中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2012年
18期
1281-1284
,共4页
谢恺庆%史伟%李东风%章斌%刘双信%周红卫
謝愷慶%史偉%李東風%章斌%劉雙信%週紅衛
사개경%사위%리동풍%장빈%류쌍신%주홍위
C反应蛋白质%肾小管%上皮细胞%受体,IgG%转化生长因子β1
C反應蛋白質%腎小管%上皮細胞%受體,IgG%轉化生長因子β1
C반응단백질%신소관%상피세포%수체,IgG%전화생장인자β1
C-reactive protein%Kidney tubules%Epithelial cells%Receptors,IgG%Transforming growth factor betal
目的 观察C反应蛋白(CRP)对肾小管上皮细胞Fcγ受体表达的影响,并探讨Fcγ受体在CRP诱导转化生长因子β1(TGFβ1)表达中的作用.方法 以CRP干预人肾小管上皮细胞株(HK-2细胞),实时定量PCR检测FcγRⅠ (CD64)、FcγRⅡa(CD32a)、FcγRⅢ(CD16)3种Fcγ受体mRNA表达.根据实验结果,采用流式细胞术和Western印迹法检测CD32a蛋白的表达.以抗CD32a抗体封闭CD32a受体,实时定量PCR检测TGFβ1 mRNA表达,酶联免疫吸附试验(ELISA)检测TGFβ1蛋白和Ⅰ型胶原蛋白、Ⅳ型胶原蛋白的分泌水平.结果 HK-2细胞无CD64和CD16mRNA表达,有CD32a mRNA表达.CD32a mRNA表达呈剂量依赖性增高(P＜0.01),CRP 10 mg/L刺激达到高峰.流式细胞术示CRP 10 mg/L刺激HK-2细胞24 h CD32a平均表达量为23.35％±7.43％,未刺激组为1.66％±0.28％,差异有统计学意义(P＜0.01).Western印迹显示CRP以剂量依赖方式上调CD32a表达,CRP10 mg/L显著性上调CD32a表达(P＜0.01).抗CD32a抗体封闭CD32a受体后,CRP作用减弱,TGFβ1 mRNA表达下调(P＜0.01),TGFβ1、Ⅰ型胶原蛋白、Ⅳ型胶原蛋白的分泌下降(均P＜0.05).结论 CRP刺激可上调肾小管上皮细胞表面的CD32a受体的表达,CRP通过肾小管上皮细胞CD32a受体介导促纤维化因子TGFβ1表达上调.
目的 觀察C反應蛋白(CRP)對腎小管上皮細胞Fcγ受體錶達的影響,併探討Fcγ受體在CRP誘導轉化生長因子β1(TGFβ1)錶達中的作用.方法 以CRP榦預人腎小管上皮細胞株(HK-2細胞),實時定量PCR檢測FcγRⅠ (CD64)、FcγRⅡa(CD32a)、FcγRⅢ(CD16)3種Fcγ受體mRNA錶達.根據實驗結果,採用流式細胞術和Western印跡法檢測CD32a蛋白的錶達.以抗CD32a抗體封閉CD32a受體,實時定量PCR檢測TGFβ1 mRNA錶達,酶聯免疫吸附試驗(ELISA)檢測TGFβ1蛋白和Ⅰ型膠原蛋白、Ⅳ型膠原蛋白的分泌水平.結果 HK-2細胞無CD64和CD16mRNA錶達,有CD32a mRNA錶達.CD32a mRNA錶達呈劑量依賴性增高(P＜0.01),CRP 10 mg/L刺激達到高峰.流式細胞術示CRP 10 mg/L刺激HK-2細胞24 h CD32a平均錶達量為23.35％±7.43％,未刺激組為1.66％±0.28％,差異有統計學意義(P＜0.01).Western印跡顯示CRP以劑量依賴方式上調CD32a錶達,CRP10 mg/L顯著性上調CD32a錶達(P＜0.01).抗CD32a抗體封閉CD32a受體後,CRP作用減弱,TGFβ1 mRNA錶達下調(P＜0.01),TGFβ1、Ⅰ型膠原蛋白、Ⅳ型膠原蛋白的分泌下降(均P＜0.05).結論 CRP刺激可上調腎小管上皮細胞錶麵的CD32a受體的錶達,CRP通過腎小管上皮細胞CD32a受體介導促纖維化因子TGFβ1錶達上調.
목적 관찰C반응단백(CRP)대신소관상피세포Fcγ수체표체적영향,병탐토Fcγ수체재CRP유도전화생장인자β1(TGFβ1)표체중적작용.방법 이CRP간예인신소관상피세포주(HK-2세포),실시정량PCR검측FcγRⅠ (CD64)、FcγRⅡa(CD32a)、FcγRⅢ(CD16)3충Fcγ수체mRNA표체.근거실험결과,채용류식세포술화Western인적법검측CD32a단백적표체.이항CD32a항체봉폐CD32a수체,실시정량PCR검측TGFβ1 mRNA표체,매련면역흡부시험(ELISA)검측TGFβ1단백화Ⅰ형효원단백、Ⅳ형효원단백적분비수평.결과 HK-2세포무CD64화CD16mRNA표체,유CD32a mRNA표체.CD32a mRNA표체정제량의뢰성증고(P＜0.01),CRP 10 mg/L자격체도고봉.류식세포술시CRP 10 mg/L자격HK-2세포24 h CD32a평균표체량위23.35％±7.43％,미자격조위1.66％±0.28％,차이유통계학의의(P＜0.01).Western인적현시CRP이제량의뢰방식상조CD32a표체,CRP10 mg/L현저성상조CD32a표체(P＜0.01).항CD32a항체봉폐CD32a수체후,CRP작용감약,TGFβ1 mRNA표체하조(P＜0.01),TGFβ1、Ⅰ형효원단백、Ⅳ형효원단백적분비하강(균P＜0.05).결론 CRP자격가상조신소관상피세포표면적CD32a수체적표체,CRP통과신소관상피세포CD32a수체개도촉섬유화인자TGFβ1표체상조.
Objective To explore the effect of C-reactive protein (CRP) on the expression of Fcγ receptors in human renal tubular epithelial cells and determine the role of Fcγ receptors in CRP-induced expression of transforming growth factor β1 ( TGFβ1 ).Methods Human renal tubular epithelial cells ( HK-2) were cultured and stimulated with recombinant human CRP.The mRNA expression of Fcγ receptors,including FcγR Ⅰ ( CD64 ),FcγR Ⅱ a ( CD32a ) and FcγR Ⅲ( CD16 ),was detected by real-time polymerase chain reaction (PCR).On the basis of real-time PCR results,CD32a was selected for further analysis:the CD32a expression in HK-2 cells incubated with 10 mg/L CRP for 24 h was determined by flow cytometry and Western blotting.HK-2 cells were preincubated with or without anti-CD32a IgG,followed by the addition of recombinant human CRP.Subsequently the biological effects of CRP were tested.TGFβ1,type Ⅰ collagen ( Col Ⅰ ) and type Ⅳ collagen ( ColⅣ ) released into media were detected by enzyme-linked immunosorbent assay.And TGFβ1 mRNA expression was measured by real-time PCR.Results On realtime PCR,CRP was found to significantly up-regulate the CD32a mRNA expression in HK-2 cells in a dosedependent manner (P ＜ 0.01 ).The peak up-regulation was observed at a dose of 10 mg/L.In contrast,mRNAs of CD16 and CD64 were not detected in HK-2 cells.Flow cytometry showed that CD32a expressed on HK-2 cells incubated with 10 mg/L recombinant human CRP for 24 h accounted for 23.35％ ± 7.43％,significantly higher than that on non-CRP-treated cells ( 1.66％ ± 0.28％,P ＜ 0.01 ).Western blottingshowed that CRP up-regulated CD32a expression in a dose-dependent manner.And 10 m/L CRP induced the peak effect.Antibodies to CD32a inhibited the stimulatory effect of CRP on the generation of TGFβ1,Col I and Col Ⅳ ( all P ＜ 0.05 ) and down-regulated the expression of TGFβ1 mRNA ( P ＜ 0.01 ).Conclusion The stimulation of CRP can significantly increase CD32a expression in renal tubular epithelial cells and up-regulate the expression of transforming growth factor TGFβ1 through CD32a receptor.
