İnsan spermatogonial kök hücrelerin iki ve üç boyutlu In vitro kültürü
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Dosyalar
Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Erkek inferilite sebeplerinden olan azospermi, semen örneğinde hiç sperm görülmemesidir. Spermatogenez erkekte puberte ile başlayıp mitoz ve mayoz bölünme aşamalarından geçerek spermatid oluşumu gerçekleşir. Bu süreci spermatogonium hücreleri mayoz aşamasını kontrol eden faktörler ile ileri farklılaşma göstererek tamamlar. Azospermi olgularında testiküler sperm ektrasiyonu (TESE) ile testisden alınan örneklerde sperm taraması yapılır. Fakat TESE örneklerinde sperm gözlenemeyen olgularda yardımcı üreme teknikleri uygulanamamaktadır. Çalışmada sperm gözlenmeyen fakat spermatogonial hücrelerin olduğu TESE örneklerinden spermatogonial kök hücrelerin farklı kültür ortamlarında farklılaştırılması ve farklılaştırılan hücrelerin karakterizasyonu amaçlanmıştır. Üç boyutlu (3B) kültür ortamlarının in vivo ortamlara benzerlikleri açısından spermatogonium hücrelerinin farklılaşması için in situ organizasyona benzer bir mikroçevre sağlayabileceği öngörüsünden yola çıkılarak, farklı 3D kültür ortamlarının bu farklılaşma sürecine olan etkilerini incelemek hedeflenmiştir. Çalışmanın özgün değeri insan spermatogonium hücrelerinden sperm benzeri hücre farklılaşmasını sağlayacak kültür sistemlerinin karşılaştırılması ve farklılaştırılan hücrelerin karakterizasyonunun hem immunhistokimyasal hem de moleküler düzeyde analiz edilerek bu hücrelerin fonksiyonel olarak spermatogenetik seriye ait hangi hücre aşamasına farklılaştırılabildiğinin analiz edilmesidir. Tez çalışmasında Manisa Celal Bayar Üniversitesi, Aile Planlaması İnfertilite Uygulama ve Araştırma Merkezine başvuran non-obstrüktif azospermi ön tanısı almış, semen örneğinde sperm olmayan ve TESE sonrasında sperm gözlenmeyen 8 hastaya ait örneklerin bir kısmının rutin ışık mikroskobik incelemesi ve DIFF Quick boyaması yapıldı ve spermatogonium hücrelerinin varlığı tespit edildi. Geriye kalan örnekler ise bir gece tripsin/EDTA solüsyonu içerisinde +40oC'de inkübe edildikten sonra jelatinli kültür kaplarında Ham's F10 içerisinde kültüre edildi ve kültür kabına yapışmayan hücreler toplandı. Toplanan hücreler CD90+ MiniMAcs seperasyonu yapıldıktan sonra elde edilen spermatogonium hücreleri, geleneksel 2B, yumuşak agar (SACS) ve kollajen köpük doku iskeleleri içerisinde farklılaştırma kültür vasatı ile 3 hafta boyunca kültüre edildi. RT-PCR gen ekspresyon analizlerinde spermatogonial kök hücrelerden elde edilen komplementer DNA'lar kullanılarak c-kit, Sirtl, Aurora-B, Aurora-C, Jmjdla ve Akrozin gen ekspresyonları belirlendi. GPR125 CD90, c-kit, α6 integrin, β1 integrin ve akrozin dağılımı indirekt immünohistokimya yöntemleri ile analiz edildi. Üç farklı kültür ortamında ileriye farklılaşmanın immunohistokimyasal verileri değerlendirildiğinde, kollajen köpük doku iskeleleri, germ hücrelerin devamlılığı ve çoğalması için uygun olduğu, agarın ise 15inci günden itibaren spermatogenezin farklılaşmasını desteklediği gösterildi. İleriye farklılaşmanın sonuçlarına göre, primer spermatosit veya erken dönem sekonder spermatosit aşamasına kadar olduğu düşünülmektedir. Agar gurubunda GPR125 (15 ve 22 inci günlerde) immunoreaktivitesi zayıf olması Akrozinin şiddetli olması agarın özellikle gerem hücrelerin ileri farklılaşmasını düşündürdü. Özellikle, Akrozin, immunoreaktivitesi agar ve kollajende 15 inci günde şiddetli pozitif iken, 22 inci günde kollajende pozitifliğin devam etmesi kollajenin özellikle germ hücrelerinin çoğalmasını ve ileri farklılaşmasını desteklediğini düşündürmekte ve hipotezi güçlendirmektedir. Matrijel ile kültüre edilen TESE örneklerinden elde edilen spermatogonium hücrelerinde kültürün 2. Haftasında c-kit ekspresyonun azalmış, özellikle Aurora-C ekspresyonun en fazla olarak gözlenmesi kültür ortamının spermatogonium hücrelerinin farklılşamasını desteklediği düşünüldü.
Azoospermia, which is one of the causes of male infertility, is no sperm count in semen. Spermatogenesis begins in the male with puberty and ocur both mitosis and meiosis stages to result for spermatid formation. During this process, the spermatogonium cells complete their progressive differentiation with the factors controlling the meiosis stage. In azoospermia patient, testicular sperm extraction (TESE) in testes samples retrieval. However, assisted reproductive techniques can not be applied in cases with no sperm in TESE specimens. In the study, it was aimed to differentiate spermatogonial stem cells in different culture media and characterize differentiated cells from TESE specimens in which spermatogonial cells are not observed. It was aimed to investigate the effects of different 3D culture media on this differentiation process by predicting that three-dimensional (3D) culture media could provide a similar microenvironment for in situ organization for the differentiation of spermatogonium cells in terms of their compatibility with in vivo media. The work analyzes the characterization of differentiated cells at both immunohistochemical and molecular levels to determine which cell stage of the spermatogenetic line can be differentiated functionally by comparing the culture systems that provide sperm-like cell differentiation from human spermatogonium cells. The fact that different culture systems are being investigated for their differentiation is also being studied extensively in terms of molecular analyzes, making the project unique from the projects already in the literature. In the project proposal, some of the samples of 8 patients who had non-obstructive azoospermia pre-diagnosis referred to Celal Bayar University, Family Planning Infertility Practice and Research Center and whose sperm were not sperm samples and after TESE were observed, should be routinely light microscopically and DIFF Qick staining and presence of spermatogonium cells. The remaining samples are incubated in a solution of trypsin / EDTA overnight at + 40 °C, then cultured in Ham's F10 on gelatinous culture dishes and cells that do not adhere to the culture chamber collected. Collected cells separated with CD90+ MiniMAcs and they will culture with differentiation culture medium in conventional 2B, Soft Agar (SACS), Collagen foam tissue scaffolds. In RT-PCR gene expression analysis, the complementary DNAs obtained from spermatogonial stem cells were used to express c-kit, Sirtl, Aurora-B, Aurora-C, Jmjdla and Akrozin gene expressions determined. GPR125 CD90, c-kit, α6 integrin, β1 integrin and akrozin distribution analyzed by indirect immunohistochemical method. According to immunohistochemical analysis of forward triggering differentiation in three different culture were evaluated, it was found that collagen foam tissue scaffolds were suitable for the maintenance and proliferation of germ cells, agar was shown to support the differentiation of spermatogenesis from the 15th day. According to the results of differentiation, it is thought to be up to the stage of primary spermatocytes or early secondary spermatocytes. In the agar group, immunoreactivity of GPR125 (days 15 and 22) were silm and Akrozin was strongly positive, suggests that agar, especially effective for triggering the differentiation. Particularly, Acrosine, immunoreactivity in agar and collagen on the 15 th day, while strongly positive positive, On the 22 day, the collagen positivity continued. It suggests that collagen, especially effective for triggering proliferation and differentiation. This positiveness strengthens the hypothesis. Spermatogonium cells obtained from TESE samples cultured with matrigel in the 2nd week of culture decreased c-kit expression, especially, the highest observation of Aurora-C expression was thought to support the differentiation of spermatogonium cells in the culture medium.
Azoospermia, which is one of the causes of male infertility, is no sperm count in semen. Spermatogenesis begins in the male with puberty and ocur both mitosis and meiosis stages to result for spermatid formation. During this process, the spermatogonium cells complete their progressive differentiation with the factors controlling the meiosis stage. In azoospermia patient, testicular sperm extraction (TESE) in testes samples retrieval. However, assisted reproductive techniques can not be applied in cases with no sperm in TESE specimens. In the study, it was aimed to differentiate spermatogonial stem cells in different culture media and characterize differentiated cells from TESE specimens in which spermatogonial cells are not observed. It was aimed to investigate the effects of different 3D culture media on this differentiation process by predicting that three-dimensional (3D) culture media could provide a similar microenvironment for in situ organization for the differentiation of spermatogonium cells in terms of their compatibility with in vivo media. The work analyzes the characterization of differentiated cells at both immunohistochemical and molecular levels to determine which cell stage of the spermatogenetic line can be differentiated functionally by comparing the culture systems that provide sperm-like cell differentiation from human spermatogonium cells. The fact that different culture systems are being investigated for their differentiation is also being studied extensively in terms of molecular analyzes, making the project unique from the projects already in the literature. In the project proposal, some of the samples of 8 patients who had non-obstructive azoospermia pre-diagnosis referred to Celal Bayar University, Family Planning Infertility Practice and Research Center and whose sperm were not sperm samples and after TESE were observed, should be routinely light microscopically and DIFF Qick staining and presence of spermatogonium cells. The remaining samples are incubated in a solution of trypsin / EDTA overnight at + 40 °C, then cultured in Ham's F10 on gelatinous culture dishes and cells that do not adhere to the culture chamber collected. Collected cells separated with CD90+ MiniMAcs and they will culture with differentiation culture medium in conventional 2B, Soft Agar (SACS), Collagen foam tissue scaffolds. In RT-PCR gene expression analysis, the complementary DNAs obtained from spermatogonial stem cells were used to express c-kit, Sirtl, Aurora-B, Aurora-C, Jmjdla and Akrozin gene expressions determined. GPR125 CD90, c-kit, α6 integrin, β1 integrin and akrozin distribution analyzed by indirect immunohistochemical method. According to immunohistochemical analysis of forward triggering differentiation in three different culture were evaluated, it was found that collagen foam tissue scaffolds were suitable for the maintenance and proliferation of germ cells, agar was shown to support the differentiation of spermatogenesis from the 15th day. According to the results of differentiation, it is thought to be up to the stage of primary spermatocytes or early secondary spermatocytes. In the agar group, immunoreactivity of GPR125 (days 15 and 22) were silm and Akrozin was strongly positive, suggests that agar, especially effective for triggering the differentiation. Particularly, Acrosine, immunoreactivity in agar and collagen on the 15 th day, while strongly positive positive, On the 22 day, the collagen positivity continued. It suggests that collagen, especially effective for triggering proliferation and differentiation. This positiveness strengthens the hypothesis. Spermatogonium cells obtained from TESE samples cultured with matrigel in the 2nd week of culture decreased c-kit expression, especially, the highest observation of Aurora-C expression was thought to support the differentiation of spermatogonium cells in the culture medium.
Açıklama
Anahtar Kelimeler
İnfertilite, Spermatogonium, 3D Kültür Sistemi, Farklılaştırma, Karakterizasyon, Infertility, 3D Culture System, Differentiation, Characterisation
