中华创伤骨科杂志
中華創傷骨科雜誌
중화창상골과잡지
CHINESE JOURNAL OF ORTHOPAEDIC TRAUMA
2014年
4期
334-339
,共6页
梁亮%许长鹏%黎润光%余斌
樑亮%許長鵬%黎潤光%餘斌
량량%허장붕%려윤광%여빈
应力,物理%间质干细胞%成脂分化%过氧化物酶体增殖物激活受体%脂联素
應力,物理%間質榦細胞%成脂分化%過氧化物酶體增殖物激活受體%脂聯素
응력,물리%간질간세포%성지분화%과양화물매체증식물격활수체%지련소
Stress,mechanical%Mesenchymal stem cells%Adipogenesis%Peroxisome proliferator-activated receptors%Adiponectin
目的 探讨周期性牵张应力对Sprague Dawley(SD)大鼠骨髓基质干细胞(BMSCs)成脂分化的影响. 方法 选取4～5周龄,体质量为80～ 100 g的SD大鼠6只,雌雄不限.体外培养SD大鼠BMSCs至第3代,采用流式细胞仪检测CD29、CD34、CD44、CD45,并利用Flexcell-5000X T牵张应力系统加载力学信号(应力拉伸幅度为5％,6 h/d,10次/min,持续3d或5d,正弦波形),采用显微镜观察BMSCs的形态.将实验对象BMSCs根据培养基不同(普通培养基和促成脂分化培养基)、是否加载牵张应力及牵张应力持续时间(3、5d)共分为8组,受力组为实验组,未受力组为对照组.油红O染色法观察各组细胞成脂分化水平.荧光定量聚合酶链式反应(Q-PCR)法检测BMSCs成脂分化指标:过氧化物酶体增殖物激活受体(PPAR-γ)、脂联素、CAAT增强子结合蛋白α(C/EBPα)的mRNA表达水平.结果 镜下观察原代BMSCs生长良好、形态排列正常.流式细胞检测结果显示第3代BMSCs的CD29、CD34、CD44、CD45的表型阳性率分别为93.6％、5.3％、91.4％和4.6％.BMSCs受成脂诱导剂诱导3、5d,细胞内出现脂滴并为油红O所染色,PPARγ-2、脂联素和C/EBPα的mRNA表达均明显增高,而在诱导过程中,加载应力信号后,未见明显脂滴出现,应力刺激3d和5d,细胞内PPARγ-2、脂联素、C/EBPα的基因表达分别下降64.80％、84.45％、67.12％和70.11％、68.12％、80.39％,3种基因的表达明显受到抑制. 结论 适当的牵张应力可抑制BMSCs向脂肪方向分化的能力.
目的 探討週期性牽張應力對Sprague Dawley(SD)大鼠骨髓基質榦細胞(BMSCs)成脂分化的影響. 方法 選取4～5週齡,體質量為80～ 100 g的SD大鼠6隻,雌雄不限.體外培養SD大鼠BMSCs至第3代,採用流式細胞儀檢測CD29、CD34、CD44、CD45,併利用Flexcell-5000X T牽張應力繫統加載力學信號(應力拉伸幅度為5％,6 h/d,10次/min,持續3d或5d,正絃波形),採用顯微鏡觀察BMSCs的形態.將實驗對象BMSCs根據培養基不同(普通培養基和促成脂分化培養基)、是否加載牽張應力及牽張應力持續時間(3、5d)共分為8組,受力組為實驗組,未受力組為對照組.油紅O染色法觀察各組細胞成脂分化水平.熒光定量聚閤酶鏈式反應(Q-PCR)法檢測BMSCs成脂分化指標:過氧化物酶體增殖物激活受體(PPAR-γ)、脂聯素、CAAT增彊子結閤蛋白α(C/EBPα)的mRNA錶達水平.結果 鏡下觀察原代BMSCs生長良好、形態排列正常.流式細胞檢測結果顯示第3代BMSCs的CD29、CD34、CD44、CD45的錶型暘性率分彆為93.6％、5.3％、91.4％和4.6％.BMSCs受成脂誘導劑誘導3、5d,細胞內齣現脂滴併為油紅O所染色,PPARγ-2、脂聯素和C/EBPα的mRNA錶達均明顯增高,而在誘導過程中,加載應力信號後,未見明顯脂滴齣現,應力刺激3d和5d,細胞內PPARγ-2、脂聯素、C/EBPα的基因錶達分彆下降64.80％、84.45％、67.12％和70.11％、68.12％、80.39％,3種基因的錶達明顯受到抑製. 結論 適噹的牽張應力可抑製BMSCs嚮脂肪方嚮分化的能力.
목적 탐토주기성견장응력대Sprague Dawley(SD)대서골수기질간세포(BMSCs)성지분화적영향. 방법 선취4～5주령,체질량위80～ 100 g적SD대서6지,자웅불한.체외배양SD대서BMSCs지제3대,채용류식세포의검측CD29、CD34、CD44、CD45,병이용Flexcell-5000X T견장응력계통가재역학신호(응력랍신폭도위5％,6 h/d,10차/min,지속3d혹5d,정현파형),채용현미경관찰BMSCs적형태.장실험대상BMSCs근거배양기불동(보통배양기화촉성지분화배양기)、시부가재견장응력급견장응력지속시간(3、5d)공분위8조,수력조위실험조,미수력조위대조조.유홍O염색법관찰각조세포성지분화수평.형광정량취합매련식반응(Q-PCR)법검측BMSCs성지분화지표:과양화물매체증식물격활수체(PPAR-γ)、지련소、CAAT증강자결합단백α(C/EBPα)적mRNA표체수평.결과 경하관찰원대BMSCs생장량호、형태배렬정상.류식세포검측결과현시제3대BMSCs적CD29、CD34、CD44、CD45적표형양성솔분별위93.6％、5.3％、91.4％화4.6％.BMSCs수성지유도제유도3、5d,세포내출현지적병위유홍O소염색,PPARγ-2、지련소화C/EBPα적mRNA표체균명현증고,이재유도과정중,가재응력신호후,미견명현지적출현,응력자격3d화5d,세포내PPARγ-2、지련소、C/EBPα적기인표체분별하강64.80％、84.45％、67.12％화70.11％、68.12％、80.39％,3충기인적표체명현수도억제. 결론 괄당적견장응력가억제BMSCs향지방방향분화적능력.
Objective To study the effect of mechanical strain on the adipogenic differentiation of bone mesenchymal stem cells (BMSCs) in Sprague Dawley (SD) rats.Methods We isolated and cultured primary BMSCs from 6 SD rats (random gender) weighing from 80 to 100 grams and aged from 4 to 5 weeks old.After the levels of CD29,CD34,CD44 and CD45 in the third passage of BMSCs were determined by flow cytometry,mechanical stretch signals (5％ strain,sine wave type,10 cycles per minute for 6 hours daily,and lasting for 3 or 5 days) were delivered to cultures using the Flexcell-5000X tension system.After microscopic observation was performed,the BMSCs were divided into 8 groups according to their medium,mechanical strain and/or duration of mechanical strain.Experimental groups received mechanical strain while control ones no mechanical strain.The adipogenic level was observed in every group by oil red O staining; the mRNA expression levels of PPARγ-2,adiponectin and C/EBPα were measured by RT-PCR.Results Microscopic observation showed that the primary MSCs were morphologically normal; flow cytometry showed that the phenotypic positive rates of CD29,CD34,CD44 and CD45 in the third generation of BMSCs were 93.6％,5.3％,91.4％ and 4.6％.After adipogenic differentiation induction for 3 and 5 days,the lipid was appreciable in BMSCs and the levels of intracellular PPARγ-2,adiponectin and C/EBPα mRNA were significantly higher.However,their expression was inhibited significantly and the cells stained by oil red O were hardly shown after the BMSCs were stimulated by mechanical strain.The expression levels of PPARγ-2,adiponectin and C/EBPoα mRNA were reduced respectively by 64.80％,84.45％ and 67.12％ after mechanical strain stimulation for 3 days and reduced by 70.11％,68.12％ and 80.39％ after mechanical strain stimulation for 5 days.Conclusion The data suggest that proper mechanical strain may inhibit the adipogenic differentiation of BMSCs in rats.
