Construction Of Recombinant Adenovirus Vector Expressing The Axonal Attractant Netrin-1 And Its Expression In Bone Mesenchymal Stem Cells

Background Netrins is one of the most important axonal attractant factor playing a key role in attracting or repulsing the axon growth according its receptor DCC or UNC5B. The netrins are the prototypical axonal attractants, first identified as extracellular factors secreted from the floor plate that attract spinal commissural axons toward the midline by Serafini . This family includes netrin-1, netrin-2 (netrin-3 in mouse), netrin-4, and the netrin-related molecules, netrin-G1 and netrin-G2, and Ntn1 is the most extensive studied factor. Ntn1 protein was purified from embryonic chick brain on the basis of their ability to mimic the outgrowth-promoting effects of floor plate cells. Many studies concluded that Ntn1 can promote the nerve repair in the central nervous system and the peripheral nervous system as well as in the vascular system. The nervous system and vascular system have the similar structure and projection in the embryonic development, so there may be a molecular crossing-talk exited. So Ntn1 has the predominance in vascular and nerve injury diseases, such as diabetes mellitus and vascular diseases. This study we constructed the recombinant adenovirus vector expressing the Ntn1 for the further research.Methods The construction of netrin-1 recombinant adenovirus was completed in three phases. Firstly, the netrin-1 was cloned by RT-PCR and the then subcloned into shuttle vector pDC316-CMV which carries the report gene EGFP. Secondly, this newly constructed plasmid pDC316-netrin, after identification by nuclease digestion analysis sequencing analysis, was transfected into human embryonic kidney cells HEK293 by lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGlox_E1.3Cre. Based on homologous recombination of two plasmids within HEK293 cells, the recombinant adenovirus vector carrying netrin-1, Ad5-netrin-CMV-EGFP, was created. Finally, Ad5-netrin-CMV-EGFP was subsequently identified by PCR, purified using repeated plaque passages, proliferated using freezing and melting with HEK293 cells, and titrated using 50% Tissue Culture Infective Dose (TCID50). Bone mesenchymal stem cells of Spage-Dawley rat(Age: 3-4 weeks)were primary cultured. MSCs were infected with Ad5-netrin-CMV-EGFP. Expression of Ntn1 was tested by Enzyme-Linked Immunosorbent Assay.Results we constructed a recombinant adenovirus vector containing the cDNA for the netrin-1, a axonal guidance factor. Bone mesenchymal stem cells were infected with adenovirus vector at low MOI (50pfu/cell). The MSCs infected with Ad5–netrin -CMV-EGFP demonstrated higher Ntn1 protein secretion into the supernatant compared with uninfected group.Conclusions These studies demonstrated that (1) the recombinant adenovirus vector can be used to deliver gene of netrin-1 into mammalian cells efficiently. (2) MSCs infected Ad5-netrin-CMV-EGFP secreted netrin-1 protein at high level. (3) Because of low immunity, self proliferation, to obtain facility and so on, MSCs may serve as a platform for gene transfer of netrin-1 to enhance the nerve repair and angiogenesis.