The Preliminary Study On Characteristics Of Sertoli Cells From Preadolescence Buffalo Testis In Vitro And Establishment Of FGF2 Overexpressing Sertoli Cells Lines

The buffalo Sertoli cells and their secretions have an important effect on the proliferation of buffalo spermatogonial stem cells.Sertoli cells encircle the spermatagonial stem cells,to provide a stable space for its growth,at the same time to participate in the formation of blood-testis barrier,which helps to provide the stability of the environment for spermatagonial stem cells and to provide physical support for the spermatagonial stem cells proliferation.GDNF and FGF2 which were secreted by Sertoli cells,played an important role in the proliferation of spermatogonial stem cells.FGF2 is an important component,in the process of stem cells' growth because it is essential for stem cells in a state of undifferentiated,and it can be the stimulants of cell division for the cells from mesoderm and ectoderm.This study explores the methods of isolation and IV purification of buffalo Sertoli cells,compares the prepubertal buffalo and adult buffalo Sertoli cells FGF2 expression differences and observes the effects of them as feeder layer cultivation for buffalo SSCs,builds the Sertoli cells FGF2 overexpression cell line,in order to establish the foundation for building up an optimal buffalo spermatagonial stem cells culture system in vitro.The research results of this dissertation are summarized as follows:Chapter 1:It researches the location and purification of the Sertoli cells of buffalo testis.The results showed that anti-DDX4 anti-DDX4 was used as a specific marker for germline cells,anti-WT1 was used as a marker for supporting cell-specific antibodies,and buffalo testis tissue was subjected to tissue immunofluorescence analysis.It was found that the cells of the prepubertal buffalo testis seminiferous tubules near the basement membrane location The cytoplasm was DDX4-positive,whereas the nucleus of cells in the testicular seminiferous tubule near the basement membrane showed WT1 positive,and these results were consistent with expectations.The differential planting method was uesed to purify the Sertoli cells,which were collected at different time periods.The real-time PCR and WB were used to analysis the transcript and protein expression level of the attached cells and suspension cells.The results showed that the expression of WT1 of suspension cells for 1h to 2h or 3h to 4h were decreased significantly,however,the expression of WT1 of attached cells for 1h was higher than the cells for 5h.The RT-PCR and WB results showed that the purified cells expressed the Sertoli cells markers such as WT1,GATA4,and SOX9 without the expression of mesenchymal cell marker 3?-HSD and peritubular cell-like cell marker a-SMA,and the protein expression of purified cells of WT1 was higher than pre-purification cells.Chapter 2:The differences in the expression of FGF2 in testicular Sertoli cells from buffalo at different ages and their effects on the culture of buffalo SSCs were studied.The real-time PCR was used to analyze the expression of transcripts in adult;buffalo testicular Sertoli cells and prepubertal buffalo testicular Sertoli cells.It was found that the expression of FGF2 and LIF in the adult testis Sertoli cells was significantly higher than that of prepubertal buffalo,the expression of GDNF and SCF difference between the prepubertal buffalo and adult buffalo testicular Sertoli cells were not significant.The adult testis Sertoli cells and prepubertal buffalo testicular Sertoli cells were co-cultured with SSCs in vitro,respectively.The expression of PLZF,NANOS2,GFRal of SSCs which were co-cultured with the prepubertal buffalo testicular Sertoli cells were higher than the SSCs which were co-cultured with the adult testis Sertoli cells.Chapter 3:The construction of FGF2 over-expressed buffalo testicular Sertoli cell line was conducted.PEGFP-N1 plasmid was used to construct PEGFP-N1-FGF2 recombinant plasmid.The recombinant plasmid was transfected into the sertoli cells by x-fect,and G418 was used for screening then FGF2 overexpression cell line was obtained.The expression of OCT4,PLZF,NANOS2,GFRal of SSCs which were co-cultured with the overexpressed FGF2 cell lines were higher than the SSCs which were co-cultured with the normal Sertoli cells.In summary,The differential planting method was uesed to purify the Sertoli cells,prepubertal buffalo testicular Sertoli cells serve as a feeder layer to facilitate the in vitro proliferation of buffalo spermatogonial stem cells,construction of FGF2 over-expressed buffalo testicular Sertoli cell line,and it serve as a feeder layer to facilitate the in vitro proliferation of buffalo spermatogonial stem cells.