性差异(P均<0.01);C组TG、TC与B组相比降低,且有显著性差异(P均<0.01);D组TG与B组相比降低,有显著性差异(P<0.01),而TC无显著性差异(P>0.05)。B、C、D组血清Scr、BUN、ALT与A组相比均升高,且有显著性差异(P<0.01, P<0.01, P<0.01; P<0.05, P<0.01, P<0.01; P<0.01, P<0.01, P<0.01);C、D组血清Scr、BUN与B组相比降低,且有显著性差异(P均<0.01),而ALT无显著性差异(P>0.05)。
5血清PEDF、TGF-β1水平测定结果显示,与A组相比,B、C、D组PEDF、TGF-β1水平较A组升高(P均<0.05);与B组相比,C、D组PEDF、TGF-β1水平降低,差异具有显著性(P均<0.05)。
6 HE及PAS染色结果显示,与A组比较,B组肾小球肥大,基底膜增厚,系膜区基质明显增生,细胞增生。少数肾小球出现轻度硬化,部分肾小管呈空泡变性。C、D组较B组有所改善。
7免疫组织化学分析结果显示,与A组相比,B组PEDF、MMP-2表达明显下降,阳性细胞的染色强度和分布范围之和明显降低且有显著性差异(P均<0.05),而TGF-β1表达上升(P<0.05)。C、D组PEDF、MMP-2表达较B组有所上升(P均<0.05),TGF-β1表达较B组降低(P<0.05)。
8逆转录PCR反应结果显示,与A组比较,B、C、D组PEDF mRNA表达明显降低(P<0.01, P<0.05, P<0.05),经人工发酵虫草菌丝体干粉及RSG干预后,C、D组PEDF mRNA表达较B组有所上调(P均<0.01)。
结论:
1大鼠经腹腔一次性注射大剂量STZ,可建立1型糖尿病大鼠模型。该模型具有持续高血糖、尿量增加、体重减轻等明显的DM症状,与人类1型糖尿病的临床特征类似。故此模型可用于1型糖尿病慢性并发症的研究。
2随着高血糖持续存在,DM大鼠肾脏出现不可逆损害,病理结构破坏,肾功能下降。形态学和实验室检查均符合糖尿病肾损害改变。糖代谢的异常对脂代谢及肝功能产生了一定的影响,共同对机体产生不利影响。
3 DM大鼠肾脏组织PEDF、MMP-2及TGF-β1之间可能存在一定的相互作用,三者表达水平的改变可能促进了糖尿病肾损害的病程进展。
4 DM大鼠血清PEDF、TGF-β1含量升高,提示肾脏病变过程中,循环中PEDF水平可能通过其他途径代偿性升高,与TGF-β1相互作用。
5经人工发酵虫草菌丝体干粉和RSG干预后,DM大鼠肾脏功能及病理改变均明显改善,表明其可能通过改善肾功能、减少尿白蛋白排泄、调节血脂、调节PEDF、MMP-2和TGF-β1的蛋白表达,发挥明显的肾脏保护作用。
Objective: Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes mellitus (DM). Long-term hyperglycaemia and metabolic disorders activates many pathways and causes renal pathological irreversible changes. DN, a principal cause of end-stage renal diseases, is characterized by glomerular sclerosis, thickening of glomerular basement membrane and extracellular matrix deposition. The pathogenesis of DN is not clear at present. Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor, that can protect neuronal nutrition, regulate vascular permeability and antiangiogiogenic activity. The synthesize and/or degradation of extracellular matrix is abnormal in the process of occurrence and development of DN. Matrix metalloproteinases (MMPs) play an important role in the metabolism of extracellular matrix. MMPs are classified into five groups based on substrate. MMP-2 belongs to gelatinases and is expressed in Mesangial cell, endotheliocyte, fibroblast and so on. It degradations typeⅣcollagen majorly. The alterations of expression and activity of MMP-2 may participate in genesis and development of diabetic nephropathy. Overexpression of TGF-β1 in diabetic glomeruli is believed to contribute to the glomerular hypertrophy, matrix accumulation, filtration barrier lesion, meanwhile inhibiting MMPs synthesis, diminishing ECM accumulation, accelerates the development of DN. Recent studies show that PEDF inhibits oxidant stress reactions, fiber production and inflammatory reaction, thus ameliorates renal hemodynamics and maintains renal homeostasis, and may be responsible for its salutary effect in DN. Cordyceps sinensis (Berk.)Sacc is a valuable traditional chinese medicine thainrich in polyoses, amino acids, fatty acid, mannitol and many kinds of microelements. Researches show that its protection may function by lowering inflammation reaction, inhibiting Mesangial cell proliferation and so on. Rosiglitazone is thiazolidinediones (TZDs) euglycemic agent and is believed to work through binding and modulating the activity of a family of nuelear transcription fators termed peorxisome proliefrator-activated receptorγ(PAPRγ). It is associated with slow improvement in glycemic control, antiinflammatory, regulation of lipid metabolism, inhibition of cell growth fators, reduction of urine protein excretion, etc.
In the study, rat model of type 1 diabetes was set up by an high-dose intraperitoneal injection of streptozotocin (STZ), and Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone was applied for intervention. Applying the methods of biochemistry, pathomorphology for detecting the changes of renal morphous and function and the expression of PEDF, MMP-2 and TGF-β1, explore the effect of PEDF and MMP-2 in the genesis and development of diabetic nephropathy and protection of Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone.
Methods: 60 eight weeks male SD rats were divided into two groups randomly: 15 normal control rats (group A) and 45 test rats. STZ (55mg/kg) was injected intraperitoneally to destroy some pancreas in order to induce hyperglycemia for test rats. 72 hours later, the rats whose blood glucose level was higher than 16.7 mmol/L in the subsequent 3 days were considered as diabetes. The diabetic rats were randomly divided into following groups: diabetic model rats (group B), diabetic model rats treated with 2.4g/kg/d Cordyceps Sinensis cultivated by Artificial Fermentation (group C), diabetic model rats treated with 4mg/kg/d rosiglitazone (group D). The experiment lasted 12 weeks. Blood glucose and body mass were measured at the beginning of the experiment and at the end of 0 weeks, 4 weeks, 8 weeks and 12 weeks respectively. After 12 weeks, 24 hour urine volume was collected with metabolic cage. Serum total cholesterol (TC), serum triglyceride (TG), blood urea nitrogen (BUN), serum creatinine (Scr), alanine aminotransferase (ALT), kindey weight index (KWI) and 24 hour urinary albumin excretion (UAE) were detected. Morphological changes were observed by microscope; The protein expression and location of PEDF, MMP-2 and TGF-β1 were examined by immunohistochemistry. Moreover, PEDF mRNA expression in kidney was analysed with reverse transcription polymerase chain reaction (RT-PCR). All the experimental data were dealt with SPSS13.0. Group comparisons adopt analysis of variance, the comparison of two sets adopt LSD test, level data adopt Kruskal-Wallis test.
Results:
1 72h after injecting STZ, blood glucose in B, C, D group, all over 16.7 mmol/L, was markedly increased compared with A group (P all<0.01). It suggested the model of type 1 diabetic mellitus was set up. At the end of 4, 8 and 12 weeks, blood glucose in B, C, D group all significantly higher than A group (P all<0.01).
2 At the end of 72h after injecting STZ and 4, 8 and 12 weeks, body mass in B, C, D group were markedly decreased compared with A group (P all<0.01). KWI in B, C, D group were markedly increased compared with A group (P all<0.01). KWI in C, D group were markedly improved compared with B group.
3 At the end of 12 weeks, urinary albumin excretion (UAE) in B、C、D group were significantly increased compared with A group (P all<0.01). UAE in both C and D group were significantly decreased compared with B group (P all<0.01).
4 Compared with A group, TG and TC in B, C, D group were increased (P all<0.01), TG and TC in C group were decreased compared with B group (P all<0.01); TG in D group were decreased compared with B group (P<0.01), while TC had no difference (P>0.05). Compared with A group, Scr, BUN and ALT in B, C, D group were increased (P<0.01, P<0.01, P<0.01; P<0.05, P<0.01, P<0.01; P<0.01, P<0.01, P<0.01); Scr, BUN in C, D group were decreased compared with B group (P all<0.01), while TC had no difference (P>0.05).
5 Compared with A, serum level of PEDF and TGF-β1 was significantly increased in B, C, D group (P all<0.05). Compared with B group, serum level of PEDF and TGF-β1 was decreased in C, D group (P all<0.05).
6 H.E. stain and PAS stain showed that compared with A, glomerular hypetrophy, thickening of glomerular basement membrance, mesangial expansion and cell proliferation in B group. A few glomerulars appeared sclerosis lightly, some renal tubules showed vacuolar degeneration. C and D group were ameliorated than B group.
7 Immunohistochemistry suggested that compared with A, expression of PEDF and MMP-2 in B group was obviously decreased, while that of TGF-β1 was significantly increased (P all<0.05). Expression of PEDF and MMP-2 in C, D group were meliorated compared with B group (P all<0.05), the expression of TGF-β1 downregulated (P<0.05).
8 Reverse transcription-polymerase chain reaction (RT-PCR) results showed that: compared with A group, the expression of PEDF mRNA was decreased significantly in B, C and D group (P<0.01, P<0.05, P<0.05); the expression of PEDF mRNA was markly upregulated in C and D group (P all<0.01).
Conclusion:
1 Rat model of type 1 diabetic mellitus can be set up by high-dose intrapenitoneal injection STZ. The model characterized by significantly diabetic symptoms analog to human, such as continuous hyperglycemia, polyuria, weight reduction and so on. So this model can satisfy the need of diabetic complication researches.
2 With the persistence of high blood glucose, irreversible damages appeared in diabetic rat kidneys, pathological structure damaged and renal function declined. Morphology and laboratory tests are in line with changes in diabetic kidney damage. Dysglycemia can cause dyslipidemia and hepatic dysfunction. These disturbances may play an adverse role in organism.
3 Interaction between PEDF, MMP-2 and TGF-β1 may exist in diabetic rat kidneys. Changes in the expression levels of the three may promote the progression of diabetic kidney damage.
4 Serum content of PEDF and TGF-β1 elevated in diabetic rats, indicating that PEDF may be compensated increased by other means and interacted with TGF-β1 in the pathogenesis of diabetic kidney damage.
5 After treated by Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone, kindey function and pathologic changes were improved noticeably. The results showed it could protect the kindey through ameliorating renal function, reducing the level of urine albuminuria, regulating the levels of serum lipids and the expression of PEDF, MMP-2 and TGF-β1.
因此,本研究通过腹腔注射大剂量链脲佐菌素(streptozotocin, STZ)建立1型糖尿病大鼠模型,并应用人工发酵虫草菌丝体干粉和RSG进行干预治疗,运用生物化学、病理形态学等方法检测肾脏形态及功能的改变和PEDF、MMP-2及TGF-β1在肾脏表达情况,探讨PEDF和MMP-2在糖尿病肾损害中的作用和人工发酵虫草菌丝体干粉及RSG在其中发挥的保护作用。
方法:雄性8周龄SD大鼠60只,随机抽取15只作为正常对照组(A组),其余45只作为实验组。将实验组大鼠给予STZ 55mg/kg腹腔注射,72小时后测尾尖血糖大于16.7 mmol/L,持续3天,确认为1型糖尿病动物模型。把成模DM大鼠随机分为糖尿病对照组(B组)、人工发酵虫草菌丝体干粉2.4g/kg/d干预治疗组(C组)、RSG 4mg/kg/d干预治疗组(D组)。实验全程共持续12周,造模前及成模后0、4、8、12周末测量大鼠体重、血糖。第12周末实验结束前,代谢笼收集24小时尿液。股静脉放血处死动物,收集血液分离血清,测定血中总胆固醇(TC)、甘油三酯(TG)、尿素氮(BUN)、血肌酐(Scr)、谷丙转氨酶(ALT)、肾重指数(KWI)和24小时尿白蛋白排泄量(UAE)。肾组织制备光镜切片,运用HE、PAS染色方法观测肾脏形态学变化;用免疫组织化学方法检测PEDF、MMP-2和TGF-β1在肾组织的表达水平及部位;以逆转录PCR方法检测肾脏PEDF mRNA的表达情况。使用SPSS13.0统计软件处理实验数据,多组间比较采用方差分析,两组间比较采用LSD检验,等级资料采用秩和检验。
结果:
1造模72小时后,B组、C组和D组都较A组大鼠血糖明显升高,差异有显著性(P均<0.01),血糖均超过16.7 mmol/L,表明已成功建立1型糖尿病大鼠模型。4周、8周、12周末,B组、C组和D组血糖均高于A组,且差异有显著性(P均<0.01)。
2造模72小时后及4周、8周及12周末实验结束时,B组、C组和D组都较A组大鼠体重明显降低,差异有显著性(P均<0.01)。B组、C组和D组肾重指数均较A组升高,差异具有显著性(P均<0.01),C组和D组肾重指数均较B组有所改善。
3 12周末实验结束时,B、C、D组UAE较A组升高,差异具有显著性(P均<0.01)。C、D组UAE较B组有所降低,差异具有显著性(P均<0.01)。
4 B、C、D组血清TG、TC与A组相比均升高,且有显著性差异(P均<0.01);C组TG、TC与B组相比降低,且有显著性差异(P均<0.01);D组TG与B组相比降低,有显著性差异(P<0.01),而TC无显著性差异(P>0.05)。B、C、D组血清Scr、BUN、ALT与A组相比均升高,且有显著性差异(P<0.01, P<0.01, P<0.01; P<0.05, P<0.01, P<0.01; P<0.01, P<0.01, P<0.01);C、D组血清Scr、BUN与B组相比降低,且有显著性差异(P均<0.01),而ALT无显著性差异(P>0.05)。
5血清PEDF、TGF-β1水平测定结果显示,与A组相比,B、C、D组PEDF、TGF-β1水平较A组升高(P均<0.05);与B组相比,C、D组PEDF、TGF-β1水平降低,差异具有显著性(P均<0.05)。
6 HE及PAS染色结果显示,与A组比较,B组肾小球肥大,基底膜增厚,系膜区基质明显增生,细胞增生。少数肾小球出现轻度硬化,部分肾小管呈空泡变性。C、D组较B组有所改善。
7免疫组织化学分析结果显示,与A组相比,B组PEDF、MMP-2表达明显下降,阳性细胞的染色强度和分布范围之和明显降低且有显著性差异(P均<0.05),而TGF-β1表达上升(P<0.05)。C、D组PEDF、MMP-2表达较B组有所上升(P均<0.05),TGF-β1表达较B组降低(P<0.05)。
8逆转录PCR反应结果显示,与A组比较,B、C、D组PEDF mRNA表达明显降低(P<0.01, P<0.05, P<0.05),经人工发酵虫草菌丝体干粉及RSG干预后,C、D组PEDF mRNA表达较B组有所上调(P均<0.01)。
结论:
1大鼠经腹腔一次性注射大剂量STZ,可建立1型糖尿病大鼠模型。该模型具有持续高血糖、尿量增加、体重减轻等明显的DM症状,与人类1型糖尿病的临床特征类似。故此模型可用于1型糖尿病慢性并发症的研究。
2随着高血糖持续存在,DM大鼠肾脏出现不可逆损害,病理结构破坏,肾功能下降。形态学和实验室检查均符合糖尿病肾损害改变。糖代谢的异常对脂代谢及肝功能产生了一定的影响,共同对机体产生不利影响。
3 DM大鼠肾脏组织PEDF、MMP-2及TGF-β1之间可能存在一定的相互作用,三者表达水平的改变可能促进了糖尿病肾损害的病程进展。
4 DM大鼠血清PEDF、TGF-β1含量升高,提示肾脏病变过程中,循环中PEDF水平可能通过其他途径代偿性升高,与TGF-β1相互作用。
5经人工发酵虫草菌丝体干粉和RSG干预后,DM大鼠肾脏功能及病理改变均明显改善,表明其可能通过改善肾功能、减少尿白蛋白排泄、调节血脂、调节PEDF、MMP-2和TGF-β1的蛋白表达,发挥明显的肾脏保护作用。
Objective: Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes mellitus (DM). Long-term hyperglycaemia and metabolic disorders activates many pathways and causes renal pathological irreversible changes. DN, a principal cause of end-stage renal diseases, is characterized by glomerular sclerosis, thickening of glomerular basement membrane and extracellular matrix deposition. The pathogenesis of DN is not clear at present. Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor, that can protect neuronal nutrition, regulate vascular permeability and antiangiogiogenic activity. The synthesize and/or degradation of extracellular matrix is abnormal in the process of occurrence and development of DN. Matrix metalloproteinases (MMPs) play an important role in the metabolism of extracellular matrix. MMPs are classified into five groups based on substrate. MMP-2 belongs to gelatinases and is expressed in Mesangial cell, endotheliocyte, fibroblast and so on. It degradations typeⅣcollagen majorly. The alterations of expression and activity of MMP-2 may participate in genesis and development of diabetic nephropathy. Overexpression of TGF-β1 in diabetic glomeruli is believed to contribute to the glomerular hypertrophy, matrix accumulation, filtration barrier lesion, meanwhile inhibiting MMPs synthesis, diminishing ECM accumulation, accelerates the development of DN. Recent studies show that PEDF inhibits oxidant stress reactions, fiber production and inflammatory reaction, thus ameliorates renal hemodynamics and maintains renal homeostasis, and may be responsible for its salutary effect in DN. Cordyceps sinensis (Berk.)Sacc is a valuable traditional chinese medicine thainrich in polyoses, amino acids, fatty acid, mannitol and many kinds of microelements. Researches show that its protection may function by lowering inflammation reaction, inhibiting Mesangial cell proliferation and so on. Rosiglitazone is thiazolidinediones (TZDs) euglycemic agent and is believed to work through binding and modulating the activity of a family of nuelear transcription fators termed peorxisome proliefrator-activated receptorγ(PAPRγ). It is associated with slow improvement in glycemic control, antiinflammatory, regulation of lipid metabolism, inhibition of cell growth fators, reduction of urine protein excretion, etc.
In the study, rat model of type 1 diabetes was set up by an high-dose intraperitoneal injection of streptozotocin (STZ), and Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone was applied for intervention. Applying the methods of biochemistry, pathomorphology for detecting the changes of renal morphous and function and the expression of PEDF, MMP-2 and TGF-β1, explore the effect of PEDF and MMP-2 in the genesis and development of diabetic nephropathy and protection of Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone.
Methods: 60 eight weeks male SD rats were divided into two groups randomly: 15 normal control rats (group A) and 45 test rats. STZ (55mg/kg) was injected intraperitoneally to destroy some pancreas in order to induce hyperglycemia for test rats. 72 hours later, the rats whose blood glucose level was higher than 16.7 mmol/L in the subsequent 3 days were considered as diabetes. The diabetic rats were randomly divided into following groups: diabetic model rats (group B), diabetic model rats treated with 2.4g/kg/d Cordyceps Sinensis cultivated by Artificial Fermentation (group C), diabetic model rats treated with 4mg/kg/d rosiglitazone (group D). The experiment lasted 12 weeks. Blood glucose and body mass were measured at the beginning of the experiment and at the end of 0 weeks, 4 weeks, 8 weeks and 12 weeks respectively. After 12 weeks, 24 hour urine volume was collected with metabolic cage. Serum total cholesterol (TC), serum triglyceride (TG), blood urea nitrogen (BUN), serum creatinine (Scr), alanine aminotransferase (ALT), kindey weight index (KWI) and 24 hour urinary albumin excretion (UAE) were detected. Morphological changes were observed by microscope; The protein expression and location of PEDF, MMP-2 and TGF-β1 were examined by immunohistochemistry. Moreover, PEDF mRNA expression in kidney was analysed with reverse transcription polymerase chain reaction (RT-PCR). All the experimental data were dealt with SPSS13.0. Group comparisons adopt analysis of variance, the comparison of two sets adopt LSD test, level data adopt Kruskal-Wallis test.
Results:
1 72h after injecting STZ, blood glucose in B, C, D group, all over 16.7 mmol/L, was markedly increased compared with A group (P all<0.01). It suggested the model of type 1 diabetic mellitus was set up. At the end of 4, 8 and 12 weeks, blood glucose in B, C, D group all significantly higher than A group (P all<0.01).
2 At the end of 72h after injecting STZ and 4, 8 and 12 weeks, body mass in B, C, D group were markedly decreased compared with A group (P all<0.01). KWI in B, C, D group were markedly increased compared with A group (P all<0.01). KWI in C, D group were markedly improved compared with B group.
3 At the end of 12 weeks, urinary albumin excretion (UAE) in B、C、D group were significantly increased compared with A group (P all<0.01). UAE in both C and D group were significantly decreased compared with B group (P all<0.01).
4 Compared with A group, TG and TC in B, C, D group were increased (P all<0.01), TG and TC in C group were decreased compared with B group (P all<0.01); TG in D group were decreased compared with B group (P<0.01), while TC had no difference (P>0.05). Compared with A group, Scr, BUN and ALT in B, C, D group were increased (P<0.01, P<0.01, P<0.01; P<0.05, P<0.01, P<0.01; P<0.01, P<0.01, P<0.01); Scr, BUN in C, D group were decreased compared with B group (P all<0.01), while TC had no difference (P>0.05).
5 Compared with A, serum level of PEDF and TGF-β1 was significantly increased in B, C, D group (P all<0.05). Compared with B group, serum level of PEDF and TGF-β1 was decreased in C, D group (P all<0.05).
6 H.E. stain and PAS stain showed that compared with A, glomerular hypetrophy, thickening of glomerular basement membrance, mesangial expansion and cell proliferation in B group. A few glomerulars appeared sclerosis lightly, some renal tubules showed vacuolar degeneration. C and D group were ameliorated than B group.
7 Immunohistochemistry suggested that compared with A, expression of PEDF and MMP-2 in B group was obviously decreased, while that of TGF-β1 was significantly increased (P all<0.05). Expression of PEDF and MMP-2 in C, D group were meliorated compared with B group (P all<0.05), the expression of TGF-β1 downregulated (P<0.05).
8 Reverse transcription-polymerase chain reaction (RT-PCR) results showed that: compared with A group, the expression of PEDF mRNA was decreased significantly in B, C and D group (P<0.01, P<0.05, P<0.05); the expression of PEDF mRNA was markly upregulated in C and D group (P all<0.01).
Conclusion:
1 Rat model of type 1 diabetic mellitus can be set up by high-dose intrapenitoneal injection STZ. The model characterized by significantly diabetic symptoms analog to human, such as continuous hyperglycemia, polyuria, weight reduction and so on. So this model can satisfy the need of diabetic complication researches.
2 With the persistence of high blood glucose, irreversible damages appeared in diabetic rat kidneys, pathological structure damaged and renal function declined. Morphology and laboratory tests are in line with changes in diabetic kidney damage. Dysglycemia can cause dyslipidemia and hepatic dysfunction. These disturbances may play an adverse role in organism.
3 Interaction between PEDF, MMP-2 and TGF-β1 may exist in diabetic rat kidneys. Changes in the expression levels of the three may promote the progression of diabetic kidney damage.
4 Serum content of PEDF and TGF-β1 elevated in diabetic rats, indicating that PEDF may be compensated increased by other means and interacted with TGF-β1 in the pathogenesis of diabetic kidney damage.
5 After treated by Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone, kindey function and pathologic changes were improved noticeably. The results showed it could protect the kindey through ameliorating renal function, reducing the level of urine albuminuria, regulating the levels of serum lipids and the expression of PEDF, MMP-2 and TGF-β1.
引文
1 Rayner S, Bringnac S, Bumeister R, et al. MerMade: an oligodeo- xyribonucleotide synthesizer for high throughput oligonucleotide production in dual 96-well plates. Genome Res, 1998; 8(7): 741-747
2 Gettins PG, Simonovic M, Volz K. Pigment epithelium-derived factor (PEDF), a serpin with potent anti-angiogenic and neurite outgrowth- promoting properties. Biol Chem, 2002, 383(11): 1677-1682
3 Barnstable CJ, Tombran-Tink J. Neuroprotective and antiangiogenic actions of PEDF in the eye: molecular target sand therapeutic potential. Prog Retin Eye Res, 2004, 23(5): 561-577
4 Abramson LP, Stellmach V, Doll JA, et al. Wilms' tumor growth is suppressed by antiangiogenic pigment epithelium-derived factor in a xenograft model. J Pediatr Surg, 2003, 38(3): 336-342
5 Joshua J Wang, Sarah X Zhang, Kangmo Lu, et al. Decreased expression of pigment epithelium-derived factor is involved in the pathogenesis of diabetic nephropathy. Diabetes, 2005(1), 54: 243-250
6 Umezono T, Toyoda M, Kato M, et al. Glomerular expression of CTGF, TGF-bete1 and typeⅣcollagen in diabetic nephropathy. Nephrol, 2006, 19(6): 751-757
7 Wang JJ, Zhang SX, Mott R, et al. Salutary effect of pigment epithelium- derived factor in diabetic nephropathy: evidence for antifibrogenic activities. Diabetes, 2006, 55(6): 1678-1685
8 Bates DO, Harper SJ. Regulation of vascular permeability by vascular endothelial growth factors. Vascular Pharmacol, 2002, 39(4/5): 225-237
9 Sho-ichi Yamagishi, Kazuo Nakamura, Takanori Matsui, et al. Pigment epithelium-derived dactor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen speciesmediated vascular endothelial growth factor expression. J Biol Chem, 2006, 281(29): 20213-20220
10 Sho-ichi Yamagishi, Kazuo Nakamura, Seiji Ueda, et al. Pigmentepithelium-derived factor (PEDF) blocks angiotensin II signaling in endothelial cells via suppression of NADPH oxidase: a novel anti-oxidative mechanism of PEDF. Cell and Tissue Research, 2005, 320(3): 437-445
11 Yoshida Yumiko, Yamagishi Sho-Ichi, Matsui Takanori, et al. Protective role of pigment epithelium-derived factor (PEDF) in early phase of experimental diabetic retinopathy. Diabetes/ metabolism research and reviews, 2009, 25(7): 678-686
12 Tsao YP, Ho TC, Chen SL, et al. Pigment epithelium-derived factor inhibits oxidative stress-induced cell death by activation of extracellular signal-regulated kinases in cultured retinal pigment epithelial cells. Life Sci, 2006, 79(6): 545-550
13 Zhang SX, Wang JJ, Gao G, et al. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor. FASEB J, 2006, 20(2): 323-325
14 Joshua J. Wang, Sarah X. Zhang, Robert Mott, et al. Antiinflammatory effects of pigment epithelium-derived factor in diabetic nephropathy. Am J Physiol Renal Physiol, 2008, 294(5): F1166-F1173
15 Celentano DC, Frishman WH. Matrix metalloproteinases and coronary artery disease: a novel therapeutic target. J Clin Pharmacol, 1997, 37(11): 991-1000
16 Singh R, Song RH, Alavi N, et al. High glucose decreases matrix metallop roteinase-2 activity in rat mesangial cells via transforming growth factor-beta1. Exp Nephrol, 2001, 9(4): 249-257
17 Han SY, Jee YH, Han KH, et al. An imbalance between matrixmetallop- roteinase-2 and tissue inhibitor of matrixmetallop roteinase-2 contributes to the development of early diabetic nephropathy. Nephrol Dial Transplant, 2006, 21(9): 2406-2416
18 Mclennan S, Fisher E, Martell S Y, et al. Effects of glucose on matrix metallop roteinase and plasmin activities in mesangial cells: possible role in diabetic nephropathy. Kideny Int, 2000, 58 (Suppl 77): S81-S87
19 Cheng J, Grande J P. Trans forming growth factor-βsignal transductionand progressive renal disease. Exp Biol Med (Maywood), 2002, 227(11): 943-956
20迟秀娥,王元松.转化生长因子-β与糖尿病肾病.中国中西医结合肾病杂志,2009,10(4):374-376
21 Tut tle KR, Anderson PW. A novelpotential therapy fordiabetic nephropathy and vascular complications: protein kinase C beta inhibition. American Journal of Kidney Diseases, 2003, 42 (3): 456-465
22 Fukami K, Yamagishi S, Ueda S, et al. Role of AGEs in diabetic nephropathy. Curr Pharm Des, 2008, 14(10): 946-952
23 Ha H, Hwang IA, Park JH, et al. Role of reactive oxygen species in the pathogenesis of diabetic nephropathy. Diabetes Res Clin Pract, 2008, 82 (Suppl 1): S42-S45
24 Nam JS, Cho MH, Lee GT, et al. The activation of NF-kappaB and AP-1 in peripheral blood mononuclear cells isolated from patients with diabetic nephropathy. Diabetes Res Clin Pract, 2008, 81(1): 25-32
25 Mclennan SV, Wang XY, Moreno V, et al. Connective tissue growth factor mediates high glucose effects on matrix degradation through tissue inhibitor of matrix metalloproteinase type1: implications for diabetic nephropathy. Endocrinology, 2004, 145(12): 5646-5655
26王战建,王书畅.冬虫夏草治疗糖尿病肾病的作用机制研究进展.中国中西医结合肾病杂志,2008,9(1):88-90
27 Panchapakesan U, Sumual S, Pollock CA, et al. PPARγagonists exert antifibrotic effects in renal tubular cells exposed to high glucouse. Am J Physiol Renal Physiol, 2005, 289(5): 1153-1158
28 Moraes LA, Piqueras L, Bishop D, et al. Peroxisome proliferator-activated receptors and inflammation. Pharmacol Ther, 2005, 14(1): 2-4
29 Schernthaner G, Forst T, Gulba D, et al. Challenge in diabetes therapy: effects of glitazones beyond blood glucose control. Dtsch Med Wochenschr, 2009, 134(18): 949-954
30 Rieusset J, Chambrier C, Bouzakri K, et al. The expression of the p85alpha subunit of phosphatidylinositol 3-kinase is induced by activation of theperoxisome proliferator-activated receptor gamma in human adipocytes. Diabetologia, 2001, 44(5): 544-554
31 Guan Y. Peroxisome proliferator-activated receptor family and its relationship to renalcomplications of themetabo ic syndrome. J Am Soc N ephrol, 2004, 15(11): 2801-2815
32 BavirtiS, Ghanaat F, TayekJ A. Peroxisomep roliferator-activated rceptor-gamma agonist increases both low-density lipoprotein cholesterol particlesize and small high-density lipoprotein cholesterol inpatients with type 2 diabetes independent of diabeticc ontrol. Endocrine Practice, 2003, 9(6): 487-493
33 Pistrosch F, HerbingK, Kindel B, et al. Rosiglitazone improves glomerular hyperfiltration , renal endothelial dysfunction , and microalbuminura of incipient diabetic nephropathy in patients. Diabetes, 2005, 54(7): 2206-2211
34 Kim MK, Ko SH, Baek KH, et al. Long-term effects of rosiglitazone on the progressive decline in renal function in patients with type 2 diabetes. Korean J Intern Med, 2009, 24(3): 227-232
35 Isshiki K, Haneda M, Koya D, et al. Pathogenetic mechanisms of diabetic nephropathy. Diabetes, 2000, 49(6): 1022-1032
36 Tang SC, Chan LY, Leung JC, et al. Bradykinin and high glucose promote renal tubular inflammation. Nephrol Dial Transplant, 2010, 25(3): 698-710
1 Gettins PG, Simonovic M, Volz K. Pigment epithelium-derived factor (PEDF), a serpin with potent antiangiogenic and neurite outgrowth- promoting properties. Biol Chem, 2002, 383(11): 1677-1682
2 Barnstable CJ, Tombran-Tink J. Neuroprotective and antiangiogenic actions of PEDFin the eye: molecular target sand therapeutic potential. Prog Retin Eye Res, 2004, 23(5): 561-577
3 Abramson LP, Stellmach V, Doll JA, et al. Wilms’tumor growth is suppressed by antiangiogenic pigmentepithelium-derived factor in axenograft model. J Pediatr Surg, 2003, 38(3): 336-342
4 Joshua J Wang, Sarah X Zhang, Kangmo Lu, et al. Decreased expression of pigment epit helium-derived factor is involved in the pathogenesis of diabetic nephropathy. Diabetes, 2005, 54(1): 243-250
5 Hua L, Ren J G, William L, et al. Identification of the antivasopermeability effect of pigment epithelium-derived factor and its active site. Proc Natl Acad Sci USA, 2004, 101(17): 6605-6610
6 Zhang SX, Wang JJ, Gao G, et al. Pigment epithelium-derived factor downregulates vascular endothelial growth factor(VEGF)expression and inhbits VEGF-VEGF receptor 2 binding in diabetic retinopathy. Mol Endocrinol, 2006, 37(1): 1-12
7 Dawson DW, Volpert OV, Gillis P, et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science, 1999, 285(5425): 245-248
8 Bouck N. PEDF: anti-angiogenic guardian of ocular function. Trends Mol Med, 2002, 8(7): 330-334
9 Wang JJ, Zhang SX, Mott R, et al. Salutary effect of pigment epithelium-derived factor in diabetic nephropathy: evidence for antifibro-genic activities. Diabetes, 2006, 55(6): 1678-1685
10 Zhang SX, Wang JJ, Gao G, et al. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor. FASEB J, 2006, 20(2): 323-325
11 Guan M, Yam HF, Su B, et al. Loss of pigment epithelium derived factor expression in glioma progression. J Clin Patho l, 2003, 56(4): 277-282
12 Cai J, Parr C, Watkins G, et al. Decreased pigment epithelium-derived factor expression in human breast cancer progression. Clin Cancer Res, 2006, 12(11 Pt1): 3510-3517
13冯智英,邵春奎,金亦,等.胃癌组织P27、MMP-9和PEDF蛋白表达及意义.中国误诊学杂志,2007,7(7):1430-1432
14钟海花,徐勇,顾峻菱,等.高糖和SB203580对大鼠肾系膜细胞色素上皮细胞衍生因子表达的影响.中国糖尿病杂志,2008,16(5):307-309
15崔静,李竞,王蜀鄂.色素上皮衍生因子和血管内皮生长因子在糖尿病大鼠肾脏的不平衡表达.中国微循环,2008,6(12):155-158
16 Umezono T, Toyoda M, Kato M, et al. Glomerular expression of CTGF, TGF-bete 1 and typeⅣcollagen in diabetic nephropathy. Nephrol, 2006, 19(6): 751-757
17 Bates DO, Harper SJ. Regulation of vascular permeability by vascular endothelial growth factors. Vascular Pharmacol, 2002, 39(4/5): 225-237
18 Sho-ichi Yamagishi, Kazuo Nakamura, Takanori Matsui, et al. Pigment epithelium-derived dactor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen speciesmediated vascular endothelial growth factor expression. Biol Chem, 2006, 281(29): 20213-20220
19 Sho-ichi Yamagishi, Kazuo Nakamura, Seiji Ueda, et al. Pigment epithelium-derived factor (PEDF) blocks angiotensin II signaling in endothelial cells via suppression of NADPH oxidase: a novel anti-oxidative mechanism of PEDF. Cell and Tissue Research, 2005, 320(3): 437-445
20 Tsao YP, Ho TC, Chen SL, et al. Pigment epithelium-derived factor inhibits oxidative stress-induced cell death by activation of extracellular signal- regulated kinases in cultured retinal pigment epithelial cells. Life Sci, 2006, 79(6): 545-550
2 Gettins PG, Simonovic M, Volz K. Pigment epithelium-derived factor (PEDF), a serpin with potent anti-angiogenic and neurite outgrowth- promoting properties. Biol Chem, 2002, 383(11): 1677-1682
3 Barnstable CJ, Tombran-Tink J. Neuroprotective and antiangiogenic actions of PEDF in the eye: molecular target sand therapeutic potential. Prog Retin Eye Res, 2004, 23(5): 561-577
4 Abramson LP, Stellmach V, Doll JA, et al. Wilms' tumor growth is suppressed by antiangiogenic pigment epithelium-derived factor in a xenograft model. J Pediatr Surg, 2003, 38(3): 336-342
5 Joshua J Wang, Sarah X Zhang, Kangmo Lu, et al. Decreased expression of pigment epithelium-derived factor is involved in the pathogenesis of diabetic nephropathy. Diabetes, 2005(1), 54: 243-250
6 Umezono T, Toyoda M, Kato M, et al. Glomerular expression of CTGF, TGF-bete1 and typeⅣcollagen in diabetic nephropathy. Nephrol, 2006, 19(6): 751-757
7 Wang JJ, Zhang SX, Mott R, et al. Salutary effect of pigment epithelium- derived factor in diabetic nephropathy: evidence for antifibrogenic activities. Diabetes, 2006, 55(6): 1678-1685
8 Bates DO, Harper SJ. Regulation of vascular permeability by vascular endothelial growth factors. Vascular Pharmacol, 2002, 39(4/5): 225-237
9 Sho-ichi Yamagishi, Kazuo Nakamura, Takanori Matsui, et al. Pigment epithelium-derived dactor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen speciesmediated vascular endothelial growth factor expression. J Biol Chem, 2006, 281(29): 20213-20220
10 Sho-ichi Yamagishi, Kazuo Nakamura, Seiji Ueda, et al. Pigmentepithelium-derived factor (PEDF) blocks angiotensin II signaling in endothelial cells via suppression of NADPH oxidase: a novel anti-oxidative mechanism of PEDF. Cell and Tissue Research, 2005, 320(3): 437-445
11 Yoshida Yumiko, Yamagishi Sho-Ichi, Matsui Takanori, et al. Protective role of pigment epithelium-derived factor (PEDF) in early phase of experimental diabetic retinopathy. Diabetes/ metabolism research and reviews, 2009, 25(7): 678-686
12 Tsao YP, Ho TC, Chen SL, et al. Pigment epithelium-derived factor inhibits oxidative stress-induced cell death by activation of extracellular signal-regulated kinases in cultured retinal pigment epithelial cells. Life Sci, 2006, 79(6): 545-550
13 Zhang SX, Wang JJ, Gao G, et al. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor. FASEB J, 2006, 20(2): 323-325
14 Joshua J. Wang, Sarah X. Zhang, Robert Mott, et al. Antiinflammatory effects of pigment epithelium-derived factor in diabetic nephropathy. Am J Physiol Renal Physiol, 2008, 294(5): F1166-F1173
15 Celentano DC, Frishman WH. Matrix metalloproteinases and coronary artery disease: a novel therapeutic target. J Clin Pharmacol, 1997, 37(11): 991-1000
16 Singh R, Song RH, Alavi N, et al. High glucose decreases matrix metallop roteinase-2 activity in rat mesangial cells via transforming growth factor-beta1. Exp Nephrol, 2001, 9(4): 249-257
17 Han SY, Jee YH, Han KH, et al. An imbalance between matrixmetallop- roteinase-2 and tissue inhibitor of matrixmetallop roteinase-2 contributes to the development of early diabetic nephropathy. Nephrol Dial Transplant, 2006, 21(9): 2406-2416
18 Mclennan S, Fisher E, Martell S Y, et al. Effects of glucose on matrix metallop roteinase and plasmin activities in mesangial cells: possible role in diabetic nephropathy. Kideny Int, 2000, 58 (Suppl 77): S81-S87
19 Cheng J, Grande J P. Trans forming growth factor-βsignal transductionand progressive renal disease. Exp Biol Med (Maywood), 2002, 227(11): 943-956
20迟秀娥,王元松.转化生长因子-β与糖尿病肾病.中国中西医结合肾病杂志,2009,10(4):374-376
21 Tut tle KR, Anderson PW. A novelpotential therapy fordiabetic nephropathy and vascular complications: protein kinase C beta inhibition. American Journal of Kidney Diseases, 2003, 42 (3): 456-465
22 Fukami K, Yamagishi S, Ueda S, et al. Role of AGEs in diabetic nephropathy. Curr Pharm Des, 2008, 14(10): 946-952
23 Ha H, Hwang IA, Park JH, et al. Role of reactive oxygen species in the pathogenesis of diabetic nephropathy. Diabetes Res Clin Pract, 2008, 82 (Suppl 1): S42-S45
24 Nam JS, Cho MH, Lee GT, et al. The activation of NF-kappaB and AP-1 in peripheral blood mononuclear cells isolated from patients with diabetic nephropathy. Diabetes Res Clin Pract, 2008, 81(1): 25-32
25 Mclennan SV, Wang XY, Moreno V, et al. Connective tissue growth factor mediates high glucose effects on matrix degradation through tissue inhibitor of matrix metalloproteinase type1: implications for diabetic nephropathy. Endocrinology, 2004, 145(12): 5646-5655
26王战建,王书畅.冬虫夏草治疗糖尿病肾病的作用机制研究进展.中国中西医结合肾病杂志,2008,9(1):88-90
27 Panchapakesan U, Sumual S, Pollock CA, et al. PPARγagonists exert antifibrotic effects in renal tubular cells exposed to high glucouse. Am J Physiol Renal Physiol, 2005, 289(5): 1153-1158
28 Moraes LA, Piqueras L, Bishop D, et al. Peroxisome proliferator-activated receptors and inflammation. Pharmacol Ther, 2005, 14(1): 2-4
29 Schernthaner G, Forst T, Gulba D, et al. Challenge in diabetes therapy: effects of glitazones beyond blood glucose control. Dtsch Med Wochenschr, 2009, 134(18): 949-954
30 Rieusset J, Chambrier C, Bouzakri K, et al. The expression of the p85alpha subunit of phosphatidylinositol 3-kinase is induced by activation of theperoxisome proliferator-activated receptor gamma in human adipocytes. Diabetologia, 2001, 44(5): 544-554
31 Guan Y. Peroxisome proliferator-activated receptor family and its relationship to renalcomplications of themetabo ic syndrome. J Am Soc N ephrol, 2004, 15(11): 2801-2815
32 BavirtiS, Ghanaat F, TayekJ A. Peroxisomep roliferator-activated rceptor-gamma agonist increases both low-density lipoprotein cholesterol particlesize and small high-density lipoprotein cholesterol inpatients with type 2 diabetes independent of diabeticc ontrol. Endocrine Practice, 2003, 9(6): 487-493
33 Pistrosch F, HerbingK, Kindel B, et al. Rosiglitazone improves glomerular hyperfiltration , renal endothelial dysfunction , and microalbuminura of incipient diabetic nephropathy in patients. Diabetes, 2005, 54(7): 2206-2211
34 Kim MK, Ko SH, Baek KH, et al. Long-term effects of rosiglitazone on the progressive decline in renal function in patients with type 2 diabetes. Korean J Intern Med, 2009, 24(3): 227-232
35 Isshiki K, Haneda M, Koya D, et al. Pathogenetic mechanisms of diabetic nephropathy. Diabetes, 2000, 49(6): 1022-1032
36 Tang SC, Chan LY, Leung JC, et al. Bradykinin and high glucose promote renal tubular inflammation. Nephrol Dial Transplant, 2010, 25(3): 698-710
1 Gettins PG, Simonovic M, Volz K. Pigment epithelium-derived factor (PEDF), a serpin with potent antiangiogenic and neurite outgrowth- promoting properties. Biol Chem, 2002, 383(11): 1677-1682
2 Barnstable CJ, Tombran-Tink J. Neuroprotective and antiangiogenic actions of PEDFin the eye: molecular target sand therapeutic potential. Prog Retin Eye Res, 2004, 23(5): 561-577
3 Abramson LP, Stellmach V, Doll JA, et al. Wilms’tumor growth is suppressed by antiangiogenic pigmentepithelium-derived factor in axenograft model. J Pediatr Surg, 2003, 38(3): 336-342
4 Joshua J Wang, Sarah X Zhang, Kangmo Lu, et al. Decreased expression of pigment epit helium-derived factor is involved in the pathogenesis of diabetic nephropathy. Diabetes, 2005, 54(1): 243-250
5 Hua L, Ren J G, William L, et al. Identification of the antivasopermeability effect of pigment epithelium-derived factor and its active site. Proc Natl Acad Sci USA, 2004, 101(17): 6605-6610
6 Zhang SX, Wang JJ, Gao G, et al. Pigment epithelium-derived factor downregulates vascular endothelial growth factor(VEGF)expression and inhbits VEGF-VEGF receptor 2 binding in diabetic retinopathy. Mol Endocrinol, 2006, 37(1): 1-12
7 Dawson DW, Volpert OV, Gillis P, et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science, 1999, 285(5425): 245-248
8 Bouck N. PEDF: anti-angiogenic guardian of ocular function. Trends Mol Med, 2002, 8(7): 330-334
9 Wang JJ, Zhang SX, Mott R, et al. Salutary effect of pigment epithelium-derived factor in diabetic nephropathy: evidence for antifibro-genic activities. Diabetes, 2006, 55(6): 1678-1685
10 Zhang SX, Wang JJ, Gao G, et al. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor. FASEB J, 2006, 20(2): 323-325
11 Guan M, Yam HF, Su B, et al. Loss of pigment epithelium derived factor expression in glioma progression. J Clin Patho l, 2003, 56(4): 277-282
12 Cai J, Parr C, Watkins G, et al. Decreased pigment epithelium-derived factor expression in human breast cancer progression. Clin Cancer Res, 2006, 12(11 Pt1): 3510-3517
13冯智英,邵春奎,金亦,等.胃癌组织P27、MMP-9和PEDF蛋白表达及意义.中国误诊学杂志,2007,7(7):1430-1432
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