Oksitosinin takrolimusa maruz kalmış adipoz doku kaynaklı mezenkimal kök hücre hattı üzerine etkilerinin incelenmesi
Yükleniyor...
Dosyalar
Tarih
2016
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Takrolimus (FK 506) organ nakillerinde alıcı bireyde immün cevabın
oluşmasını engellemek için kullanılan ve bu etkiyi kalsinörin sinyal yolağı üzerinden
T hücrelerinin uyarılmasını engelleyerek yapan kemoterapotik bir kimyasaldır.
Metabolik olarak incelendiğinde vücutta temel olarak karaciğerde sitokrom P450
enzim sistemi tarafından metabolize edilen takrolimusun farklı hücreler üzerinde
yüksek toksik etkilerinin olduğu bilinmektedir.
Mezenkimal kök hücreler doku yenilenmesi ve hücresel tedavide
kullanılabilmeleri sebebiyle son zamanlarda oldukça önem kazanmışlardır. Bazı
klinik vakalarda organ nakli sonrasında hastaya destekleyici amaçla mezenkimal kök
hücreler nakledilmektedir. Mezenkimal kök hücreler immünomodülatör etkileri ve
rejenerasyona yardımcı olmaları sebebiyle günümüzde gittikçe yaygınlaşan bir
kullanıma sahiptir.
Oksitosin, primer olarak insanda nöromodülatör olarak işlev gören ve hipofizin
nörohipofiz kısmından salgılanan peptit bir hormondur. Oksitosinin hücreler
üzerinde oksidatif strese karşı direnç kazandırmak, proliferasyonu arttırmak ve
rejenerasyona yardımcı olmak gibi etkileri vardır.
Yapılan çalışmada takrolimus adlı etken maddenin, insan adipoz doku kaynaklı
mezenkimal kök hücreler üzerindeki sitotoksik etkilerinin araştırılması
hedeflenmiştir. Bunun yanında yine aynı şekilde nöral bir peptit hormon olan
oksitosinin, adipoz doku kaynaklı mezenkimal hücreler üzerinde takrolimus kaynaklı
oluşan toksisiteyi inhibe edip etmediği araştırılmıştır.
İnsan adipoz doku kaynaklı mezenkimal kök hücre hattı üzerinde takrolimusun
sitotoksik etkisi WST-1 analizi kullanılarak zaman ve doza bağımlı olarak
değerlendirilmiştir. Belirlenen zaman ve doz ile yapılan izobologram analizleri
sonucunda madde kombinasyonlarının endeks değerleri belirlenmiştir.
Takrolimus ve oksitosinin IC50 dozu (72. saat) insan adipoz doku kaynaklı
mezenkimal kök hücreler için sırasıyla 17.44 μM ve 13.43 μM olarak belirlenmiştir.
İzobologram analizleri sonucu antagonistik olarak belirlenen iki etken maddenin
kombinasyonlarının endeks değerleri ED50: 1.418, ED75: 1.25719, ED90: 1.23533
olarak kaydedilmiştir.
Apoptotik ve nekrotik ölüm oranlarını belirlemek amacıyla anneksin V
yöntemi uygulanmıştır. Analiz sonucunda apoptotik olmayan, erken apoptotik, geç
apoptotik, toplam apoptotik ve ölü hücreler hem yüzde olarak hem de hücre/ml
olarak hesaplanmıştır.
Oksidatif stres ile birlikte oluşan ve reaktif oksijen metabolitlerinin verdiği
zararı belirlemek amacıyla lipit peroksidasyonu, süperoksit dismutaz (SOD)
aktivitesi, katalaz (CAT) aktivitesi, glutatyon peroksidaz (GPX) aktivitesi
ölçülmüştür.
Ayrıca antijen-antikor bağlanma prensibine göre çalışan immünofloresan
boyama yöntemi ile hücrelerin içerisinde bulunan yapıtaşları ve meydana gelen
kimyasal reaksiyonlar gösterilmiştir.
Yapılan çalışmada elde edilen veriler doğrultusunda takrolimusun adipoz doku
kaynaklı mezenkimal kök hücreler üzerinde oksidatif stres yaratarak hücrelerin erken
apoptoza gitmelerine neden olduğu görülmüştür. Oksitosine maruz bırakılan
hücrelerde proliferasyonun arttığı gözlemlenirken takrolimus ve oksitosin
kombinasyonlarında takrolimusun hücreler üzerindeki sitotoksik etkisinin azaldığı
belirlenmiştir. Çalışma sonunda belirlenen dozun alıcıya modifiye edilmesiyle, hem
immünosupresif ilacın hem de mezenkimal kök hücrelerin birbirlerinden negatif
yönde etkilenmeden immün sistem üzerinde gerekli baskıyı oluşturacakları
düşünülmektedir. Bu duruma bağlı olarak organ nakilleri sonrasında uygulanan
tedavi protokollerinde destekleyici olarak oksitosinin kullanımına ışık tutacağı da
öngörülen sonuçlar içerisindedir.
Tacrolimus (FK 506) is a chemotherapeutic agent, which uses calcineurin pathway via inhibiting the stimulation of T cells to prevent the formation of immune response in the recipient individual in organ transplants. Tacrolimus is mainly metabolized in the liver by cytochrome P450 enzyme system and is known to be of high toxic effects on different cells. Mesenchymal stem has recently gained importance because they can be used in cellular therapy and tissue regeneration. In some clinical cases mesenchymal stem cells are transferred into the patient after the organ transplantations in order to support the treatment. Because of their immunomodulatory effects and to assist the regeneration, mesenchymal stem cells has an increasingly prevalent use today. Oxytocin is primarily acting as neuromodulators in humans and is a peptide hormone secreted by the pituitary gland of the neurohypophysis. Oxytocin has such effects on cells as to confer resistance against oxidative stress on cells and to increase the proliferation and help regeneration. Studies on the active substance of tacrolimus were aimed to investigate the cytotoxic effects on human adipose tissue derived mesenchymal stem cells. Besides, likewise a peptide hormone oxytocin neural adipose tissue was investigated in order to reveal the inhibition of the tacrolimus-induced toxicity comprising the mesenchymal cells Cytotoxic effects of tacrolimus on human adipose tissue derived mesenchymal stem cell line were evaluated by WST- 1 assay as time and dose dependent. Isobologram analysis was conducted using the assessed dose and time revealed the index values of substance combinations. IC50 dose of tacrolimus and oxytosin (72 hours) for human adipose tissue derived mesenchymal stem cell were designated 17.44 μM and 13.43 μM, respectively. The results of isobologram analysis for two substance determined as antagonistic and the combination indexes were recorded as ED50: 1.418, ED75: 1.25719, ED90: 1.23533, respectively. In order to determine the apoptotic and necrotic death rate, annexin V method was used. By the help of the result of the analysis, non apoptotic, early apoptotic, late apoptotic, apoptotic and dead cells, both in total and percent cells / ml was calculated. In order to determine the damage caused by the active metabolites that formed with oxidative stress, superoxide dismutase activity (SOD), catalase activity (CAT), glutathione peroxidase activity (GPX) were measured. Also immunofluorescence staining, which works by the the antigen-antibody binding principle was conducted to the show the cell constituents and chemical reactions that take in place. The data obtained in the present study suggest that tacrolimus has been shown to cause early apoptosis on human by creating oxidative stress. Proliferation was observed in cells that exposed to oxytocin and the combination of tacrolimus and oxytocin also decreased cytotoxic effects on cells. By the modification of the determined dose to recipient’s will generate the necessary pressure on the immune system without being affected by both immunosuppressive drug and mesenchymal stem cells. Depending on this case, the results obtained in this study will enlighten the use of oxytocin in addition to treatment protocols that applied after the transplantation.
Tacrolimus (FK 506) is a chemotherapeutic agent, which uses calcineurin pathway via inhibiting the stimulation of T cells to prevent the formation of immune response in the recipient individual in organ transplants. Tacrolimus is mainly metabolized in the liver by cytochrome P450 enzyme system and is known to be of high toxic effects on different cells. Mesenchymal stem has recently gained importance because they can be used in cellular therapy and tissue regeneration. In some clinical cases mesenchymal stem cells are transferred into the patient after the organ transplantations in order to support the treatment. Because of their immunomodulatory effects and to assist the regeneration, mesenchymal stem cells has an increasingly prevalent use today. Oxytocin is primarily acting as neuromodulators in humans and is a peptide hormone secreted by the pituitary gland of the neurohypophysis. Oxytocin has such effects on cells as to confer resistance against oxidative stress on cells and to increase the proliferation and help regeneration. Studies on the active substance of tacrolimus were aimed to investigate the cytotoxic effects on human adipose tissue derived mesenchymal stem cells. Besides, likewise a peptide hormone oxytocin neural adipose tissue was investigated in order to reveal the inhibition of the tacrolimus-induced toxicity comprising the mesenchymal cells Cytotoxic effects of tacrolimus on human adipose tissue derived mesenchymal stem cell line were evaluated by WST- 1 assay as time and dose dependent. Isobologram analysis was conducted using the assessed dose and time revealed the index values of substance combinations. IC50 dose of tacrolimus and oxytosin (72 hours) for human adipose tissue derived mesenchymal stem cell were designated 17.44 μM and 13.43 μM, respectively. The results of isobologram analysis for two substance determined as antagonistic and the combination indexes were recorded as ED50: 1.418, ED75: 1.25719, ED90: 1.23533, respectively. In order to determine the apoptotic and necrotic death rate, annexin V method was used. By the help of the result of the analysis, non apoptotic, early apoptotic, late apoptotic, apoptotic and dead cells, both in total and percent cells / ml was calculated. In order to determine the damage caused by the active metabolites that formed with oxidative stress, superoxide dismutase activity (SOD), catalase activity (CAT), glutathione peroxidase activity (GPX) were measured. Also immunofluorescence staining, which works by the the antigen-antibody binding principle was conducted to the show the cell constituents and chemical reactions that take in place. The data obtained in the present study suggest that tacrolimus has been shown to cause early apoptosis on human by creating oxidative stress. Proliferation was observed in cells that exposed to oxytocin and the combination of tacrolimus and oxytocin also decreased cytotoxic effects on cells. By the modification of the determined dose to recipient’s will generate the necessary pressure on the immune system without being affected by both immunosuppressive drug and mesenchymal stem cells. Depending on this case, the results obtained in this study will enlighten the use of oxytocin in addition to treatment protocols that applied after the transplantation.
Açıklama
Anahtar Kelimeler
Takrolimus, Oksitosin, Adipoz Doku Kaynaklı Mezenkimal Kök Hücre, Oksidatif Stres, Tacrolimus, Oxytocin, Adipose Derived Mesenchymal Stem Cells, Oxidative Stress
