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Growth factor and neurotrophic factor mRNA in human lacrimal gland
D H Nguyen1, R W Beuerman, H W Thompson
1Department of Ophthalmology, LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans 70112, USA.
Purpose:
This study examined the expression of important growth factor and receptor messenger RNA (mRNA) in human lacrimal gland.
Method:
Lacrimal gland tissue was obtained from six women, aged 31-85 years, who were undergoing surgery. The specimens were frozen immediately in liquid nitrogen. Total cellular RNA was collected by cesium chloride centrifugation, and the integrity of the RNA was analyzed by gel electrophoresis and spectrophotometry. For the reverse-transcription polymerase chain reaction (RT-PCR) procedure, 1 microgram of total cellular RNA was used for the first strand synthesis and amplified in separate reactions for 34 cycles by using primers specific for transforming growth factor-beta 1, -beta 2, and -beta 3 (TGF beta 1, TGF beta 2, TGF beta 3), TGF beta 3 receptor (TGF beta 3R), basic fibroblast growth factor (bFGF), fibroblast growth factor receptor-1 (FGF-R1), nerve growth factor (beta-NGF), low-affinity NGF receptor (p75NGF-R), and platelet-derived growth factor-AA (PDGF-AA; two of the six cases) and -BB (PDGF-BB; three of the six cases). Product identity was confirmed by restriction endonuclease digestion, by the "hot blot" technique, by DNA sequencing, or by a combination of these.
Results:
All the lacrimal gland specimens were positive for all growth factors, neurotrophic factors, and receptors except for two specimens that were negative for bFGF. The patients from whom the bFGF-negative specimens were obtained were 68 and 74 years old, and both were identified as having dry eyes-one severe and the other moderate, respectively. These specimens were also analyzed for PDGF-AA and PDGF-BB mRNA, and both were positive.
Conclusions:
This study demonstrates that RT-PCR is a rapid and sensitive method for analyzing rare mRNA transcripts in small amounts of tissue. The results suggest that these growth and neurotrophic factors may have autocrine and paracrine roles in modulating the lacrimal gland, and their absence may play a role in pathologic states such as fibrosis and dry eyes.