Subcloning of human neurotrophin-3 gene and construction of its genetically engineered cell model

Abstract

Abstract: Objective To subclone human neurotrophin-3 gene (NT3) and transfer this gene into human bone marrow mesenchymal stem cells (BM-MSCs) to construct genetically engineered cells that produce NT3 in vitro. Methods Human BM-MSCs were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum and 10 ng/ml epidermal growth factor. Flow cytometry (FCM) was used to examine the phenotypes of the cells. The eukaryotic expression vector pcDNA3.1 (+)/NT3 was constructed and transferred into human BM-MSCs in vitro via liposomes. The genetically engineered BM-MSCs were selected several times with G418 and the clones were obtained and then amplified, followed by extraction of the RNA for detection of NT3 gene expression by reverse transcriptional (RT) PCR. The biological activity of the genetically engineered cells was examined by the collecting the supernatant of the culture medium for incubation of guinea pig cochlea hair cells. Results The cultured cells expressed CD13, CD29 and CD59, but no7 CD11, CD14, CD31, CD34, CD45, CD80, CD86, CD117 or HLA-DR. The BM-MSCs genetically modified with pcDNA3.1(+)/NT3 not only expressed and produced NT3, but also promoted the survival of the guinea pig cochlea hair cells in vitro. Conclusion It is possible to construct the genetically engineered BM-MSCs that excrete NT3 in vitro.

CLC Number:

Q343

CHANG Hong¹, GUO Meng-he¹, GUO Kun-yuan², LI Yong-he¹. Subcloning of human neurotrophin-3 gene and construction of its genetically engineered cell model[J]. Journal of Southern Medical University, 2004, 24(07): 805-808.

References

[1] Ernfors P, Van DW, Loring J, et al. Complementary roles of BDNF and NT-3 in vestibular and auditory development[J]. Neuron, 1995,14(6): 1153-64.
[2] Morayma R, Troy L, Todd L, et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells [J].Blood, 2001,98(9): 2615-25.
[3] 汪谦.现代医学实验方法[M].北京:人民卫生出版社,1997.143.
[4] 萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯著T著.金冬雁,黎孟枫,译.分子克隆实验指南[M].第2版,北京:科学出版社,1992.
[5] Ruan RS, Leong SK, Mark I, et al. Effects of BDNF and NT-3 on hair cell survival in guinea pig cochlea damaged by kanamycin treatment[J]. Neuroreport, 1999, 10(10): 2067-71.
[6] Agerman K, Canlon B, Duan M, et al. Neurotrophins, NMDA receptors, and nitric oxide in development and protection of the auditory system[J]. Ann N Y Acad Sci, 1999, 28, 884: 131-42.
[7] Staecker H, Gabaizadeh R, Federoff H, et al. Brain-derived neurotrophic factor gene therapy prevents spiral ganglion degeneration after hair cell loss[J]. Otolaryngol Head Neck Surg, 1998, 119(1):7-13.
[8] Jukka Y, Ulla P, Maxim M, et al. Expression patterns ofneurotrophin and their receptor mRNAs in the rat inner ear[J]. Hear Res, 1993,65(1): 69-78.
[9] van DW, Staecker H, Halterman MW, et al. Gene therapy in the inner ear. Mechanisms and clinical implication [J]. Ann N Y Acad Sci, 1999, 884: 345-60.
[10] Cornetta K, Morgan RA, Anderson WF. Safety issues related to retroviral-mediated gene transfer in human[J]. Human Gene Ther, 1991,2(1): 5-14.
[11] Li K J, Dilber MS, Abedi MR, et al. Retroviral-mediated gene transfer into human bone marrow stromal cells: studies of efficiency and in vivo survival in SCID mice[J]. Eur J Haematol, 1995, 55(5):302-6.
[12] Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells [J]. J Cell Physiol,1999, 181(1): 67-73.
[13] 路艳蒙,傅文玉,朴英杰.人骨髓间充质干细胞的培养及性质鉴定[J].第一军医大学学报,2001,21(8):571-3.Lu YM, Fu WY, Piao YJ. Culture and identification of human mesenchymal stem cells [J]. J First Mil Med Unir/Di Yi Jun Yi Da Xue Xue Bao, 2001, 21(8): 571-3.
[14] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science[J]. 1999, 284(541 1):143-7.
[15] 金丹,裴国献,王珂,等.hBMP-7基因转染对骨髓间充质干细胞增殖和碱性磷酸酶合成的影响[J].中国医学科学院学报(Acta Acad Med Sin),2003,25(1):22-5.
[16] 金丹,曾位森,裴国献,等.经转染人骨形态发生蛋白7基因后BMSc mRNA的表达[J].第一军医大学学报.2002,22(3):22-4.Jin D, Zeng WS, Pei GX, et al. Expression of human bone morphogenetic protein 7 mRNA after the gene transfection in rabbit bone marrow-derived mesenchymal stem cells [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(3): 22-4.