Glukagon benzeri peptid-1'in adipoz doku kökenli mezenkimal kök hücrelerin kardiyomiyosite dönüşümüne etkisi
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Dosyalar
Tarih
2020
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Amaç: Kardiyovasküler hastalıklar (KVH) dünya çapında en yaygın ölüm nedenlerinden biri olmaya devam etmektedir. Mevcut farmakolojik ve girişimsel tedavi prosedürlerinde önemli ilerlemelere rağmen, kalp hastalıkları uzun süreli ve kötü prognozla seyrederler. Mevcut yaklaşımlar her ne kadar semptomları iyileştirip kalp dokusunda geri dönüşü olmayan hasarı yavaşlatsa da, altta yatan problemi tamamen ortadan kaldırmazlar. Son yıllarda, hasarı önlemeye ve tedaviye yönelik tıbbi ve cerrahi yaklaşımların yanısıra hücresel tedaviler giderek önem kazanmaktadır. Mezenkimal kök hücreler (MKH), çeşitli protokoller kullanılarak in vitro koşullarda kardiyomiyositlere kolayca farklılaşabilir. Bununla birlikte, bu protokollerde kullanılan ajanların hücreler proliferasyonu üzerinde bazı olumsuz etkilere sahip olduğu bildirilmiştir.
Gereç ve Yöntem: Bu çalışmada kullanılan insan MKH'leri ticari olarak elde edildi. MKH'lerin kardiyomiyojenik farklılaşması, 10 μM azasitidin ile indüklendi. İlk olarak, azasitidin ve eksenatidin (10-250 nM) hücre canlılığı ve MKH'lerin proliferasyonu üzerindeki etkileri sitotoksisite deneyi ile analiz edildi. Daha sonra MKH'ler, farklılaşma prosedürü için 4 hafta boyunca azasitidin ve eksenatid ile inkübe edildi. Çalışma grupları, kontrol (DMEM), azasitidin (10 μM), eksenatid (100 nM) ve azasitidin (10 μM) + eksenatid (100 nM) şeklinde oluşturuldu. MKH'lerin morfolojik değişiklikleri 4 hafta boyunca izlendi. Daha sonra cTnI, GATA4 ve MYH7 gibi kardiyomiyojenik farklılaşma belirteçlerinin ekspresyonları immünohistokimya ile değerlendirildi. Ayrıca, hücre kültürlerindeki cTnI düzeyleri ELISA yöntemi ile ölçüldü. Veriler ortalama ± standart ortalama hatası olarak ifade edildi ve tek yönlü varyans analizi (ANOVA) ve post-hoc test ile değerlendirildi.
Bulgular: MKH‟lere azasitidin uygulamasının hücre canlılığını kontrol grubuna kıyasla (%54.4) anlamlı ölçüde azaltırken, azasitidin + eksenatid ile hücre ölümünün doza bağımlı bir şekilde azaldığı görüldü. Faz kontrast mikroskobu ile yapılan incelemede azasitidin ve eksenatid uygulaması yapılan kültürlerde kardiyomiyojenik farklılaşma ile uyumlu morfolojik değişiklikler gözlendi. Kardiyomiyojenik belirteçlerin immünohistokimyasal analizi, azasitidin ve eksenatid ile muamele hücrelerde cTnI, GATA4 ve MYH7 ekspresyonunda önemli bir artış olduğunu ortaya koydu. Ayrıca, azasitidin ve eksenatid ile ayrı ayrı ve birlikte muamele edilmiş kültürlerde cTnI seviyeleri, kontrole kıyasla daha yüksek bulundu (p<0.005 ve p<0.0005).
Sonuç: Sonuç olarak, bu çalışmanın bulguları, GLP-1 reseptör agonisti eksenatidin, azasitidinin neden olduğu hücre hasarını azaltarak ve hücre canlılığının devamını sağlayarak, insan MKH‟lerinin kardiyomiyojenik farklılaşması üzerinde faydalı olabileceğini düşündürmektedir.
Aim: Cardiovascular diseases (CVD) remain one of the most common causes of death worldwide. Despite important advances in current pharmacological and interventional treatment protocols, heart disease has a long-term and poor prognosis. Although existing approaches improve symptoms and slow down irreversible damage to heart tissue, they do not completely eliminate the underlying problem. In recent years, medical and surgical approaches to prevent and treat damage as well as cellular therapies are gaining importance. Mesenchymal stem cells (MSCs) can easily differentiate to cardiomyocytes in vitro conditions using various protocols. However, the agents used in these protocols have been reported to have some adverse effects on cell proliferation. Glucagon-like peptide-1 (GLP-1) is an incretin and plays a significant role in the physiological control of glucose metabolism and energy homeostasis. Therefore, GLP-1 receptor agonists, such as exenatide and liraglutide, are clinically use for the treatment of diabetes mellitus and obesity. In addition to its metabolic effects, it has been shown that GLP-1 receptor agonists have cytoprotective, antiapoptotic, antioxidant and proliferative effects on several types of cells and tissues such as neurons and cardiomyocytes. The aim of the present study was to investigate the effects of exenatide, a GLP-1 receptor agonist, on differentiation of human adipose derived MSCs into cardiomyocytes. Materials and Methods: Human MSCs utilized in this study were obtained commercially. Cardiomyogenic differentiation of MSCs was induced with 10 μM azacytidine. First, the effects of azacytidine and exenatide (10-250 nM) on cell viability and proliferation of MSCs were analyzed with cytotoxicity assay. Then, for differentiation procedure, MSCs were incubated with azacytidine and exenatide through 4 weeks The study groups were conducted as follows; control (DMEM), azacytidine (10 μM), exenatide (100 nM), and azacytidine (10 μM) + exenatide (100 nM). The morphological alterations of MSCs were monitored through 4 weeks. Finally, the expressions of cardiomyogenic differentiation markers, such as cTnI, GATA4 ve MYH7, were evaluated with immunohistochemistry. Alsa, the levels of cTnI in the cell cultures were measured using ELISA. Data were evaluated by one way analysis of variance (ANOVA) and post-hoc test. Data were expressed as mean ± standart error of mean. Results: Treatment of MSCs with azacytidine significantly decreased cell viability (54.4%) compared to control group whereas treatment of cells with azacytidine+exenatide prevented cell death in a dose-dependent manner. Examination with phase contrast microscopy revealed significant morphological alterations in azacytine and exenatide treated cultures consistent with cardiyomyogenic differentiation. Immunohistochemical analysis of cardiomyogenic markers demonstrated significant increase in expression of cTnI, GATA4 ve MYH7 in azacytine and exenatide treated cells. In addition, cTnI levels were found significantly higher in cultures treated separately and together with azascytidine and exenatide compared to control (p<0.005 and p<0.0005). Conclusion: Overall, the findings of the present study suggested that GLP-1 receptor agonist exenetide may have beneficial effects on cardiomyogenic differentiaon of human MSCs by reducing cell damage caused by azacytidine.
Aim: Cardiovascular diseases (CVD) remain one of the most common causes of death worldwide. Despite important advances in current pharmacological and interventional treatment protocols, heart disease has a long-term and poor prognosis. Although existing approaches improve symptoms and slow down irreversible damage to heart tissue, they do not completely eliminate the underlying problem. In recent years, medical and surgical approaches to prevent and treat damage as well as cellular therapies are gaining importance. Mesenchymal stem cells (MSCs) can easily differentiate to cardiomyocytes in vitro conditions using various protocols. However, the agents used in these protocols have been reported to have some adverse effects on cell proliferation. Glucagon-like peptide-1 (GLP-1) is an incretin and plays a significant role in the physiological control of glucose metabolism and energy homeostasis. Therefore, GLP-1 receptor agonists, such as exenatide and liraglutide, are clinically use for the treatment of diabetes mellitus and obesity. In addition to its metabolic effects, it has been shown that GLP-1 receptor agonists have cytoprotective, antiapoptotic, antioxidant and proliferative effects on several types of cells and tissues such as neurons and cardiomyocytes. The aim of the present study was to investigate the effects of exenatide, a GLP-1 receptor agonist, on differentiation of human adipose derived MSCs into cardiomyocytes. Materials and Methods: Human MSCs utilized in this study were obtained commercially. Cardiomyogenic differentiation of MSCs was induced with 10 μM azacytidine. First, the effects of azacytidine and exenatide (10-250 nM) on cell viability and proliferation of MSCs were analyzed with cytotoxicity assay. Then, for differentiation procedure, MSCs were incubated with azacytidine and exenatide through 4 weeks The study groups were conducted as follows; control (DMEM), azacytidine (10 μM), exenatide (100 nM), and azacytidine (10 μM) + exenatide (100 nM). The morphological alterations of MSCs were monitored through 4 weeks. Finally, the expressions of cardiomyogenic differentiation markers, such as cTnI, GATA4 ve MYH7, were evaluated with immunohistochemistry. Alsa, the levels of cTnI in the cell cultures were measured using ELISA. Data were evaluated by one way analysis of variance (ANOVA) and post-hoc test. Data were expressed as mean ± standart error of mean. Results: Treatment of MSCs with azacytidine significantly decreased cell viability (54.4%) compared to control group whereas treatment of cells with azacytidine+exenatide prevented cell death in a dose-dependent manner. Examination with phase contrast microscopy revealed significant morphological alterations in azacytine and exenatide treated cultures consistent with cardiyomyogenic differentiation. Immunohistochemical analysis of cardiomyogenic markers demonstrated significant increase in expression of cTnI, GATA4 ve MYH7 in azacytine and exenatide treated cells. In addition, cTnI levels were found significantly higher in cultures treated separately and together with azascytidine and exenatide compared to control (p<0.005 and p<0.0005). Conclusion: Overall, the findings of the present study suggested that GLP-1 receptor agonist exenetide may have beneficial effects on cardiomyogenic differentiaon of human MSCs by reducing cell damage caused by azacytidine.
Açıklama
Anahtar Kelimeler
Kök Hücre, Mezenkimal Kök Hücre, Yağ Dokusu, Kardiyomiyosit, Glukagon Benzeri Peptid-1, Stem Cell, Mesenchymal Stem Cell, Adipose Tissue, Cardiomyocyte, Glucagon-Like Peptide-1
