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Isolation and Cultivation of Calf Sertoli Cells and Their Effection on Lymphocytes

Author: WuHaiJun
Tutor: JinYaPing
School: Northwest University of Science and Technology
Course: Clinical Veterinary Medicine
Keywords: Calves Sertoli cells FasL PBMC Inhibition
CLC: Q813
Type: Master's thesis
Year: 2007
Downloads: 65
Quote: 1
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Abstract


This test uses a different method of calf testicular Sertoli cells isolated and cultured in vitro , and to analyze the best separation and purification methods . Recovery after cryopreservation trials, observational support cryopreservation . Detect and support cells and their conditioned medium transformation of of calf PBMC and mouse splenocytes . Obtain the following results: 1 respectively I, IV collagenase and trypsin combined sequential digestion of bovine testis proved to 1g / L of type IV collagenase digestion for 10 min , 2.5 g / L trypsin digestion 8 min digestion method digested calf the support cells shortest , obtained in a large number of cells , cell viability above 95% . The 2 calves Sertoli cells adherent cultured for 3 h ~ 4 h after start , 5 h the medium was changed to remove most of spermatogonia , 24 h after , with 20 mmol / L Tris-HCl buffer ( pH 7.0 ) 3 min ~ 5 min hypotonic treatment , after 48 h to remove residual spermatogonia cells , the cells basically paved the monolayer . Isolated calf support cells 21 d after liquid nitrogen cryopreservation , remove the cultured cells to normal growth . The 3 calves Sertoli cells cultured 3 d ~ 4 d, closely connected to adjacent cells , the cells integrated into the film , and paved the membrane monolayer , the cytoplasm seen more particulate matter or vacuoles . After Feulgen staining , visible the intranuclear satellite nucleosome SABC immunohistochemical staining support cytoplasmic membrane was stained yellow , FasL expression . 4 calves Sertoli cells of lymphocyte activity experiments show support cells significantly inhibited the calf and mouse lymphocyte transformation . At the same time proved the supporting cells may directly or paracrine role . SHOP agarose between the two types of cells , to establish a new co - culture method . Through the test , a simple , easy calf Sertoli cell isolation and culture methods , which laid the foundation for in-depth study of the support cells . Inhibition of lymphocyte culture medium by measuring the Sertoli cells and its conditions , provided the experimental basis to reveal the role of Sertoli cells in the regulation of local immune privilege . The establishment of a new co-culture conducive to co-culture of research in the future .

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CLC: > Biological Sciences > Bioengineering ( Biotechnology ) > Cell Engineering
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