Sertoli' S Cell Culture And The Establishment Of GDNF Gene Tm4 Cell Line

Study on the in vitro culture and differentiation of spermatogonial stem cells (SSCs) is the research hot topic of reproductive biology recently and the new way to elucidate the mechanism of spermatogenesis. Previous studies have been revealed that the proliferation of SSCs in vitro is supported by feeder cell layers and a lot of growth factors. In seminiferous tubule of testis, Sertoli cells, the only somatic cells, provide nutrients for spermatogenic cells and secrete a lot of kinds of growth factors including glial cell line-derived neurotrophic factor(GDNF) and fibroblast growth factor (FGF). Studies have been also suggested that GDNF was mainly secreted by Sertoli cells, and that the high concentration of GDNF enhance the proliferation of SSCs but the the low concentration of GDNF promote the differentiation of SSCs in virro culture. Therefore, in present study, we firstly tried to gain the pure cultures of Sertoli cells by isolation and culture of mouse Sertoli cells. Then, we cloned the gdnf gene from the cultured Sertoli cells, and transfected into cells of Sertoli cell line TM4. The aim of this study is to gain the TM4 cells over-expressing GDNF as the feeder cells for SSC culture in vitro.In order to obtain high purified Sertoli cell cultures, we improved the preparation method of Sertoli cells reported by previous researchers. Our improved method mainly consisted of two-step enzymatically digestion, gradual centrifugation and differential adherent selection. The experimental animal was KM pup mice of 0-5 days after birth. The purification of Sertoli cell cultures was examined through morphological observation and immunocytochemical analysis of Sertoli cells marker vimentin. For the molecular biology operating, the total RNA was extracted from cultured Sertoli cells by using Trizol reaget, the gdnf gene was cloned by RT-PCR method, and the cloned gdnf gene was recombined into pMD-T vector by T4 DNA ligase. And then the recombined vectors were transformed into competent E.coli cells of DH5, and the positive cloning of recombinant was screened. After gene sequencing, the gdnf gene was cloned again from pMD-T vector into eukaryotic expression vector pcDNA3.1, the recombinant plasmid was transfected into TM4 cells by lipidosome transfection method, and the positive cells were screened out by using G418. In order to examined the expression profile of gdnf gene in positive TM4 cells, we conducted immunocytochemcal analysis and Western bloting for GDNF protein.The morphological observation of testis cell cultures indicated that the characteristics of cultured cells were in accordance with that of Sertoli cells reported previously. The result of immnocytochemical analysis revealed that more than 98% of cultured cells were positive cells of vimentin, namely, the cultures were made up of high purification of Sertoli cells. We also observed that Sertoli cells was not long-term cultured in vitro, and only could be passaged for 3-5 times about 20 days. The result of denatural agarose elec-trophoresis indicated that the total RNA of Sertoli cells was free degradation. The result of agarose elec-trophoresis for cloned gdnf gene by RT-PCR method indicated that the band was consistent with expected length. The sequencing result indicated that the cloned gdnf gene was exactly correct, suggesting that the gdnf gene have been amplified. The result of double enzyme digestion for pcDNA3.1-gdnf plasmid indicated that the eukaryotic expression vector of gdnf was constructed exactly. The result of RT-PCR by using the total RNA form cultured positive TM4 cells and gene specific primes for gdnf indicated that the GDNF was expressed in positive TM4 cells. The results of immnocytchemical detection of GDNF and Western Blot all indicated that the GDNF was high expressed in positive TM4 cells at protein level.In conclusion, the present study reported that we isolated and cultured mouse Sertoli cells from pup mice purely and successfully, and that the gdnf gene was cloned from cultured Sertoli cells. Importantly, we obtained the transgenic TM4 cells high-expressing GDNF.