【作者】 谢敏；

【导师】 周建华；

【作者基本信息】 华中科技大学 ， 儿科学， 2007， 硕士

【摘要】 目的观察单侧输尿管梗阻(unilateral ureteral obstruction,UUO)诱导的大鼠肾间质纤维化模型中,肾间质纤维化病变及管周毛细血管的丢失情况,同时用新型免疫调节剂FTY720对其进行干预,观察其对肾间质纤维化程度及管周毛细血管丢失的影响及防治作用,并探讨其作用机制。方法将45只SD大鼠随机分为三组,即假手术组(SHAM),单侧输尿管梗阻模型组(UUO)和FTY720治疗组(FTY720),每组15只。模型组和FTY720治疗组大鼠行单侧输尿管梗阻术,假手术组开腹后不结扎输尿管,缝合腹腔,作为对照组。治疗组于术前三天给予FTY720 0.5mg·kg-1·d-1灌胃,模型组和假手术组给予等量生理盐水,三组灌胃直至处死动物。每组分别于术后第7﹑14﹑21天各处死大鼠五只,每组大鼠分别于处死前放入代谢笼留取24小时尿液,进行尿蛋白定量。并通过心脏采血,取血清测定血Scr、BUN。处死大鼠后留取梗阻侧肾组织做HE和MASSON染色观察肾间质纤维化程度,同时用Tunel法检测肾小管细胞凋亡情况,用免疫组化方法测定肾组织中CD3、ED1、α-平滑肌肌动蛋白(α-SMA)、胶原-Ⅲ(COL-Ⅲ)、CD141、VEGF的表达水平。结果24h尿蛋白定量,术后14d模型组为(15.48±1.52 )mg·d-1, FTY720治疗组为(9.7±1.74)mg·d-1,较模型组明显减低,差异有统计学意义(P<0.05)。术后7、14、21天,模型组血肌酐(Scr)水平分别为(123±15.76)μmol·L-1、(65.88±6.08)μmol·L-1、(51.38±6.9)μmol·L-1,FTY720治疗组血肌酐相应为(41.76±4.51)μmol·L-1、(39.54±7.94)μmol·L-1、(38.5±4.52)μmol·L-1,均明显降低(P<0.05),FTY720治疗组与假手术组比较差异无统计学意义。术后7、14天,模型组BUN分别为(16.14±3.21)mmol·L-1和(10.7±1.66)mmol·L-1,FTY720治疗组BUN分别为(8.08±0.96 )mmol·L-1和(6.98±0.80)mmol·L-1,均有一定程度下降,差异有统计学意义(P<0.05)。病理学检查显示,FTY720治疗组肾间质纤维化程度较模型组明显减轻。免疫组化结果显示,假手术组肾间质各时间点均未见炎性细胞浸润,模型组术后第7天肾间质出现大量CD3+、ED1+细胞浸润,第14、21天明显增多,与模型相比,FTY720治疗组肾间质CD3+、ED1+细胞数有一定程度地降低(P<0.01)。α-SMA表达在假手术组中仅限于血管,而在FTY720治疗组和UUO模型组中,表达明显增多,可见于肾小管和间质细胞,但FTY720治疗组中表达要弱于模型组。假手术组中各个时间点肾间质中可见少量Ⅲ型胶原表达,模型组手术后7、14和21dⅢ型胶原表达明显增强,主要集中于肾小管间质病变明显区域,FTY720治疗组中表达则明显减少。假手术组肾间质微血管未见明显病变,术后7、14、21天CD141阳性毛细血管密度分别为158.6±8.29、155.2±5.89、157±4.47,模型组肾间质微血管病变严重,主要集中于肾间质小管损伤明显区域, CD141阳性毛细血管数进行性减少,术后7、14、21天分别为116.8±8.92、98.8±3.96、62.2±7.01。FTY720治疗可明显减轻毛细血管病变及丢失,术后7、14、21天毛细血管密度分别为132.5±8.32、119.4±12.21、85±2.59,均显著高于模型组(P<0.01)。免疫组化结果显示,假手术组各时间点均未见明显凋亡,模型组术后第7天已见肾小管上皮细胞凋亡,随梗阻时间延长,凋亡细胞呈明显增多趋势,而FTY720治疗组各时间段肾小管上皮细胞凋亡数均低于模型组(P<0.01)。假手术组肾小管上皮细胞可见较多VEFG蛋白表达,模型组术后第7、14、21天VEFG蛋白表达量呈逐渐降低趋势,炎性细胞浸润明显处几乎无VEGF表达。与模型组相比,FTY720治疗组肾小管上皮细胞VEGF蛋白表达量高于模型组(P<0.01),但低于假手术组(P<0.05)。结论新型免疫调节剂FTY720能明显减少UUO大鼠肾间质的T淋巴细胞和巨噬细胞的浸润,从而减轻肾小管上皮细胞调亡,上调VEGF表达,减少肾间质毛细血管丢失,并且FTY720在一定程度上可以抑制肾小管上皮细胞转分化和细胞外基质的积聚,明显减轻单侧输尿管梗阻后大鼠肾间质纤维化的程度,对肾功能有一定的保护作用。更多还原

【Abstract】 【Objective】FTY720 is a novel immune modulating drug which obtained through structural modification of ISP-1, an immunosuppressive substance newly purified from Cordyceps sinensis , a Chinese herb traditionally used in the treatment of renal diseases. In recent years, the role of FTY720 in the immune mediated glomerulonephritis has been demonstrated, but its effects on renal interstitial fibrosis, a major determinant for prognosis, remain uncertain.. The present study is aimed to examine the inhibitory effect of FTY720 on renal interstitial fibrosis and peritubular cappilary loss in unilateral ureteral obstructic rats and to study the underlying mechanism .【Methods】Fouty-five males SD rats were randomly divided into sham-operated(SHAM) , unilateral ureteral obstruction (UUO) and UUO treated with FTY720 (UUO+FTY720) group . 0.5mg.kg-1.d-1 of FTY720 or vehicle was administrated through daily gavage and begun from three days before the operation till being sacrificed. 5 rats in each groups were sacrificed at day 7, day 14 and day 21 afte UUO or Sham-operation. In order to document if renal fibrosis closely correlates with renal insufficiency, 24-hour urine protein, blood urea nitrogen (BUN) and plasma creatinine were determined .Tubulointerstitial injury and interstitial fibrosis were examined by morphological analysis, apoptosis of tubular epithelial cells was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling , the expression of CD3,ED1 ,α-SMA ,COL-Ⅲ,CD141and VEGF was detected by immunohistochemical nethod and scored semi-quantitively. The number of CD141 positive PTC was quantitated in an area of 100 renal tubules.【Results】The amount of 24 hours urine protein was ((9.7±1.74)mg·d-1 at 14d after operation in FTY720 treated group, much lower than that in UUO group, which was (15.48±1.52 )mg·d-1 at same time .(P<0.05). Serum creatinine were (123±15.76)μmol·L-1,(65.88±6.08)μmol·L-1(,51.38±6.9)μmol·L-1 in UUO groups respectively at 7,14,21d, and (41.76±4.51)μmol·L-1,(39.54±7.94)μmol·L-1,(38.5±4.52)μmol·L-1 in FTY720 treated group at 7,14,21d, the levels in FTY720 treated group were significantly lower than those in UUO group. The Blood urea nitrogen was (16.14±3.21)mmol·L-1(,10.7±1.66)mmol·L-1 in UUO group and (8.08±0.96 )mmol·L-1(,6.98±0.80)mmol·L-1 in FTY720 treated group at 7,14d respectively, the levels were significantly lower in FTY720 group than that in UUO group (P<0.05). The renal pathological evaluation indicated that the scores of renal interstitial fibrosis were lower in FTY720 group than that in UUO group. Obvious lymphocyte and macrophage infiltration were found in tubular interstitial area in UUO group but significantly less in FTY720 treated group (P<0.01). Immunohistochemistry study revealed thatα-SMA expression was limited to vessels in SHAM group, but extended to renal tubule and interstitium in UUO and FTY720 treated group, while relatively weaker expression was observed in FTY720 group than in UUO group . Some collagenⅢexpression was found in SHAM group, which was much enhanced in UUO group at 7,14,21d and mainly distributed in renal interstitium, the expression in FTY720 group was also increased compared to SHAM group, but much lower than that in UUO group. In the SHAM group , there were no significant injury in peritubular capillary, and the numbers of CD141 positive PTC were 158.6±8.29, 155.2±5.89, 157±4.47 at 7, 14, 21 day. In the UUO group, peritubular capillary injuried severely ,and peritubular capillary density decreased markedly, the numbers of CD141 positive PTC were 116.8±8.92, 98.8±3.96, 62.2±7.01 at the 7th,14th,21st day. FTY720 could relieve the impairment and loss of peritubular capillary, as demonstrated in FTY720 treated group,the numbers of CD141 positive PTC were 132.5±8.32, 119.4±12.21, 85±2.59 at the 7th,14th,21st day ,significantly higher than those in UUO group(P<0.01)., No tubular epithelial cells apoptosis was observed in SHAM group, apoptosis was found in UUO group at day 7 after operation but was decreased in FTY720 treated group (P<0.01). The immunohistochemistry study revealed that the expression of VEGF protein was higher in FTY720 treated group than in UUO group(P<0.01),but still lower than in SHAM group(P<0.05).【Conclusions】Novel immunomodulator FTY720 can obviously inhibite lymphocyte and macrophage infiltration, renal tubule cell transdifferentiation, extracellular matrix accumulation and inhibit the tubular epithelial cell apoptosis, increase the expression of VEGF protein ,and prevent the loss of peritubular capillary interstitial fibrosis, thus ameliorate renal interstitial fibrosis and prevent renal disease progression.更多还原