补肾活血汤石油醚提取物对BMSCs迁移过程中Wnt5a/PKC通路的影响
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摘要
目的探讨补肾活血汤(熟地黄、菟丝子、补骨脂,等)石油醚提取物对大鼠骨髓间充质干细胞(BMSCs)的迁移作用及相关机制。方法全骨髓贴壁法培养BMSCs,取第3代细胞用于实验,采用流式细胞仪检测细胞表面抗原,细胞划痕、Transwell实验观察补肾活血汤石油醚提取物0、25、50、100、200μg/m L剂量组的细胞迁移能力,Western blot和RT-PCR检测Wnt5a、蛋白激酶C(PKC)表达情况。结果第3代BMSCs表面抗原CD29、CD90、CD44表达阳性,而CD45表达阴性;细胞划痕6 h及12 h后,药物各剂量组细胞迁移速度明显高于空白组(P<0.05,P<0.01);Transwell实验中100μg/m L补肾活血汤石油醚提取物为促细胞迁移最佳剂量,差异有统计学意义(P<0.01);RT-PCR结果显示与空白对照组比较,药物各剂量组的Wnt5a mRNA水平均升高(P<0.05,P<0.01),而与空白对照组的PKC mRNA水平比较,药物只有100μg/m L剂量mRNA表达水平升高(P<0.01);Western blot结果表明药物组各剂量的Wnt5a、PKC蛋白表达较空白对照组都明显升高(P<0.01),均且以100μg/m L剂量最高(P<0.01)。结论补肾活血汤石油醚提取物可以促进BMSCs体外迁移,其作用机制可能与Wnt5a/PKC信号通路有关。
AIM To investigate the effect and mechanism of petroleum ether extract from Bushen Huoxue Decoction(BSHXD,Rehmanniae Radix Praeparata,Cuscutae Semen,Psoraleae Fructus,etc.) on rat BMSCs migration. METHODS Bone marrow mesenchymal stem cells isolated from whole bone marrow of rats were amplified by adherent culture in vitro. Flow cytometry was employed to detect the cell surface antigens of the third generation cells. Migration examination of cells treated with petroleum ether extract from BSHXD at concentrations of 25,50,100 and 200 μg/m L was accomplished by cell scratch test and Transwell assay. Expressions of Wnt5 a and PKC protein were traced by Western blot,and so were the mRNA expressions of Wnt5 a and PKC by RT-PCR.RESULTS Positive expressions of CD29,CD44 and CD90 in the passage 3 of BMSCs but negative expression of CD45 were observed. The cells given different concentrations of petroleum ether extract from BSHXD groups displayed a significantly higher wound healing speed than the control group after 6 h and 12 h cell scratch(P <0. 05). 100 μg/m L BSHXD was shown to be the most active concentration for promoting cell migration by Transwell migration experiments,with a statistically significant difference(P < 0. 01). Compared with the control group,the levels of Wnt5 a mRNA in the BSHXD groups were significantly higher(P < 0. 05),and the PKC mRNA level in 100 μg/m L petroleum ether extract from BSHXD group was significantly increased as well(P < 0. 01)by RT-PCR. Although BSHXD groups brought forth significant improvement in both the expressions of Wnt5 a and PKC protein(P < 0. 01),the 100 μg/m L concentration conferred the highest(P < 0. 01). CONCLUSION Petroleum ether extract from BSHXD can promote BMSCs migration in vitro,and the mechanism may lie in its association with Wnt5 a signaling pathway.
AIM To investigate the effect and mechanism of petroleum ether extract from Bushen Huoxue Decoction(BSHXD,Rehmanniae Radix Praeparata,Cuscutae Semen,Psoraleae Fructus,etc.) on rat BMSCs migration. METHODS Bone marrow mesenchymal stem cells isolated from whole bone marrow of rats were amplified by adherent culture in vitro. Flow cytometry was employed to detect the cell surface antigens of the third generation cells. Migration examination of cells treated with petroleum ether extract from BSHXD at concentrations of 25,50,100 and 200 μg/m L was accomplished by cell scratch test and Transwell assay. Expressions of Wnt5 a and PKC protein were traced by Western blot,and so were the mRNA expressions of Wnt5 a and PKC by RT-PCR.RESULTS Positive expressions of CD29,CD44 and CD90 in the passage 3 of BMSCs but negative expression of CD45 were observed. The cells given different concentrations of petroleum ether extract from BSHXD groups displayed a significantly higher wound healing speed than the control group after 6 h and 12 h cell scratch(P <0. 05). 100 μg/m L BSHXD was shown to be the most active concentration for promoting cell migration by Transwell migration experiments,with a statistically significant difference(P < 0. 01). Compared with the control group,the levels of Wnt5 a mRNA in the BSHXD groups were significantly higher(P < 0. 05),and the PKC mRNA level in 100 μg/m L petroleum ether extract from BSHXD group was significantly increased as well(P < 0. 01)by RT-PCR. Although BSHXD groups brought forth significant improvement in both the expressions of Wnt5 a and PKC protein(P < 0. 01),the 100 μg/m L concentration conferred the highest(P < 0. 01). CONCLUSION Petroleum ether extract from BSHXD can promote BMSCs migration in vitro,and the mechanism may lie in its association with Wnt5 a signaling pathway.
引文
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[14]李静,陈军,钱彦方,等.红花苷对模拟微重力下细胞迁移的作用[J].中医杂志,2013,54(1):55-58.
[15]施剑明,邬亚华,耿书国,等.间充质干细胞增殖、衰老及分化:Wnt信号通路经典与非经典的调控作用[J].中国组织工程研究2014,18(41):6719-6724.
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[18]Oishi I,Suzuki H,Onishi N,et al.The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway[J].Genes Cells,2003,8(7):645-654.
[19]Shen Y L,Luo Q,Guo Y X,et al.Bone marrow mesenchymal stem cell-derived Wnt5a inhibits leukemia cell progression in vitro via activation of the non-canonical Wnt signaling pathway[J].Oncol Lett,2014,8(1):85-90.
[20]Binda E,Visioli A,Giani F,et al.Wnt5a drives an invasive phenotype in human glioblastoma stem-like cells[J].Cancer Res,2017,77(4):996-1007.
[2]Dhinsa B S,Adesida A B.Current clinical therapies for cartilage repair,their limitation and the role of stem cells[J].Curr Stem Cell Res Ther,2012,7(2):143-148.
[3]Chamberlain G,Fox J,Ashton B,et al.Concise review:mesenchymal stem cells:their phenotype,differentiation capacity,immunological features,and potential for homing[J].Stem Cells,2007,25(11):2739-2749.
[4]Rattigan Y,Hsu J M,Mishra P J,et al.Interleukin 6 mediated recruitment of mesenchymal stem cells to the hypoxic tumor milieu[J].Exp Cell Res,2010,316(20):3417-3424.
[5]Zou C,Luo Q,Qin J,et al.Osteopontin promotes mesenchymal stem cell migration and lessens cell stiffness via integrinβ1,FAK,and ERK pathways[J].Cell Biochem Biophys,2013,65(3):455-462.
[6]Ling L,Nurcombe V,Cool S M.Wnt signaling controls the fate of mesenchymal stem cells[J].Gene,2009,433(1-2):1-7.
[7]黄永铨,罗毅文,王斌,等.补肾活血汤提取物促进大鼠骨髓间充质干细胞增殖的研究[J].广州中医药大学学报,2015,32(1):86-91.
[8]罗毅文,王斌,吴志方,等.补肾活血汤提取物促进大鼠骨髓间充质干细胞体外迁移及CXCR4表达的研究[J].中药新药与临床药理,2016,27(3):356-361.
[9]黄永铨,罗毅文,王斌,等.补肾活血汤治疗老年桡骨远端骨折的临床疗效观察[J].中国中医骨伤科杂志,2015,23(3):5-8.
[10]林桂涛,李荣伟,耿子凯,等.补骨脂水及乙醇提取物雌激素样作用的比较[J].中成药,2016,38(12):2688-2691.
[11]韩艳,温利平,刘娜,等.补肾活血方对去卵巢大鼠骨代谢及骨密度的影响[J].中医正骨,2015,27(12):7-11,15.
[12]方健康,周轶平,李玛琳.中药对骨髓间充质干细胞增殖、凋亡及分化的影响研究进展[J].中国中药杂志,2014,39(15):2834-2837.
[13]欧阳长生,王云开,苏海,等.丹参及血府逐瘀汤含药血清与兔骨髓间充质干细胞的体外迁移:促进还是抑制?[J].中国组织工程研究与临床康复,2009,13(40):7847-7851.
[14]李静,陈军,钱彦方,等.红花苷对模拟微重力下细胞迁移的作用[J].中医杂志,2013,54(1):55-58.
[15]施剑明,邬亚华,耿书国,等.间充质干细胞增殖、衰老及分化:Wnt信号通路经典与非经典的调控作用[J].中国组织工程研究2014,18(41):6719-6724.
[16]Kikuchi A,Yamamoto H,Sato A,et al.Wnt5a:its signalling,functions and implication in diseases[J].Acta Physiol(Oxf),2012,204(1):17-33.
[17]Cheng C W,Yeh J C,Fan T P,et al.Wnt5a-mediated noncanonical Wnt signalling regulates human endothelial cell proliferation and migration[J].Biochem Biophys Res Commun,2008,365(2):285-290.
[18]Oishi I,Suzuki H,Onishi N,et al.The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway[J].Genes Cells,2003,8(7):645-654.
[19]Shen Y L,Luo Q,Guo Y X,et al.Bone marrow mesenchymal stem cell-derived Wnt5a inhibits leukemia cell progression in vitro via activation of the non-canonical Wnt signaling pathway[J].Oncol Lett,2014,8(1):85-90.
[20]Binda E,Visioli A,Giani F,et al.Wnt5a drives an invasive phenotype in human glioblastoma stem-like cells[J].Cancer Res,2017,77(4):996-1007.
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