β-神经生长因子的诱导表达及其对角膜缘干细胞的作用
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摘要
目的运用Tet-on 3G四环素诱导表达系统探讨β-神经生长因子(β-NGF)在人胚肾(HEK293FT)细胞中的过表达情况及其对角膜缘干细胞(LSCs)的作用。方法将p LVX-TRE3G-IRES-β-NGF和p LVX-Tet3G慢病毒在空白的HEK293FT细胞进行扩增,收获慢病毒,再共转染HEK293FT细胞,同时用不同剂量强力霉素(Dox)诱导β-NGF表达(分为未转染组、1000、100和1000μg/L Dox诱导表达组)。48 h后收集细胞,免疫印迹法(Western blotting)检测各组细胞内β-NGF表达情况。体外分离培养LSCs,慢病毒共转染LSCs,分为对照组(未转染组)和诱导表达组[实验组A(慢病毒载体Dox 1000μg/L)、实验组B(慢病毒载体Dox 100μg/L)、实验组C(慢病毒空载体Dox 1000μg/L)],诱导表达48 h后细胞免疫荧光染色检测NGF及相关蛋白(p63、p38、Trk A)的表达情况。结果NGF在转染细胞内被诱导表达,随着Dox剂量增加,表达量增强,差异有统计学意义(P<0.05)。与对照组及实验组C相比,实验组A的p63表达降低,Trk A表达降低,p38表达增加,差异有统计学意义(P<0.05);与对照组及实验组C相比,实验组B的p63表达增加,Trk A表达增加,p38表达降低,差异有统计学意义(P<0.05)。结论运用Tet-on 3G四环素诱导表达系统,可成功诱导表达不同剂量β-NGF蛋白,低剂量Dox诱导下NGF产生的微环境可促进LSCs的体外扩增,并能维持LSCs的干细胞特性。
Objective To construct recombinant lentiviral vector carrying rat β-nerve growth factor( β-NGF)gene by inducing expression of the Tet-on 3G tetracycline induced expression system,and to observe its overexpression in HEK293 FT cells and effects on limbal stem cells( LSCs). Methods After transfection,β-NGF protein was detected to express on HEK293 FT cells following the increase of doxycycline( Dox) dose. After primary cultured,LSCs were allocated to four groups with different doses of Dox: control group( non-transfection group),1000,100 and 1000μg/L of Dox inducing groups( A,B,C). The expressions of p63,NGF,p38,Trk A were observed. Results After transfection,β-NGF protein was detected to express on HEK293 FT cells and LSCs following the increase of Dox dose. The experimental groups expressed different quantities of p63,p38,and Trk A,which was statistically significant. Conclusion The β-NGF gene could be expressed successfully on LSCs by Tet-on 3G induced expression system. These result highlight that NGF is benefit LSCs amplification in vitro,and maintain their stemness.
Objective To construct recombinant lentiviral vector carrying rat β-nerve growth factor( β-NGF)gene by inducing expression of the Tet-on 3G tetracycline induced expression system,and to observe its overexpression in HEK293 FT cells and effects on limbal stem cells( LSCs). Methods After transfection,β-NGF protein was detected to express on HEK293 FT cells following the increase of doxycycline( Dox) dose. After primary cultured,LSCs were allocated to four groups with different doses of Dox: control group( non-transfection group),1000,100 and 1000μg/L of Dox inducing groups( A,B,C). The expressions of p63,NGF,p38,Trk A were observed. Results After transfection,β-NGF protein was detected to express on HEK293 FT cells and LSCs following the increase of Dox dose. The experimental groups expressed different quantities of p63,p38,and Trk A,which was statistically significant. Conclusion The β-NGF gene could be expressed successfully on LSCs by Tet-on 3G induced expression system. These result highlight that NGF is benefit LSCs amplification in vitro,and maintain their stemness.
引文
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[21]Wang J,Luo Ch J,Ran XZ,et al.The role of p38 on the differentiation of MSCs to myoblasts[J].Chinese Journal of Applied Physiology,2004,20(4):345-348.(in Chinese)王劲,罗成基,冉新泽,等.p38通路对骨髓间充质干细胞成肌分化作用研究[J].中国应用生理学杂志,2004,20(4):345-348.
[22]Yin TT.The effect of p38 signaling pathway on the proliferation and differentiation of limbal stem cells and skin epithelial stem cells in vitro[D].Xiamen University(Electonic Theses and Dissertations Database),2011:73.(in Chinese)殷婷婷.p38信号通路对角膜缘干细胞和皮肤上皮干细胞体外培养过程中增殖与分化的影响[D].厦门:厦门大学(眼科研究所),2011:73.
[23]Idgides D,Salvador R,Pereira Perrin M.Trypanosoma cruzi coaxes cardiac fibroblasts into preventing cardiomyocyte death by activating nerve growth factor receptor Trk A[J].PLo S One,2013,8(2):e57450.
[24]Yuan J,Huang G,Xiao Z,et al.Overexpression of b-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway[J].Mol Cell Biochem,2013,383(1-2):201-211.
[2]Kwitko S,Mariuho D,Barcaro S,et al.Allograft conjunctival transplantation for bilateral ocular surface disorders[J].Ophtha Imo Iogy,1995,1029(7):1020-1025.
[3]Lambiase A,Mantelli F,Sacchetti M,et al.Clinical applications of NGF in ocular diseases[J].Arch Ital Biol,2011,149(2):283-292.
[4]Maness LM,Kastin AJ,Weber JT,et al.The neurotrophins and their receptors:structure,function,and neuropathology[J].Neurosci Biobehav Rev,1994 Spring,18(1):143-159.
[5]Qi H,Li DQ,Shine HD,et al.Nerve growth factor and its receptor Trk A serve as potential markers for human corneal epithelial progenitor cells[J].Exp Eye Res,2008,86(1):34-40.
[6]You L,Kruse FE,Volcker HE.Neurotrophic factors in the human cornea[J].Invest Ophthalmol Vis Sci,2000,41(3):692-702.
[7]Touhami A,Grueterich M,Tseng SC.The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture[J].Invest Ophthalmol Vis Sci,2002,43(4):987-994.
[8]Gao J,Yan HW,Li H,et al.Inducing expression ofβ-neural growth factor and effect on PC12 cells[J].Acta Anatomica Sinica,2016,47(4)):476-481.(in Chinese)高杰,严会文,李红,等.β-神经生长因子基因克隆和诱导表达及其对PC12细胞的诱导分化作用[J].解剖学报,2016,47(4):476-481.
[9]Liu Q,Su M,Hu R,et al.Effect of nerve growth factor on the proliferation of the limbal stem cells in vitro[J].Journal of Regional Anatomy and Operative Surgery,2013,22(2):127-130.(in Chinese)刘强,苏敏,胡蓉,等.NGF对角膜缘干细胞体外扩增的影响[J].局部手术学杂志,2013,22(2):127-130.
[10]Lee SK,Teng Y,Wong HK,et al.MicroRNA-145 regulates human corneal epithelial differentiation[J].PLo S One,2011,6(6):212-230.
[11]Saghizadeh M,Soleymani S,Harounian A,et al.Alterations of epithelial stem cell marker patterns in human diabetic corneas and effects of c-met gene therapy[J].Mol Vis,2011,17:2177-2190.
[12]Yoshida S,Yasuda M,Miyashita H,et al.Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells[J].PLo S One,2011,6(12):e28856.
[13]Dua HS,Joseph A,Shanmuganathan VA,et al.Stem cell differentiation and the effects of deficiency[J].Eye,2003,17(8):877-885.
[14]Hemandez GEE,Theiss C,Steuhl KP,et al.Expression of Delta NP63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane[J].Invest Ophthalmal Vis Sci,2003,44(7):2959-2965.
[15]Su M,Xiao L,Xu TL.Insulin promotes the proliferation of rabbit iimbal stem cells in vitro[J].Acta Anatomica Sinica,2010,41(4)):550-553.(in Chinese)苏敏,肖莉,许庭良.胰岛素促进体外兔角膜缘干细胞的增殖[J].解剖学报,2010,41(4):550-553.
[16]Lambiase A,Coassin M,Sposato V,et al.NGF topical application in patients with corneal ulcer does not generate circulating NGF antibodies[J].Pharmacol Res,2007,56(1):65-69.
[17]Palazzo E,Marconi A,Truzzi F,et al.Role of neurotrophins on dermal fibroblast survival and differentiation[J].J Cell Physiol,2012,227(3):1017-1025.
[18]Dashti SR,Efimova T,Eckert RL.MEK7-dependent activation of p38 MAP kinase in keratinocytes[J].J Biol Chem,2001,276(11):8059-8063.
[19]Watt FM,Estrach S,Ambler CA.Epidermal Notch signaling:differentiation,cancer and adhesion[J].Curr Opin Cell Biol,2008,20(2):171-179.
[20]Jaiswal RK,Jaiswal N,Bruder SP,et al.Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-axtivated protein kinase[J]J Biol Chem,2000,275(13):9645-9652.
[21]Wang J,Luo Ch J,Ran XZ,et al.The role of p38 on the differentiation of MSCs to myoblasts[J].Chinese Journal of Applied Physiology,2004,20(4):345-348.(in Chinese)王劲,罗成基,冉新泽,等.p38通路对骨髓间充质干细胞成肌分化作用研究[J].中国应用生理学杂志,2004,20(4):345-348.
[22]Yin TT.The effect of p38 signaling pathway on the proliferation and differentiation of limbal stem cells and skin epithelial stem cells in vitro[D].Xiamen University(Electonic Theses and Dissertations Database),2011:73.(in Chinese)殷婷婷.p38信号通路对角膜缘干细胞和皮肤上皮干细胞体外培养过程中增殖与分化的影响[D].厦门:厦门大学(眼科研究所),2011:73.
[23]Idgides D,Salvador R,Pereira Perrin M.Trypanosoma cruzi coaxes cardiac fibroblasts into preventing cardiomyocyte death by activating nerve growth factor receptor Trk A[J].PLo S One,2013,8(2):e57450.
[24]Yuan J,Huang G,Xiao Z,et al.Overexpression of b-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway[J].Mol Cell Biochem,2013,383(1-2):201-211.