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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 investigate the overexpression of β-nerve growth factor (β-NGF) in human embryonic kidney (HEK293FT) cells and its effect on limbal stem cells (LSCs) using Tet-on 3G tetracycline-induced expression system. Methods Lentiviral plasmids p LVX-TRE3G-IRES-β-NGF and p LVX-Tet3G were amplified in blank HEK293FT cells and lentivirus was harvested. The lentivirus was co-transfected into HEK293FT cells and induced with different doses of doxorubicin (Dox) β-NGF expression (divided into untransfected group, 1000, 100 and 1000 μg / L Dox induced expression group). After 48 h, the cells were collected and the expression of β-NGF in each group was detected by Western blotting. LSCs were co-transfected with lentivirus in vitro and divided into control group (untransfected group) and induced expression group (experimental group A (lentiviral vector Dox 1000μg / L), experimental group B (lentiviral vector Dox 100μg / L and Dox at 1000μg / L in experimental group). The expression of NGF and related proteins (p63, p38, Trk A) were detected by immunofluorescence staining 48 h after induction. Results NGF was induced in transfected cells. With the increase of dose of Dox, the expression of NGF increased, with statistical significance (P <0.05). Compared with control group and experimental group C, the expression of p63, Trk A decreased and p38 increased in experimental group A (P <0.05) B increased p63 expression, Trk A expression increased, p38 decreased, the difference was statistically significant (P <0.05). CONCLUSION: Tet-on 3G tetracycline-induced expression system can successfully induce the expression of different doses of β-NGF protein. Low-dose Dox-induced microenvironment of NGF can promote the in vitro expansion of LSCs and maintain the stem cell characteristics of LSCs.