骨髓间充质干细胞促进结肠癌细胞HT29增殖和迁移
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摘要
目的探讨骨髓来源的间充质干细胞(bone marrowe-derived mesenchymal stem cells,BD-MSCs)对结肠癌细胞HT29的增殖与迁移能力的影响及其可能的机制。方法将BD-MSCs与结肠癌细胞系HT29共培养,并观察共培养后对结肠癌细胞的增殖和迁移能力的影响。通过Transwell实验检测共培养组与对照组细胞在迁移能力方面的差异;CCK-8实验、克隆形成和成球实验检测HT29细胞的体外增殖能力;体内成瘤实验比较共培养组和对照组细胞的成瘤能力差异;机制的阐明是通过荧光定量PCR和Western blot分别在mRNA和蛋白水平检测共培养组与对照组细胞间E-Cadherin、N-Cadherin、Vimentin、Fibronectin、Snail1、Snail2、Twist1和Twist2的表达差异。结果结肠癌细胞系HT29在与BD-MSCs共培养后,增殖能力显著增强(P<0.05)。与对照组相比,与BD-MSCs共培养后的HT29细胞的迁移能力[(259.6±19.23)个细胞/视野vs(81.6±15.74)个细胞/视野]显著增强(P<0.05)。克隆形成率[(37±2.94)%vs(15.33±2.87)%]和成球率[(31±3.27)%vs(10.33±2.62)%]均显著提高(P<0.05)。在体内成瘤实验中,与BD-MSCs共同注射的实验组所成肿瘤的体积是显著大于对照组的,并且所成肿瘤的重量也是具有显著差异的[(1.73±0.62)g vs(0.54±0.81)g,P<0.05]。最终在mRNA和蛋白水平检测到,与对照组细胞相比,与BD-MSCs共培养的HT29细胞中上皮样标记物E-Cadherin出现显著下调表达,而间质样标记物Vimentin和Twist1则显著上调表达。结论骨髓来源的间充质干细胞促进结肠癌细胞HT29的增殖与迁移。
Objective To investigate the effect of bone marrow-derived mesenchymal stem cells( BDMSCs) on the proliferation and migration of colorectal cancer cell line HT29 and to study the possible mechanism. Methods HT29 cells co-cultured with BD-MSCs were defined as a co-culture group,and HT29 cells cultured alone were selected as a control group. The cell migration was detected by Transwell assay.CCK-8 assay,clone formation and sphere formation were applied to determine in vitro proliferation capacity of the HT29 cells. The tumorigenicity of the co-culture group and the control group was compared by in vivo test.Fluorescence quantitative PCR and Western blotting were used for detecting the expressions of E-cadherin,N-cadherin,vimentin,fibronectin,Snail1,Snail2,Twist1 and Twist2 in the 2 groups. Results After co-culture with BD-MSCs,the proliferation of the HT29 cells was significantly increased( P < 0. 05). Compared with the control group,the cell migration of the co-culture group( 259. 6 ± 19. 23 vs 81. 6 ± 15. 74 cells / field)was significantly enhanced( P < 0. 05),and the clone formation percentage [( 37 ± 2. 94) % vs( 15. 33 ±2. 87) %]and the sphere formation percentage [( 31 ± 3. 27) % vs( 10. 33 ± 2. 62) %] were significantly improved( P < 0. 05). The tumor formed in the experimental by injection of HT29 cells and BD-MSCs was significantly larger than that formed in the control group by injection of HT29 cells only,and the tumor weight was also statistically different( 1. 73 ± 0. 62 vs 0. 54 ± 0. 81 g,P < 0. 05). The co-culture group exhibited significant down-regulation of E-cadherin and up-regulation of vimentin and Twist1 compared with the control group. Conclusion BD-MSCs promote the proliferation and migration in colorectal cancer cell line HT29.
Objective To investigate the effect of bone marrow-derived mesenchymal stem cells( BDMSCs) on the proliferation and migration of colorectal cancer cell line HT29 and to study the possible mechanism. Methods HT29 cells co-cultured with BD-MSCs were defined as a co-culture group,and HT29 cells cultured alone were selected as a control group. The cell migration was detected by Transwell assay.CCK-8 assay,clone formation and sphere formation were applied to determine in vitro proliferation capacity of the HT29 cells. The tumorigenicity of the co-culture group and the control group was compared by in vivo test.Fluorescence quantitative PCR and Western blotting were used for detecting the expressions of E-cadherin,N-cadherin,vimentin,fibronectin,Snail1,Snail2,Twist1 and Twist2 in the 2 groups. Results After co-culture with BD-MSCs,the proliferation of the HT29 cells was significantly increased( P < 0. 05). Compared with the control group,the cell migration of the co-culture group( 259. 6 ± 19. 23 vs 81. 6 ± 15. 74 cells / field)was significantly enhanced( P < 0. 05),and the clone formation percentage [( 37 ± 2. 94) % vs( 15. 33 ±2. 87) %]and the sphere formation percentage [( 31 ± 3. 27) % vs( 10. 33 ± 2. 62) %] were significantly improved( P < 0. 05). The tumor formed in the experimental by injection of HT29 cells and BD-MSCs was significantly larger than that formed in the control group by injection of HT29 cells only,and the tumor weight was also statistically different( 1. 73 ± 0. 62 vs 0. 54 ± 0. 81 g,P < 0. 05). The co-culture group exhibited significant down-regulation of E-cadherin and up-regulation of vimentin and Twist1 compared with the control group. Conclusion BD-MSCs promote the proliferation and migration in colorectal cancer cell line HT29.
引文
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[13]de-Herreros A G,Peiro S,Nassour M,et al.Snail family regulation and epithelial mesenchymal transitions in breast cancer progression[J].J Mammary Gland Biol Neoplasia,2010,15(2):135-147.
[14]Lander R,Nordin K,La Bonne C.The F-box protein Ppa is a common regulator of core EMT factors Twist,Snail,Slug,and Sip1[J].J Cell Biol,2011,194(1):17-25.
[15]Roussos E T,Keckesova Z,Haley J D,et al.AACR special conference on epithelial-mesenchymal transition and cancer progression and treatment[J].Cancer Res,2010,70(19):7360-7364.
[2]Qiao L,Xu Z,Zhao T,et al.Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model[J].Cell Res,2008,18(4):500-507.
[3]Zhu W,Xu W,Jiang R,et al.Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo[J].Exp Mol Pathol,2006,80(3):267-274.
[4]Shinagawa K,Kitadai Y,Tanaka M,et al.Mesenchymal stem cells enhance growth and metastasis of colon cancer[J].Int J Cancer,2010,127(10):2323-2333.
[5]Ohlsson L B,Varas L,Kjellman C,et al.Mesenchymal progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatin matrix[J].Exp Mol Pathol,2003,75(3):248-255.
[6]朱传琳,姚超,钱程,等.应用Lv-p Nanog-GFP慢病毒载体探讨Nanog在调控结直肠癌细胞的自我更新与转移能力中的作用[J].第三军医大学学报,2014,36(1):29-32.
[7]Tian L L,Yue W,Zhu F,et al.Human mesenchymal stem cells play a dual role on tumor cell growth in vitro and in vivo[J].J Cell Physiol,2011,226(7):1860-1867.
[8]Le-Blanc K,Frassoni F,Ball L,et al.Mesenchymal stem cells for treatment of steroid-resistant,severe,acute graft-versus-host disease:a phase II study[J].Lancet,2008,371(9624):1579-1586.
[9]庞一琳,张斌,陈虎.间充质干细胞与肿瘤细胞相互作用:促进还是抑制[J].中华细胞与干细胞杂志:电子版,2013,3(1):30-37.
[10]Lin J T,Wang J Y,Chen M K,et al.Colon cancer mesenchymal stem cells modulate the tumorigenicity of colon cancer through interleukin 6[J].Exp Cell Res,2013,319(14):2216-2229.
[11]Nieto M A.Epithelial-Mesenchymal Transitions in development and disease:old views and new perspectives[J].Int J Dev Biol,2009,53(8/10):1541-1547.
[12]Lim J,Thiery J P.Epithelial-mesenchymal transitions:insights from development[J].Development,2012,139(19):3471-3486.
[13]de-Herreros A G,Peiro S,Nassour M,et al.Snail family regulation and epithelial mesenchymal transitions in breast cancer progression[J].J Mammary Gland Biol Neoplasia,2010,15(2):135-147.
[14]Lander R,Nordin K,La Bonne C.The F-box protein Ppa is a common regulator of core EMT factors Twist,Snail,Slug,and Sip1[J].J Cell Biol,2011,194(1):17-25.
[15]Roussos E T,Keckesova Z,Haley J D,et al.AACR special conference on epithelial-mesenchymal transition and cancer progression and treatment[J].Cancer Res,2010,70(19):7360-7364.