红景天苷对诱导的成骨细胞在低氧环境中的保护作用
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摘要
目的:探讨红景天苷对诱导的成骨细胞在低氧环境中的保护作用及其机制。方法:组织块法分离培养小鼠成纤维细胞,转染重编程为多能诱导干细胞,进行Nanog、Oct-4、Rexl、Sox-2的QT-PCR检测。诱导性多潜能干细胞(IPS细胞)诱导分化为成骨细胞,进行茜素红染色鉴定。将诱导后的成骨细胞分为红景天苷预处理组、正常组和低氧组,用Annexin V-FITC/PI染色检测细胞凋亡率,Western blot检测HIF-1α和VEGF蛋白表达量。结果:多能诱导干细胞的内源性基因Nanog、Oct-4、Rexl、Sox-2的表达量均明显高于小鼠成纤维细胞(均P<0.05)。茜素红染色显示诱导后的成骨细胞染色阳性。红景天苷预处理组细胞凋亡率明显低于低氧组,差异有统计学意义(P<0.05)。红景天苷预处理组的HIF-1α和VEGF表达量高于低氧组(0.93±0.05 vs. 0.81±0.02,0.95±0.03 vs. 0.79±0.04;均P<0.05)。结论:红景天苷对诱导后成骨细胞在低氧环境中有保护作用,其机制可能是通过提高细胞HIF-1α和VEGF的表达来抑制成骨细胞的凋亡。
Objective: To investigate the protective effect of salidroside on induced osteoblasts in hypoxia environment and its mechanism. Methods: the mouse fibroblasts were separated and cultured by tissue block method. The transfected induced pluripotent stem cells were reprogrammed and the QT-PCR of Nanog, Oct-4,Rexl and Sox-2 were detected to the induced osteoblasts differentiated from the induced pluripotent stem cells and identified by alizarin red staining. The induced osteoblasts were divided into three groups: salidroside preconditioning group, normal oxygen group and hypoxia group. Annexin V-FITC/PI staining was used to detect the apoptosis rate, and Western-blot was used to detect VEGF, HIF-1 alpha. Results: The expression of endogenous gene Nanog, Oct-4, Rexl and Sox-2 of induced pluripotent stem cells was higher than that of mouse fibroblasts cells. The result is Nanog(783±42 vs. 308±23), Oct-4(774±37 vs. 319±31), Rexl(742±28 vs. 382±31), Sox-2(831±31 vs. 394±12). Alizarin red staining showed that the induced osteoblasts cells had positive staining results.The apoptotic rate of salidroside group was significantly lower than that of hypoxia group [(35.65±3.92)% vs.(95.20±1.03)%]. There was significant difference between the two groups(P<0.05). The expression of HIF-1 a and VEGF in salidroside group was higher than that in hypoxic group(0.93±0.05 vs. 0.81±0.02, 0.95±0.03 vs.0.79±0.04, P<0.05). Conclusion: Salidroside has protective effect on hypoxic environment of osteoblasts.
Objective: To investigate the protective effect of salidroside on induced osteoblasts in hypoxia environment and its mechanism. Methods: the mouse fibroblasts were separated and cultured by tissue block method. The transfected induced pluripotent stem cells were reprogrammed and the QT-PCR of Nanog, Oct-4,Rexl and Sox-2 were detected to the induced osteoblasts differentiated from the induced pluripotent stem cells and identified by alizarin red staining. The induced osteoblasts were divided into three groups: salidroside preconditioning group, normal oxygen group and hypoxia group. Annexin V-FITC/PI staining was used to detect the apoptosis rate, and Western-blot was used to detect VEGF, HIF-1 alpha. Results: The expression of endogenous gene Nanog, Oct-4, Rexl and Sox-2 of induced pluripotent stem cells was higher than that of mouse fibroblasts cells. The result is Nanog(783±42 vs. 308±23), Oct-4(774±37 vs. 319±31), Rexl(742±28 vs. 382±31), Sox-2(831±31 vs. 394±12). Alizarin red staining showed that the induced osteoblasts cells had positive staining results.The apoptotic rate of salidroside group was significantly lower than that of hypoxia group [(35.65±3.92)% vs.(95.20±1.03)%]. There was significant difference between the two groups(P<0.05). The expression of HIF-1 a and VEGF in salidroside group was higher than that in hypoxic group(0.93±0.05 vs. 0.81±0.02, 0.95±0.03 vs.0.79±0.04, P<0.05). Conclusion: Salidroside has protective effect on hypoxic environment of osteoblasts.
引文
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[2]POSRITONG S, HONG J M, ELENISTE P P, et al. Pyk2deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene[J]. Mol Cell Endrcrinol, 2018, 474:35-47.
[3]ALTMAYER N C, GALATA V, WARSCHBURGER N, et al.Gene amplification in mesenchymal stem cells and during differentiation towards adipocytes or osteoblasts[J]. Oncotarget, 2017, 9(2):1803-1812.
[4]SHEYN D, BEN-DAVID S, SHAPIRO G, et al. Human inducedpluripotentstemcellsdifferentiateintofunctional mesenchymal stem cells and repair bone defects[J]. Stem Cells Transl Med, 2016, 5(11):1447-1460.
[5]KATO H, OCHIAI-SHINO H, ONODERA S, et al. Promoting effect of 1,25(OH)2 vitamin D3 in osteogenic differentiation from induced pluripotent stem cells to osteocyte-like cells[J]. Open Biol, 2015, 5(2):140201.
[6]MA M S, KANNAN V, DE VRIES A E, et al. Characterization and comparison of osteoblasts derived from mouse embryonicstemcellsandinducedpluripotentstemcells[J].J Bone Miner Metab, 2017, 35(1):21-30.
[7]TOKITA R, NAKAJIMA K, INOUE K, et al. Differentiation behavior of iPS cells cultured on PLGA with osteoinduction medium[J]. Dent Mater J, 2017, 36(1):103-110.
[8]祁琳,王川,梅国华,等.低氧状态下红景天苷经HIF-1α信号通路调节人成骨样MG-63细胞表型表达的研究[J].中国药理学通报, 2017, 33(6):836-843.
[9]OKAMOTO H, MATSUMI Y, HOSHIKAWA Y, et al. Involvement of microRNAs in regulation ofosteoblastic differentiation in mouse induced pluripotent stemcells[J]. PLoS One, 2012, 7(8):e43800.
[10]钱浩,徐增琦,李杨,等,小鼠诱导性多能干细胞诱导成骨分化的初步研究[J].口腔生物医学, 2015, 6(4):193-197.
[11]GENG Y J. Molecular mechanisms for cardiovascular stem cell apoptosis and growth in the hearts with atherosclerotic coronary disease and ischemic heart failure[J]. Ann N Y Acad Sci, 2003, 687-697.
[12]FOLLMAR K E, DECROOS F C, PRICHARD H L, et al.Effects of glutamine, glucose, and oxygen concentration on the metabolism and proliferation of rabbit adipose-derived stem cells[J]. Tissue Eng, 2006, 12(12):3525-3533.
[13]TAN C B, GAO M, XU W R, et al. Protective effects of salidroside on endothelial cell apoptosis induced by cobalt chloride[J]. Biol Pharm Bull, 2009, 32(8):1359-1363.
[14]李庆勇,钱志远,卞中国,等.创伤性颅脑损伤后缺氧诱导因子-1α的表达及其在血管修复中的作用[J].中华实验外科杂志, 2015, 32(4):926.
[15]STEGEN S, VAN GASTEL N, EELEN G, et al. HIF-1αpromotes glutamine-mediated redox homeostasis and glycogendependentbioenergeticstosupportpostimplantationbone cell[J]. Cell Metab, 2016, 23(2):265-279.
[16]LIN Y, ZHAI E, LIAO B, et al. Autocrine VEGF signaling promotes cell proliferation through a PLC-dependent pathway and modulates Apatinib treatment efficacy in gastric cancer[J]. Oncotarget, 2017, 8(7):11990-12002.
[17]郦铮铮,林以诺,郭宴会,等, VEGF经PI3K-Akt-Bad通路降低冻存后EOCs凋亡率[J].温州医科大学学报, 2014,44(12):882-886.
[18]KLUCK R M, BOSSY-WETZEL E, GREEN D R, et al.Thereleaseofcytochromecfrommitochondria:Aprimary site for bcl-2 regulation of apoptosis[J]. Science, 1997,275(5303):1132-1136.