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Донецкий национальный медицинский университет им. М.Горького
Donetsk National Medical University n. a. M. Gorky
83003, Украина, г. Донецк, пр. Ильича 16
Illicha Av. 16, Donetsk, 83003, Ukraine
Introduction. Diabetes mellitus (DM) presently behaves to one of the most considerable medical problems, which is widespread around the world.
In obedience to the conclusion of WHO Assembly, DM is the third reason of death rate after cardiovascular and oncologic diseases [1,2].
Amount of patients with this pathology each 12-15 years increased on the average in 2 times, and according to WHO a prognosis, to 2025 year the increase of number of DM patients is expected to 300 million persons [4,5].
Diabetic retinopathy (DR), being the leading reason of blindness and poor vision, results in the decline of quality of life of patients and frequent work disability in yet young age [ 3,4].
Research of pathogenesis, clinical displays of diabetic retinopathy, possibilities of its treatment and prophylaxis is the theme of special attention of scientists of the whole world [2,6,7,9,14,15]. A certain role in development DR belongs to the retinal pigment epithelium. A retinal pigment epithelium and Bruch’s membrane limit the retinal metabolism’s pathological products outflow (lactic acid, growth factors, disintegrated hemorrhage and fibrin) in choroid circulation [12,13].
In this connection there is scientific interest to study of specificity of violations in fabric of retina, resulting in DR development.
The purpose of this research was a study, in the conditions of experimental diabetes, states of membrane structures of retinal pigment epithelium on the level of lysosomal enzyme marker - acid phosphatase.
Material and methods of research.
Researches were conducted on 25 white rats of «Vistar» line of weight 180-220g, which was contained on the standard ration of vivarium. 8 from them were in control group (intact rats).
Conducted the design of diabetes on 17 rats, 8 from them eliminated from
the experiment on 10 day of design and 9 - on 28 day of experiment.
Caused experimental diabetes created by intraperitoneal injection of streptozotocin (60 mg /1 kg of weight of rat), here the day before during night, rats did not get food [11,12].
Control’s rats got the injection of solvent (10 mM of citrate buffer, рН 4,5).
On completion of the stages of experiment (10 and 28 days after introduction of streptozotocin), rats were decapitated with preceding anesthesia of thiopental sodium (50 mgs / 1 kg of weight). Eyes were enucleated on ice at the temperature of 0-5ºС.
Principle of method of determination of activity of hydrophosphotase is based on determination of concentration of free organic component of material -paranitrophenilphosphate, appearing as a result action of enzyme .
For determination of activity of hydrophosphotase in test tubes consistently mixed up 0,1 ml of blood plasma or extract of tissure and 1,0 ml of substrate- buffer solution (0,127 % paranitrophenilphosphate solution is in an acetate buffer, рН 5,0).
Test tubes with reactionary solution were incubated exactly 30 mines at a temperature 37,0 ± 0,5ºС.
Reaction was stopping with addition 1,0 ml of sodium hydroxide solution at a temperature 0ºС .
Measuring of optical dense of the probed solutions was conducted on the spectrophotometer of «Specol - 210» in 1-sm cuvette and wave-length of 410 nm.
Expected of hydrophosphotase activity with the use of molar coefficient of extinction, found by extrapolation on the preliminary built chart and expressed in nkat/ml of blood plasma or nkat/g fabric. Coefficient of variation - 7,8 %.
Results and discussion.
Information, got at the study of different forms of lysosomal enzyme marker - acid phosphatase in a retinal pigment epithelium and blood plasma of experimental rats, is presented in figure 1.
Hydrophosphotase activity in blood plasma (nkat/ml) and in retina (nkat/g) of experimental rats at experimental streptozotocin diabetes (M ± m)
Note. P - is a level of meaningfulness of distinctions in relation to a control group.
It was set in this research, that at development of streptozotocin diabetes in white rats, retinal violation of membrane’s structures of such intracellular organoids as lysosomes of retinal pigment epithelium are marked in the early terms of experiment (10-28 days).
To it the found out the changes of acid phosphatase level testify in our experiment. The damage of ultra-structure elements of organs and tissues registers in these terms, that, in same queue, results in an output in blood of this lysosomal enzyme marker and increase of his activity in plasma on 22,9 % from 3,85 ± 0,24 to 4,73 ± 0,30 to 28 day of experiment.
Retinal acid phosphatase level for certain differed from a level in the control group of rats already on the early terms of development of experimental diabetes (14 days) and to completion of experiment rose on 37,8 % .
At the same time general amount of acid phosphatase in a retinal pigment epithelium, determined after influence on the last by a detergent, practically did not change in times during experiment.
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