The Promotion Of Tissue Transglutaminase By Androgen May Be Related To Pathogenesis Of POAG

[PURPOSE]: To study the level of testosterone in the male patients blood and aqueous humor who suffer with POAG. Then to study whether bovine trabecular meshwork cells (BTMC) are capable of expressing and secreting tissue transglutaminase (tTG), an enzyme cross-linking extracellular matrix (ECM) proteins, and whether tTG are increased after treatment of BTMC with testosterone. To study the expression of tissue transglutaminase (tTG) in the patients'angle of anterior chamber who suffer with primary open angle glaucoma (POAG). And to compare with the patients'angle of chamber who suffer with primary angle closure glaucoma (PACG) and the tissue of normal people.[METHODS]: 1. Obtained venous blood 2 ml, and human aqueous humor 0.1 ml from the operations. Using radio-immunity technique detected the level of testosterone.2. The human angle of chambers was obtained 1 hour from the trabeculectomys. Among the total there are 26 POAG and 17 PACG. Besides we obtain 10 normal samples from the donor of corneal transplantation. The expression of tissue transglutaminase protein was detected by immunohistochemical technique respecticely with antibodies to tTG. Using image analysis system to calculate them, gained optical density value (OD value). Utilize SPSS system to deal with the OD values.3. Using image analysis system to calculate them, Utilized SPSS system to deal with the data.4. BTMC was primary cultured and subcultured from tissue pieces of new born bovine eyes. After identified with the shape and immunohistochemical technique, the cells from the third to fifth generation were used in this experiment. 5. BTMC was treated with 0 ng/l, 5ng/l, 10ng/l, 15ng/l, 20ng/l testosterone for 24 hours. 6. Induction of tTgase was investigated by PT-PCR and Western blot analysis.[RESULTS]: 1. Compared with normal people, the content of T (testosterone) was higher in the group of POAG's blood and aqueous humor.2. The result of immunohistochemical technique demonstrated that the normal group's and the PACG group's angle of chamber had no different in the expression of tTG. The optical density value (OD value) are 0.180±0.032 and 0.212±0.019, P>0.05. But the group of POAG expressed tTG far more than the other groups. The OD value is 0.325±0.052,compare with the normal group's P<0.001. And compare with the PACG group's P<0.05. The resoults show that the level of tissue transglutaminase of POAG is increased significantly compared to that of the PACG and normal people. But the levels of tTG have no different in PACG and normal people.3. Accroding to growth characteristics, morphology features and staining features (Fn, Ln and CollagenⅣ) determine cells cultured were BTMC. Cultured BTMCs expressed tTG intra- and extracellularly. Treatment of cultured BTMCs with testosterone in different concentration increased the tTgase mRNA and protein levels. The result of RT-PCR showed that a single and bright band for tTG at approximately 433bp was detected, but the expression of tTG mRNA wasmarkedly upgrade in the group of T-treated. Theβ-actin product, an internalhousekeeping control for PCR amplification, was appearance in all specimens at approximately 242bp. Through scaned by image analysis system, the result showed that the ratios of tTG were (2.111±0.016),(2.536±0.032) and (3.176±0.026) in the group of T-treated groups. Respectively and significant difference compared with the normal group (1.487±0.013, P<0.05). However there was no difference between the group of untreated group and the group which density is 10ng/l (1.461±0.021).[CONCLUSIONS]: Sex hormone metabolic disturbance may effect in the beginning and development of POAG. Further more, this experiment demonstrated that the enzyme tTG is expressed in the BTMC and is inducible by testosterone. Tissue trasglutaminase may play a pathogenetic role in the development and progression of POAG via cross-linking extracellulae matrix protein, leading to accumulation of ECM in human trabecular meshwork with POAG. Increased levels of testosterone in the aqueous humor may lead to an increase of tTG expression and activity in the HTM, causing an increase of irreversibly cross-linked ECM proteins. This mechanism might play a role for the increased outflow resistance seen in glaucomatous eyes.