不同代次大鼠骨髓间充质干细胞Wnt/β-catenin信号通路表达分析
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摘要
目的观察不同代次大鼠骨髓间充质干细胞(MSCs)体外增殖能力及其纤维分化倾向,为临床治疗及组织工程种子细胞的选择提供依据。方法采用贴壁培养法分离培养大鼠MSCs,流式细胞术鉴定细胞表面标记; MTT法检测不同代次MSCs的增殖情况;蛋白质印迹法(Western Blot)检测MSCs Wnt通路相关蛋白的表达情况;实时聚合酶链式反应(Realtime-PCR)法检测不同代次MSCs中成纤维细胞分子标记表达。结果流式细胞术鉴定结果证实我们培养的大鼠MSCs符合间充质干细胞的特征,且纯度高; MTT法检测显示P3代之后细胞的增殖能力明显高于P1、P2代,Western Blot检测显示细胞内Wnt/β-catenin信号通路蛋白表达随培养代次增强,Realtime-PCR法检测结果证实P9代细胞开始出现成纤维分化倾向。结论 MSCs作为种子细胞不宜超过P9代,P3～P7代具有纯度高、倍增快等优点,是能够满足组织工程细胞植入的种子细胞。
Objective To observe the proliferation ability of rat bone marrow mesenchymal stem cells(MSCs) and the tendency of fibrous differentiation in vitro; and to provide basis for clinical treatment and tissue engineering seed cells selection. Methods The rat MSCs were isolated and cultured by the adherent culture method; The cell surface markers were identified by flow cytometry; The proliferation of different generations of MSCs was detected by MTT method; The expression of Wnt pathway related proteins of MSCs was detected by Western Blot analysis; The molecular markers of fibroblasts in different generations of MSCs were detected by realtime-PCR method. Results Flow cytometry analysis confirmed that rat MSCs cultured met the characteristics of MSCs with high purity. The MTT assay showed that the proliferation ability of the cells after P3 generation was significantly higher than that of P1 and P2 generation. The Western Blot analysis showed that the expression of Wnt/β-catenin signaling protein in the cells was enhanced with the culture generation; While realtime-PCR assay confirmed that the P9 generation cells of MSCs demonstrated tendency of fibroblast differentiation.Conclusions The results in the study show that the seed cells should not be cultured for more than P9 generation,and the P3 to P7 generations shows advantages in high purity and rapid multiplication,which satisfies requirements of seed cells for engineering cells implantation in tissue.
Objective To observe the proliferation ability of rat bone marrow mesenchymal stem cells(MSCs) and the tendency of fibrous differentiation in vitro; and to provide basis for clinical treatment and tissue engineering seed cells selection. Methods The rat MSCs were isolated and cultured by the adherent culture method; The cell surface markers were identified by flow cytometry; The proliferation of different generations of MSCs was detected by MTT method; The expression of Wnt pathway related proteins of MSCs was detected by Western Blot analysis; The molecular markers of fibroblasts in different generations of MSCs were detected by realtime-PCR method. Results Flow cytometry analysis confirmed that rat MSCs cultured met the characteristics of MSCs with high purity. The MTT assay showed that the proliferation ability of the cells after P3 generation was significantly higher than that of P1 and P2 generation. The Western Blot analysis showed that the expression of Wnt/β-catenin signaling protein in the cells was enhanced with the culture generation; While realtime-PCR assay confirmed that the P9 generation cells of MSCs demonstrated tendency of fibroblast differentiation.Conclusions The results in the study show that the seed cells should not be cultured for more than P9 generation,and the P3 to P7 generations shows advantages in high purity and rapid multiplication,which satisfies requirements of seed cells for engineering cells implantation in tissue.
引文
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[2] Pierro MT,Soll R.Mesenchymal stem cells for the prevention and treatment of bronchopulmonary dysplasia in preterm infants[Z].2017:CD011932.
[3] Pedrazza L,Cubillos-Rojas M,De Mesquita FC,et al.Mesenchymal stem cells decrease lung inflammation during sepsis,acting through inhibition of the MAPK pathway[J]. Stem Cell Res Ther,2017,8(1):289.
[4] Yao YZ,Song Q. Mesenchymal stem cells:A double-edged sword in radiation-induced lung injury[J].Thoracic Cancer,2018,9(2):208-217.
[5] Huang X,Zhong L,Hendriks J,et al.The effects of the WNTSignaling modulators BIO and PKF118-310 on the chondrogenic differentiation of human mesenchymal stem cells[J].Int J Mol Sci,2018,19(2):561.
[6] Wang Z. IL-17A inhibits osteogenic differentiation of bone mesenchymal stem cells via Wnt signaling pathway[Z].2017:4095-4101.
[7] Li XY,Luo Z. Mesenchymal stem cells in idiopathic pulmonary fibrosis[J]. Oncotarget, 2017, 8(60):102600-102616.
[8] Zhang J,Shao Y,He D,et al.Evidence that bone marrow-derived mesenchymal stem cells reduce epithelial permeability following phosgene-induced acute lung injury via activation of wnt3a protein-induced canonical wnt/β-catenin signaling[J].Inhal Toxicol,2016,28(12):572-579.
[9] Wagner W,Horn P,Castoldi M,et al.Replicative senescence of mesenchymal stem cells:a continuous and organized process[J].PLoS One,2008,3(5):e2213.
[10] Polo JM,Liu S,Figueroa ME,et al.Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells[J]. Nat Biotechnol,2010,28(8):848-855.
[11] Kim K,Doi A,Wen B,et al.Epigenetic memory in induced pluripotent stem cells[J]. Nature,2010,467(7313):285-290.
[12] Narcisi R,Arikan OH,Lehmann J,et al.Differential effects of small molecule WNT agonists on the multilineage differentiation capacity of human mesenchymal stem cells[J]. Tissue Eng Part A,2016,22(21/22):1264-1273.
[13] Feng Y,Ren J,Gui Y,et al.Wnt/β-Catenin-Promoted macrophage alternative activation contributes to kidney fibrosis[J].J Am Soc Nephrol,2018,29(1):182-193.