Proteozom inhibitörü bortezomibin tip 1 diyabetin tedavisinde kullanılabilirliğinin in vivo koşullarda araştırılması
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Tarih
2016
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Yayıncı
Aksaray Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Tip 1 Diyabet, pankreasın langerhans adacıklarına inflamatuar hücrelerin infiltrasyonu ve sonrasında insülin salgılayan ? hücrelerinin kaybı ile karakterize edilen otoimmün bir hastalıktır. İn vitro ve in vivo olarak yapılmış birçok çalışma, IL-1?, TNF-? ve IFN-? gibi proinflamatuar sitokinlerin neden olduğu ? hücre ölümünde Nüklear Faktör Kappa-B (NF-?B) sinyal yolağının rol aldığını göstermiştir. NF-?B'nin hücre büyümesi, hücre canlılığı, apoptozis ve inflamasyon gibi önemli biyolojik işlemlerde rol alan birçok genin ifadesini düzenleyen bir transkripsiyonel faktör olduğu bilinmektedir. Bu tez çalışmasının amacı, günümüzde multiple myeloma tedavisinde kullanılan ve klinikteki etkinliği NF-kB inhibisyonuna bağlı olan proteozom inhibitörü bortezomib´in, tip 1 diyabetin tedavisinde kullanılabilirliğini in vivo koşullarda araştırmaktır. Bunun için dişi Sprague Dawley cinsi rat bulunan beş farklı çalışma grubu (n=5-7) oluşturuldu. Bunlar, kontrol grubu, diyabet grubu (grup II), bortezomib verilen grup (grup III), STZ ve bortezomibin eş zamanlı verildiği grup (grup IV) ve STZ sonrası diyabetik olduğu doğrulanan ratlara bortezomib verilen tedavi grubudur (grup V). Bortezomib klinikte kullanıldığı şekilde 3 gün ara ile toplamda üç (0, 3, ve 7, gün) doz uygulanmıştır. İlaç uygulaması sonrası 24 saat kültüre edilmiş tüm adacıklarda hücre canlılığı, fluoresans mikroskopta saptanmıştır. Sonrasında kültür ortamından alınan adacıklardan RNA izolasyonu ve cDNA sentezi yapılarak apoptotik genlerden bcl-2, bax ve kaspaz 3 genlerinin ifade düzeyleri real time RT-PCR yöntemi kullanılarak araştırılmıştır. Bu tez çalışması sonucunda, STZ'nin pankreasın insülin salgılayan beta hücrelerinde apoptozise neden olduğu kan şekerlerindeki artış, adacıkların % canlılıklarındaki azalma ve apoptotik genlerin ifade düzeylerine bakılarak doğrulandı (bax/bcl-2=3.14 /1.99). Tek başına uygulanan bortezomib dozunun pankreasın ? hücrelerinde sitotoksik olmadığı (%91) kontrol grubuna göre değişiklik göstermemesi ile saptandı. Bu grupta ayrıca apoptotik gen ifade düzeylerine (bax/bcl-2=0.83/1.69) bakıldığında Bor'in antiapoptotik etkiye neden olduğu gözlendi. STZ ve Bor'in eş zamanlı uygulandığı grup IV de, STZ uygulaması ile kan şekeri düzeyleri artmasına karşın, Bor'in ? hücrelerinde anti-apoptotik etkiye sahip olduğu bax/bcl-2 (3.84/4.68) oranına bakılarak saptandı. STZ sonrası Bor verilen grup V'de Bor'in hem kan şekeri hem de adacık sayısı açısından bir iyileşmeye neden olmadığı apoptotik gen ifade düzeylerine bakıldığında (bax/bcl-2=4.45/3.30) proapoptotik etki gözlendi. Sonuç olarak, adacıkları tamamen harap olmamış diyabetik ratlarda Bor'in NF-kB sinyal yolağını bloke ederek hücrelerin apoptozise girmesini engelleyebileceği sonucuna varılmıştır.
Tip 1 diabet is a otoimmüne disease that is characterizied as the infiltration of inflamatuar cells to pancreas langerhans islets and destruction of the insulin secreting ? cells. The leukocytes, which is infiltrated to inner parts of islet, secrete proinflamatuar cytokines like IL-?, TNF-? and IFN-? that causes death of the ? cells. In most studies both in vitro and in vivo, it is showed that Nuclear Factor Kappa-B (NF-?B) pathway has a role in ? cell death that is triggered by those cytokines. The objective of this these is investigate usability of bortezomib, a Proteozome inhibitor, whose effectiveness in clinical practise depends on NF-kB inhibition and which is nowadays used in treatment of multiple myeloma, in treatment of Type-1 Diabetes Mellitus in vivo. For this purpose, female Sprague Dawley rats which were not obese were comprised. These were the control group, the diabetes group for which streptozotocin (STZ) was injected (Group II), the group for which bortezomib was administered (Group III), the group for which STZ and bortezomib were administered simultaneously (Group IV) and the treatment group in which bortezomib was administered to the rats which were confirmed to be diabetic following STZ administration (Group V). Bortezomib was administered totally as three (0th, 3rd and 7th days) with 3-day interval, as it is used in clinical practice. Following drug administration, all islets which were cultured for 24 hours, cell viability was determined under fluorescence microscopy. In conclusion of this study, the fact that STZ causes apoptozis in insulin-secreting beta cells of pancreas was confirmed by increase in blood glucose levels, decrease in viability of islets and investigation of expression levels of apoptotic genes (bax/bcl-2=3.14 /1.99). The fact that bortezomib dosage that was administered alone was not cytotoxic for ? cells (91%) did not demonstrate any alteration compared to the control group. When apoptotic gene expression levels were investigated in this group, it was determined that Bortezomib had anti-apoptotic effect. In group IV, in which STZ and BOR were administered simultaneously, although blood glucose increased with administration of STZ, the fact that Bortezomib had anti-apoptotic effect on ? cells was determined by evaluating bax/bcl-2 (3.84/4.68) ratio. In group V, in which Bortezomib was administered following STZ, it was determined that there was no improvement in terms of neither blood glucose nor number of islets. In group V, when apoptotic gene expression levels were investigated (bax/bcl-2=4.45/3.30), as is in the Group II, it pro-apoptotic effect was determined. In conclusion, it was concluded that in diabetic rats whose islets were not completely destructed, Bortezomib may prevent cells to undergo apoptozis by inhibiting NF-kB signalling pathway.
Tip 1 diabet is a otoimmüne disease that is characterizied as the infiltration of inflamatuar cells to pancreas langerhans islets and destruction of the insulin secreting ? cells. The leukocytes, which is infiltrated to inner parts of islet, secrete proinflamatuar cytokines like IL-?, TNF-? and IFN-? that causes death of the ? cells. In most studies both in vitro and in vivo, it is showed that Nuclear Factor Kappa-B (NF-?B) pathway has a role in ? cell death that is triggered by those cytokines. The objective of this these is investigate usability of bortezomib, a Proteozome inhibitor, whose effectiveness in clinical practise depends on NF-kB inhibition and which is nowadays used in treatment of multiple myeloma, in treatment of Type-1 Diabetes Mellitus in vivo. For this purpose, female Sprague Dawley rats which were not obese were comprised. These were the control group, the diabetes group for which streptozotocin (STZ) was injected (Group II), the group for which bortezomib was administered (Group III), the group for which STZ and bortezomib were administered simultaneously (Group IV) and the treatment group in which bortezomib was administered to the rats which were confirmed to be diabetic following STZ administration (Group V). Bortezomib was administered totally as three (0th, 3rd and 7th days) with 3-day interval, as it is used in clinical practice. Following drug administration, all islets which were cultured for 24 hours, cell viability was determined under fluorescence microscopy. In conclusion of this study, the fact that STZ causes apoptozis in insulin-secreting beta cells of pancreas was confirmed by increase in blood glucose levels, decrease in viability of islets and investigation of expression levels of apoptotic genes (bax/bcl-2=3.14 /1.99). The fact that bortezomib dosage that was administered alone was not cytotoxic for ? cells (91%) did not demonstrate any alteration compared to the control group. When apoptotic gene expression levels were investigated in this group, it was determined that Bortezomib had anti-apoptotic effect. In group IV, in which STZ and BOR were administered simultaneously, although blood glucose increased with administration of STZ, the fact that Bortezomib had anti-apoptotic effect on ? cells was determined by evaluating bax/bcl-2 (3.84/4.68) ratio. In group V, in which Bortezomib was administered following STZ, it was determined that there was no improvement in terms of neither blood glucose nor number of islets. In group V, when apoptotic gene expression levels were investigated (bax/bcl-2=4.45/3.30), as is in the Group II, it pro-apoptotic effect was determined. In conclusion, it was concluded that in diabetic rats whose islets were not completely destructed, Bortezomib may prevent cells to undergo apoptozis by inhibiting NF-kB signalling pathway.
Açıklama
Anahtar Kelimeler
Tip I Diyabet, Bortezomib, NF-?B Sinyal Yolağı, Apoptozis, NF-?B, Diabetes Mellitus, Cytokine
