Пятница, 29.03.2024, 16:55
Вітаю Вас Гость | Реєстрація | Вхід

і

Архив статей

Головна » Файли » 2014 » 3 (123)

Пасєчнікова Н. В., Зборовська О. В., Чорнобай М. О. Вивчення впливу метиленового синього в комбінації із низько енергетичним лазером
27.06.2014, 17:28

Резюме
Пасєчнікова Н. В., Зборовська О. В.1, Чорнобай М. О.2 Вивчення впливу метиленового синього в комбінації із низько енергетичним лазером на культуру Enterobacter aerogenes in vitro.
Метою роботи було встановити ефективність методу фотохіміотерапії - метиленовий синій із комбінацією низько енергетичного лазерного випромінювання, на культуру Enterobacter aerogenes in vitro. Відмічається пригнічення росту культури Enterobacter aerogenes під впливом МС у 2 рази. При використанні в якості провідника метиленового синього ДМСО відбувається пригнічення росту культури в 2 рази, але статистично значимої різниці в порівнянні з групою з МС немає. Низько енергетичне лазерне випромінювання протягом 1,5 хв. і 3 хв. не впливає на ріст Enterobacter aerogenes, що підтверджено статично не значимою різницею показників р=0,183 і р=0,422 відповідно. Метиленовий синій призводить до пригнічення росту Enterobacter aerogenes, при тривалості НЕЛВ як 1,5 хв. так і 3 хв., майже в 2 рази. Застосування ДМСО, як провідника для МС, в комбінації з НЕЛВ тривалістю як 1,5 хв. так і 3 хв. призводить до значного пригнічення росту культури Enterobacter aerogenes на 2 порядки, з максимальним ефектом при експозиції НЕЛВ 3 хвилини.
Ключові слова: фотохіміотерапія, метиленовий синій, Enterobacter aerogenes.
Резюме
Пасечникова Н.В., Зборовская А.В., Чорнобай М.А. Изучение влияния метиленового синего в комбинации с низкоэнергетическим лазером на культуру Enterobacter aerogenes in vitro.
Цель данного исследования изучить эффективность метода фотохимиотерапии – комбинация метиленового синего (МС) и низкоэнергетического лазерного излучения (НЭЛИ с длиной волны 630-670 нм)на культуру Enterobacter aerogenes in vitro. МС снижает рост Enterobacter aerogenes в два раза. ДМСО в качетсве проводника МС снижает рост культуры Enterobacter aerogenes в два раза, но статистически значимой разницы по сравнению с группой с МС не отмечено. НЭЛИ при облучении 1,5 и 3 мин. Не изменил рост Enterobacter aerogenes, что подтверждено статистически не значимыми показателями p=0,183 и p=0,422. МС в качестве фотосенсибилизатора ингибирует рост Enterobacter aerogenes при НЭЛИ 1,5 и 3 мин в два раза. Применение ДМСО как проводника МС, в комбинации НЭЛИ 1,5 и 3 мин. приводит к значительному снижению роста культуры Enterobacter aerogenes на два порядка, с максимальным эффектом облучения НЭЛИ 3 минуты.
Ключевые слова: фотохимиотерапия, метиленовый синий, Enterobacter aerogenes.
Summary
Pasyechnikova N.V., Zborovska O.V., Chornobai M.O. The influence of methylene blue in combination with low- energy laser on the culture of Enterobacter aerogenes in vitro.
The purpose of study was to set the effectiveness of the method photochemotherapy - methylene blue (MB) with a combination of low power laser irradiation (LPLI with a wavelength 630-670 nm) to the culture of Enterobacter aerogenes in vitro. Inhibition of growth of culture Enterobacter aerogenes marked under the influence of MB by half. DMSO as a conductor of MB inhibits culture growth twice less, but no statistically significant difference compared to a group with MB. LPLI with duration for 1,5 min. and 3 min. have no effect on the growth of Enterobacter aerogenes, which confirmed statistically not significant difference indices p=0,183 and p=0,422. MB as a photosensitizer, shows growth inhibition of Enterobacter aerogenes for the duration LPLI 1.5 min. and 3 min. by half. Use of DMSO as a conductor for MB, in combination with LPLI 1,5 min. and 3 min. leads to a significant inhibition of growth of culture of Enterobacter aerogenes by two numbers of magnitude, with a maximum effect at exposure LPLI 3 minutes.
Key words: photochemotherapy, methylene blue, Enterobacter aerogenes.

Рецензент: д.мед.н., проф. А.М. Петруня

УДК 579.842+615.849.19+547.869

ДУ «Інститут очних хвороб і тканинної терапії ім. В. П. Філатова НАМН України» (Одеса)

ГУ "Институт глазных болезней и тканевой терапии им. В.П.Филатова НАМН Украины"

ГУ "Институт глазных болезней и тканевой терапии им. В.П.Филатова НАМН Украины"
SI “The Filatov Institute of Eye Diseases and Tissue Therapy of the National Academy of Medical Sciences of Ukraine”

65061, Ukraine, Odessa, Frantsuzskii Bulvar, 49/51

1filatoveye@mail.ru

2z_mariya@hotmail.com

Introduction. Infectious- inflammatory diseases continue to occupy one of the leading places in the structure of morbidity. Despite the wide arsenal of pharmacological agents, the progress achieved by the treatment of inflammation, the incidence of pathology has a clear downward trend [3,4].  Severities in the treatment of infectious eye diseases related to a number of reasons, among them are: antibiotic resistance, reducing mechanisms of general and local immunity, long duration, or the inability to identify the pathogen [2]. In the study of the etiology of keratitis defined reduction percentage in Gram -positive bacteria and percent increase in Gram - negative bacteria [2, 3, 11]. One of the treatments is a method of photodynamic therapy showing photo destroying   effect on a wide range of pathogenic organisms without suppressing saprophytic, shows no toxic effect on the structure macro organism and does not contribute to the selection of resistant strains [1, 3, 7]. Photodynamic therapy - a phototherapy unit in which to achieve a therapeutic effect using light of a specific wavelength with photo sensitizer and oxygen. One of photo sensitizers, which have been used long in ophthalmology, as an antiseptic, is methylene blue. Methylene blue has the greatest effect when illuminated with red light range of 665 nm with a maximum absorption within 600-900 nm in the therapeutic safety for human [8, 10, 9].
Methylene blue proved to be a phototoxic drug for many bacteria. It has been proven its effectiveness in a number of Gram- positive bacteria, showing high photo toxicity to microorganisms and therapeutically acceptable level [5]. It is noted the sensitivity of pathogenic strains of Ps. aeruginosa i C. albicans to the combined use of methylene and laser radiation, and, this sensitivity is increased according to the increase in the concentration of methylene blue [6].
Experience of clinical observations and research suggests - treatments of infectious and inflammatory diseases do not always meet the needs of the treatment of this disease. So important is the search for new alternative antimicrobial agents that allow to effective treatment of infectious diseases without identification of the pathogen. The aim is to determine the efficacy of the method photo chemotherapy - methylene blue with a combination of low power laser irradiation (LPLI), to the culture of Enterobacter aerogenes in vitro.

Materials and methods

  1. Determining the influence of methylene blue on the growth of the culture of the microorganism Enterobacter aerogenes without laser irradiation. Preparation of the test strain of Enterobacter aerogenes for work. Test strain Enterobacter aerogenes were grown in an incubator at 37 ± 10C on solid media MPA (Petri dishes and test tubes PK- 16) for 18 ± 6 hours. Then prepare the bacterial inoculums, determining the density of bacterial suspensions on a scale McFarland using densitometer DENSIMAT BioMerieux. To control used of 6 standards of MacFarlane. Prepared a working bacterial suspension density of 1 unit. The concentration of bacterial cells according to the intestinal microbial groups was 1 U – 0.08 × 109/ml. We used a working solution of 0.1% MC. The experiment was performed three reps for the dilution of MB. In a test tube with 1 ml of bacterial suspension with a concentration of microorganism cells corresponding 1.0 unit MacFarlane added 9 ml of 0.1% solution of methylene blue, thus sought a concentration of 0.1 IU MacFarlane concentration of bacterial cells which was 8.0 × 105 mg / ml. After 30 minutes of exposure at room temperature prepared a series of serial dilutions then made ​​hanging on tight environment MPA, followed by incubation in an incubator at 37 ± 10 C for 18 ± 6 hours. Sowing dose was 0.01 ml. After 24 hours counted the number of colonies, that grew up on cups.
  2. Determining the impact of low power laser (LPLI with wavelength 630-670 nm) on the growth of the culture of the microorganism Enterobacter aerogenes. To operate used the bacterial suspension Enterobacter aerogenes density of 0.1 OD. In a test tube with 1 ml of bacterial suspension with a concentration of microorganism cells corresponding 1 U MacFarlane added 9 ml of 0.1% solution of methylene blue, thus sought a concentration of 0.1 U MacFarlane concentration of bacterial cells which was 8.0 × 105 mg / ml. After 30 minutes of exposure at room temperature experiment was performed three repetitions for each time the laser action at 1.5 minutes and 3 minutes. After each exposure performed a series of serial dilutions of the working suspension in sterile 0.9 % sodium chloride solution, and then hung in the quantity produced 0.01 ml dense culture media for further quantification of surviving microorganisms. Crops were incubated in an incubator at 37 ± 10C for 18 ± 6 hours. After 24 hours counted the number of colonies, which grew up on cups.
  3. Determining the influence of methylene blue activated laser light (wavelength 630-670 nm) as a photo sensitizer to the growth of the microorganism culture Enterobacter aerogenes. To operate using the bacterial suspension Enterobacter aerogenes density OD 1 and 0.1 % solution of methylene blue. Prepared by a working bacterial suspension density of 1 unit. We used a working solution of 0.1% methylene blue. The experiment was conducted in three repeated for the dilution MB. To do this in a test tube with 1 ml of bacterial suspension with a concentration of bacterial cells 1 U MacFarlane added 9 ml of 0.1% solution of methylene blue, thus sought a concentration of 0.1 U MacFarlane concentrations of bacterial cells which was 8.0 × 105 mg / ml. After 30 minutes of exposure at room temperature experiment was performed three repetitions for each time the laser action at 1.5 minutes, 3 minutes. After each exposure performed a series of serial dilutions of the working suspension in sterile 0.9 % sodium chloride solution, and then hung to produce a dense nutrient medium for subsequent assay of surviving microorganisms. Sowing dose was 0.01 ml. Crops were incubated in an incubator at 37 ± 10C for 18 ± 6 hours. After 24 hours counted the number of colonies, that grew up on cups.
  4. Determining the influence of methylene blue solution and Dimethylsulfoxide (DMSO) on the growth of the microorganism culture Enterobacter aerogenes. To operate used the bacterial suspension Enterobacter aerogenes density OD 1, a solution of 0.1% methylene blue and 10% DMSO. Prepared a working bacterial suspension density of 1 unit. We used a working solution of 0.1% methylene blue and 10% DMSO. Again the experiment was conducted three times. In a test tube with 1 ml of bacterial suspension with a concentration of bacterial cells 1 U MacFarlane added 9 ml of 0.1% solution of methylene blue and 10% DMSO thus sought a concentration of 0.1 U MacFarlane concentration cell microorganism Enterobacter aerogenes in which was 8.0 × 105 mg / ml. After 30 minutes of exposure at room temperature prepared a series of serial dilutions, and then hung produced in dense environments MPA, followed by incubation in an incubator at 37 ± 10C for 18 ± 6 hours. Sowing dose was 0.01 ml. After 24 hours counted the number of colonies, that grew up on cups.
  5. Determining the influence of methylene blue solution and DMSO, activated by laser radiation (wavelength 630-670 nm) as a photo sensitizer for the growth of the microorganism culture Enterobacter aerogenes. To operate used the bacterial suspension Enterobacter aerogenes density OD 1, a solution of 0.1% methylene blue and 10% DMSO (eye drops). Prepared a working bacterial suspension density of 1 unit. We used a working solution of 0.1% methylene blue and 10% DMSO. In a test tube with 1 ml of bacterial suspension with a concentration of bacterial cells 1 IU MacFarlane added 9 ml of 0.1% solution of methylene blue and 10% DMSO thus sought a concentration of 0.1 U MacFarlane concentrations of bacterial cells of Enterobacter aerogenes which was 8.0 × 105 mg / ml. After 30 minutes of exposure at room temperature experiment was performed in three repeated each time the laser action at 1.5 minutes, 3 minutes. After each exposure performed a series of serial dilutions working suspensions in sterile 0.9 % sodium chloride solution, and then hung to produce a dense nutrient medium for subsequent assay of surviving microorganisms. Crops were incubated in an incubator at 37 ± 10C for 18 ± 6 hours. Sowing dose was 0.01 ml. After 24 hours counted the number of colonies, that grew up on cups.

Results and discussion

As a result of 0.1% methylene blue on the growth of the culture of the microorganism Enterobacter aerogenes without laser irradiation, a decrease in the number of live microbial cells Enterobacter aerogenes in one order (4.0 ± 1.7) × 104 compared with control 8.0 × 105 ( p ≤ 0.0001). Any reduction in the number of microbial cells Enterobacter aerogenes (7.93 ± 0.06) × 105 compared with control 8.0 × 105, as a result of laser irradiation ( LPLI of wavelength 630-670 nm) of 1.5 min. and 3 min. were noted : p = 0.183 and p = 0.422 , respectively.
After incubation of 0.1% methylene blue and microbial cultures for 30 min, followed by laser irradiation of 1.5 min and 3 min, a decrease in the number of colonies that grew on the cups on average twice: (4.36 ± 1.0) × 105, p = 0.013 and (4.13 ± 0.5) × 105, p = 0.005, respectively, compared with control 8.0 × 105, however, remained the same degree as in control - 105 mg / ml. After incubation of 0.1% methylene blue and microbial cultures for 30 min, a decrease in the number of live microbial cells Enterobacter aerogenes in one order (3.87 ± 1.6) × 104 compared with control 8.0 × 105, p  ≤ 0 , 0001. After incubation of 0.1% methylene blue, 10% dimexide and microbial cultures for 30 min without laser irradiation, a decrease in the number of live microbial cells Enterobacter aerogenes in one order (3.87 ± 1.6) × 104 compared with control 8.0 × 105 (p ≤ 0,0001). After incubation of 0.1% methylene blue, 10% DMSO and microbial cultures for 30 min, followed by laser irradiation of 1.5 min and 3 min, a decrease in the number of live microbial cells Enterobacter aerogenes two orders : (4.0 ± 1.7) × 103 and (2.0 ± 0.9) × 103 , respectively, compared with the control – 8.0 × 105 mg / ml , p ≤ 0.0001.

The results are presented in Table 1.

Table 1.

Quantifying the survival test culture Enterobacter aerogenes ( x ± σ), for various treatments in vitro

Method

Time

Group

P*

The number of bacterial cells of the main group ( mg / ml )

Number of bacterial cells in the control group ( mg / ml)

1

МB 30 min

 

(4.0±1.7)× 104

8.0 × 105

≤0.0001

2

LPLI 1.5 min

 

(7.93±0.06) ×105

8.0× 105

0.183

LPLI 3 min

 

(7.93±0.06) ×105

8.0× 105

0.422

3

LPLI 1.5 min+МB 30 min

 

(4.36±1.0) ×105

8.0× 105

0.013

LPLI 3 min+МB 30 min

 

(4.13±0.5) ×105

8.0× 105

0.005

4

МB+DMSO 30 min

 

(3.87±1.6) ×104

8.0× 105

≤0.0001

5

LPLI 1.5min+МB+DMSO 30 min

(4.0±1.7) ×103

8.0× 105

≤0.0001

LPLI 3 min+МB+DMSO 30 min

(2.0±0.9) ×103

8.0× 105

≤0.0001

Where p * - evaluation of the significance difference with Mann - Whitney.

Conclusions
Growth suppression is noted Enterobacter aerogenes culture influenced MB 2 times. When used as a conductor of methylene blue DMSO culture growth inhibition occurs 2 times, but no statistically significant difference when compared with a group of MB. Low power laser irradiation for 1.5 min. and 3 min. have no effect on the growth of Enterobacter aerogenes; which confirmed no statistically significant difference indices p = 0.183 and p = 0.422, respectively. When using MB, as a photo sensitizer, this results in growth inhibition for Enterobacter aerogenes, for the duration LPLI 1.5 min. and 3 min. almost 2 times. Application of DMSO, as a conductor for MB, in combination with LPLI duration 1.5 min. and 3 min. leads to a significant inhibition of growth of culture of Enterobacter aerogenes by 2 orders of magnitude, with a maximum effect at exposure LPLI 3 minutes. Further study dynamic photo therapy will help expand the ability to treat different etiological types of keratitis with more positive anatomical and functional visual performance at the exit of the disease.

Література

1. Аветисов С.Э. Фотодинамическая терапия: перспективы применения в офтальмологи / С.Э. Аветисов, М.В. Будзинская, В.Г. Лихванцева // Вестник офтальмологии: Науч.-практ. журн. – М., 2005. - № 5. - C. 3-6.
2. Белий Ю.А. Фотодинамические эффекты в лечении гнойной язвы роговицы / Ю.А. Белий, А.В. Терещенко, М.А. Плахотний [и др.] // Рефракционная хирургия и офтальмология. – 2008. - № 2. – С. 28-33.
3. Доленко О.В. Показники імуноглобулінів та лізоциму церві кального слизу при фото динамічній терапії неспецифічних бактеріальних вульвовагінітів і цервіцитів / О.В. Доленко // Експериментальна і клінічна медицина. – 2006. - № 2. - С. 141-143.
4. Майчук Ю.Ф. Терапевтические алгоритмы при инфекционных язвах роговицы / Ю.Ф. Майчук // Вестн. Офтальмол. - 2000. - № 3. - С. 35-37.
5. Пасєчнікова Н.В. Фотодинамічна терапія інфекційних агентів (огляд літератури) / Н.В. Пасєчнікова, О.В. Зборовська // Ліки. - 2002. - № 5-6. - C. 43-47.
6. Пасєчнікова Н.В. Фотодинамічний вплив гелій-неонового лазера на staphylococcus aureus I streptococcus pyogenes in vitro / Н.В. Пасєчнікова, О.В. Зборовська, В.А. Піотрович, Т.В. Таран // Одеський медичний журнал. - 2003. - № 3. - C. 14-16.
7. Странадко Е.Ф. Фотодинамическая терапия при гнойных заболеваниях мягких тканей / Е.Ф. Странадко, У.М. Корабоев, М.П. Толстых // Хирургия. – 2000. - № 9. - С. 67–70.
8. Соболев А.С. Подходы к направленной внутриклеточной доставке фотосенсибилизаторов для увеличения их эффективности и придания клеточной специфичности / А.С. Соболев, А.А. Розенкранц, Д.Г. Гилязова // Биофизика. – 2004. - № 49 (3). – С. 351-379.
9. Современный взгляд на механизм фотодинамической терапии. Фотосенсибилизаторы и их биодоступность / Д.М. Ягудаев, А.Е. Сорокатый, А.В. Гейниц // Урология. - 2006. - № 5. - C. 94-98.
10. Peloi L.S. Biondo Photodynamic effect of light-emitting diode light on cell growth inhibition induced by methylene blue / L.S. Peloi, R.S. Soares, E.G. Biondo // J. Biosci. - 2008. - № 33(2). - Р. 231-237.
11. Shifting trends in bacterial keratitis in Toronto / A. Lichtinger, S. Yeung, P. Kim [at al.] // Ophthalmology. - 2012. - Vol. 119. - P. 1785-1790.

Категорія: 3 (123) | Додав: siderman
Переглядів: 726 | Завантажень: 0 | Рейтинг: 0.0/0
Всього коментарів: 0
Додавати коментарі можуть лише зареєстровані користувачі.
[ Реєстрація | Вхід ]
RSS

Форма входу

Категорії розділу

1 (121) [47] 2 (122) [36]
3 (123) [28] 4 (124) [34]
5 (125) [0] 6 (126) [0]

ПОИСК

НАШ ОПРОС

Оцените наш сайт
Всего ответов: 55

ДРУЗЬЯ САЙТА

Статистика


Онлайн всего: 1
Гостей: 1
Пользователей: 0