| BACKGROUND Male infertility has increased year by year, research onspermatogonial stem cell will show broad application in the field of malereproductive health. Spermatogonial stem cell transplantation has very largepotential clinical value to treat non-obstructive azoospermia, especially for theadolescents who have suffered from infertility caused by high-dose chemotherapyor radiotherapy.The spermatogonial stem cell transplantation has brought the hopeto many male patients with infertility and their families.Establishing an effectiveand long-term culture system of spermatogonial stem cell in vitro in order toobtain spermatogonial stem cells with the sufficient number and high purity,whichis an essential approach to ensure the spermatogonial stem cells to be transplantedeffectively. It is still the hot point to find a simple and effective culture method invitro of spermatogonial stem cell. In recent years, research on culture method invitro of spermatogonial stem cells have made some progress, but have not sought asimple and effective method yet. Therefore, how to establish a rapid and effectiveculture mehod of spermatogonial stem cells in vitro is still be one of the problemsto be resolved in the spermatogonial stem cell research.OBJECTIVE To investigate the effect of lycium barbarum polysaccharides onproliferation of spermatogonial stem cells cultured in vitro.METHODS Spermatogonial stem cells and sertoli cells were separated fromtestes of4-6days postpartum C57BL/6male mice by mechanical and two-stepenzyme digestion method. Sertoli cells served as feeder layer, Spermatogonial stem cells were seeded on the sertoli cells feeder layer and cocultured in vitro.Lycium barbarum polysaccharides and cytokines were added into cell culturemedium according to the difference of experimental groups.According to thedifference of cell culture medium,we established three groups,DMEM/F12－C ingroup A, DMEM/F12－C with100ug/ml LBP in group B,DMEM/F12－C with100ug/ml lycium barbarum polysaccharide（s LBP）,10ng/ml glial cell line-derivedneurotrophic factor（GDNF） and1000U/ml leukemia inhibitory factor（LIF）in group C.After one-week coculture,the cell activity rate and cell cycle ofspermatogonial stem cells were determined by flow cytometry.We used GFRa-1,Thy-1,c-kit as cell markers respectively, the positive rate of spermatogonial stemcells were detected by flow cytometry in each group.RESULTS The cell activity rate were detected by flow cytometry, experimentaldata were analysed through the SPSS11.5statistical software,t-test and ANOVAanalysis.The cell activity rate of spermatogonial stem cells was（63.42±4.24）%ingroup A,（87.16±0.88）%in group B and（97.27±1.59）%in group C（all P <0.05）respectively.The quantity of chromosome in S phase was （11.61±0.99）%in groupA,（16.38±0.78）%in group B and（16.59±0.74）%in group C,the value in groupA was less than that in group B and group C（both P <0.05）,but there is nosignificant difference between group B and group C （P＞0.05）.Using GFRa-1as the cell marker,the positive rate was （6.43±1.63）%in group A,（11.07±1.31）%in group B and （22.0±0.41）%in group C （all P <0.05） respectively.UsingThy-1as the cell marker, the positive rate was （29.89±0.53）%in group A,（36.85±0.64）in group B and （43.42±1.71）%in group C（all P <0.05）respectively.Using c-kit as the cell marker,the positive rate was （7.97±0.32）%ingroup A,（9.74±2.23）%in group B and （16.77±1.71）%in group C,the value ingroup C was greater than that in group A and group B（both P <0.05）,but there is no significant difference between group A and group B （P＞0.05）.CONCLUSION While the spermatogonial stem cells and sertoli cells arecocultured,adding LBP into the culture medium,or adding LBP,GDNF and LIF intothe culture medium together,both can promote the proliferation of spermatogonialstem cells in vitro.This culture system can achieve the purpose of expansion ofspermatogonial stem cells in vitro effectively. |
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