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Abstract This study aimed to compare the in vitro effects of laminin, fibronectin, collagen, and coculture with sertoli cells on the efficiency of chicken (Gallus gallus) spermatogonial stem cells (SSCs) proliferation. Chicken SSCs were isolated by two-step enzyme digestion and purified by gelatin differential adherent. Three passaged SSCs were inoculated on laminin, fibronectin and collagen coated culture dishes and co-cultured with sertoli cells. The efficiency of cell proliferation under each culture condition was detected by alkaline phosphatase activity(AKP), EDU(5-ethynyl-2'-deoxyuridine) staining and quantitative polymerase chain reaction (qPCR). Results showed that the number of SSCs AKP positive clones was highest in sertoli cells group with (32±3) clones each vision, while that of laminin group, fibronectin group and collagen group were (26±3), (24±2) and (23±2), respectively, and the later three groups showed no significant difference (P>0.05); EDU staining showed cell proliferation rate in sertoli cell group reached (92.82±2.15)%, which was significantly higher than that of other groups (P<0.01); qRT-PCR results showed that the expression of proliferation marker genes such as c-myc(myconcogene), Klf4(kruppel-like factor 4) and PCNA(proliferating cell nuclear antigen) was highest in sertoli cells, followed by laminin group, the expression of c-myc and Klf4 in fibronectin group were higher than that of collagen group, while PCNA was on the contrary. The results indicated that both matrix proteins and sertoli cells could promote the proliferation of chicken SSCs in vitro, and sertoli cells was identified as the most appropriate factor for in vitro cell proliferation, followed by laminin, fibronectin and collagen showed no significant difference (P>0.05). The results provide theoretical and experimental references for the further optimization of chicken SSCs culture system and understanding of SSCs proliferation mechanism in vitro.
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Received: 23 January 2014
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