中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2010年
9期
633-637
,共5页
唐功耀%杨敏%谌贻璞%王艳艳
唐功耀%楊敏%諶貽璞%王豔豔
당공요%양민%심이박%왕염염
脂联素%肿瘤坏死因子-α%肾小球%内皮细胞%DNA测序
脂聯素%腫瘤壞死因子-α%腎小毬%內皮細胞%DNA測序
지련소%종류배사인자-α%신소구%내피세포%DNA측서
Adiponectin%Tumor ncerosis factor-α%Glomerulus%Endothelial%DNA
目的 探讨人肾小球微血管内皮细胞(HRGEC)是否合成脂联素,及肿瘤坏死因子α(TNF-α)对HRGEC合成脂联素的影响.方法 用免疫组织化学染色检测脂联素在人肾组织表达;分别用逆转录多聚酶链反应(RT-PCR)和免疫印迹法检测体外培养的原代HRGEC对脂联素mRNA及蛋白的表达,并对PCR产物进行DNA测序鉴定.用TNF-α刺激HRGEC,分别用实时荧光定量PCR和时间分辨荧光法检测HRGEC脂联素mRNA及蛋白表达变化.结果 (1)免疫组织化学染色显示人肾小球毛细血管壁脂联素染色强阳性.(2)RT-PCR检测发现原代培养的HRGEC表达脂联素mRNA;PCR产物经DNA测序与基因库中的脂联素DNA序列完全一致;蛋白质印迹检测证实HRGEC培养上清中含有脂联素蛋白.(3)不同浓度TNF-α刺激HRGEC 24 h,与未刺激组比较,实时荧光定量PCR显示.250 IU/ml和500 IU/ml组的脂联素mRNA表达量显著上调(P<0.05和P<0.01);时间分辨荧光检测显示,250 IU/ml和500 IU/ml组的脂联素蛋白含量显著增加(P<0.05和P<0.01).(4)500 U/ml浓度TNF-α刺激HRGEC 6、12、24和48 h,与未刺激组比较,6、12和24 h组的脂联索mRNA表达均显著上调(P<0.05,P<0.05和P<0.01);24 h和48 h组的脂联素蛋白含量显著增加(P<0.01).结论 HRGEC能合成脂联素,TNF-α可上调HRGEC的脂联素mRNA和蛋白表达.
目的 探討人腎小毬微血管內皮細胞(HRGEC)是否閤成脂聯素,及腫瘤壞死因子α(TNF-α)對HRGEC閤成脂聯素的影響.方法 用免疫組織化學染色檢測脂聯素在人腎組織錶達;分彆用逆轉錄多聚酶鏈反應(RT-PCR)和免疫印跡法檢測體外培養的原代HRGEC對脂聯素mRNA及蛋白的錶達,併對PCR產物進行DNA測序鑒定.用TNF-α刺激HRGEC,分彆用實時熒光定量PCR和時間分辨熒光法檢測HRGEC脂聯素mRNA及蛋白錶達變化.結果 (1)免疫組織化學染色顯示人腎小毬毛細血管壁脂聯素染色彊暘性.(2)RT-PCR檢測髮現原代培養的HRGEC錶達脂聯素mRNA;PCR產物經DNA測序與基因庫中的脂聯素DNA序列完全一緻;蛋白質印跡檢測證實HRGEC培養上清中含有脂聯素蛋白.(3)不同濃度TNF-α刺激HRGEC 24 h,與未刺激組比較,實時熒光定量PCR顯示.250 IU/ml和500 IU/ml組的脂聯素mRNA錶達量顯著上調(P<0.05和P<0.01);時間分辨熒光檢測顯示,250 IU/ml和500 IU/ml組的脂聯素蛋白含量顯著增加(P<0.05和P<0.01).(4)500 U/ml濃度TNF-α刺激HRGEC 6、12、24和48 h,與未刺激組比較,6、12和24 h組的脂聯索mRNA錶達均顯著上調(P<0.05,P<0.05和P<0.01);24 h和48 h組的脂聯素蛋白含量顯著增加(P<0.01).結論 HRGEC能閤成脂聯素,TNF-α可上調HRGEC的脂聯素mRNA和蛋白錶達.
목적 탐토인신소구미혈관내피세포(HRGEC)시부합성지련소,급종류배사인자α(TNF-α)대HRGEC합성지련소적영향.방법 용면역조직화학염색검측지련소재인신조직표체;분별용역전록다취매련반응(RT-PCR)화면역인적법검측체외배양적원대HRGEC대지련소mRNA급단백적표체,병대PCR산물진행DNA측서감정.용TNF-α자격HRGEC,분별용실시형광정량PCR화시간분변형광법검측HRGEC지련소mRNA급단백표체변화.결과 (1)면역조직화학염색현시인신소구모세혈관벽지련소염색강양성.(2)RT-PCR검측발현원대배양적HRGEC표체지련소mRNA;PCR산물경DNA측서여기인고중적지련소DNA서렬완전일치;단백질인적검측증실HRGEC배양상청중함유지련소단백.(3)불동농도TNF-α자격HRGEC 24 h,여미자격조비교,실시형광정량PCR현시.250 IU/ml화500 IU/ml조적지련소mRNA표체량현저상조(P<0.05화P<0.01);시간분변형광검측현시,250 IU/ml화500 IU/ml조적지련소단백함량현저증가(P<0.05화P<0.01).(4)500 U/ml농도TNF-α자격HRGEC 6、12、24화48 h,여미자격조비교,6、12화24 h조적지련색mRNA표체균현저상조(P<0.05,P<0.05화P<0.01);24 h화48 h조적지련소단백함량현저증가(P<0.01).결론 HRGEC능합성지련소,TNF-α가상조HRGEC적지련소mRNA화단백표체.
Objective To investigate the expression and regulation of adiponectin in human renal glomerular endothelial cells (HRGEC). Method The adiponectin expression in human renal tissue was examined with immunohistochemistry assay, and the adiponectin mRNA and protein expression of HRGECwas confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively.PCR product was sequenced. To investigate the regulation action of TNF-α, the adiponectin produced by HRGEC was semi-quantitatively determined with real-time PCR in mRNA level and quantitatively measured with dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) in protein level, respectively.Results (1) Positive adiponectin staining was observed on human glomerular capillary wall with immunohistochemistry assay. (2) Adiponectin mRNA expression in HRGEC was detected by RT-PCR, and the DNA sequence of this PCR product is consistent with adiponectin DNA sequence in Gene Bank.Adiponectin protein was also found in the supernatant of cultured HRGEC by Western blot. (3) Compared with control groups, the adipouectin mRNA expression in HRGEC determined by real-time PCR was significantly up-regulated after 250 IU/ml and 500 IU/ml TNF-α stimulation for 24 h ( P < 0. 05 and P <0. 01 ), and the adiponectin protein levels in culture supernatant measured by DELFIA were also significantly increased in these two groups ( P < 0. 05 and P < 0. 01 ). (4) Compared with control groups, the adiponectin mRNA expression in HRGEC was significantly up-regulated after 500 IU/ml TNF-α stimulation for 6 h, 12 h and 24 h (P<0.05, P<0. 05 and P<0. 01), and the adiponectin protein levels in culture supernatant were also significantly increased after stimulation for 24 h and 48 h ( P < 0. 01 ). ConclusionsHRGEC is able to synthesize adiponectin, and TNF-α can up-regulate its mRNA and protein expression.
