Expression of recombinant retroviral vector carrying nerve growth factor gene in neural stem cells

doi:10.3969/j.issn.2095-4344.2014.19.024

Abstract

Abstract:

BACKGROUND: Nerve growth factor (NGF) belongs to a biological macromolecule that is difficult to pass through the blood-brain barrier. However, a retroviral vector carrying exogenous gene can be stably inserted and integrated into the host cell genome, which is suitable for gene therapy.
OBJECTIVE: To study the gene expression of recombinant retroviral vector carrying rat NGF gene in neural stem cells.
METHODS: The rat NGF gene was inserted into a retroviral vector pLEGFP-N1, which was transferred into packaging cells PT67 by Lipofectamine 2000. The positive clones in virus supernatant were collected by G418 selection and used to infect neural stem cells. After that, the NGF expression was tested by enzyme linked immunosorbent assay and the biological competence by PC12 cells, and then morphological change of neural stem cells and cell typing were examined by fluorescent microscope.
RESULTS AND CONCLUSION: The neural stem cells could express extrinsic source NGF protein after the recombinant plasmid was infected into neural stem cells. The PC12 cells increased in the experimental group and stretched out long neurites. And the NGF protein could maintain the neural stem cell survival and stimulate their differentiation. These findings suggest that the neural stem cells carrying extrinsic source NGF gene could express NGF successfully, and the NGF protein induced the survival and differentiation of neural stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: nerve growth factor, neural stem cells, PC12 cells, retroviridae, rats, Sprague-Dawley

CLC Number:

R394.2

He Jun-feng, Gu Guo-mei, Long Da-hong. Expression of recombinant retroviral vector carrying nerve growth factor gene in neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(19): 3100-3104.

Figures/Tables 1

2.1 pLEGFP-NGF的包装及在神经干细胞中表达选择培养的第4代神经干细胞，采用Nestin免疫荧光染色对神经干细胞进行鉴定，结果呈阳性(图1A)。阴性对照组：未感染病毒上清的神经干细胞在碱性成纤维细胞生长因子的作用下，保持分裂球状，没有绿色荧光蛋白的表达(图1B，C)。空载体对照组：感染空载体pLEGFP-N1病毒上清液的神经干细胞保持分裂球状，24 h后出现绿色荧光蛋白的表达(图1D，E)。实验组：感染重组pLEGFP-NGF反转录病毒上清液的神经干细胞于24 h后出现绿色荧光蛋白的表达，在荧光显微镜下可见绿色荧光，48 h后开始分化，向四周长出放射状突起(图1F，G)。取病毒上清感染NIH3T3细胞，于荧光显微镜下计算绿色荧光蛋白阳性细胞数，估算病毒滴度为5×107 pfu/L。 2.2 神经干细胞培养液对PC12细胞的影响对照组：未感染病毒上清的神经干细胞培养液(对照组培养液)加入PC12细胞的培养瓶中培养3 d，大部分细胞胞体呈圆形，没有明显的突起。实验组：感染重组pLEGFP-NGF反转录病毒液的神经干细胞培养液(条件培养液)加入PC12细胞24 h后可见部分细胞长出突起，48 h后不少细胞呈梭形、三角形或多角形，胞体出现突起，72 h后突起生长明显，某些视野中细胞之间的突起呈网络状(图2)。表明神经生长因子基因修饰后的神经干细胞能表达神经生长因子，且具有的生物学活性。 2.3 ELISA检测培养液上清中的神经生长因子水平用大鼠神经生长因子定量ELISA试剂盒进行检测，根据所得标准曲线测得各组神经生长因子量，阴性对照组为(60.1± 4.72) ng/L，空载体对照组为(59.8±4.58) ng/L，实验组为(116.2±5.44) ng/L。用SPSS 17.0统计软件进行方差分析，P < 0.01，提示各组间神经生长因子的量差异有非常显著性意义；分别进行3组样本间的两两比较，结果显示实验组神经生长因子的表达量明显高于阴性对照组(P < 0.01)及空载体对照组(P < 0.01)，而阴性对照组与空载体对照组之间神经生长因子表达量无明显差别(P > 0.05)。 2.4 已转染神经生长因子基因的神经干细胞分化的鉴定对已转染神经生长因子基因的神经干细胞进行抗NF-m和抗GFAP特异性免疫荧光双染色，结果显示抗NF-m和抗GFAP均为阳性(图3)，提示神经生长因子转导入神经干细胞后，能促进神经干细胞向神经元细胞和神经胶质细胞分化。"

References

[1] Cruz JC, Tsai LH.Cdk5 deregulation in the pathogenesis of Alzheimer's disease.Trends Mol Med. 2004;10(9):452-458.
[2] Steele M, Stuchbury G, Münch G.The molecular basis of the prevention of Alzheimer's disease through healthy nutrition. Exp Gerontol. 2007;42(1-2):28-36.
[3] Ferri CP, Prince M, Brayne C, et al.Global prevalence of dementia: a Delphi consensus study.Lancet. 2005;366(9503): 2112-2117.
[4] Zhang ZX, Zahner GE, Román GC,et al. Dementia subtypes in China: prevalence in Beijing, Xian, Shanghai, and Chengdu. Arch Neurol. 2005;62(3):447-453.
[5] Baskin DS, Browning JL, Pirozzolo FJ,et al.Brain choline acetyltransferase and mental function in Alzheimer disease. Arch Neurol. 1999;56(9):1121-1123.
[6] Clive Holmes. Molecular pathology of Alzheimer's disease. Psychiatry. 2005; 4(1): 37-40.
[7] Barrio JR,Kepe V,Satyamurthy N,et al.Brain pathology and neuronal losses in the living brain of Alzheimer's patients. International Congress Series.2006;1290(4):150-155.
[8] 龙大宏,李沃棠,许孟杰,等.神经生长因子对老年痴呆鼠学习记忆能力的影响[J].中国行为医学科学,2001,10(1): 4-6.
[9] Frielingsdorf H, Simpson DR, Thal LJ,et al.Nerve growth factor promotes survival of new neurons in the adult hippocampus.Neurobiol Dis. 2007;26(1):47-55.
[10] 何峻峰,龙大宏,李佳楣,等.神经生长因子基因重组逆转录病毒载体的构建与鉴定[J].解剖学研究,2006,28(4):254-257.
[11] 黄宗海,杨文宇,龚小卫,等. 靶向性双自杀基因系统对脐静脉内皮细胞的杀伤作用[J]. 第一军医大学学报, 2004,24(2): 139-143.
[12] F.M.奥斯伯著.马学军,等译.精编分子生物学实验指南[M].北京:科学出版社,2005.
[13] Petruska JC, Mendell LM.The many functions of nerve growth factor: multiple actions on nociceptors.Neurosci Lett. 2004; 361(1-3):168-171.
[14] Mok SA, Campenot RB.A nerve growth factor-induced retrograde survival signal mediated by mechanisms downstream of TrkA.Neuropharmacology. 2007;52(2): 270-278.
[15] Tuszynski MH, Blesch A.Nerve growth factor: from animal models of cholinergic neuronal degeneration to gene therapy in Alzheimer's disease.Prog Brain Res. 2004;146:441-449.
[16] Martínez-Serrano A, Björklund A.Immortalized neural progenitor cells for CNS gene transfer and repair.Trends Neurosci. 1997;20(11):530-538.
[17] Garbossa D, Fontanella M, Fronda C,et al.New strategies for repairing the injured spinal cord: the role of stem cells.Neurol Res. 2006;28(5):500-504.
[18] Villa A, Snyder EY, Vescovi A,et al.Establishment and properties of a growth factor-dependent, perpetual neural stem cell line from the human CNS.Exp Neurol. 2000;161(1): 67-84.
[19] Martino G, Pluchino S.The therapeutic potential of neural stem cells.Nat Rev Neurosci. 2006;7(5):395-406.
[20] Lynch WP, Sharpe AH, Snyder EY.Neural stem cells as engraftable packaging lines can mediate gene delivery to microglia: evidence from studying retroviral env-related neurodegeneration.J Virol. 1999;73(8):6841-6851.
[21] Henderson CE.Role of neurotrophic factors in neuronal development.Curr Opin Neurobiol. 1996;6(1):64-70.
[22] Chiou SH, Kao CL, Chang YL,et al.Evaluation of anti-Fas ligand-induced apoptosis and neural differentiation of PC12 cells treated with nerve growth factor using small interfering RNA method and sampling by microdialysis.Anal Biochem. 2007;363(1):46-57.