TLN1-PI3K/AKT通路调控骨髓间充质干细胞成骨分化
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摘要
目的探讨Talin1在骨髓间充质干细胞(Mesenchymal stem cell,MSC)成骨分化中的作用及其调控机制。方法诱导MSC成骨分化后通过qRT-PCR及Western blot检测TLN1的表达变化,在MSC中敲低TLN1后采用CCK-8检测细胞增殖能力改变,利用ALP活性检测及茜素红染色观察成骨分化变化情况。通过Western blot实验研究TLN1在MSC成骨分化过程中参与的分子机制。结果 qPCR及Western blot结果提示,TLN1在成骨分化过程中表达上升;siRNA敲低TLN1表达后,MSC增殖无明显差异,ALP活性及矿化结节定量较对照组下降(P<0.05)。Western blot结果显示敲低TLN1表达后p-AKT表达下降,回补实验证明激活PI3K-AKT表达可回复MSC的成骨分化能力。结论诱导MSC成骨分化后TLN1表达逐渐上调,敲低TLN1后通过PI3K-AKT通路抑制骨髓间充质干细胞成骨分化。
Objective To investigate the role and regulatory mechanism of Talin 1 in osteogenic differentiation of bone marrow mesenchymal stem cells. Methods The expression level of TLN1 in osteogenic differentiation of mesenchymal stem cells was detected by qRT-PCR and western blot. The proliferation of MSC was measured by CCK-8 assay after TLN1-siRNA intervention. The osteogenic differentiation capacity was determined through alizarin red S and alkaline phosphatase assays. Western blotting was used to study the molecular mechanism of TLN1 in the osteogenic differentiation of MSC.Results qRT-PCR and Western blot results suggested that TLN1 expression increased during osteogenic differentiation. The osteogenic differentiation capacity was decrease compared with the negative group after siRNA transfection. After transfection with siRNA,the expression of p-AKT was decreased by Western blot. Furthermore,the rescue experiment showed that active PI3 K/AKT pathway can reserve osteogenic differentiation capacity. Conclusion Talin1 expression was gradually up-regulated after induced osteogenic differentiation of MSC. TLN1 knockdown inhibited osteogenic differentiation of MSC through PI3 K/AKT pathway.
Objective To investigate the role and regulatory mechanism of Talin 1 in osteogenic differentiation of bone marrow mesenchymal stem cells. Methods The expression level of TLN1 in osteogenic differentiation of mesenchymal stem cells was detected by qRT-PCR and western blot. The proliferation of MSC was measured by CCK-8 assay after TLN1-siRNA intervention. The osteogenic differentiation capacity was determined through alizarin red S and alkaline phosphatase assays. Western blotting was used to study the molecular mechanism of TLN1 in the osteogenic differentiation of MSC.Results qRT-PCR and Western blot results suggested that TLN1 expression increased during osteogenic differentiation. The osteogenic differentiation capacity was decrease compared with the negative group after siRNA transfection. After transfection with siRNA,the expression of p-AKT was decreased by Western blot. Furthermore,the rescue experiment showed that active PI3 K/AKT pathway can reserve osteogenic differentiation capacity. Conclusion Talin1 expression was gradually up-regulated after induced osteogenic differentiation of MSC. TLN1 knockdown inhibited osteogenic differentiation of MSC through PI3 K/AKT pathway.
引文
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[12]Fujita T,Azuma Y,Fukuyama R,et al.Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling[J].J Cell Biol,2004,166(1):85-95.
[13]Song B,Estrada KD,Lyons KM.Smad signaling in skeletal development and regeneration[J].Cytokine Growth Factor Rev,2009,20(5-6):379-388.
[14]Baron R,Kneissel M.WNT signaling in bone homeostasis and disease:from human mutations to treatments[J].Nat Med,2013,19(2):179-192.2019-06-21
[2]Wong SP,Rowley JE,Redpath AN,et al.Pericytes,mesenchymal stem cells and their contributions to tissue repair[J].PharmacolTher,2015,151:107-120.
[3]Steward AJ,Kelly DJ.Mechanical regulation of mesenchymal stem cell differentiation[J].J Anat,2015,227(6):717-731.
[4]Nayal A,Webb DJ,Horwitz AF.Talin:an emerging focal point of adhesion dynamics[J].CurrOpin Cell Biol,2004,16(1):94-98.
[5]Tadokoro S,Shattil SJ,Eto K,et al.Talin binding to integrin beta tails:a final common step in integrin activation[J].Science,2003,302(5642):103-106.
[6]Zou W,Izawa T,Zhu T,et al.Talin1 and Rap1 Are Critical for Osteoclast Function[J].Mol Cell Biol,2013,33(4):830-844.
[7]Devlin MJP,Rosen CJP.The bone-fat interface:basic and clinical implications of marrow adiposity[J].Lancet Diabetes Endocrinol,2015,3(2):141-147.
[8]Berebichez-Fridman R,Gómez-García R,Granados-Montiel J,et al.The Holy Grail of Orthopedic Surgery:Mesenchymal Stem Cells-Their Current Uses and Potential Applications[J].Stem Cells Int,2017,2017:1-14.
[9]Trappmann B,Gautrot JE,Connelly JT,et al.Extracellularmatrix tethering regulates stem-cell fate[J].Nat Mater,2012,11(7):642-649.
[10]Marie PJ,Ha?E,Saidak Z.Integrin and cadherin signaling in bone:role and potential therapeutic targets[J].Trends Endocrinol Metab,2014,25(11):567-575.
[11]Hu H,Yang L,Wang Z,et al.Overexpression of integrin a2promotes osteogenic differentiation of hBMSCs from senile osteoporosis through the ERK pathway[J].Int J Clin Exp Pathol,2013,6(5):841.
[12]Fujita T,Azuma Y,Fukuyama R,et al.Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling[J].J Cell Biol,2004,166(1):85-95.
[13]Song B,Estrada KD,Lyons KM.Smad signaling in skeletal development and regeneration[J].Cytokine Growth Factor Rev,2009,20(5-6):379-388.
[14]Baron R,Kneissel M.WNT signaling in bone homeostasis and disease:from human mutations to treatments[J].Nat Med,2013,19(2):179-192.2019-06-21
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