中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
Chinese Journal of Nephrology
2015年
9期
686-692
,共7页
马雯%陈晓岚%徐玉音%陆飞%范亚平
馬雯%陳曉嵐%徐玉音%陸飛%範亞平
마문%진효람%서옥음%륙비%범아평
受体,前列腺素E%RNA,小分子干扰%转化生长因子β1%肾小球系膜细胞%细胞增殖%MAP激酶信号系统
受體,前列腺素E%RNA,小分子榦擾%轉化生長因子β1%腎小毬繫膜細胞%細胞增殖%MAP激酶信號繫統
수체,전렬선소E%RNA,소분자간우%전화생장인자β1%신소구계막세포%세포증식%MAP격매신호계통
Receptors%prostaglandin E%RNA%small interfering%Transforming growth factor beta1%Mesangial cells%Cell proliferation%MAP kinase signaling system
目的 探讨前列腺素E2(PGE2)受体3亚型(EP3)对转化生长因子?β1(TGF?β1)诱导的小鼠肾小球系膜细胞的损伤及可能机制. 方法 体外培养原代野生型系膜细胞,采用脂质体LipofectamineTM 2000化学合成法将siRNA转染至系膜细胞中沉默EP3受体,筛选干扰效率最大的EP3?siRNA片段.实验分组:(1)正常对照组;(2)TGF?β1(10 μg/L)组;(3)NC?siRNA+TGF?β1(10μg/L)组;(4)EP3?siRNA组;(5)EP3?siRNA+TGF?β1(10μg/L)组.CCK?8法检测TGF?β1对细胞增殖的影响;ELISA法检测细胞PGE2和cAMP的表达;实时荧光定量PCR法检测纤维连接蛋白(FN)、结缔组织生长因子(CTGF)、环氧化酶2(COX2)、膜结合型前列腺素E2合酶1(mPGES1)mRNA的表达;Western印迹检测FN、CTGF、COX2、mPGES1蛋白表达及ERK1/2、p38 MAPK活性的变化. 结果 与正常对照组相比,TGF?β1组系膜细胞增殖明显增加(P<0.05),PGE2和cAMP表达增加(均P<0.05),FN、CTGF、COX2、mPGES1的mRNA和蛋白的表达均上调(均P<0.05);与TGF?β1组相比,EP3?siRNA+TGF?β1组系膜细胞增殖减少(P<0.05),FN、CTGF、COX2、mPGES1的mRNA和蛋白的表达均下调(均P<0.05).与正常组比较,TGF?β1组ERK1/2、p38 MAPK磷酸化水平明显增强(均P<0.05);而EP3?siRNA+TGF?β1组的ERK1/2、p38 MAPK磷酸化水平较TGF?β1组明显下调(均P<0.05). 结论 EP3?siRNA可能通过增加cAMP的产生,抑制ERK1/2、p38 MAPK磷酸化,反馈抑制COX2及PGE2 ,从而下调FN、CTGF的表达,减轻TGF?β1诱导的小鼠系膜细胞损伤.
目的 探討前列腺素E2(PGE2)受體3亞型(EP3)對轉化生長因子?β1(TGF?β1)誘導的小鼠腎小毬繫膜細胞的損傷及可能機製. 方法 體外培養原代野生型繫膜細胞,採用脂質體LipofectamineTM 2000化學閤成法將siRNA轉染至繫膜細胞中沉默EP3受體,篩選榦擾效率最大的EP3?siRNA片段.實驗分組:(1)正常對照組;(2)TGF?β1(10 μg/L)組;(3)NC?siRNA+TGF?β1(10μg/L)組;(4)EP3?siRNA組;(5)EP3?siRNA+TGF?β1(10μg/L)組.CCK?8法檢測TGF?β1對細胞增殖的影響;ELISA法檢測細胞PGE2和cAMP的錶達;實時熒光定量PCR法檢測纖維連接蛋白(FN)、結締組織生長因子(CTGF)、環氧化酶2(COX2)、膜結閤型前列腺素E2閤酶1(mPGES1)mRNA的錶達;Western印跡檢測FN、CTGF、COX2、mPGES1蛋白錶達及ERK1/2、p38 MAPK活性的變化. 結果 與正常對照組相比,TGF?β1組繫膜細胞增殖明顯增加(P<0.05),PGE2和cAMP錶達增加(均P<0.05),FN、CTGF、COX2、mPGES1的mRNA和蛋白的錶達均上調(均P<0.05);與TGF?β1組相比,EP3?siRNA+TGF?β1組繫膜細胞增殖減少(P<0.05),FN、CTGF、COX2、mPGES1的mRNA和蛋白的錶達均下調(均P<0.05).與正常組比較,TGF?β1組ERK1/2、p38 MAPK燐痠化水平明顯增彊(均P<0.05);而EP3?siRNA+TGF?β1組的ERK1/2、p38 MAPK燐痠化水平較TGF?β1組明顯下調(均P<0.05). 結論 EP3?siRNA可能通過增加cAMP的產生,抑製ERK1/2、p38 MAPK燐痠化,反饋抑製COX2及PGE2 ,從而下調FN、CTGF的錶達,減輕TGF?β1誘導的小鼠繫膜細胞損傷.
목적 탐토전렬선소E2(PGE2)수체3아형(EP3)대전화생장인자?β1(TGF?β1)유도적소서신소구계막세포적손상급가능궤제. 방법 체외배양원대야생형계막세포,채용지질체LipofectamineTM 2000화학합성법장siRNA전염지계막세포중침묵EP3수체,사선간우효솔최대적EP3?siRNA편단.실험분조:(1)정상대조조;(2)TGF?β1(10 μg/L)조;(3)NC?siRNA+TGF?β1(10μg/L)조;(4)EP3?siRNA조;(5)EP3?siRNA+TGF?β1(10μg/L)조.CCK?8법검측TGF?β1대세포증식적영향;ELISA법검측세포PGE2화cAMP적표체;실시형광정량PCR법검측섬유련접단백(FN)、결체조직생장인자(CTGF)、배양화매2(COX2)、막결합형전렬선소E2합매1(mPGES1)mRNA적표체;Western인적검측FN、CTGF、COX2、mPGES1단백표체급ERK1/2、p38 MAPK활성적변화. 결과 여정상대조조상비,TGF?β1조계막세포증식명현증가(P<0.05),PGE2화cAMP표체증가(균P<0.05),FN、CTGF、COX2、mPGES1적mRNA화단백적표체균상조(균P<0.05);여TGF?β1조상비,EP3?siRNA+TGF?β1조계막세포증식감소(P<0.05),FN、CTGF、COX2、mPGES1적mRNA화단백적표체균하조(균P<0.05).여정상조비교,TGF?β1조ERK1/2、p38 MAPK린산화수평명현증강(균P<0.05);이EP3?siRNA+TGF?β1조적ERK1/2、p38 MAPK린산화수평교TGF?β1조명현하조(균P<0.05). 결론 EP3?siRNA가능통과증가cAMP적산생,억제ERK1/2、p38 MAPK린산화,반궤억제COX2급PGE2 ,종이하조FN、CTGF적표체,감경TGF?β1유도적소서계막세포손상.
Objective To investigate the effects and mechanisms of prostaglandin E2 receptor subtype 3 (EP3) on transforming growth factor β1 (TGF-β1)-induced mouse mesangial cells damage. Methods Primary mouse mesangial cells were separated and cultured. Three siRNAs were synthesized and transfected into mesangial cells for silencing EP3 by LipofectamineTM 2000 and the best one was chosen. MCs were grouped into: (1)control group; (2)TGF-β1 (10 μg/L) group; (3)NC-siRNA plus TGF-β1 (10 μg/L) group; (4) EP3-siRNA group; (5)EP3-siRNA plus TGF-β1 (10 μg/L). Then the proliferation of MCs was evaluated by CCK-8 assay. The expression of PGE2 and cAMP in cell supernatant were detected by ELISA. The mRNA and protein expression of fibronectin (FN), connective tissue growth factor (CTGF), cyclooxygenase-2 (COX2), membrane-bound prostaglandin E2 synthase 1 (mPGES1) were detected by real - time quantitative PCR and Western blotting. The phosphorylation of p38 MAPK and ERK1/2 was decected by Western blotting. Results Compared with control group, the cell proliferation induced by TGF-β1 was increased (P<0.05), the expression of PGE2 and cAMP were improved, mRNA and protein expression of FN, CTGF, COX2 and mPGES1 were up-regulated (all P<0.05). Compared with TGF-β1 group, the cell proliferation in EP3-siRNA plus TGF-β1 group was reduced, the expression of FN, CTGF, COX2 and mPGES1 mRNA and protein were downregulated (all P<0.05), the phosphorylation of ERK1/2, p38 MAPK were also declined (P<0.05). Conclusion EP3-siRNA may reduce TGF-β1-induced cell damage through upregulating the expression of cAMP, repressing the activity of ERK1/2 and p38 MAPK, inhibiting the expression of COX2 mPGES1 and PGE2 by feedback, then decreased the expression of FN and CTGF.
