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20.11.2009 16:23 - Дипломна работа: ЕКСПЕРИМЕНТАЛНО ИЗСЛЕДВАНЕ НА НЯКОИ БИОХИМИЧНИ МАРКЕРИ ПРИ НОРМАЛНО ЗАРАСТВАЩИ И УСЛОЖНЕНИ ФРАКТУРИ ПРИ КУЧЕТА
Автор: drjeliazkov Категория: Лични дневници   
Прочетен: 589113 Коментари: 44 Гласове:
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Последна промяна: 27.04.2018 09:55


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В хуманната медицина тези маркери се използват широко за контрол на различните терапевтични схеми и мониторинг на клетъчната активност при метаболитни заболявания на костите, както и други заболявания даващи отражение върху костите (Okazaki, R и кол., 1997). Те се явяват добра алтернатива и при изследване върху животински модели на остеопороза, костни метастази на тумори и някои форми на артрити ( Kippo, K и кол., 1998; Tamura, H и кол., 1999; Chavassieux, P и кол., 2001).
 Независимо, че при човека е установена строга зависимост между костните маркери в кръвта и урината и хистоморфометричните показатели на костното ремоделиране (Eastell, R и кол., 1988), окончателната (дефинитивна) диагноза при остеопорозата и болеста на Paget се поставя след дензитометрия и/или биопсия.
При животните костните маркери са използвани за установяване разликите в нивата на костното формиране и костната резорбция при различни възрасти коне (Lepage, OM и кол., 1991; Price, JS и кол., 1995), и котки (DeLaurier, A и кол., 2002, 2004). При кучето костните маркери са проучвани на фундаментално ниво, но по отношение на възрастови, породни и др. различия (Аllen, MJ и кол., 1998, 2000; Аllen, LC и кол., 2000; Ladlow, JF и кол., 2002; Breur, G J и кол., 2004). Има единични публикации по използването им в клиничната практика при остеосаркоми и остеоартрити (Ehrhart, N и кол., 1998; Garzotto, CK и кол., 2000; Fox, DB и Cook, JL, 2001), при остектомия на радиуса (Francis, DA и Millis, DL, 2002), за следене на дистракционната остеогенеза (Lammens, J и кол., 1998), за мониторинг на забавено костно зарастване и незарастване на фрактури при тръбести кости (Komnenou, A. и кол., 2005). Маркерите за костната резорбция, които сега се използуват в клиничната практика, са или неспецифични, като хидроксипролина в урината, или изискват продължителен и сложен анализ с течна хроматография, както е с колагеновите пиридинолинови кръстосани връзки (Delmas P.D. и кол., 1983).  Тези методи обаче се считат като неспецифични за колагена от тип І, който съставлява около 90% от костния органичен матрикс (Uebelhart D., 1992). Един нов маркер за разграждането на колагена от тип І, който бе скоро разработен (Risteli J. и кол., 1993), е карбокси-терминалния телопептид на колагена от тип І (ICTP). Има данни, че неговите серумни концентрации у човека са увеличени при редица заболявания, характеризиращи се с генерализирана или локална костна деструкция, като мултиплена миелома (Elomaa I. и кол., 1992), ревматиоден артрит (Risteli J. и кол., 1993) и др. В достъпната ни литература данните за измененията на биохимичните костни маркери при зарастването на фрактурите и при развитието на постравматичен остеомиелит при кучета са оскъдни или такива липсват.
 Целта на настоящето проучване е да проследим промените в серумните нива на някои биохимични маркери на костния метаболизъм, които настъпват в ранния период (до 1 месец) на нормалното фрактурно зарастване, както и тези при експериментален модел на остеомиелит при кучета.
 За постигане на поставената цел трябваше да изпълним следните конкретни задачи:
 1. Експериментално възпроизвеждане на фрактури на бедрена кост и интрамедуларно фиксиране с Кюнчеров гвоздей.
2. Експериментално  възпроизвеждане на фрактури на бедрена кост, интрамедуларно инокулиране на микробни суспензии и интрамедуларно фиксиране с Кюнчеров гвоздей.
 3. Проследяване на някои костни биохимични маркери в кръвния серум на кучетата при нормално зарастваща фрактура и при остеомиелит.
 В проучванията бяха използвани 12 бр. кучета, смесена порода, на възраст 1-3 год. от мъжки пол. Те бяха разделени на две групи от по 6 кучета както следва: І група, при която беше възпроизведена нормално зарастваща фрактура (група НЗФ) и ІІ група, при която беше възпроизведен посттравматичен (постфрактурен) остеомиелит (група ОСМ). Кучетата се отглеждаха в индивидуални боксове, хранеха се със суха храна за възрастни кучета (Jambo-dog®, Gallisman-94 S.A., Bulgaria) и имаха свободен достъп до вода. Преди експеримента всички кучета бяха третирани срещу външни с Fipronil (FRONTLINE®, MERIAL, France) и вътрешни с praziquantel/pyrantel pamoate (Azipyrin®, Balkanpharma, Bulgaria) паразити.
 Оперативен протокол
 Обезболяването беше направено по следната схема: Премедикация - Atropinum sulfuricum (Sopharma, Sofia-Bulgaria)-0.02 mg/kg м. s.c. и Acepromazin maleas (NEUROTRANQ®, Alfasan, Woerden-Holand)-0.05 mg/kg м., i.m.; Въвеждане в анестезия след 15 min с Thiopenton sodium (Thiopental®, Biochemie GmbH, Kundl-Austria) - 2.5%, 6 mg/kg м., i.v.; Интубация и поддържане на анестезията с 2.5 об.% Halothane (Narcotan®, Leciva, Praha-Czech Republic).
При всички кучета след асептично подготвено операционно поле и латерален оперативен достъп беше извършена транс­периостална остеотомия на едната бедрена кост в диафизарната и част.
 При групата НЗФ през fossa intertrochanterica в костно-мозъчния канал вкарвахме Кюнчеров гвоздей.  При групата ОСМ през fossa intertrochanterica в костно-мозъчния канал също вкарвахме Кюнчеров гвоздей. Преди фиксирането на двата фрагмента във всеки един от тях аплицирахме по 0.5 ml смесена култура от равни части бактериални суспензии на E.coli 026 и Ps.aeruginosa с гъстота 1.105 б.к./ml.
 И при двете групи меките тъкани зашивахме по рутинния начин. В следоперативния период, в продължение на 2 месеца провеждахме клинични (пулс, температура, дишане) и рентгенологични изследвания, за да следим дали процеса на костно зарастване върви нормално (при група НЗФ) или дали се развива остеомиелит (при група ОСМ). Параклинични изследвания извършвахме ежеседмично до края на четвъртата седмица.
 За елиминиране влиянието на денонощните колебания върху резултатите, кръвните проби бяха вземани между 7 и 8 ч. сутринта преди операцията и в края на първата, втората, третата и четвъртата седмица след операцията. В деня на вземане на кръвните проби бяха правени и рентгенографии в медиолатерална проекция на съответната бедрена област.
 Параклиничните изследвания обхващаха определяне на серумните или плазмени концентрации на следните показатели:
 - общ калций и йонизиран калций -  чрез колориметричен тест на Biotrol.
- неорганичен фосфор – чрез колориметричен тест на Biotrol.
-обща и костна алкална фосфатаза – чрез колориметричен тест на Biotrol.
- серумен остеокалцин - беше определен с радиоимунологичен тест (Henning-Berlin GmbH, Германия). Всяка проба беше дублирана, а резултатът е средноаритметичната стойност от двете измервания.
 - серумните нива на карбокситерминалния телопептид на колаген тип I (ICTP) - бяха определени с радиоимунологичен тест (Оrion Diagnostica, Финландия), точност на метода 0.2 ng/ml. Всяка от пробите бе анализирана двукратно, а получената стойност е средно аритметична на двете измервания.
 Получените резултати бяха обработени вариационно-статистически с помощта на ANOVA-метода.
 Kurdy, NM, (2000) проследява ролята на пропептидите на колаген тип I и III и костната алкална фосфатаза (КАФ) при бавнозарастващи диафизарни фрактури на тибията при хора и установява, че до десетата седмица от травмата не се установяват статистически значими намаления в нивата на КАФ и пропептидите. Дори пропептидите на колаген тип III са статистически повишени. Данни за недостатъчен остеобластичен отговор обаче съществуват след двадесетата седмица. Противно на това при кучето в нашите изследвания, макар че фрактурите зарастват нормално КАФ се понижава значително без това да е статистически достоверно,

СНИМКИТЕ :
http://drjivkojelyazkov.snimka.bg/daily/eksperimentalen-osteomielit-ustanovyavane-na-kostniya-marker-ictp-diplomna-rabota.430278.15302210
   
 Въз основа на данни за стойностите на костната алкална фосфатаза, остеокалцина и телопептидите Emami, A. и кол., (1999) установяват, че при бавнозарастващи фрактури на тибията при човека костната резорбция до четвъртата седмица е еднаква с нормално зарастващите, докато костното формиране се измества по-късно (около 10-16 седмица). При нашите проучвания върху фрактури на бедрената кост при кучета маркерът на костната резорбция (ICTP) е статистически повишен през втората седмица, и до края на четвъртата седмица се връща към началните стойности. Това съвпада с процесите на остеолиза около костните краища след травмата, които вероятно при кучето протичат по рано. Потвърждават се обаче данните за костната алкална фосфатаза и остеокалцина, които през този период или намаляват (КАФ) или не се променят (остеокалцина), въпреки че фрактурите зарастват без усложнения. В тази връзка резултатите ни се доближават до изводите на Akesson, K и кол., 2005, които доказват, че при клиновидни остектомии на тибията костния метаболизъм се разстройва сериозно и това дори може да затрудни мониторинга на остеопорозата при тези пациенти. Чрез костните маркери в серума и урината те доказват, че костната резорбция е по-силно изразена от костното формиране в периода след остектомията (фрактурата).
 Според Herrmann, M и кол., (2002) при бавнозарастващи фрактури на хора остеокалцина е може би по-ранен маркер защото остава непроменен след четвъртата седмица, докато при нормалнозарастващите се увеличава статистически достоверно. При подобно проучване Nyman и кол., (1991) установяват статистическо значимо повишаване на остеокалцина и КАФ на 6 седмица и при двете групи. След това при групата на нормалнозарастващите фрактури стойностите намаляват, а при другата група остават високи, но без статистическа разлика между двете групи. Това за нормалнозарастващите фрактури се потвърждава и от нашите резултати защото колебанията в нивата на остеокалцина са незначителни, а КАФ намалява почти наполовина без това да е статистически достоверно. В тази връзка Herrmann и кол., (2002) използват Mann-Whitney U–тест, при което достоверността се повишава.
 Komnenou, A. и кол. (2005) остановяват връзка между нивата на общата алкална фосфатаза (ОАФ) в серума на кучета и хода на процеса на зарастване на случайни фрактури при дългите кости. Според тях общата алкална фосфатаза се покачва статистически достоверно още на десетия ден и до тридесетия ден се връща към началните стойности. Според нас по-правилно е да се следи не само общата, а и КАФ, която е неин изоензим и е по-чувствителен маркер на костния метаболизъм. Това е така защото при нашето изследване при запазени нива на ОАФ нивата на КАФ започват да намаляват още на първата седмица и тази тенденция се запазва до края на наблюдавания период. Нашите резултати съвпадат и с тези на Akesson, K и кол., (2005), според които след остектомии на тибията в началото костната резорбция е по-силно изразена от костното формиране.
 В групата с експериментален остеомиелит  след първата седмица беше установена трайна тенденция на повишаване на ICTP до края на четвъртата седмица придружено с усилено костообразуване през първата и втората седмица (достоверно повишени нива на КАФ и остеокалцин). За разлика от промените при остеомиелита, костната резорбция изразена чрез нивата на ICTP при групата с нормално зарастваща фрактура е най-висока през първата и втората седмица и бързо се връща към норма след това. Тези разлики вероятно се дължат на задълбочаващите се остеолитични процеси при остеомиелита придружени с бурна реакция на периоста в началото, докато при нормалнозарастващите фрактури резорбтивната фаза е кратка при сравнително постоянно костообразуване.
 Остеомиелитът е инфекциозно възпалително заболяване на костната тъкан, с хронично и прогресивно развитие без тенденция за спонтанно оздравяване (Hulin, 1970). Характерните изменения в костта, резултат от инфекцията, са локални деструкции и реактивно образуване на нова костна тъкан.
 Динамиката на изменение на резултатите показва най-общо значително увеличаване на стойностите на параметрите, характерни за костната формация в края на 1-вата до 2-рата седмица, съпроводено със силно понижаване на серумните нива на йонизирания калций и неорганичния фосфат. Този факт се обяснява с ускоряване на биологични процеси в костта или с т.нар. местно явление на акцелерация (RAP) (Woodard и Riser, 1991). Характерното за остеомиелита разрушаване на костта се визуализира на рентгенограмите едва в края на опитния период, т.е. при резорбция на около 30% от минералните вещества на костта (Waldvogel et al., 1970), докато данните за нивата на серумния телопептид показват статистически доказано рязко повишение още в края на 2-та седмица, след което за една седмица те почти се удвояват. Според Gillespie (1990) вероятно е костната лезия при остеомиелита да се дължи на освобождаването на лизозомни ензими от полиморфоядрените левкоцити, но е възможно и активизиране на костните макрофаги, каквито са остеокластите. Рязкото повишаване на нивата на серумния телопептид, който е продукт на разграждането на колаген от тип I, или на костния колаген, потвърждава втората хипотеза.
 Прави впечатление и факта, че пиковете в нивата на маркерите на костното формиране и костната резорбция не съвпадат. Най-усиленото костно формиране (т.е. най-висока активност на костната АФ) се наблюдава около седмица преди най-ускорената резорбция (отчетена по високите нива на ICTP). Тези редуващи се периодични колебания в темповете на двата основни процеса на костното ремоделиране обясняват и характерната мозаечна структура на костните гредици.
  При обсъждане на промените в параклиничните показатели, характерни за костното ремоделиране при развитието на един остеомиелит, би трябвало да се има предвид още един фактор, чиято роля не е още напълно изяснена, а именно обездвижването вследствие операцията и костната инфекция и резултатната остеопороза. Съществуват данни при човека (Eastell и кол., 1988; Akesson и кол., 2005), че остеопорозата също води до повишаване на серумните нива на С-терминалния телопептид. В случая с акутния остеомиелит обаче ние считаме, че ролята на обездвижването не е толкова голяма. За нейното изясняване са необходими допълнителни проучвания, които не бяха предмет на нашето изследване.
 
ИЗВОДИ 1. При нормално зарастваща фрактура на тръбести кости при кучета серумните нива на С-терминалния телопептид се повишават статистически значимо на първата седмица, запазват се до края на втората, след което се връщат към началните си стойности. 2. При експериментален остеомиелит на кучета серумните нива на С-терминалния телопептид се повишават статистически значимо на втората седмица и се запазват високи до края на изследвания период. Началното повишение е доста преди процеса да се прояви рентгенологично. Тъй като ранната рентгенова диагноза на остеомиелита е трудна или невъзможна, при съмнение за костна инфекция или следоперативни усложнения освен рентгенологично изследване е добре да се изследват и биохимичните маркери за костната резорбция. 3. При остеомиелита през първата и втората седмица се повишават достоверно и стойностите на костната алкална фосфатаза и остеокалцина, но бързо се връщат към изходните нива.     /със съкращения/     

ТРАКИЙСКИ УНИВЕРСИТЕТ,

ВЕТЕРИНАРНОМЕДИЦИНСКИ ФАКУЛТЕТ,
Д-Т: ХИРУРГИЯ НА ДРЕБНИТЕ ЖИВОТНИ.

 

 

 

   Научен ръководител: Доц. Михаил Паскалев  
                Дипломант: Д-р Живко Димитров Желязков, д.в.м.



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