中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2012年
5期
392-396
,共5页
基质金属蛋白酶9%金属蛋白酶1组织抑制剂%成纤维细胞%罗格列酮%环孢素A
基質金屬蛋白酶9%金屬蛋白酶1組織抑製劑%成纖維細胞%囉格列酮%環孢素A
기질금속단백매9%금속단백매1조직억제제%성섬유세포%라격렬동%배포소A
Matrix metalloproteinase 9%Tissue inhibitor of metalloproteinase-1%Fibroblasts%Rosiglitazone%Cyclosporine A
目的 观察罗格列酮(RGZ)对环孢素A(CsA)作用下大鼠肾脏成纤维细胞(NRK)过氧化物酶体增生物激活受体γ(PPARγ)和基质金属蛋白酶-9(MMP-9)表达的影响,探讨罗格列酮对环孢素A肾毒性保护作用的分子机制.方法 构建、筛选和扩增PPARγ基因的siRNA载体,并将载体转染至体外培养的NRK细胞.体外培养NRK细胞,随机分组.(1)对照组:不加处理；(2)RGZ组:加RGZ( 10 μmol/L)；(3)CsA组:加CsA( 1.0 mg/L)；(4)CsA+RGZ 组:同时加CsA( 1.0 mg/L)及RGZ(10 μmol/L)；(5)CsA+RGZ+siRNA组:质粒pRNAT- U6.2/LentiPPARγ-236转染NRK细胞,然后同时加入CsA( 1.0 mg/L)及RGZ(10 μmol/L).培养24 h后,用实时荧光定量和RT-PCR法检测各组细胞中PPARγ、MMP-9、金属蛋白酶1组织抑制剂(TIMP-1) mRNA表达,用Western印迹法检测纤连蛋白(FN)的蛋白表达.结果 CsA明显上调PPARγ、MMP-9和TIMP-1 mRNA的表达(均P＜0.05);RGZ与CsA合用后,PPARγ、MMP-9和TIMP-1 mRNA表达下降(均P＜0.05)；应用PPARγ siRNA后,与RGZ+ CsA组相比,PPARγ mRNA表达显著下降(P＜0.05),MMP-9 mRNA和TIMP-1 mRNA表达有所增加(均P＜ 0.05).CsA明显上调FN蛋白表达(P＜0.05)；RGZ与CsA合用后,FN蛋白表达下降(P＜0.05)；应用含PPARγ siRNA后,FN蛋白表达有所增加(P＜0.05).结论 罗格列酮可以显著减轻CsA诱导NRK细胞分泌的FN蛋白及MMP-9、TIMP-1 mRNA的表达,构建的siRNA质粒转染NRK细胞,能够有效地阻断NRK中PPARγ mRNA的表达,部分阻断罗格列酮对CsA毒性的改善作用.
目的 觀察囉格列酮(RGZ)對環孢素A(CsA)作用下大鼠腎髒成纖維細胞(NRK)過氧化物酶體增生物激活受體γ(PPARγ)和基質金屬蛋白酶-9(MMP-9)錶達的影響,探討囉格列酮對環孢素A腎毒性保護作用的分子機製.方法 構建、篩選和擴增PPARγ基因的siRNA載體,併將載體轉染至體外培養的NRK細胞.體外培養NRK細胞,隨機分組.(1)對照組:不加處理；(2)RGZ組:加RGZ( 10 μmol/L)；(3)CsA組:加CsA( 1.0 mg/L)；(4)CsA+RGZ 組:同時加CsA( 1.0 mg/L)及RGZ(10 μmol/L)；(5)CsA+RGZ+siRNA組:質粒pRNAT- U6.2/LentiPPARγ-236轉染NRK細胞,然後同時加入CsA( 1.0 mg/L)及RGZ(10 μmol/L).培養24 h後,用實時熒光定量和RT-PCR法檢測各組細胞中PPARγ、MMP-9、金屬蛋白酶1組織抑製劑(TIMP-1) mRNA錶達,用Western印跡法檢測纖連蛋白(FN)的蛋白錶達.結果 CsA明顯上調PPARγ、MMP-9和TIMP-1 mRNA的錶達(均P＜0.05);RGZ與CsA閤用後,PPARγ、MMP-9和TIMP-1 mRNA錶達下降(均P＜0.05)；應用PPARγ siRNA後,與RGZ+ CsA組相比,PPARγ mRNA錶達顯著下降(P＜0.05),MMP-9 mRNA和TIMP-1 mRNA錶達有所增加(均P＜ 0.05).CsA明顯上調FN蛋白錶達(P＜0.05)；RGZ與CsA閤用後,FN蛋白錶達下降(P＜0.05)；應用含PPARγ siRNA後,FN蛋白錶達有所增加(P＜0.05).結論 囉格列酮可以顯著減輕CsA誘導NRK細胞分泌的FN蛋白及MMP-9、TIMP-1 mRNA的錶達,構建的siRNA質粒轉染NRK細胞,能夠有效地阻斷NRK中PPARγ mRNA的錶達,部分阻斷囉格列酮對CsA毒性的改善作用.
목적 관찰라격렬동(RGZ)대배포소A(CsA)작용하대서신장성섬유세포(NRK)과양화물매체증생물격활수체γ(PPARγ)화기질금속단백매-9(MMP-9)표체적영향,탐토라격렬동대배포소A신독성보호작용적분자궤제.방법 구건、사선화확증PPARγ기인적siRNA재체,병장재체전염지체외배양적NRK세포.체외배양NRK세포,수궤분조.(1)대조조:불가처리；(2)RGZ조:가RGZ( 10 μmol/L)；(3)CsA조:가CsA( 1.0 mg/L)；(4)CsA+RGZ 조:동시가CsA( 1.0 mg/L)급RGZ(10 μmol/L)；(5)CsA+RGZ+siRNA조:질립pRNAT- U6.2/LentiPPARγ-236전염NRK세포,연후동시가입CsA( 1.0 mg/L)급RGZ(10 μmol/L).배양24 h후,용실시형광정량화RT-PCR법검측각조세포중PPARγ、MMP-9、금속단백매1조직억제제(TIMP-1) mRNA표체,용Western인적법검측섬련단백(FN)적단백표체.결과 CsA명현상조PPARγ、MMP-9화TIMP-1 mRNA적표체(균P＜0.05);RGZ여CsA합용후,PPARγ、MMP-9화TIMP-1 mRNA표체하강(균P＜0.05)；응용PPARγ siRNA후,여RGZ+ CsA조상비,PPARγ mRNA표체현저하강(P＜0.05),MMP-9 mRNA화TIMP-1 mRNA표체유소증가(균P＜ 0.05).CsA명현상조FN단백표체(P＜0.05)；RGZ여CsA합용후,FN단백표체하강(P＜0.05)；응용함PPARγ siRNA후,FN단백표체유소증가(P＜0.05).결론 라격렬동가이현저감경CsA유도NRK세포분비적FN단백급MMP-9、TIMP-1 mRNA적표체,구건적siRNA질립전염NRK세포,능구유효지조단NRK중PPARγ mRNA적표체,부분조단라격렬동대CsA독성적개선작용.
Objective To investigate the effects of rosiglitazone on the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and matrix metalloproteinase-9 (MMP-9) in renal interstitial fibroblasts induced by cyclosporine A, and discuss renal protective effect of rosiglitazone on renal toxicity of cyclosporine A. Methods Construction, screening and amplification of the target siRNA vector for PPARγ were carried out.The inhibitory effect of siRNA on the expression of PPARγ in normal rat kidney (NRK) cells was evaluated.NRK cells were cultured in the routine way.Experimental groups: (1)control group:single NRK cells without treatment; (2)RGZ group:NRK cells with RGZ (10 μmol/L); (3)CsA group:NRK cells with CsA (1.0 mg/L); (4)CsA+RGZ group:NRK cells with CsA (1.0 mg/L) plus RGZ (10 μ mol/L); (5)CsA+RGZ+siRNA group:pRNAT-U6.2/Lenti-PPARγ-236 plasmid transfected into NRK cells,then CsA (1.0 mg/L) plus RGZ (10 μmol/L).The mRNA expression of PPARγ was detected by real-time RCR.The mRNA expressions of MMP-9 and TIMP-1 were detected by RT-RCR.The protein expression of FN was detected by Western blotting. Results CsA up-regulated the mRNA level of PPARγ,MMP-9 and TIMP-1 (P＜0.05),and the up-regulation was inhibited by RGZ significantly (P＜0.05).The application of PPARγ siRNA resulted in the decreasing of PPARγ mRNA (P＜0.05),and partly reversed the inhibition effect of RGZ on MMP-9 and TIMP-1 mRNA (all P＜0.05).CsA up-regulated the protein level of FN (P＜0.05),and this effect was significantly inhibited by RGZ (P＜0.05).The application of PPARγ siRNA could reverse the inhibition effect of RGZ on FN protein expression (P＜0.05). Conclusion RGZ can inhibit the expressions of FN,MMP-9 and TIMP-1 induced by CsA which may be the mechanism of the protective effect of RGZ on renal interstitial fibrosis induced by CsA.
