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

doi:10.16098/j.issn.0529-1356.2016.04.007

摘要/Abstract

摘要：

目的构建β-神经生长因子（β-NGF）慢病毒表达载体pLVX-TRE3G-IRES-β-NGF，应用Tet-on 3G四环素诱导表达系统，探讨β-NGF在人胚肾（HEK293FT）细胞中的过表达情况，及其对大鼠肾上腺嗜铬细胞瘤（PC12）细胞分化的影响。方法从SD大鼠脑组织提取总RNA，反转录成cDNA，利用分子生物学技术，克隆鼠β-NGF基因完整编码区，将β-NGF基因定向插入载体pLVX-TRE3G-IRES中。将重组载体pLVX-TRE3G-IRES-β-NGF和载体pLVXTet3G分别转染空白HEK293FT细胞，制备收获慢病毒，共转染HEK293FT细胞，同时用不同剂量强力霉素（Dox）诱导NGF表达（分为未转染组、0、100、500和1000μg/L Dox诱导表达组）。48h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。同时收集培养上清，1. ELISA检测各组上清内β-NGF的分泌量； 2. 制作条件培养基，加入PC12细胞培养基中进行诱导培养，观察PC12细胞诱导分化情况。结果双酶切和测序鉴定重组质粒pLVX-TRE3G-IRES-β-NGF序列和方向正确；β-NGF在转染细胞内被诱导表达，随着Dox剂量增加，表达量增强；β-NGF在培养基的上清内表达，表达量随Dox剂量增加而增多。诱导表达的条件培养基可诱导PC12细胞形态改变，表达神经元特异性烯醇化酶（NSE）蛋白。结论成功重组慢病毒载体 pLVX-TRE3G-IRES-β-NGF, 转染后β-NGF基因能够在HEK293FT细胞中表达和分泌，且该蛋白具有诱导PC12细胞向神经元样细胞分化的能力；运用Tet-On 3G四环素诱导表达系统，可成功诱导表达获得不同剂量β-NGF蛋白。

关键词: β-神经生长因子, PC12细胞, 强力霉素, 酶联免疫吸附法, 免疫印迹法, 大鼠

Abstract:

Objective To construct recombinant lentiviral vector carrying rat β-neural growth factor (β-NGF) gene by inducing expression of the Tet-on 3G tetracycline，and to observe its expression in HEK293FT cells and effects on PC12 cells. Methods Total RNA was extracted from rat brain as the template. By gene recombination technique，rat β-NGF gene was inserted into lentiviral vector as pLVX-TRE3G-IRES-β-NGF. The supernatant of virus including the recombinant vector of pLVX-TRE3G-IRES-β-NGF and pLVX-Tet3G was collected from packaging cells, respectively, and then cotransfected blank HEK293FT cells. The expression of β-NGF was induced with different doses of doxycycline (Dox) (divided into non-transfection group, 0, 100, 500 and 1000μg/L of Dox inducing group). Cells were collected and analyzed by Western blotting assay, 48 hours after. The supernatant was detected by ELISA assay for the secretion and used as conditional medium for PC12 cells. Results A band of β-NGF gene was about 726bp. After transfection, β-NGF protein was detected to express on 293FT cells and supernatant following the increase of Dox dose. The neuron-specific enolase (NSE) was expressed on PC12 cells after induced by conditional media. Conclusion That theβ-NGF gene was be expressed successfully on HEK 293FT cells by Tet-on 3G induced expression system, and the protein has effected on inducing PC12 cells differentiation into neuron-like cells.

Key words: β-Nerve growth factor, PC12 cell, Doxycycline, ELISA, Western blotting, Rat

高杰严会文李红黄悦胡蓉苏敏. 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.

GAO Jie YAN Hui-wen LI Hong HUANG Yue HU Rong SU Min. Inducing expression of β-neural growth factor and effect on PC12 cells[J]. Acta Anatomica Sinica, 2016, 47(4): 476-481.

参考文献

［1］Kruse FE,Tseng SCG.Growth factors modulate clonal growth and differentiation of cultured rabbit limbal and corneal epithelium［J］.Invest Ophthalmol Vis Sci,l993,34(6): 1963-1976.

［2］Lambiase A, Mantelli F, Sacchetti M, et al. Clinical applications of NGF in ocular diseases ［J］. Archives Italiennes de Biologie, 2011,149(2): 283-292.

［3］Aloe L, Rocco ML, Bianchi P, et al. Nerve growth factor: from the early discoveries to the potential clinical use ［J］.J Transl Med, 2012, 29(10):239.

［4］Lewin GR, Lechner SG, Smith ES.Nerve growth factor and nociception: from experimental embryology to new analgesic therapy ［J］. Handb Exp Pharmacol,2014, 220(1): 251-282.

［5］Aloe L,Levi-Montalcini: the discovery of nerve growth factor and modern neurobiology ［J］.Trends Cell Biol, 2004,14(7):395-399.

［6］Kaihola H, Olivier J, Poromaa IS, et al. The effect of antenatal depression and selective serotonin reuptake inhibitor treatmenton nerve growth factor signalingin human placenta［J］.PLoS One, 2015, 10 (1): e0116459.

［7］Capsoni S. From genes to pain: nerve growth factor and hereditary sensory and autonomic neuropathy type V［J］. Eur J Neurosci,2014,39(3):392-400.

［8］Fink DM, Connor AL,Kelley PM, et al. Nerve growth factor regulates neurolymphatic remodeling during corneal inflammation and resolution［J］.PLoS One,2014,9 (11):e112737.

［9］Vera C, Tapia V, Vega M, et al. Role of nerve growth factor and its TRKA receptor in normal ovarian and epithelial ovarian cancer angiogenesis［J］. J Ovarian Res, 2014,1(7):82.

［10］Wang W, Chen J, Guo X.The role of nerve growth factor and its receptors in tumorigenesis and cancer pain ［J］. Biosci Trends,2014, 8(2):68-74.

［11］He JF,Gu GM,Long DH. Expression of recombinant retroviral vector carrring nerve growth factor gene in neural stem cells ［J］. Chinese Journal of Tissue Eengineering Research, 2014, 18(19):3100-3104.(in Chinese)

何峻峰,顾国妹,龙大宏.神经生长因子重组反转录病毒载体在神经干细胞中的表达［J］.中国组织工程研究,2014, 18(19):3100-3104.

［12］Hu W,Li YX,Ling Y, et al.Construction and identification of a new Tet-on gene expression system with lower basal expression ［J］Bulletin of the Academy of Military Medical Sciences, 2008,32（4）:309-327.(in Chinese)

胡伟,李玉霞,凌焱,等.新型低基础表达Tet-on 基因表达系统的构建与鉴定［J］.军事医学科学院院刊, 2008,32（4）:309-327.

［13］Kwitko S,Mariuho D,Barcaro S,et al. AIIograft conjunctivaI transpIantation for DiIateraI ocuIar surface disorder［J］.Ophthaimoiogy, 1995,102（7）:1020-1025.

［14］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.

［15］Liu YR，Liu Q. Meta-analysis of mNGF therapy for peripheral nerve injury：a systematic review［J］. Chin J Traumatol，2012，15（2）：86-91.

［16］Lambiase A，Aloe L，Centofanti M，et al. Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops：Implications for glaucoma［J］. Proc Natl Acad Sci USA，2009，106（32）：13469-13474.

［17］Qi H,Li DQ,Pflugfelder SC, et al. Nerve growth factor and its receptor TrkA serve as potential markers for human corneal epithelial progenitor cells ［J］.Exp Eye Res, 2008,86(1):34-40.

［18］Di Girolamo N,Sarris M,Chui J,et al.Localization of the low-affinity nerve growth factor receptor p75 in human limbal epithelial cells ［J］. Cell Mol Med, 2008, 12(6B):2799-2811.［19］Liu Z， CarvajaI M， Carraway CA， et al. Expression of the receptor tyrosine kinases， epidermaI growth factor receptor， ErDB2， and ErDB3， in human ocuIar surface epitheIi［J］. Cornea， 2001,2（1）：81-85.

［20］Li QX,Pei XL,Liu Y, et al.Expression and secretion of nerve growth factor in esophageal squamous cell cancer cell line Eca109［J］Acta Anatomica Sinica, 2015,45（3）:348-353. (in Chinese)

李秋霞,裴学莲,刘洋,等.神经生长因子在食管鳞癌细胞中的表达及分泌［J］.解剖学报，2015,45（3）:348-353.

［21］Yuan J, Huang G, Xiao Z, et al. Overexpression of β-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.

［22］Wu Y, Chu R, Zhou X, et al. Determination of the nerve growth factor level in the central cornea after LASIK and Epi-LASIK treatment in a rabbit model system ［J］. Cornea,2009, 28(10):1144-1148.

［23］Meng LX, Ding ZhJ, Chen XP, et al.Nerve growth factor promotes proliferation of human pancreatic cancer line MIA PaCa-2 ［J］. Chinese Journal of Cancer Biotherapy, 2010,17( 1):88-92. (in Chinese)

孟令新,丁兆军,陈希平,等. 神经生长因子促进 MIA PaCa-2人胰腺癌细胞的增殖. ［J］中国肿瘤生物治疗杂志, 2010,17(1):88-92.

［24］Lambiase A, Aloe L, Centofanti M, et al. Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops: Implications for glaucoma ［J ］.Proc Natl Acad Sci USA， 2009， 106(32)： 13469-13474.

［25］Zhang LY,Yang LY，Wang XH, et al. The influence of carbon tetrach loride upon the expression of low-affinity neural growth factor receptor-p75 in hipocampal cal area neueons ［J］.Acta Anatomica Sinica, 2008,39（1）:23-26. (in Chinese)

张立英，杨立元，王晓红,等. 四氯化碳对大鼠海马CAI区神经元低亲和力神经生长因子受体p75表达的影响［J］.解剖学报，2008,39（1）:23-26.

［26］Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters ［J］. Proc Natl Acad Sci USA, 1992, 89(12): 5547-5551.

［27］Chen H, Xia HB. Research progress and application of Tet inducible regulated expression system ［J］. Chemistry of Life, 2011，31(2)：285-291. (in Chinese)

陈皓, 夏海滨. 四环素诱导调控表达系统的研究与应用［J］,生命的化学， 2011，31(2)：285-291.

［28］Smith JR, Verwaerde C, Rolling F, et al. Tetracycline-inducible viral interleukin-10 intraocular gene transfer, using adeno-associated virus in experimental autoimmune uveoretinitis［J］. Hum Gene Ther, 2005,16(9): 1037-1046.

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