Heat Shock Protein 72 Expression Into the Human Trabecular Meshwork Cells and the Rat Retinal Ganglion Cells Through Recombinant Replication-Deficient Adenovirus

Abstract

Purpose:: To determine whether Heat shock protein 72 (HSP72) gene can be expressed in the human trabecular meshwork cells in vitro and rat ganglion cells in vivo through means of a recombinant replication-deficient adenovirus.

Methods:: Total human HSP72 RNA was extracted from the HeLa cell and HSP72 cDNA was obtained using a reverse transcriptase. The HSP72 gene was inserted into pShuttle-CMV vector, and a recombinant adenoviral vector was constructed by adding pAdEacy-1 vector to the pShuttle-CMV vector. The HSP72 gene was transferred into human trabecular meshwork cells in vitro and rat retinal ganglion cells in vivo. The expression of HSP72 in those cells was evaluated via Western blot technique, immunocytochemistry, and immunohistochemistry.

Results:: A singe protein band of 72 kDa was observed in the Western blot analysis of human trabecular meshwork cells, which was previously infected with the HSP72-containing adenoviral vectors. Immunocytochemistry also demonstrated cytoplasmic fluorescence of much stronger density in the infected human trabecular meshwork cells than in the control. After intravitreal injection of HSP72-containing adenoviral vectors, immunohistochemistry revealed much stronger brownish color signals in the rat retinal ganglion cells than in the control.

Conclusions:: The HSP72 gene was successfully transferred into human trabecular meshwork cells in vitro and rat retinal ganglion cells in vivo. This HSP72-containing adenoviral vector would play an important role in future studies on the unique neuroprotective effects of HSP72.

Keywords: gene transfer/gene therapy