β-神经生长因子的诱导表达及其对角膜缘干细胞的作用

发布时间：2018-03-29 14:22

本文选题：β-神经生长因子　切入点：Tet-on　出处：《解剖学报》2017年04期

【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(P0.05)。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(P0.05);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(P0.05)。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。
[Abstract]:Objective to investigate the overexpression of 尾 -NGF in human embryonic kidney cell line HEK293FTs and its effect on limbal stem cells by using Tet-on 3G tetracycline induced expression system. Methods pLVX-TRE3G-IRES- 尾 -NGF and p LVX-Tet3G lentivirus were amplified in blank HEK293FT cells. Lentivirus was harvested and co-transfected into HEK293FT cells. 尾 -NGF expression was induced by doxycycline in different doses. The expression of 尾 -NGF was detected by Western blotting. LSCswere isolated and cultured in vitro. Lentivirus co-transfected LSCswere divided into two groups: control group (untransfected group) and induced expression group [experimental group A (lentivirus vector Dox 1000 渭 g / L), experimental group B (lentivirus). The expression of NGF and its associated protein, p63, p38, Trk A, was detected by cell immunofluorescence staining 48 h after induction and expression of lentivirus empty vector Dox (1000 渭 g / L). Results the expression of NGF was induced in transfected cells. Compared with the control group and the experimental group C, the expression of p63 in experimental group A decreased and the expression of TrkA decreased, and the expression of p38 increased. Compared with the control group and the experimental group C, the expression of p63 in experimental group B increased, and the expression of p38 decreased. Conclusion the expression system of tetracycline induced by Tet-on 3G was used. 尾 -NGF protein was successfully induced and expressed in different doses. The microenvironment of NGF induced by low dose Dox could promote the expansion of LSCs in vitro and maintain the stem cell character of LSCs.
【作者单位】：贵州医科大学组织学胚胎学教研室;贵州医科大学基础医学科学研究中心;贵州医科大学贵州省细胞工程生物医药技术国家地方联合工程实验室贵州省再生医学重点实验室;
【基金】：贵州省科技厅基金[黔科合J字(2015)2011号] 贵州省优秀科技教育人才省长专项基金[黔省专合字(2012)41号]
【分类号】：R77

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