中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
3期
500-503
,共4页
邓仲磊%徐东亮%谭若芸%鲁佩%张炜%顾民
鄧仲磊%徐東亮%譚若蕓%魯珮%張煒%顧民
산중뢰%서동량%담약예%로패%장위%고민
骨桥蛋白%肾小管%上皮细胞%间质细胞%脱噬作用
骨橋蛋白%腎小管%上皮細胞%間質細胞%脫噬作用
골교단백%신소관%상피세포%간질세포%탈서작용
Osteopontin%Proximal tubular%Epithelial cell%Mesenchymal cell%Apoptosis
目的 观察骨桥蛋白(OPN)对人近端肾小管上皮细胞(HK-2)向间质细胞转化(EMT)的影响.方法 依据加入的重组人骨桥蛋白(rhOPN)的浓度,将HK-2细胞分成6组,rhOPN的浓度依次为:0.00、0.04、0.08、0.12、0.16、0.20 mg/L,刺激时间为48h.采用共聚焦显微镜观察细胞形态学变化；实时定量聚合酶链反应(PCR)、Western blot检测E-钙黏素(E-cad)、α-平滑肌肌动蛋白(α-SMA)、纤连蛋白(FN)的表达；应用流式细胞仪检测各组细胞的凋亡.结果 在形态学上,rhOPN能够刺激HK-2细胞向间质细胞转变,通过实时定量PCR、Western blot分析发现上皮细胞的表面标记E-cad随着刺激浓度的增加而逐渐降低,间质细胞的标记物α-SMA、FN的表达逐渐升高,实时定量PCR检测E-cad依次为:1947.0±301.0、1114.0±187.4、668.7±180.4、436.7±121.8、242.7±105.2、223.5±73.4; α-SMA依次为:0.01465±0.003 38、0.034 28±0.01200、0.06034±0.005 65、0.09205±0.010 68、0.11360±0.018 92、0.186 30±0.021 75;FN依次为:0.000 818±0.000103、0.001 341±0.000203、0.001 708±0.000128、0.002 170±0.000161、0.003 369±0.000314、0.004961±0.000364.Western blot检测E-cad依次为:1.719±0.159、1.370±0.016、1.238±0.031、0.916±0.056、0.520±0.065、0.456±0.073;α-SMA依次为:0.623±0.127、0.640±0.031、0.865±0.045、1.015±0.107、1.290±0.103、1.381±0.040; FN依次为:0.475±0.044、0.499±0.062、0.904±0.066、0.981 ±0.052、1.272±0.093、1.614±0.056,各组之间差异有统计学意义(均P＜0.05),且随着OPN刺激浓度的增加,细胞的抗凋亡能力也逐渐增强.结论 OPN能够刺激HK-2细胞向间质细胞转化,且呈现出明显的剂量依赖性.
目的 觀察骨橋蛋白(OPN)對人近耑腎小管上皮細胞(HK-2)嚮間質細胞轉化(EMT)的影響.方法 依據加入的重組人骨橋蛋白(rhOPN)的濃度,將HK-2細胞分成6組,rhOPN的濃度依次為:0.00、0.04、0.08、0.12、0.16、0.20 mg/L,刺激時間為48h.採用共聚焦顯微鏡觀察細胞形態學變化；實時定量聚閤酶鏈反應(PCR)、Western blot檢測E-鈣黏素(E-cad)、α-平滑肌肌動蛋白(α-SMA)、纖連蛋白(FN)的錶達；應用流式細胞儀檢測各組細胞的凋亡.結果 在形態學上,rhOPN能夠刺激HK-2細胞嚮間質細胞轉變,通過實時定量PCR、Western blot分析髮現上皮細胞的錶麵標記E-cad隨著刺激濃度的增加而逐漸降低,間質細胞的標記物α-SMA、FN的錶達逐漸升高,實時定量PCR檢測E-cad依次為:1947.0±301.0、1114.0±187.4、668.7±180.4、436.7±121.8、242.7±105.2、223.5±73.4; α-SMA依次為:0.01465±0.003 38、0.034 28±0.01200、0.06034±0.005 65、0.09205±0.010 68、0.11360±0.018 92、0.186 30±0.021 75;FN依次為:0.000 818±0.000103、0.001 341±0.000203、0.001 708±0.000128、0.002 170±0.000161、0.003 369±0.000314、0.004961±0.000364.Western blot檢測E-cad依次為:1.719±0.159、1.370±0.016、1.238±0.031、0.916±0.056、0.520±0.065、0.456±0.073;α-SMA依次為:0.623±0.127、0.640±0.031、0.865±0.045、1.015±0.107、1.290±0.103、1.381±0.040; FN依次為:0.475±0.044、0.499±0.062、0.904±0.066、0.981 ±0.052、1.272±0.093、1.614±0.056,各組之間差異有統計學意義(均P＜0.05),且隨著OPN刺激濃度的增加,細胞的抗凋亡能力也逐漸增彊.結論 OPN能夠刺激HK-2細胞嚮間質細胞轉化,且呈現齣明顯的劑量依賴性.
목적 관찰골교단백(OPN)대인근단신소관상피세포(HK-2)향간질세포전화(EMT)적영향.방법 의거가입적중조인골교단백(rhOPN)적농도,장HK-2세포분성6조,rhOPN적농도의차위:0.00、0.04、0.08、0.12、0.16、0.20 mg/L,자격시간위48h.채용공취초현미경관찰세포형태학변화；실시정량취합매련반응(PCR)、Western blot검측E-개점소(E-cad)、α-평활기기동단백(α-SMA)、섬련단백(FN)적표체；응용류식세포의검측각조세포적조망.결과 재형태학상,rhOPN능구자격HK-2세포향간질세포전변,통과실시정량PCR、Western blot분석발현상피세포적표면표기E-cad수착자격농도적증가이축점강저,간질세포적표기물α-SMA、FN적표체축점승고,실시정량PCR검측E-cad의차위:1947.0±301.0、1114.0±187.4、668.7±180.4、436.7±121.8、242.7±105.2、223.5±73.4; α-SMA의차위:0.01465±0.003 38、0.034 28±0.01200、0.06034±0.005 65、0.09205±0.010 68、0.11360±0.018 92、0.186 30±0.021 75;FN의차위:0.000 818±0.000103、0.001 341±0.000203、0.001 708±0.000128、0.002 170±0.000161、0.003 369±0.000314、0.004961±0.000364.Western blot검측E-cad의차위:1.719±0.159、1.370±0.016、1.238±0.031、0.916±0.056、0.520±0.065、0.456±0.073;α-SMA의차위:0.623±0.127、0.640±0.031、0.865±0.045、1.015±0.107、1.290±0.103、1.381±0.040; FN의차위:0.475±0.044、0.499±0.062、0.904±0.066、0.981 ±0.052、1.272±0.093、1.614±0.056,각조지간차이유통계학의의(균P＜0.05),차수착OPN자격농도적증가,세포적항조망능력야축점증강.결론 OPN능구자격HK-2세포향간질세포전화,차정현출명현적제량의뢰성.
Objective To study the effects of osteopontin (OPN) on human proximal tubular epithelial cells (HK-2) to the transition of mesenchymal cells (EMT).Methods The HK-2 cells were divided into six groups depending on different concentrations of added recombinant human OPN (rhOPN).The concentrations of OPN were 0.00,0.04,0.08,0.12,0.16,0.20 mg/L.After 48 h,the cells were collected to observe the morphological changes under the confocal microscopy.The expression of E-cadherin,α-smooth muscle actin (α-SMA) and fibronectin (FN) was detected by using real-time polymerase chain reaction (PCR) and Western blotting.The apoptosis was assayed by using flow cytometry.Results OPN could change the shape of HK-2 cells.Real-timePCR and Western blotting revealed that epithelial cell surface marker E-cadherin was reduced and mesenchymal cell markers α-SMA and fibronectin were gradually increased with the increase in the concentration of OPN.The results of E-cadherin detected by real-time PCR were:1947.0 ± 301,1114.0 ± 187.4,668.7 ± 180.4,436.7 ± 121.8,242.7 ± 105.2,223.5 ±73.4; α-SMA:0.014 65 ± 0.003 38,0.034 28 ±0.012 00,0.060 34 ±0.005 65,0.092 05 ± 0.010 68,0.11360± 0.01892,0.18630± 0.021 75; FN:0.000817± 0.000 103,0.001 341± 0.000203,0.001 708±0.000 128,0.002 170 ±0.000 161,0.003 369 ±0.000 314,0.004 961 ± 0.000 364.The expression levels of E-cadherin detected by Western blotting were as follows:1.719 ± 0.159,1.370 ±0.016,1.238 ± 0.031,0.916 ± 0.056,0.520 ± 0.065,0.456 ± 0.073; α-SMA:0.623 ± 0.127,0.640±0.031,0.865 ±0.045,1.015 ±0.107,1.290±0.103,1.381 ±0.040; Fibronectin:0.475 ±0.044,0.499 ±0.062,0.904 ±0.066,0.981 ±0.052,1.272 ±0.093,1.614 ±0.056 (all P＜0.05).The capacity of resistance to death was increased gradually with the increase in the concentration of OPN.Conclusion OPN could induce HK-2 cells to EMT in a concentration-dependent manner.
