雷帕霉素激活自噬对钛颗粒诱导下骨髓间充质干细胞成骨能力的影响
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摘要
目的探讨雷帕霉素激活细胞自噬对钛颗粒诱导下骨髓间充质干细胞(BMSCs)成骨能力的影响。方法常规培养C57小鼠BMSCs,经0、1、5、10、20、50、100 nM雷帕霉素分别处理12、24、36、48h后采用CCK-8法检测细胞活力,筛选雷帕霉素最佳的实验条件;Western blot检测最佳条件下雷帕霉素对自噬相关蛋白5(Atg5)、微管相关蛋白轻链3(LC3)表达的影响。实验分为A组:空白对照组(单纯诱导成骨分化),B组:钛颗粒处理组(150 mg·L-1),C组:雷帕霉素处理组(10 nM),D组:钛颗粒+雷帕霉素处理组,各组均诱导分化培养21 d。分别于诱导第7、14和21天采用比色法检测各组碱性磷酸酶(ALP)活性。诱导分化结束后采用茜素红染色法检测各组的成骨分化能力并定量分析细胞钙沉积量,Western blot检测成骨细胞标志物Runt相关转录因子2(Runx2)和骨钙蛋白(OCN)的表达变化。结果 1)10 nM的雷帕霉素处理C57小鼠BMSCs24 h时,细胞活力最佳,该条件下细胞Atg5、LC-3Ⅰ/Ⅱ表达量较处理前增高(P均<0.05)。2)与A组相比,B组ALP活性、钙沉积量、Runx2和OCN表达水平降低(P<0.05);联合雷帕霉素后,D组细胞ALP活性、钙沉积量、Runx2和OCN表达水平较B组升高(P均<0.05)。C组ALP活性、钙沉积量、Runx2和OCN表达水平高于A、B、D组(P均<0.05)。结论钛颗粒可抑制BMSCs向成骨的分化能力,雷帕霉素可有效减轻钛颗粒对BMSCs成骨能力的抑制,这可能与其增强干细胞自噬水平有关。
Objective To investigate the effect of rapamycin activated autophagy on bone formation of bone marrow mesenchymal stem cells(BMSCs)induced by titanium particles. Methods The C57 mice BMSCs were cultured routinely and treated with rapamycin at 0,1,5,10,20,50 and 100 nM for 12,24,36 and 48 hours,respectively. CCK-8 method was used to detect cell viability and the best experimental conditions for rapamycin was selected. Western blot assay was applied to detect autophagy related protein 5(Atg5),microtubule-associated protein light chain 3(LC-3)Ⅰ/Ⅱexpression under the best condition of rapamycin. The experiment was divided into four groups: group A: blank control group(induced osteogenic differentiation only),group B: titanium granule treatment group(150 mg·L-1),group C: rapamycin treatment group(10 nM),group D: titanium granule + rapamycin treatment group. All groups were induced to differentiate for 21 days.The alkaline phosphatase(ALP)activity of each group was detected by colorimetry on the 7 th,14 th and 21 st days of induction respectively. After the induction of differentiation,the osteogenic differentiation ability of each group was detected by the pigment red staining method,and the amount of cell calcium deposition was quantitatively analyzed. The expression changes of osteoblast markers Runt-related transcription factor 2(Runx2)and Osteocalcin(OCN)were detected by Western blot. Results When rapamycin 10 nM was used to treat BMSCs of C57 mice for 24 hours,the cell viability was the best. Under this condition,the expression of Atg5 and LC-3 I/II was higher than that before treatment(P<0.05). Compared with group A,ALP activity,calcium deposition,Runx2 and OCN expression levels in group B decreased(P<0.05);after combined rapamycin,ALP activity,calcium deposition,Runx2 and OCN expression levels in group D were higher than those in group B(P<0.05). ALP activity,calcium deposition,Runx2 and OCN expression in group C were higher than those in group A,B and D(P<0.05). Conclusion Titanium granules can inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells,rapamycin can effectively reduce the inhibition of titanium granules on BMSCs osteogenic ability,which may be related to the enhancement of autophagy level of stem cells.
Objective To investigate the effect of rapamycin activated autophagy on bone formation of bone marrow mesenchymal stem cells(BMSCs)induced by titanium particles. Methods The C57 mice BMSCs were cultured routinely and treated with rapamycin at 0,1,5,10,20,50 and 100 nM for 12,24,36 and 48 hours,respectively. CCK-8 method was used to detect cell viability and the best experimental conditions for rapamycin was selected. Western blot assay was applied to detect autophagy related protein 5(Atg5),microtubule-associated protein light chain 3(LC-3)Ⅰ/Ⅱexpression under the best condition of rapamycin. The experiment was divided into four groups: group A: blank control group(induced osteogenic differentiation only),group B: titanium granule treatment group(150 mg·L-1),group C: rapamycin treatment group(10 nM),group D: titanium granule + rapamycin treatment group. All groups were induced to differentiate for 21 days.The alkaline phosphatase(ALP)activity of each group was detected by colorimetry on the 7 th,14 th and 21 st days of induction respectively. After the induction of differentiation,the osteogenic differentiation ability of each group was detected by the pigment red staining method,and the amount of cell calcium deposition was quantitatively analyzed. The expression changes of osteoblast markers Runt-related transcription factor 2(Runx2)and Osteocalcin(OCN)were detected by Western blot. Results When rapamycin 10 nM was used to treat BMSCs of C57 mice for 24 hours,the cell viability was the best. Under this condition,the expression of Atg5 and LC-3 I/II was higher than that before treatment(P<0.05). Compared with group A,ALP activity,calcium deposition,Runx2 and OCN expression levels in group B decreased(P<0.05);after combined rapamycin,ALP activity,calcium deposition,Runx2 and OCN expression levels in group D were higher than those in group B(P<0.05). ALP activity,calcium deposition,Runx2 and OCN expression in group C were higher than those in group A,B and D(P<0.05). Conclusion Titanium granules can inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells,rapamycin can effectively reduce the inhibition of titanium granules on BMSCs osteogenic ability,which may be related to the enhancement of autophagy level of stem cells.
引文
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[12]Zhou C,Xu AT,Wang DD,et al.The effects of Srincorporated micro/nano rough titanium surface on rBMSC migration and osteogenic differentiation for rapid osteointegration[J].Biomater Sci,2018,6(7):1946-1961.
[13]Shao D,Wang C,Sun Y,et al.Effects of oral implants with miR-122-modified cell sheets on rat bone marrow mesenchymal stem cells[J].Mol Med Rep,2018,17(1):1537-1544.
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[15]吴江,王艳霞,任弘毅,等.钛颗粒负荷对成骨细胞释放细胞因子的影响[J].生物医学工程学杂志,2011,28(4):748-752.
[16]Wang ML,Nesti LJ,Tuli R,et al.Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells[J].J Orthop Res,2002,20(6):1175-1184.
[17]Wan Y,Zhuo N,Li Y,et al.Autophagy promotes osteogenic differentiation of human bone marrow mesenchymal stem cell derived from osteoporotic vertebrae[J].Biochemical&Biophysical Research Communications,2017,488(1):46-52.
[18]Li C,Liu Y,Liu J,et al.Rapamycin inhibits human glioma cell proliferation through down-regulating mammalian target of rapamycin pathway and upregulating microRNA-143[J].Head&Neck Oncology,2012,4(3):66.
[19]李军,王倩,林小星,等.自噬活性改变对骨髓间充质干细胞成骨分化能力的影响[J].华中科技大学学报(医学版),2012,41(6):675-679.
[20]王昕,马凤霞,卢士红,等.雷帕霉素对再生障碍性贫血患者骨髓间充质干细胞生物学功能的影响[J].中国实验血液学杂志,2014,22(3):762-766.
[21]张广宇,贾延劼,王军,等.氯化锂通过调节自噬通路促进大鼠骨髓间充质干细胞向神经细胞分化[J].中国病理生理杂志,2017,33(12):2128-2133.
[22]刘宇佳,楼斯悦,章燕棋,等.低氧激活自噬促进人间充质干细胞成骨分化的研究[J].中国现代应用药学,2016,33(3):284-289.
[2]李伟,王翠喆,许彭,等.大鼠骨髓间充质干细胞向脂肪细胞、成骨细胞诱导分化及破骨细胞培养方法的建立[J].石河子大学学报(自然科学版),2016,34(2):181-186.
[3]Bozic KJ,Kurtz SM,Lau E,et al.The epidemiology of revision total knee arthroplasty in the United States[J].Clin Orthop Relat Res,2010,468(1):45-51.
[4]Bozic KJ,Kurtz SM,Lau E,et al.The epidemiology of revision total hip arthroplasty in the United States[J].J Bone Joint Surg(Am),2009,91(1):128-133.
[5]Inzunza D,Covarrubias C,Von MA,et al.Synthesis of nanostructured porous silica coatings on titanium and their cell adhesive and osteogenic differentiation properties[J].Journal of Biomedical Materials Research Part A,2014,102(1):37.
[6]Swer PB,Lohia R,Saran S.Analysis of rapamycin induced autophagy in dictyostelium discoideum[J].Indian Journal of Experimental Biology,2014,52(4):295-304.
[7]Bronietzki AW,Schuster M,Schmitz I.Autophagy in T-cell development,activation and differentiation[J].Immunol Cell Biol,2015,93(1):25-34.
[8]Botbol Y,Guerrero-Ros I,Macian F.Key roles of autophagy in regulating T-cell function[J].European Journal of Immunology,2016,46(6):1326-1334.
[9]He MX,Mcleod IX,Wei J,et al.Macroautophagy in Tlymphocyte development and function[J].Frontiers in Immunology,2012,3:22.
[10]Qi M,Zhang L,Ma Y,et al.Autophagy maintains the function of bone marrow mesenchymal stem cells to prevent estrogen deficiency-induced osteoporosis[J].Theranostics,2017,7(18):4498-4516.
[11]张磊,龚跃昆,赵学凌,等.低氧对成骨生长肽诱导的小鼠间充质干细胞成骨分化的影响[J].中国骨质疏松杂志,2016,22(11):1380-1385.
[12]Zhou C,Xu AT,Wang DD,et al.The effects of Srincorporated micro/nano rough titanium surface on rBMSC migration and osteogenic differentiation for rapid osteointegration[J].Biomater Sci,2018,6(7):1946-1961.
[13]Shao D,Wang C,Sun Y,et al.Effects of oral implants with miR-122-modified cell sheets on rat bone marrow mesenchymal stem cells[J].Mol Med Rep,2018,17(1):1537-1544.
[14]Song W,Wu K,Yan J,et al.MiR-148b laden titanium implant promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells[J].Rsc Advances,2013,3(28):11292-11300.
[15]吴江,王艳霞,任弘毅,等.钛颗粒负荷对成骨细胞释放细胞因子的影响[J].生物医学工程学杂志,2011,28(4):748-752.
[16]Wang ML,Nesti LJ,Tuli R,et al.Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells[J].J Orthop Res,2002,20(6):1175-1184.
[17]Wan Y,Zhuo N,Li Y,et al.Autophagy promotes osteogenic differentiation of human bone marrow mesenchymal stem cell derived from osteoporotic vertebrae[J].Biochemical&Biophysical Research Communications,2017,488(1):46-52.
[18]Li C,Liu Y,Liu J,et al.Rapamycin inhibits human glioma cell proliferation through down-regulating mammalian target of rapamycin pathway and upregulating microRNA-143[J].Head&Neck Oncology,2012,4(3):66.
[19]李军,王倩,林小星,等.自噬活性改变对骨髓间充质干细胞成骨分化能力的影响[J].华中科技大学学报(医学版),2012,41(6):675-679.
[20]王昕,马凤霞,卢士红,等.雷帕霉素对再生障碍性贫血患者骨髓间充质干细胞生物学功能的影响[J].中国实验血液学杂志,2014,22(3):762-766.
[21]张广宇,贾延劼,王军,等.氯化锂通过调节自噬通路促进大鼠骨髓间充质干细胞向神经细胞分化[J].中国病理生理杂志,2017,33(12):2128-2133.
[22]刘宇佳,楼斯悦,章燕棋,等.低氧激活自噬促进人间充质干细胞成骨分化的研究[J].中国现代应用药学,2016,33(3):284-289.