中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2009年
7期
548-554
,共7页
孙明姝%郭永平%顾乐怡%戴慧莉%严玉澄%倪兆慧%钱家麒
孫明姝%郭永平%顧樂怡%戴慧莉%嚴玉澄%倪兆慧%錢傢麒
손명주%곽영평%고악이%대혜리%엄옥징%예조혜%전가기
白细胞介素6%脐动脉%肌细胞,平滑肌%核心结合因子α1亚基%血管钙化%骨形态发生蛋白质类
白細胞介素6%臍動脈%肌細胞,平滑肌%覈心結閤因子α1亞基%血管鈣化%骨形態髮生蛋白質類
백세포개소6%제동맥%기세포,평활기%핵심결합인자α1아기%혈관개화%골형태발생단백질류
Interleukin-6%Umbilical arteries%Myocytes,smooth muscle%Core binding factor alpha 1 subunit%Angiosteosis%Bone morphogenetic proteins
目的 研究白细胞介素6(IL-6)对体外培养人脐动脉平滑肌细胞(HUASMC)成骨样转化、钙化的作用及可能的信号通路.方法 组织植块法原代培养HUASMC.培养基加入不同浓度重组人IL-6(rhIL-6)孵育细胞,设空白对照组.茜素红S钙沉积染色及甲氧.酚酞络合酮法榆测细胞层钙盐含量.实时定量PCR、荧光定量法以及Western印迹法分别检测骨特异性碱性磷酸酶(BAP)、骨桥蛋白(OPN)、骨形成蛋白2(BMP2)和骨保护素(OPG)基因以及蛋白表达.凝胶迁移滞后实验(EMSA)检测核心结合因子α1亚基(Cbfα1)的结合活力,以及分别应用p38丝裂原活化蛋白激酶(p38MAPK)抑制剂SB203580和蛋白激酶C二氢神经鞘氨醇(DHC)后Cbfα1的结合活力.结果 rhlL-6 50μg/L诱导12 d,细胞基质层茜素红S染色阳性.与对照组相比,细胞层钙盐含量在rhIL-6 10 μg/L组刺激9 d[(0.76+0.02)mmol/g蛋白]和12 d[(1.54±0.11)mmol/g蛋白]升高,50μg/L组刺激6 d[(1.81±0.03)mmol/g蛋白]、9 d[(2.08±0.10)mmol/g蛋白]和12 d[(3.22±0.18)mmol/g蛋白]升高,并呈时间、剂量依赖地增加.rhIL-6 10μg/L刺激12 h,BMP2 mRNA(3.04±0.07)和蛋白(8.14±0.41)及BAP mRNA(2.51±0.11)和蛋白(3.96±0.54)表达上调;刺激72 h,OPN mRNA(3.14±0.32)和蛋白(2.57±0.43)水平及OPG mRNA(4.06±0.24)和蛋白(3.46±0.34)水平上调.rhIL-6刺激6 h,Cbfα1结合活力增加;DHC能够部分抑制rhIL-6诱导的Cbfα1结合活力增加,SB203580没有明显作用.结论 IL-6体外能够诱导HUASMC发生钙化和成骨样转分化,这可能是临床观察到IL-6与血管钙化相关的机制之一.IL-6的这一作用可能与细胞内蛋白激酶C通路的活化有关.
目的 研究白細胞介素6(IL-6)對體外培養人臍動脈平滑肌細胞(HUASMC)成骨樣轉化、鈣化的作用及可能的信號通路.方法 組織植塊法原代培養HUASMC.培養基加入不同濃度重組人IL-6(rhIL-6)孵育細胞,設空白對照組.茜素紅S鈣沉積染色及甲氧.酚酞絡閤酮法榆測細胞層鈣鹽含量.實時定量PCR、熒光定量法以及Western印跡法分彆檢測骨特異性堿性燐痠酶(BAP)、骨橋蛋白(OPN)、骨形成蛋白2(BMP2)和骨保護素(OPG)基因以及蛋白錶達.凝膠遷移滯後實驗(EMSA)檢測覈心結閤因子α1亞基(Cbfα1)的結閤活力,以及分彆應用p38絲裂原活化蛋白激酶(p38MAPK)抑製劑SB203580和蛋白激酶C二氫神經鞘氨醇(DHC)後Cbfα1的結閤活力.結果 rhlL-6 50μg/L誘導12 d,細胞基質層茜素紅S染色暘性.與對照組相比,細胞層鈣鹽含量在rhIL-6 10 μg/L組刺激9 d[(0.76+0.02)mmol/g蛋白]和12 d[(1.54±0.11)mmol/g蛋白]升高,50μg/L組刺激6 d[(1.81±0.03)mmol/g蛋白]、9 d[(2.08±0.10)mmol/g蛋白]和12 d[(3.22±0.18)mmol/g蛋白]升高,併呈時間、劑量依賴地增加.rhIL-6 10μg/L刺激12 h,BMP2 mRNA(3.04±0.07)和蛋白(8.14±0.41)及BAP mRNA(2.51±0.11)和蛋白(3.96±0.54)錶達上調;刺激72 h,OPN mRNA(3.14±0.32)和蛋白(2.57±0.43)水平及OPG mRNA(4.06±0.24)和蛋白(3.46±0.34)水平上調.rhIL-6刺激6 h,Cbfα1結閤活力增加;DHC能夠部分抑製rhIL-6誘導的Cbfα1結閤活力增加,SB203580沒有明顯作用.結論 IL-6體外能夠誘導HUASMC髮生鈣化和成骨樣轉分化,這可能是臨床觀察到IL-6與血管鈣化相關的機製之一.IL-6的這一作用可能與細胞內蛋白激酶C通路的活化有關.
목적 연구백세포개소6(IL-6)대체외배양인제동맥평활기세포(HUASMC)성골양전화、개화적작용급가능적신호통로.방법 조직식괴법원대배양HUASMC.배양기가입불동농도중조인IL-6(rhIL-6)부육세포,설공백대조조.천소홍S개침적염색급갑양.분태락합동법유측세포층개염함량.실시정량PCR、형광정량법이급Western인적법분별검측골특이성감성린산매(BAP)、골교단백(OPN)、골형성단백2(BMP2)화골보호소(OPG)기인이급단백표체.응효천이체후실험(EMSA)검측핵심결합인자α1아기(Cbfα1)적결합활력,이급분별응용p38사렬원활화단백격매(p38MAPK)억제제SB203580화단백격매C이경신경초안순(DHC)후Cbfα1적결합활력.결과 rhlL-6 50μg/L유도12 d,세포기질층천소홍S염색양성.여대조조상비,세포층개염함량재rhIL-6 10 μg/L조자격9 d[(0.76+0.02)mmol/g단백]화12 d[(1.54±0.11)mmol/g단백]승고,50μg/L조자격6 d[(1.81±0.03)mmol/g단백]、9 d[(2.08±0.10)mmol/g단백]화12 d[(3.22±0.18)mmol/g단백]승고,병정시간、제량의뢰지증가.rhIL-6 10μg/L자격12 h,BMP2 mRNA(3.04±0.07)화단백(8.14±0.41)급BAP mRNA(2.51±0.11)화단백(3.96±0.54)표체상조;자격72 h,OPN mRNA(3.14±0.32)화단백(2.57±0.43)수평급OPG mRNA(4.06±0.24)화단백(3.46±0.34)수평상조.rhIL-6자격6 h,Cbfα1결합활력증가;DHC능구부분억제rhIL-6유도적Cbfα1결합활력증가,SB203580몰유명현작용.결론 IL-6체외능구유도HUASMC발생개화화성골양전분화,저가능시림상관찰도IL-6여혈관개화상관적궤제지일.IL-6적저일작용가능여세포내단백격매C통로적활화유관.
Objective To investigate the role of recombinant human interleukin 6 (rhlL-6) in calcification and osteogenic transition of cultured human umbilical artery smooth muscle cells (HUASMC), and the possible cell signal transduction way. Methods HUASMCs were isolated by the explant method. HUASMCs were treated with (treatment groups) or without (control group) rhIL-6. Alizarin Red S stain was applied for calcium deposition in extracellular matrix of control ceils and the cells treated with rhIL-6 50 μg/L at day 12. Calcium concentration in cell layer of control group and treatment group (treated with rhIL-6 10 μg/L and 50 μg/L, respectively) was determined calorimetrically by the o-cresolphthalein complexone method at day 3, 6, 9 and 12, and corrected by total cell proteins. The mRNA expressions of bone-specific alkaline phosphatase (BAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP2) and osteoprotegerin (OPG) were estimated by real-time PCR in 12, 24 and 72 hours. OPN, BMP2 and OPG expressions were assessed by Western blotting and the BAP concentration at the same time was checked by fluorometry method . Electrophoretie mobility shift assays (EMSA) was used to detect the binding activity of transcription factor Cbfα1 with or without inhibitors of p38-MAPK (SB203580) and PKC (DHC) after 6 hours stimulation by rhIL-6 10 μg/L. Results rhIL-6 induced a positive Alizarin Red S stain and a time-dose-dependent increasing of cell layer calcium deposition.Compared with control group, rhIL-6 10 μg/L enhanced gene expression and protein levels of BAP and BMP2 at the early time (12 and 24 hours), and of OPN and OPG at later hours (24 and 72 hours). RhIL-6 still induced an increasing of binding activity of Cbfα1, which could be partially blocked by DHC but not SB203580. Conclusions rhIL-6 induces HUASMCs calcification and osteogenie transition in vitro, which may be one of the mechanism involved in IL-6 associated vascular calcification as observed in clinical studies. The role of IL-6 in HUASMCs may partially achieved through the PKC cell signal transduction way.
