机构地区:[1]山西医科大学第二医院肾内科山西省肾脏病研究所,太原030001
出 处:《中华肾病研究电子杂志》2015年第1期29-33,共5页Chinese Journal of Kidney Disease Investigation(Electronic Edition)
基 金:国家自然科学基金青年科学基金项目(编号:81100531)
摘 要:目的观察上调肾组织intermedin(IMD)表达对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的影响。方法健康雄性Wistar大鼠随机分为假手术组、UUO组、IMD+UUO组、空质粒+UUO组。IMD+UUO组和空质粒+UUO组在输尿管结扎前分别将IMD-pc DNA3.1真核表达质粒和空质粒转入肾组织,real-time RT-PCR及免疫组化法检测转染效率。各组分别于术后7 d、14 d留取梗阻侧肾组织。HE、Masson染色观察肾组织病理变化;real-time RT-PCR检测肾组织中转化生长因子-β1(TGF-β1)、纤连蛋白(Fn1)的mRNA表达;Western印迹法检测Fn1的蛋白表达;免疫组化法检测TGF-β1的蛋白表达。结果与假手术组相比,UUO组肾脏出现明显的病理改变,肾间质纤维化程度随梗阻时间延长加重(与假手术组比较,7 d,t=11.927,P=0.0003;14 d,t=8.891,P=0.0009);IMD+UUO组肾脏病理改变及肾间质纤维化程度较同时间点UUO组明显减轻(7 d,t=3.892,P=0.018;14 d,t=4.047,P=0.016),而空质粒+UUO组与UUO组无显著差别(7 d,t=0.562,P=0.604;14 d,t=0.035,P=0.974)。与同时间点假手术组相比,UUO组TGF-β1、Fn1的表达明显升高(TGF-β1 mRNA水平7 d,t=4.432,P=0.011;14 d,t=4.873,P=0.006;蛋白质水平7 d,t=5.312,P=0.006;14 d,t=4.482,P=0.011;Fn1 mRNA水平7 d,t=6.053,P=0.004;14 d,t=7.345,P=0.002;蛋白质水平7 d,t=8.791,P=0.009;14 d t=8.027,P=0.001);转染IMD质粒后Fn1的表达较同时间点UUO组明显下降(mRNA水平7 d,t=3.103,P=0.036;14 d,t=2.913,P=0.044;蛋白质水平7 d,t=2.955,P=0.042;14 d,t=2.991,P=0.040);而转染空质粒后Fn1的表达无明显变化(mRNA水平7 d,t=0.095,P=0.929;14 d,t=0.158,P=0.882;蛋白质水平7 d,t=0.159,P=0.881;14 d,t=0.170,P=0.874)。转染IMD和空质粒对TGF-β1的表达均无明显影响(转染IMD质粒mRNA水平7 d,t=0.176,P=0.869;14 d,t=0.126,P=0.906;蛋白质水平7 d,t=0.198,P=0.853;14 d,t=0.196,P=0.854;转染空质粒mRNA水平7 d,t=0.100,P=0.925;14 d,t=0.097,P=0.928;蛋白质水平7 d,t=0.042,P=0.968;14 d,t=0.060,P=0.955)。结论上调肾组织IMD的表Objective To investigate the effects of intermedin (IMD) overexpression on renal interstitial fibrosis in the obstructed kidney of rats with unilateral ureteral obstruction (UUO). Methods Male Wistar rats were randomly divided into sham-operated group, UUO group, IMD + UUO group, and empty plasmid + UUO group. For IMD + UUO group or empty plasmid + UUO group, pcDNA3. 1-IMD plasmid or control empty vector was transfected into the left kidney via the renal artery by an uhrasound- microbubble-mediated system before the ureter was obstructed. The transfection rate was detected by real- time RT-PCR and immunohistoehemistry. Groups of six animals were killed at 7 d and 14 d after operation. Kidneys were harvested for further analysis. Paraffin-embedded transverse kidney slices were stained with hematoxylin and eosin. For analyzing the degree of tubulointerstitial collagen deposition, sections were stained with Masson trichrome, mRNA expression levels of TGF-~I and fibronectin (Fnl) were detected by real-time RT-PCR. Protein expression of TGF-~I was detected by immunohistochemical staining. Protein expression of Fnl was examined by Western blot analysis. Results The ultrasound- microbubble- mediated delivery system yielded high expression of IMD in kidney cells. IMD overexpression remarkably attenuated UUO-induced tubular injury, and blunted fibrotic response as shown by decreased interstitial collagen deposition (7 d, t =3. 892, P =0. 018 vs UUO group; 14 d, t =4. 047, P = 0. 016 vs UUO group) and downregulation of fibronectin ( mRNA 7 d, t = 3. 103, P = 0. 036 vs UUO group ; 14 d, t = 2. 913, P = 0. 044 vs UUO group; Protein 7 d, t = 2. 955, P = 0. 042 vs UUO group; 14 d, t = 2. 991, P = 0. 040 vs UUO group), whereas TGF-β upregulation was not affected (mRNA 7 d, t =0. 176, P =0. 869 vs UUO group; 14 d, t =0. 126, P =0. 906 vs UUO; Protein 7 d, t =0. 198, P =0. 853 vs UUO; 14 d, t =0. 196, P = 0. 854 vs UUO group). Conclusion Our results indicated that kidney-specific IMD gene delivery
关 键 词:肾脏 间质纤维化 INTERMEDIN 转化生长因子-β1 纤连蛋白
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