中华肾病研究电子杂志
中華腎病研究電子雜誌
중화신병연구전자잡지
2015年
1期
29-36
,共8页
乔晞%赵宁%王利华%张瑞婧%韩伟霞
喬晞%趙寧%王利華%張瑞婧%韓偉霞
교희%조저%왕리화%장서청%한위하
肾脏%间质纤维化%Intermedin%转化生长因子-β1%纤连蛋白
腎髒%間質纖維化%Intermedin%轉化生長因子-β1%纖連蛋白
신장%간질섬유화%Intermedin%전화생장인자-β1%섬련단백
Kidney%Interstitial fibrosis%Intermedin%Transforming growth factor-beta 1%Fibronectin
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-pcDNA3.1真核表达质粒和空质粒转入肾组织,real-timeRT-PCR及免疫组化法检测转染效率。各组分别于术后7d、14d留取梗阻侧肾组织。HE、Masson染色观察肾组织病理变化;real-timeRT-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,7d,t=11.927,P=0.0003;14d,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(7d,t=3.892,P=0.018;14d,t=4.047,P=0.016),而空质粒+UUO组与UUO组无显著差别(7d,t=0.562,P=0.604;14d,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-β1mRNA水平7d,t=4.432,P=0.011;14d,t=4.873,P=0.006;蛋白质水平7d,t=5.312,P=0.006;14d,t=4.482,P=0.011;Fn1mRNA水平7d,t=6.053,P=0.004;14d,t=7.345,P=0.002;蛋白质水平7d,t=8.791,P=0.009;14dt=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7d,t=3.103,P=0.036;14d,t=2.913,P=0.044;蛋白质水平7d,t=2.955,P=0.042;14d,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7d,t=0.095,P=0.929;14d,t=0.158,P=0.882;蛋白质水平7d,t=0.159,P=0.881;14d,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7d,t=0.176,P=0.869;14d,t=0.126,P=0.906;蛋白质水平7d,t=0.198,P=0.853;14d,t=0.196,P=0.854;转染空质粒mRNA水平7d,t=0.100,P=0.925;14d,t=0.097,P=0.928;蛋白质水平7d,t=0.042,P=0.968;14d,t=0.060,P=0.955)。结论上调肾组织IMD的表达能明显减轻肾间质纤维化,但其作用不是通过直接抑制TGF-β1的表达实现的。
目的:觀察上調腎組織intermedin(IMD)錶達對單側輸尿管梗阻(UUO)大鼠腎間質纖維化的影響。方法健康雄性Wistar大鼠隨機分為假手術組、UUO組、IMD+UUO組、空質粒+UUO組。IMD+UUO組和空質粒+UUO組在輸尿管結扎前分彆將IMD-pcDNA3.1真覈錶達質粒和空質粒轉入腎組織,real-timeRT-PCR及免疫組化法檢測轉染效率。各組分彆于術後7d、14d留取梗阻側腎組織。HE、Masson染色觀察腎組織病理變化;real-timeRT-PCR檢測腎組織中轉化生長因子-β1(TGF-β1)、纖連蛋白(Fn1)的mRNA錶達;Western印跡法檢測Fn1的蛋白錶達;免疫組化法檢測TGF-β1的蛋白錶達。結果與假手術組相比,UUO組腎髒齣現明顯的病理改變,腎間質纖維化程度隨梗阻時間延長加重(與假手術組比較,7d,t=11.927,P=0.0003;14d,t=8.891,P=0.0009);IMD+UUO組腎髒病理改變及腎間質纖維化程度較同時間點UUO組明顯減輕(7d,t=3.892,P=0.018;14d,t=4.047,P=0.016),而空質粒+UUO組與UUO組無顯著差彆(7d,t=0.562,P=0.604;14d,t=0.035,P=0.974)。與同時間點假手術組相比,UUO組TGF-β1、Fn1的錶達明顯升高(TGF-β1mRNA水平7d,t=4.432,P=0.011;14d,t=4.873,P=0.006;蛋白質水平7d,t=5.312,P=0.006;14d,t=4.482,P=0.011;Fn1mRNA水平7d,t=6.053,P=0.004;14d,t=7.345,P=0.002;蛋白質水平7d,t=8.791,P=0.009;14dt=8.027,P=0.001);轉染IMD質粒後Fn1的錶達較同時間點UUO組明顯下降(mRNA水平7d,t=3.103,P=0.036;14d,t=2.913,P=0.044;蛋白質水平7d,t=2.955,P=0.042;14d,t=2.991,P=0.040);而轉染空質粒後Fn1的錶達無明顯變化(mRNA水平7d,t=0.095,P=0.929;14d,t=0.158,P=0.882;蛋白質水平7d,t=0.159,P=0.881;14d,t=0.170,P=0.874)。轉染IMD和空質粒對TGF-β1的錶達均無明顯影響(轉染IMD質粒mRNA水平7d,t=0.176,P=0.869;14d,t=0.126,P=0.906;蛋白質水平7d,t=0.198,P=0.853;14d,t=0.196,P=0.854;轉染空質粒mRNA水平7d,t=0.100,P=0.925;14d,t=0.097,P=0.928;蛋白質水平7d,t=0.042,P=0.968;14d,t=0.060,P=0.955)。結論上調腎組織IMD的錶達能明顯減輕腎間質纖維化,但其作用不是通過直接抑製TGF-β1的錶達實現的。
목적:관찰상조신조직intermedin(IMD)표체대단측수뇨관경조(UUO)대서신간질섬유화적영향。방법건강웅성Wistar대서수궤분위가수술조、UUO조、IMD+UUO조、공질립+UUO조。IMD+UUO조화공질립+UUO조재수뇨관결찰전분별장IMD-pcDNA3.1진핵표체질립화공질립전입신조직,real-timeRT-PCR급면역조화법검측전염효솔。각조분별우술후7d、14d류취경조측신조직。HE、Masson염색관찰신조직병리변화;real-timeRT-PCR검측신조직중전화생장인자-β1(TGF-β1)、섬련단백(Fn1)적mRNA표체;Western인적법검측Fn1적단백표체;면역조화법검측TGF-β1적단백표체。결과여가수술조상비,UUO조신장출현명현적병리개변,신간질섬유화정도수경조시간연장가중(여가수술조비교,7d,t=11.927,P=0.0003;14d,t=8.891,P=0.0009);IMD+UUO조신장병리개변급신간질섬유화정도교동시간점UUO조명현감경(7d,t=3.892,P=0.018;14d,t=4.047,P=0.016),이공질립+UUO조여UUO조무현저차별(7d,t=0.562,P=0.604;14d,t=0.035,P=0.974)。여동시간점가수술조상비,UUO조TGF-β1、Fn1적표체명현승고(TGF-β1mRNA수평7d,t=4.432,P=0.011;14d,t=4.873,P=0.006;단백질수평7d,t=5.312,P=0.006;14d,t=4.482,P=0.011;Fn1mRNA수평7d,t=6.053,P=0.004;14d,t=7.345,P=0.002;단백질수평7d,t=8.791,P=0.009;14dt=8.027,P=0.001);전염IMD질립후Fn1적표체교동시간점UUO조명현하강(mRNA수평7d,t=3.103,P=0.036;14d,t=2.913,P=0.044;단백질수평7d,t=2.955,P=0.042;14d,t=2.991,P=0.040);이전염공질립후Fn1적표체무명현변화(mRNA수평7d,t=0.095,P=0.929;14d,t=0.158,P=0.882;단백질수평7d,t=0.159,P=0.881;14d,t=0.170,P=0.874)。전염IMD화공질립대TGF-β1적표체균무명현영향(전염IMD질립mRNA수평7d,t=0.176,P=0.869;14d,t=0.126,P=0.906;단백질수평7d,t=0.198,P=0.853;14d,t=0.196,P=0.854;전염공질립mRNA수평7d,t=0.100,P=0.925;14d,t=0.097,P=0.928;단백질수평7d,t=0.042,P=0.968;14d,t=0.060,P=0.955)。결론상조신조직IMD적표체능명현감경신간질섬유화,단기작용불시통과직접억제TGF-β1적표체실현적。
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 ultrasound-microbubble-mediated system before the ureter was obstructed.The transfection rate was detected by real-time RT-PCR and immunohistochemistry.Groups of six animals were killed at 7 d and 1 4 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-β1 and fibronectin (Fn1 )were detected by real-time RT-PCR.Protein expression of TGF-β1 was detected by immunohistochemical staining.Protein expression of Fn1 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 =0.01 8 vs UUO group;1 4 d,t =4.047,P =0.01 6 vs UUO group)and downregulation of fibronectin (mRNA 7 d,t =3.1 03,P =0.036 vs UUO group;1 4 d,t =2.91 3,P =0.044 vs UUO group;Protein 7 d,t =2.955,P =0.042 vs UUO group;1 4 d,t =2.991 ,P =0.040 vs UUO group),whereas TGF-β1 upregulation was not affected (mRNA 7 d,t =0.1 76,P =0.869 vs UUO group;1 4 d,t =0.1 26,P =0.906 vs UUO;Protein 7 d,t =0.1 98,P =0.853 vs UUO;1 4 d,t =0.1 96,P =0.854 vs UUO group).Conclusion Our results indicated that kidney-specific IMD gene delivery inhibited renal fibrosis induced by UUO.The inhibitory effect of IMD on renal fibrosis was not achieved by directly inhibiting TGF-β1 expression.