Stromelysin gene transfer into cultured human trabecular cells and rat trabecular meshwork in vivo.
OBJECTIVE
To determine whether stromelysin gene can be introduced into and expressed in the cultured human trabecular cells as well as in the rat eye in vivo through means of a recombinant replication-deficient adenovirus.


METHODS
Stromelysin cDNA was obtained by reverse transcription-polymerase chain reaction with mRNA extracted from the cultured human trabecular cells after induction with interleukin 1alpha. Adenovirus vector that contains stromelysin cDNA was constructed by cotransfection of pJM17 and pDeltaA.CMV-str into the 293 cells. The expression of stromelysin in the cultured human trabecular cells was assayed by Western blot and zymography. The expression of stromelysin in the trabecular meshwork of the rat eyes was detected by in situ hybridization and immunohistochemistry.


RESULTS
The constructed adenovirus vector contained stromelysin cDNA, but no E1 region. Western blot and zymogram revealed that the stromelysin could be expressed and that it possessed enzymatic activity in cultured human trabecular cells. In situ hybridization and immunostaining of the stromelysin showed that the complete form of stromelysin was expressed in the trabecular meshwork, the iris, and the uveoscleral outflow pathway of the rat eye.


CONCLUSIONS
Stromelysin, a functional gene, can be transferred in vivo into rat eyes and in vitro into cultured human trabecular cells using a replication-deficient adenovirus vector. This shows the possibility of gene therapy in glaucoma.
Kee
C
C
Department of Ophthalmology, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul, Korea. cwkee@ophthalmology.org
Sohn
S
S
Hwang
J M
JM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003673:Defective Viruses; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D019278:Matrix Metalloproteinase 3 / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000166:cytology / Q000201:enzymology*
D012333:RNA, Messenger; D019278:Matrix Metalloproteinase 3