【作者】 吕伟；

【导师】 关广聚；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2014， 博士

【摘要】 研究目的糖尿病肾病(diabetic nephropathy, DN)以早期肾脏体积增大、晚期肾小球硬化为主要特征,肾脏细胞的增殖、肥大与凋亡参与了DN的发生、发展。本研究采用体内动物实验和体外细胞培养相结合的方法,通过观察糖尿病大鼠及高糖刺激下的肾小球足细胞肥大、凋亡状态,测定细胞周期蛋白激酶抑制剂(cyclin-kinase inhibitors, CKIs)p27kip1、p21cipt及凋亡相关基因bcl-2、bax、cleaved caspase-3的表达,观察霉酚酸酯(mycophenolate mofetil, MMF)对足细胞肥大、凋亡的影响,为治疗DN提供科学的理论依据。研究方法体内动物实验：32只雄性健康Wistar大鼠,腹腔内注射链脲佐菌素(STZ),另外8只作为对照组(NC)。注射STZ72小时后剪尾采血,测血糖>16.7mmol/L,尿糖阳性,表明糖尿病模型成功建立。将糖尿病造模成功的32只大鼠,随机分为糖尿病组(DM)、霉酚酸酯组(DM+M)、缬沙坦组(DM+V)及霉酚酸酯+缬沙坦联合治疗组(DM+M.V),每组8只。成模2天后,治疗组分别给予MMF15mg.kg-1.d-1,缬沙坦40mg.kg-1.-1,联合治疗给予MMF15mg.kg-1.d-1+缬沙坦40mg.kg-1.d-1,1mg/d灌胃；而NC组和DM组每日予等量溶媒灌胃,共16周(w)。分别检测五组大鼠4w、8w及16w的左肾重/体重、血糖、血压、内生肌酐清除率及24小时尿蛋白排泄量；光学显微镜和电子显微镜观察糖尿病大鼠肾组织形态学的变化；免疫组织化学法检测肾皮质nephrin.WT1蛋白的表达；TUNEL法检测足细胞的凋亡率；Western-blot半定量及荧光实时定量PCR的分析方法,检测肾组织中p27kip1、p21cipt及bcl-2.bax.cleaved caspase-3的表达。体外细胞实验：订购的小鼠肾足细胞,按培养液中葡萄糖、霉酚酸(mycophenolic acid,MPA).缬沙坦的含量不同分为：①正常对照组NG(葡萄糖浓度5.6mmol/L+2.5%FBS的DMEM);②高糖组HG(葡萄糖浓度25mmol/L);③HG+M组：HG(葡萄糖浓度25mmol/L)+MPA2.5×10-7mol/L;④HG+V组:HG(葡萄糖浓度25mmol/L)+缬沙坦10-7mol/L;作用72小时(h)。采用流式细胞仪测定12h、24h、48h、72h各时间点足细胞的肥大指数及凋亡率,运用Western-blot半定量及荧光实时定量PCR的分析方法,检测肾小球足细胞中p27kip1、p21cip1及bcl-2、bax、cleaved caspase-3的表达。结果1.临床参数与NC组比较,DM大鼠血糖明显升高、体重减轻、肥大指数增加,24小时尿蛋白排泄量及肌酐清除率均明显增高(P<0.01)。16周末,DM大鼠肾重/体重指数较NC组明显升高(8.49±1.21vs4.57±0.82,P<0.01)；而霉酚酸酯和/或缬沙坦治疗16w后,肾重/体重指数明显降低,差异有显著性(P<O.01)；但各药物治疗组间无统计学意义(P>0.05)。16w末,DM大鼠尿蛋白量较NC组明显升高(55.37±2.33vs10.52±0.66,P<0.05);同样药物治疗16w后,24小时尿蛋白排泄量明显降低,差异有显著性(P<0.01)；各给药组间无统计学差异(P>0.05)。2.肾脏组织形态学观察光镜：4周前糖尿病大鼠无明显变化。此后,随着实验周期的延长,糖尿病大鼠肾小球的体积逐渐增大、系膜区逐渐增宽,且基底膜增厚、毛细血管腔受压、变窄；与NC组比较,16周末DM组肾小球的硬化指数(1.15±0.12vs0.15±0.05,P<0.01)及间质纤维化积分(0.58±0.08vs0.12±0.04,P<0.01)明显增加,其差异有显著性；各药物治疗组较DM组均有不同程度的改善。电镜：糖尿病大鼠肾小球毛细血管基底膜均质性增厚和系膜基质增多,伴足细胞足突的广泛融合,并可见裸露的基底膜及脱落的足细胞。MMF和/或缬沙坦治疗后,足突的融合减轻,系膜基质及足细胞的脱落均明显减少。3.肾组织中nephrin、WT-1蛋白及mRNA的表达免疫组织化学染色显示：正常大鼠nephrin、WT1沿肾小球基底膜呈棕黑色线形分布,而糖尿病大鼠的线样沉积变淡、变细、甚至消失,经MMF和/或缬沙坦治疗后,其线样沉积又恢复至粗线状。从第4周开始糖尿病组大鼠肾组织中nephrin蛋白(2.08±0.42vs2.63±0.51,P<0.05)及WT1蛋白(1.26±0.2vs1.59±0.26,P<0.05)的表达减少,且随着时间的推移nephrin、WT1蛋白的表达进一步降低。各治疗组与DM组比较,nephrin、WT1的表达均有不同程度的升高(P<0.05)；且16w末nephrin蛋白的表达DM+M组(1.83±0.43)及DM+M. V组(1.75±0.31)疗效优于DM+V组(1.39±0.28),有显著性差异(P<0.05)；而各药物治疗组间WT1蛋白的表达无明显差异(P>0.05)。从第4周开始DM组大鼠肾组织中nephrin mRNA (0.87±0.13vs0.95±0.091, P<0.05)、WT1mRNA (0.92±0.095vs1.04±0.079, P<0.05)的表达下降,且随着时间的推移其mRNA的表达进一步降低。而经霉酚酸酯和/或缬沙坦治疗后,nephrin、WT1mRNA的表达均有不同程度的上调(P<0.05),16周末WT1mRNA的表达霉酚酸酯联合治疗组(1.30±0.25)优于缬沙坦组(1.04±0.23),(P<0.05),而nephrin mRNA的表达三治疗组间比较无明显差异(P>0.05)。4.足细胞肥大指数高糖组足细胞肥大从24小时开始(0.828±0.23vs0.678±0.16,P<0.05),且随着时间的推移足细胞进一步肥大。各治疗组与高糖组比较,霉酚酸治疗组足细胞肥大从24小时开始减轻,而缬沙坦治疗组从48小时开始明显减轻,且随着时间推移,疗效逐渐增强(P<0.05)。5. p27kip1、p21cip1蛋白及mRNA的表达体内动物实验：从第4周开始DM组大鼠肾组织中p27kip1、p21cipl蛋白及mRNA的表达增强,与正常对照组相比有显著性差异(P<0.05),16周末达高峰。各治疗组与DM组比较,p27kip1、p21cipl蛋白及mRNA的表达从第8周开始降低(P<0.05),且随着实验周期的延长,p27kip1、p21cip1蛋白及mRNA的表达进一步降低；16w末p27kipl蛋白、mRNA及p21cip1mRNA的表达,霉酚酸酯+缬沙坦联合治疗组优于缬沙坦组(P<0.05)。体外足细胞培养：p27kip1、p21cipl蛋白及mRNA的表达：从24小时开始,HG组小鼠足细胞p27kip1、p21cip1蛋白及mRNA水平开始升高,与NG组相比差异有显著性(P<0.05)。且随着时间的推移,p27kipl和p21cipl蛋白及mRNA的表达进一步升高。霉酚酸及缬沙坦可明显降低p27kip1和p21cip1蛋白的表达,且72小时末p27kip1蛋白的表达霉酚酸的疗效优于缬沙坦(P<0.05)；霉酚酸和缬沙坦亦可明显降低p27kip1mRNA的表达,而p21cip1mRNA的表达仅霉酚酸起到明显的抑制作用。6.足细胞凋亡率体内动物实验：实验第8周糖尿病大鼠足细胞凋亡率开始增高(2.63±0.31vs0.84±0.26,P<0.01),且随着实验的延长,足细胞凋亡率进一步增高。各治疗组与DM组相比,均有不同程度的下降；16周末霉酚酸酯单一治疗组(1.81±0.27)及联合治疗组(1.24±0.19)优于缬沙坦组(2.25±0.33),差异有显著性(P<0.05)。体外足细胞实验：高糖培养48小时后,足细胞凋亡率明显高于NG组(4.97±0.58vs2.77±0.31,P<0.05)。两治疗组,仅霉酚酸治疗72小时后足细胞凋亡率降低(P<0.05),而缬沙坦治疗组无变化(P>0.05),且72小时末霉酚酸的疗效优于缬沙坦,差异有统计学意义(P<0.05)。7. bax、bcl-2、cleaved caspase-3蛋白及mRNA的表达体内动物实验：糖尿病大鼠的bax、cleaved caspase-3蛋白及mRNA的表达从实验第8周起明显升高,而bcl-2的表达下降,差异有显著性(P<0.05)。各治疗组与DM组比较,其表达均有不同程度的改善；且16周末bax/bcl-2蛋白的表达,DM+M组(1.42±0.2)、DM+M. V组(1.57±0.26)与DM+V(1.95±0.24)比较差异有显著性,(P<0.01)。体外足细胞培养：高糖培养下,足细胞的bax、cleaved caspase-3蛋白及mRNA的表达从实验第48小时起升高,而bcl-2的表达下降,差异有显著性(P<0.05),且随着时间的推移比值进一步升高。经霉酚酸或缬沙坦治疗后,其比值较HG组下降,差异有显著性(P<0.01)；且72小时末bax/bcl-2、cleaved caspase-3蛋白的表达,HG+M组较HG+V组明显降低,差异有显著性(P<0.05)。结论：1.足细胞的肥大与凋亡参与了糖尿病肾病的发生、发展。糖尿病早期,足细胞的肥大伴随着细胞周期蛋白激酶抑制剂p27kip1、p2cip1表达的增高。随着糖尿病病程的进展,足细胞的凋亡率增高,晚期的肾小球硬化与其过度凋亡有关,并伴随着凋亡相关基因bax、cleaved caspase-3表达的升高及bc1-2表达的降低。2.霉酚酸酯和缬沙坦可有效的抑制肾小球足细胞的肥大与凋亡,调节细胞周期相关蛋白(p27kip1、p21cip1)和凋亡相关基因(bax、bcl-2、cleaved caspase-3)的表达,起到一定的保护作用。因此,推测霉酚酸酯可能通过调节细胞周期负性调控蛋白p27kip1、p21cip及凋亡相关基因bax、bcl-2及cleaved caspase-3的表达,抑制和阻断肾小球足细胞的肥大与凋亡,从而起到降低蛋白尿、延缓糖尿病肾病进展的作用。更多还原

【Abstract】 ObjectiveDiabetic nephropathy (DN), one of the most serious microvascular complications of diabetes mellitus(DM), is a major cause of end-stage renal disease. Renal hypertrophy in the early stage and glomerulosclerosis in the end stage are the characteristics of DN. Both hypertrophy and apoptosis of renal cells are thought to be involved in the pathogenesis of DN. The present study aimed to examine the effect of mycophenolate mofetil (MMF), a new immunosuppressive agent, on the hypertrophy and apoptosis of podocyte, and investigate the underlying mechanisms.MethodsIn vivo studies:Forty male rats were randomly divided into two groups:healthy control group (NC, n=8) and diabetes mellitus group (DM, n=32). Diabetic rat models were induced by streptozotocin(STZ) injected intraperitoneally. After the DM model was established successfully, DM group was subdivided into four groups:group treated with the dissolvent (DM), group treated with mycophenolate mofetil (DM+M), group treated with valsartan (DM+V) and group treated with mycophenolate mofetil and valsartan (DM+M.V). After16weeks of treatment, the weight of kidney(Kw) and body(Bw), serum glucose, blood pressure, serum creatinine and24hours urinary protein excretion(UP) was detected. Histomorphology of renal tissue was observed by an optical microscope and electron microscope. The expressions of nephrin and Wilm’s tumor suppressor gene (WT1) were detected by immunohistochemistry. Apoptosis of podocytes were determined by transferase-mediated dUTP nick-end labeling (TUNEL) test. The protein and mRNA expressions of p27kip1,p21cip1, bax and bcl-2were examined by Western blot and competitive reverse transcription-polymerase chain reaction (RT-PCR). In vitro studies:Cultured rat podocyte were exposed to5.6mmol/L normal glucose (NG),25mmol/L high glucose (HG) with mycophenolic acid(MPA)(HG+M) or Valsartan (HG+V) for72hours. Hypertrophy and apoptosis of podocytes was determined by flow cytometry. The protein and mRNA expressions of p27kip1, p21cip1, bax and bcl-2were examined by Western blot and RT-PCR.Results1. Animal dataAfter16weeks, the serum glucose in DM group was higher than that in NC group, but it couldn’t be reduced by MMF or/and Valsartan. The systolic blood pressure and serum creatinine in DM group were not different from those in NC group.The ratio of kidney to body weight and24hours urinary protein excretion were significantly higher in DM (Kw/Bw:8.49±1.21vs4.57±0.82,P<0.01),(UP:55.37±2.33vs10.52±0.66, P<0.05), these increase were rescued by treatmeat with MMF or/and valsartan.2. Renal morphological changes of ratsLight microscope:After16weeks, rat glomerular volume increased, the glomerular basement membrane thickened, mesangial area expanded and glomerular capillary lumen narrowed by compression in DM group. The glomerulosclerosis and interstitial fibrotic lesion in DM group increased dramatically as compared with those in NC group (1.15±0.12vs0.15±0.05, P<0.01),(0.58±0.08vs0.12±0.04, P<0.01),but they could be reduced in different degrees by treatment with MMF or/and valsartan.Electron microscope:In DM group, the foot process of podocytes fused and flattened. Glomerular basement membrane exposed and some podocytes ablated. The glomerular basement membrane thickened and the mesangial matrix increased. However, in the three treatment groups, glomerular foot process fusion was reduced, the ablated podocytes decreased and the extracellular matrix was also reduced.3. The expressions of nephrin and WT1in the renal cortex of ratsNephrin and WT1expressed at the glomerular basement membrane in normal renal tissue. But in the DM renal tissue, the intensity and the positive cells number decreased or even disappeared. However, in the treatment groups, it was restored significantly.The protein expressions of nephrin and WT1were reduced significantly in the DM group at4weeks (nephrin:2.08±0.42vs2.63±0.51, P<0.05),(WT1:1.26±0.2vs1.59±0.26, P<0.05), and continued to decrease with the prolongation of time. This decrease was also inhibited by MMF or/and valsartan. In addition, the effect of MMF(1.83±0.43) and MMF+valsartan (1.75±0.31) on the protein expressions of nephrin better than valsartan(1.39±0.28) after16weeks (P<0.05).The mRNA expressions of nephrin and WT1were also markedly decreased in the DM group at4weeks (nephrin:0.87±0.13vs0.95±0.091, P<0.05),(WT1:0.92±0.095vs1.04±0.079, P<0.05), and with a gradual decrease thereafter. But in the treatment groups, it was restored significantly (P<0.05). In addition, the effect of MMF+valsartan (1.30±0.25) on the mRNA expressions of WT1better than valsartan(1.04±0.23) after16weeks (P<0.05).4. Celluar hypertrophyIn vitro results:The ratio of proterin/cell numbers as a well-established hypertrophy index revealed a significant increase in podocytes exposed to high glucose (0.828±0.23vs0.678±0.16, P<0.05) for24hours, and the increment in the ratio of protein/cell numbers by high glucose was inhibited with MPA or Valsartan in podocytes(P<0.05).5. The protein and mRNA expressions of p27kip1, p21cip1In vivo results:P27kip1and p21cip1protein expression were significantly increased in DM compared to NC rats at4weeks (p27kip1:1.83±0.25vs1.0±0.08, P<0.05; p21cipl:1.62±0.24vs0.96±0.05, P<0.05), and continued to increase with the prolongation of time. In additoin, the mRNA exprossion of these cyclin-kinase inhibitors showed similar patterns to their protein expression (P<0.05). This increase was also inhibited by MMF or/and valsartan. Moreover, Combination of MMF with Valsartan had more significantly inhibitory effect on the protein and mRNA expressions of P27kip1than valsartan after16weeks (P<0.05).In vitro results:P27kipl and p21cipl protein expression were significantly increased in podocytes exposed to HG compared to NG for24hours (p27kip1:1.24±0.13vs0.97±0.09, P O.05; p21cipl:1.27±0.140vs0.97±0.09, P<0.05), and with a gradual increase thereafter. In additoin, the mRNA exprossion of these cyclin-kinase inhibitors showed similar patterns to their protein expression (P<0.05). These changes induced by HG were significantly abrogated by MPA.6. Apoptosis of podocytesIn vivo results:Then number of apoptotic podocytes was significantly increased in DM (2.63±0.31) compared to NC(0.84±0.26)(P<0.01) at8weeks, and continued to increase with the prolongation of time. It could be inhibited by MMF and Valsartan. MMF with/or Valsartan had more significantly inhibitory effect on the apoptosis of podocytes in the glomerulus of rats with diabetic nephropathy (P<0.05). In addition, the effect of MMF(1.81±0.27) and MMF+valsartan (1.24±0.19) on the apoptosis of podocytes better than valsartan(2.25±0.33) after16weeks (P<0.05).In vitro results:Exposure to HG for48hours resulted in a remarkable increase in apoptotic podocytes(4.97±0.58vs2.77±0.31, P<0.05), and these increments in apoptosis were markedly ameliorated by the administreation of MPA after72hours. There were no differences in the apoptotic podocytes between HG and HG+V groups.7. The protein and mRNA expressions of of bax, bcl-2and cleaved caspase-3In vivo results:Bax and cleaved caspase-3protein and mRNA expressions were significantly increased,while bcl-2protein and mRNA expressions were markedly decreased in the DM compared to NC at8weeks (P<0.05). Administration of MMF or/and valsartan significantly ameliorated the increases in the radio of bax/bcl-2(P<0.05) and cleaved caspase-3protein and mRNA expressions in DM glomeruli(.P<0.05). In addition, MMF with/or Valsartan had more significantly inhibitory effect on the radio of bax/bcl-2protein expressions in DM glomeruli after16weeks (P<0.05).In vitro results:HG significantly increased bax and cleaved caspase-3protein and mRNA expressions and significantly reduced bcl-2protein and mRNA expressions in cultured podocytes for48h(P<0.05). These changes induced by HG was significantly abrogated by MPA or valsartan. Moreover, MPA had more significantly inhibitory effect than valsartan on the radio of bax/bcl-2and cleaved caspase-3protein expressions in HG after72h (P<0.05).Conclusion1. These results demonstrated hypertrophy and apoptosis of podocytes may play important roles in pathogenesis of diabetic nephropathy. It was proved that the expression of cyclin-kinase inhibitors such as p27kip1, p21cip1was significantly increased followed by the hypertrophy of podocyte. With the progression of diabetic nephropathy, the number of apoptotic podocytes increased gradually and the balance between proliferation and apoptosis was broken in diabetic kidney. Apoposis which was regulated by apoptosis related genes such as bax, bcl-2and cleaved caspase-3.2. MMF and valsartan can inhibit abnormal hypertrophy and apoptosis of podocytes in diabetes, partly by regulating the expression of cell cycle related protein p27kip1, p21cip1and apoptosis related genes,sueh as bax, bcl-2and cleaved caspase-3. These suggest that the protective effects of MMF on renal function maybe partly through inhibiting abnormal renal cell growth by regulating cell cycle related protein or apoptosis related genes.更多还原