中国骨质疏松杂志
中國骨質疏鬆雜誌
중국골질소송잡지
CHINESE JOURNAL OF OSTEOPOROSIS
2015年
5期
537-540,549
,共5页
姜旭%吴成爱%王莹%赵丹慧
薑旭%吳成愛%王瑩%趙丹慧
강욱%오성애%왕형%조단혜
膝关节骨性关节炎%软骨细胞%WISP-1%信号传导%细胞外基质
膝關節骨性關節炎%軟骨細胞%WISP-1%信號傳導%細胞外基質
슬관절골성관절염%연골세포%WISP-1%신호전도%세포외기질
Osteoarthritis%Cartilage%WISP-1%Signaling%Extracellular matrix
目的：使用膝关节骨性关节炎（ OA）临床组织标本与人软骨细胞株（ NHAC），以分子生物学手法探讨WISP-1在骨关节软骨基质合成和分泌中的调控机理，明确其在骨性关节炎中的作用。方法 NHAC细胞以1.4×106／mL密度播种于6孔板（直径3．5 cm）后培养48 h，于含有0．2％牛血清白蛋白（ BSA）的DF中继续培养12 h，使细胞处于静止状态。向含0．02％BSA的DF培养基中加入10、50和100 ng／mL的 Wnt 刺激细胞6 h。对照 siRNA 与β-catenin siRNA，使用 EntranserTM-R （ Engreen）转染试剂转染至NHAC。转染24 h后使用PBS清洗细胞两次。向含0.02％BSA的DF培养基中加入50 ng／mL的Wnt刺激细胞。向NHAC负荷不同剂量WISP-1，24 h后观察MMPs和ADAMTS4。利用总RNA提取试剂盒提取组织标本和细胞RNA，反转录产物cDNA作为PCR扩增模板进行定量PCR；使用蛋白提取试剂盒提取蛋白。结果在膝关节组织标本中发现，在骨性关节炎关节软骨中WISP-1、Wnt和b-catenin基因表达及蛋白水平明显增高。在NHAC细胞，Wnt诱导下WISP-1转录水平显著提高，与Wnt呈现剂量依赖关系。经敲除内源性β-catenin表达后，Wnt诱导WISP-1作用明显降低。 WISP-1以量依存性的诱导细胞外基质蛋白分解酶MMP3、MMP9、MMP13和ADAMTS4基因表达在软骨细胞中Wnt诱导WISP-1信号途径通过β-catenin的可能性很大。结论 WISP-1与骨性关节炎的发生具有一定的相关性；在软骨细胞中Wnt诱导WISP-1的信号途径通过b-catenin。
目的：使用膝關節骨性關節炎（ OA）臨床組織標本與人軟骨細胞株（ NHAC），以分子生物學手法探討WISP-1在骨關節軟骨基質閤成和分泌中的調控機理，明確其在骨性關節炎中的作用。方法 NHAC細胞以1.4×106／mL密度播種于6孔闆（直徑3．5 cm）後培養48 h，于含有0．2％牛血清白蛋白（ BSA）的DF中繼續培養12 h，使細胞處于靜止狀態。嚮含0．02％BSA的DF培養基中加入10、50和100 ng／mL的 Wnt 刺激細胞6 h。對照 siRNA 與β-catenin siRNA，使用 EntranserTM-R （ Engreen）轉染試劑轉染至NHAC。轉染24 h後使用PBS清洗細胞兩次。嚮含0.02％BSA的DF培養基中加入50 ng／mL的Wnt刺激細胞。嚮NHAC負荷不同劑量WISP-1，24 h後觀察MMPs和ADAMTS4。利用總RNA提取試劑盒提取組織標本和細胞RNA，反轉錄產物cDNA作為PCR擴增模闆進行定量PCR；使用蛋白提取試劑盒提取蛋白。結果在膝關節組織標本中髮現，在骨性關節炎關節軟骨中WISP-1、Wnt和b-catenin基因錶達及蛋白水平明顯增高。在NHAC細胞，Wnt誘導下WISP-1轉錄水平顯著提高，與Wnt呈現劑量依賴關繫。經敲除內源性β-catenin錶達後，Wnt誘導WISP-1作用明顯降低。 WISP-1以量依存性的誘導細胞外基質蛋白分解酶MMP3、MMP9、MMP13和ADAMTS4基因錶達在軟骨細胞中Wnt誘導WISP-1信號途徑通過β-catenin的可能性很大。結論 WISP-1與骨性關節炎的髮生具有一定的相關性；在軟骨細胞中Wnt誘導WISP-1的信號途徑通過b-catenin。
목적：사용슬관절골성관절염（ OA）림상조직표본여인연골세포주（ NHAC），이분자생물학수법탐토WISP-1재골관절연골기질합성화분비중적조공궤리，명학기재골성관절염중적작용。방법 NHAC세포이1.4×106／mL밀도파충우6공판（직경3．5 cm）후배양48 h，우함유0．2％우혈청백단백（ BSA）적DF중계속배양12 h，사세포처우정지상태。향함0．02％BSA적DF배양기중가입10、50화100 ng／mL적 Wnt 자격세포6 h。대조 siRNA 여β-catenin siRNA，사용 EntranserTM-R （ Engreen）전염시제전염지NHAC。전염24 h후사용PBS청세세포량차。향함0.02％BSA적DF배양기중가입50 ng／mL적Wnt자격세포。향NHAC부하불동제량WISP-1，24 h후관찰MMPs화ADAMTS4。이용총RNA제취시제합제취조직표본화세포RNA，반전록산물cDNA작위PCR확증모판진행정량PCR；사용단백제취시제합제취단백。결과재슬관절조직표본중발현，재골성관절염관절연골중WISP-1、Wnt화b-catenin기인표체급단백수평명현증고。재NHAC세포，Wnt유도하WISP-1전록수평현저제고，여Wnt정현제량의뢰관계。경고제내원성β-catenin표체후，Wnt유도WISP-1작용명현강저。 WISP-1이량의존성적유도세포외기질단백분해매MMP3、MMP9、MMP13화ADAMTS4기인표체재연골세포중Wnt유도WISP-1신호도경통과β-catenin적가능성흔대。결론 WISP-1여골성관절염적발생구유일정적상관성；재연골세포중Wnt유도WISP-1적신호도경통과b-catenin。
Objective To explore the regulation mechanism of WISP-1 in the formation and secretion of the joint cartilage matrix by using human osteoarthritis ( OA) tissue sample and human chondrocyte cell line ( NHAC) , and to confirm the role of WISP-1 in the joint degenerative disease.Methods NHAC cells were seeded in the 6-well plates (3.5cm in diameter) with a density of 1.4 ×106/ml and cultured for 48 h.Then they were cultured in DF with 0.2%BSA for further 12 h.When the cells were stable, they were treated with 10, 50, and 100 ng/ml of Wnt for 6h, respectively.Control siRNA and β-catenin siRNA were transfected into NHAC using the EntraserTM-R kit ( Engreen) .The cells were washed with PBS twice after 24 h transfection.50 ng/ml of Wnt was additioned to DF with 0.02%BSA to stimulate the cells.Different concentrations of WISP-1 were loaded.MMPs and ADAMTS4 were observed after 24h.Total RNA was extracted from the cells using a commercial kit.Quantitative RT-PCR was performed. Protein was extracted with an extraction kit.Results Protein and mRNA expression of WISP-1, Wnt, and β-catenin in human OA cartilage were higher than those in control cartilage.WISP-1 expression increased significantly in a dose dependent manner under the Wnt stimulation.After knocking down the endogenous β-catenin, the expression of WISP-1 was significantly down-regulated. The gene expressions of MMP-3, -9, -13, and ADAMTS4 were induced dose-dependently by WISP-1.Conclusion WISP-1 is related to the development of OA.In the cartilage cells WISP-1 can be induced by Wnt throughβ-catenin signal pathway.
