Sıçan Kuyruğundan Ultra Hızlı Ekstraksiyon Yöntemiyle Tip I Kolajenin Eldesi ve Saflaştırılması
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Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Kolajen, çok hücreli hayvanlarda en çok bulunan proteindir ve lifli bir yapıya sahiptir.
Kozmetik cerrahi, dermal enjeksiyonlar, kemik aşılama, rekonstrüktif cerrahi gibi
uygulamalarda yaygın olarak kullanılmaktadır. Bu uygulamalarda kullanılan kolajen,
mevcut kaynaklardan geleneksel kolajen ekstraksiyon yöntemleri ile elde edilmekte veya
ticari kolajen kaynaklarından temin edilebilmektedir. Geleneksel ekstraksiyon
yöntemlerinin uygulanması yaklaşık on gün sürmektedir ve ticarileşmiş kolajen
kaynakları ise ekonomik olarak maliyetlidir. Bu durum geleneksel ekstraksiyon
metotlarına ilave olarak güçlü çalkalama ve santrifüj filtrasyon teknikleriyle geleneksel
ekstraksiyon metoduna nispeten yaklaşık 3 saatte tamamlanabilen ultra hızlı kolajen
ekstraksiyon yöntemlerinin önemini ortaya çıkarmıştır. Bu çalışmada erişilebilirliği ve
kolajen verimliliği yüksek olan sıçan kuyruğu kolajen kaynağı olarak seçilmiştir.
Geleneksel ekstraksiyon tamponları ile çalkalama adımları birleştirilerek ekstraksiyon
süresi azaltılmaya çalışılan bu yöntemle, az miktardaki (yaklaşık 5 g) numuneden yüksek
oranda saf, çözünür kolajen elde edilmiştir. Sıçan kuyruğu üzerindeki kıl kökleri
temizlenmiş ve 4o
C’deki dH2O ile dengelenmiştir. Daha sonra numune, kolajenöz
olmayan maddelerden sodyum asetat tamponu ile arındırılmış ve ekstraksiyon aşaması
için sodyum sitrat tamponu ile muamele edilmiştir. Elde edilen süpernatan, santrifüj
filtrasyon tüpü ile saflaştırıldıktan sonra SDS PAGE tayin metodu kullanılarak kolajen
varlığı gösterilmiştir. Jel görüntüleme sisteminde elde edilen görüntülerdeki bantlar
üzerinde yürüme mesafelerinin ölçümleri yapılarak moleküler ağırlık standart eğrisi
oluşturulmuştur. Yapılan literatür araştırması sonucu bant değerlerinin karşılıkları hangi
zinciri temsil ettiği bulunmuş, standart eğrideki denklem aracılığıyla görünen bant
değerleri, α1 (≅ 135 kDa), α2 (≅ 125 kDa), β (≅ 280 kDa), γ (≅ 300 kDa) olarak
hesaplanmıştır. İki α, bir β ve bir γ zincirine sahip çözünebilir tip I kolajen elde edilmiştir.
Sonuç olarak elde edilen kolajenin, birçok alanda katma değeri yüksek biyomedikal
malzeme olarak kullanılabileceği öngörülmüştür.
Collagen is the most abundant protein in multicellular animals and has a fibrous structure. It is commonly used in cosmetic surgery, skin injections, bone grafting, and reconstructive surgery. The collagen used for these applications is obtained from existing sources by conventional collagen extraction methods or from commercial collagen sources. Conventional extraction procedures take about ten days, and commercial collagen sources are very expensive. This situation has demonstrated the importance of ultra-rapid collagen extraction procedures, which include strong shaking and centrifugal filtration techniques in addition to conventional extraction procedures, which can be performed in about 3 hours compared to conventional extraction procedures. Rat tail was chosen as the collagen source, which has high accessibility and collagen efficiency. Using this method, which attempts to reduce extraction time by combining conventional extraction buffers with shaking steps, a high rate of pure soluble collagen was obtained from a small sample size (approximately 5 g). All hairs and hair follicles of the rat tail were cleaned and equilibrated with dH2O at 4o C. The sample was then freed from non collagenous substances with sodium acetate buffer. The sample freed from non collagenous substances was treated with sodium citrate buffer for the extraction step. The resulting supernatants were purified using centrifugal filters that yield highly pure collagen. The presence of collagen was detected by the determination method of the purified sample SDS PAGE. The molecular weight standard curve was constructed by measuring the migration distances of the bands in the gel images. As a result of the literature review, it was determined which chain the band values correspond to, and using the equation in the standard curve, the band values were calculated as α1 (≅ 135 kDa), α2 (≅ 125 kDa), β (≅ 280 kDa), γ (≅ 300 kDa). Soluble type I collagen was obtained having two α, one β and one γ chain. It has been shown that the collagen obtained can be used as a biomedical material with high added value in many fields.
Collagen is the most abundant protein in multicellular animals and has a fibrous structure. It is commonly used in cosmetic surgery, skin injections, bone grafting, and reconstructive surgery. The collagen used for these applications is obtained from existing sources by conventional collagen extraction methods or from commercial collagen sources. Conventional extraction procedures take about ten days, and commercial collagen sources are very expensive. This situation has demonstrated the importance of ultra-rapid collagen extraction procedures, which include strong shaking and centrifugal filtration techniques in addition to conventional extraction procedures, which can be performed in about 3 hours compared to conventional extraction procedures. Rat tail was chosen as the collagen source, which has high accessibility and collagen efficiency. Using this method, which attempts to reduce extraction time by combining conventional extraction buffers with shaking steps, a high rate of pure soluble collagen was obtained from a small sample size (approximately 5 g). All hairs and hair follicles of the rat tail were cleaned and equilibrated with dH2O at 4o C. The sample was then freed from non collagenous substances with sodium acetate buffer. The sample freed from non collagenous substances was treated with sodium citrate buffer for the extraction step. The resulting supernatants were purified using centrifugal filters that yield highly pure collagen. The presence of collagen was detected by the determination method of the purified sample SDS PAGE. The molecular weight standard curve was constructed by measuring the migration distances of the bands in the gel images. As a result of the literature review, it was determined which chain the band values correspond to, and using the equation in the standard curve, the band values were calculated as α1 (≅ 135 kDa), α2 (≅ 125 kDa), β (≅ 280 kDa), γ (≅ 300 kDa). Soluble type I collagen was obtained having two α, one β and one γ chain. It has been shown that the collagen obtained can be used as a biomedical material with high added value in many fields.
Açıklama
Anahtar Kelimeler
Çözünür kolajen, doku mühendisliği, hücre dışı matris, kolajen ekstraksiyonu, sıçan kuyruğu, tip I kolajen, Collagen extraction, extracellular matrix, rat tail, soluble collagen, type I collagen, tissue engineering
Kaynak
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Scopus Q Değeri
Cilt
Sayı
Künye
Erharman, A., (2022). Sıçan Kuyruğundan Ultra Hızlı Ekstraksiyon Yöntemiyle Tip I Kolajenin Eldesi ve Saflaştırılması. (Yüksek Lisans Tezi). Selçuk Üniversitesi, Fen Bilimleri Enstitüsü, Konya.
