Transfection of Schwann cells with retrovirus pLNCX2-GDNF and its expression / 中华显微外科杂志

ABSTRACT

Objective To examine the effectiveness of a gene transfer of rat glial cell-line derived neurotrophic factor(GDNF)into Schwann cells Methods Rat GDNFcDNA was amplified from newly-born rat sciatic nerve by RT-PCR and ligated into the retroviral vector pLNCX-2 A fter being packaged by the amphotropic packaging cell line PT67,the viral supernatants from these anti-clonal producing lines were titred on fibroblast N IH3T3 The highest tire recombinant retrovirus was used to infect deviding populations of newly-born rat SCs The level of GDNF mRNA in GDNF-SCs and normal SCs was tested by RT-PCR The GDNF protein in genetically modified SCs and normal SCs was assayed by immunocytochemistry The amounts of GDNF in conditioned medium of GDNF-SCs in different phrase and normal SCs were detected by enzyme-linked immunoassay sensitive assay Biological activity of the secreted GDNF was tested using motoneuron bioassay by MTT method Results (1)The GDNF cDNA band of GDNF-SCs was more prominent than that of SCs (2)Cells in both groups were immunohistochemically positive for GDNF expression Staining for GDNF was more prominent in the Scs infected with pLNCX2-GDNF (3)The rate of GDNF secreted by GDNF-Scs was 5 1-fold compared with normal SCs (4)The bioassay comfirmed that the secreted GDNF from GDNF-Scs was biologically active,and more motoneurons survived than normal SCs group Conclusion With the help of retrovirus,GDNF gene can be transferred and stably expressed in SCs,and it′s may be a better way to graft SCs promoing regeneration of PNS injuries.