β-神经生长因子的诱导表达及其对角膜缘干细胞的作用  

Inducing expression of β-nerve growth factor and its effect on limbal stem cells

作　　者：高杰[1,2] 丁玲[1] 胡蓉[1] 黄悦[1] 苏敏[1,3] 李红[1] 

机构地区：[1]贵州医科大学组织学胚胎学教研室,贵阳550004 [2]贵州医科大学基础医学科学研究中心,贵阳550004 [3]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,贵阳550004

出　　处：《解剖学报》2017年第4期421-427,共7页Acta Anatomica Sinica

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]

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.

关 键 词：Β-神经生长因子 TET-ON 3G四环素诱导表达系统 强力霉素 角膜缘干细胞 免疫印迹法 免疫荧光 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]