Kordon kanı kökenli mezankimal kök hücrelerin kahverengi adipositlere farklılaştırılması
Küçük Resim Yok
Tarih
2016
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Mitokondriden zengin olan kahverengi yağ hücreleri "depolanmış trigliseritleri" yakarak onların içindeki enerjiyi ısıya dönüştürerek enerji tüketimini sağlayan ve bu özellikleri nedeniyle de diyabet, obezite, ve obeziteyle ilişkili çeşitli metabolik hastalıkların tedavisinde yararlı olma potansiyali olan hücrelerdir. Kordon kanı Mezenkimal Kök Hücre (KK-MKH)'leri Kemik İliği kökenli mezenkimal Kök Hücre (Kİ-MKH)'leri gibi diğer KH kaynaklarından daha az immünojenik olup allojenik ve otolog transplantasyonlarda kullanılabilirler. Şu ana kadar literatürde kordon kanından elde edilen mezankimal kök hücrelerin kahverengi adipositlere farklılaştırılmasını gösteren bir çalışma mevcut değildir. Bu projede kordon kanından elde edilen mezankimal kök hücrelerin in vitro koşullarda kahverengi adipositlere farklılaştırılması ve onların farklılaşma kapasitesi ve etkinliğinin adipoz doku kökenli MKH (AD-MKH)'ler ile kıyaslanması amaçlanmıştır. Çalışmamızda KK-MKH'leri Ege Üniversitesi Tıp Fakültesi Kadın Hastalıkları ve Doğum Ana Bilim Dalında normal doğum sonrası rutinde atılan 10 adet plasenta ve kordon kanından toplanmıştır. AD-MKH'leri ise Ege Üniversitesi Tıp Fakültesi Plastik ve Rekonstrüktif Cerrahi Kliniğinde liposakşın/lipektomi ameliyatı olan 6 adet gönüllü hastadan izole edilmiştir. KK-MKH'lerinin beyaz ve kahverengi adipositlere farklılaşma yeteneği AD-MKH'leri ile kıyaslanmıştır. İzole edilmiş MKH'ler akış sitometri analizi ile karakterize edilmiştir. Akış sitometri sonuçlarına göre hem KK-MKH'leri hem de AD-MKH'leri CD34 ve CD45 yüzey markerleri için negatif iken, CD90 ve CD105 yüzey markerleri için yüksek oranda pozitiflik gösterdiler. 3. pasaj MKH'ler, beyaz ve kahverengi olmak üzere iki farklı farklılaşma besi ortamında kültüre edilerek beyaz ve kahverengi adipositlere farklılaştırılmıştır. Kahverengi adipositleri elde etmek için MKH'ler rekombinant bone morphogenic protein 7 (BMP7) içeren farklılaşma medyumu içinde kültüre edilmiştir. Elde edilen beyaz ve kahverengi yağ hücrelerinin karakterize edilebilmesi için yapılan Oil Red O boyaması sonuçlarına göre, AD-MKH'lerden elde edilen beyaz ve kahverengi adipositler sırasıyla %85 ve %61 kırmızı pikseli gösterirken, KK-MKH'lerden elde edilen beyaz ve kahverengi adipositler ise sadece %3 ve %1,9 oranında kırmızı pikseli göstermiştir. Kahverengi adipositlere özgü UCP1 protein miktarı, Western blot ve floresan mikroskop analizleri ile incelenmiştir. Floresan analiz sonuçlarına göre, hem KK-MKH'lerden hem de AD-MKH'lerden elde edilen kahverengi adipositler farklılaştırılmamış kontrol hücrelerine göre daha fazla UCP1 proteini içermektedirler ancak sadece AD-MKH'lerden elde edilen kahverengi adipositlerin sitoplazmalarında yuvarlak parlayan cisimcikler görülmüştür. Bu cisimcikler literatüre göre bir araya gelen çok fazla sayıda kümeleşmiş mitokondriler olabilir. Western blot sonuçlarına göre KK-MKH ve AD-MKH'lerden elde edilen kahverengi adipositler farklılaştırılmamış MKH'lere göre sırasıyla 4 ve 10.64 kat daha fazla UCP1 proteini eksprese etmektedir (P<0.004). Sonuç olarak, AD-MKH'ler hem beyaz hem kahverengi adipositlere yüksek bir verimlilik ile farklılaşırken, KK-MKH'leri adipositlere çok az bir farklılaşma eğilimi göstermiştir. Bu çalışmadan elde edilen sonuçlara göre, KK-MKH'leri hem beyaz hem de kahverengi adipositlere farklılaştırılmak için uygun KH tipleri değidir. Böylece adiposit hücrelerin transplantasyonu ile yapılan tedavilerde yada rekonstrüktif cerrahilerde kullanımı AD-MKH'ler gibi olumlu sonuç vermeyebilir. Bunun aksine AD-MKH'ler yüksek bir verimlilik ile kahverengi adipositlere farklılaştırılabildiği için diyabet, obezite ve obeziteyle ilişkili metabolik hastalıkların tedavisinde de yeni umut vaat edici bir tedavi yaklaşımı olabilirler. Bu konuda ileri in vivo ve ardından klinik çalışmalara gereksinim vardır
Mitochondria-enriched brown fat cells provides energy consumption by burning "stored triglycerides" and converting th energy to heat, therefore they are potentially useful cells for the treatment of diabetes, obesity and various metabolic diseases associated with obesity. Umbilical Cord Blood derived-Mesenchymal Stem Cells (UCB-MSC) are less immunogenic than other SC sources such as Bone Marrow derived- Mesenchymal Stem Cells (BM-MSC), and can be used for allogenic and autologous transplantation. There is not any study in the literature available to demonstrate differentiation of UCB-MSC to brown adipocytes until now. In this study we aimed to perform in vitro differentiation of UCB-MSC to brown adipocytes and compare their differentiation capacity and efficiency with AD-MSCs. In this study, the UCB-MSCs were collected from 10 placenta and umbilical cord blood samples which are routinely discarded after a normal birth in Ege University Faculty of Medicine, Department of Gynecology and Obstetrics, AD-MSCs were collected from 6 voluntary patients who undergo Liposuction / lipectomy surgery at Ege University Faculty of Medicine Department of Plastic and Reconstructive Surgery. Differentiation ability of UCB-MSCs to white and brown adipocytes was compared with AD-MSCs. Isolated MSCs were characterized by flow cytometry analysis. According to the results of flow cytometry both UCB-MSCs and AD-MSCs showed high rate of positivity for CD90 and CD105 surface markers while they were negative for CD34 and CD45. MSCs in the 3rd passage were differentiated to white and brown adipocytes by culturing them in two different differentiation mediums, for white and brown adipocyte differentiation. MSCs were cultured in differentiation medium containing recombinant bone morphogenic protein7 (BMP7) to give brown adipocytes. The results of Oil Red O staining used for characterizing white and brown adipocytes demonstrated that white and brown adipocytes obtained from AD-MSCs showing 85% and 61% of red pixels, respectively, while it was 3% and 1.9%, respectively for white and brwon adipocytes obtained from UCB-MSCs. Brown adipocyte-specific UCP1 protein content, was determined by fluorescence microscopy and Western blot analysis. According to the results of the fluorescence analysis, brown adipocytes obtained from both UCB-MSCs and AD-MSCs contain more UCP1 protein than undifferentiated control cells but round shining bodies were only seen in cytoplasm of brown adipocytes obtained from AD-MSCs. According to the literature, they can be clustered mitochondria formed from large numbers of mitochondria that come together. The results of Western blot analysis show that brown adipocytes obtained from UCB-MSCs and AD-MSCs express UCP1 protein 4 and 10.64 times, respectively, more than undifferentiated MSCs (P<0.004). As a result, while AD-MSCs differentiated to white and brown adipocytes abundantly and efficiently, UCB-MSCs showed only a little differentiation tendency to adipocytes. According to the results obtained from this study, UCB-MSCs are not appropriate SC type to be differentiated to both white and brown adipocytes. So, they may not give positive results similar to AD-MSCs during treatment with adipocyte cell transplantation or in reconstructive surgery. In contrast, AD-MSCs may be a new promising therapeutic approach for treatment of diabetes, obesity and its related metabolic disorders because of their high efficiency in differentiation capacity to brown adipocytes. Further in vivo and clinical studies are needed in this regard.
Mitochondria-enriched brown fat cells provides energy consumption by burning "stored triglycerides" and converting th energy to heat, therefore they are potentially useful cells for the treatment of diabetes, obesity and various metabolic diseases associated with obesity. Umbilical Cord Blood derived-Mesenchymal Stem Cells (UCB-MSC) are less immunogenic than other SC sources such as Bone Marrow derived- Mesenchymal Stem Cells (BM-MSC), and can be used for allogenic and autologous transplantation. There is not any study in the literature available to demonstrate differentiation of UCB-MSC to brown adipocytes until now. In this study we aimed to perform in vitro differentiation of UCB-MSC to brown adipocytes and compare their differentiation capacity and efficiency with AD-MSCs. In this study, the UCB-MSCs were collected from 10 placenta and umbilical cord blood samples which are routinely discarded after a normal birth in Ege University Faculty of Medicine, Department of Gynecology and Obstetrics, AD-MSCs were collected from 6 voluntary patients who undergo Liposuction / lipectomy surgery at Ege University Faculty of Medicine Department of Plastic and Reconstructive Surgery. Differentiation ability of UCB-MSCs to white and brown adipocytes was compared with AD-MSCs. Isolated MSCs were characterized by flow cytometry analysis. According to the results of flow cytometry both UCB-MSCs and AD-MSCs showed high rate of positivity for CD90 and CD105 surface markers while they were negative for CD34 and CD45. MSCs in the 3rd passage were differentiated to white and brown adipocytes by culturing them in two different differentiation mediums, for white and brown adipocyte differentiation. MSCs were cultured in differentiation medium containing recombinant bone morphogenic protein7 (BMP7) to give brown adipocytes. The results of Oil Red O staining used for characterizing white and brown adipocytes demonstrated that white and brown adipocytes obtained from AD-MSCs showing 85% and 61% of red pixels, respectively, while it was 3% and 1.9%, respectively for white and brwon adipocytes obtained from UCB-MSCs. Brown adipocyte-specific UCP1 protein content, was determined by fluorescence microscopy and Western blot analysis. According to the results of the fluorescence analysis, brown adipocytes obtained from both UCB-MSCs and AD-MSCs contain more UCP1 protein than undifferentiated control cells but round shining bodies were only seen in cytoplasm of brown adipocytes obtained from AD-MSCs. According to the literature, they can be clustered mitochondria formed from large numbers of mitochondria that come together. The results of Western blot analysis show that brown adipocytes obtained from UCB-MSCs and AD-MSCs express UCP1 protein 4 and 10.64 times, respectively, more than undifferentiated MSCs (P<0.004). As a result, while AD-MSCs differentiated to white and brown adipocytes abundantly and efficiently, UCB-MSCs showed only a little differentiation tendency to adipocytes. According to the results obtained from this study, UCB-MSCs are not appropriate SC type to be differentiated to both white and brown adipocytes. So, they may not give positive results similar to AD-MSCs during treatment with adipocyte cell transplantation or in reconstructive surgery. In contrast, AD-MSCs may be a new promising therapeutic approach for treatment of diabetes, obesity and its related metabolic disorders because of their high efficiency in differentiation capacity to brown adipocytes. Further in vivo and clinical studies are needed in this regard.
Açıklama
Anahtar Kelimeler
Biyokimya, Biochemistry, Biyoloji, Biology, Biyoteknoloji, Biotechnology
