To Discuss About Separation And Purification Methods Of The Rat Spermatogonial Stem Cells

With the socio-economic development and the change of people's lives, the incidence of male infertility has increased year by year .Because the pathogenesis of male infertility and some links aren't understood,the treatments of male infertility are faced with the situation of a single and ineffective means. At present, Azoospermia, caused by the decreased testicular function in male infertility has no precisely effective treatment.Spermatogonial stem cells (SSCs) are precursor in the formation process of sperm, which has eternal life ,many potential differentiation and the capacity of proliferation at testicular microcirculation. Available information confirmed that SSCs may continue to differentiate after transplanted in the allogeneic testis,which has immune exemption. The technology of SSCs transplantation make patients of testicular azoospermia produce sperm and obtain fertility,then through the common mode of couples to conceive naturally. In line with the ethics and traditional beliefs,it is easier conceived by patients.SSCs transplantation could be applied to the pre-adolescent and adolescence tumor patients,who need to radiotherapy and chemotherapy.Pre-adolescence,adolescence and middle-aged men's common malignancies includ Hodgkin's disease, testicular cancer, leukemia and so on.because of the increasing development of diagnostic technology and surgery, improving radiotherapy and chemotherapy,long-term survival or even cure of these patients become possible.However,spermatogenic function of these patients is also subject to serious damage in the treatment process.In view of this we could collect spermatogonial stem cells with cryopreservation to preserve their fertility before their treatment. SSCs transplantation also applies to patients with bilateral cryptorchidism,only found in adults. The spermatogenic function of these patients is serious damaged under the influence of high temperature,which manifest as less serious asthenospermia,even azoospermia.but the patients's testes still have the spermatogonial stem cells and support cells,which is not sensitive to hot. The treatment technology of SSCs transplantation can be used to restore fertility .At the same time,it applies to serious spermatogenic dysfunction patients. Recently, the study shows that autologous stem cells (adipose-derived stem cells,skin-derived stem cells,etc.)can be induced and differentiated to reproductive stem cells before transplantation. The application are conceived by couples in the common way.1 ObjectiveThe purpose of this study: 1.To approach the method of purification of spermatogonial stem cells (SSCs).2.To explore a simple and practical cultural method of SSCs, and reduce the training costs of SSCs.It is a system study,which autologous adult stem cells(adipose stem cells,skin stem cells,etc.) transplantation into the testes treatment asthenospermia,azoospermia. The study,as a pre-exploration,would provide valuable experiences for follow-up research to eventually achieve the purpose of curing testicular infertility.2 Materials and methods2.1 Experimental animalswe Selected 10 days after the birth of healthy Sprague-Dawley(SD) male rats, weighed 19 25g,and then divided randomly into three groups:Groupâ… :â‘ Cell suspension was isolated through mechanical method+two-step enzymatic method;Groupâ…¡:â‘¡Cell suspension was isolated through mechanical method+two-step enzymatic method + percoll separation method;Groupâ…¢:â‘¢Cell suspension was isolated through mechanical method+two-step enzymatic method+percoll separation method+differential attachment method.2.2 Experimental methods2.2.1 We got the 10 days after birth of the SD rats,killed from neck to death,then asepticly collected bilateral testes, prepared SSCs suspension throughâ‘ mechanical method+two-step enzymatic method;â‘¡mechanical method+two-step enzymatic method + percoll separation method;â‘¢mechanical method+two-step enzymatic method+percoll separation method+differential attachment method.2.2.2 Detection of c-kit expressionDealed with the received single cell suspension ,then added rabbit anti-mouse c-kit polyclonal antibody , added goat anti-rabbit IgG-Fluorescein Isothiocyanate(FITC). Took phosphate buffered saline(PBS) in place of the first anti in contrast,detected c-kit expression under fluorescence microscopy.2.2.3 Detection ofα₆-Integrin expressionDealed with the purified cell suspension ,then added rabbit anti-mouseα₆-Integrin polyclonal antibody, dropped ready to use biotinylated goat anti-rabbit (the secondary antibody)IgG,rinsed by PBS,dropped streptavidin- biotin- peroxidase compl (SABC), rinsed by PBS again,dropped diaminobenzidine(DAB)chromogenic solution,dropped three distilled water to terminate staining reaction,and then immersed in thanol to dehydrate,dropped xylene to transparent,neutral rubber mount,took PBS in place of the first anti in contrast,detectedα₆-Integrin expression under microscopy.2.2.4 Trypan blue exclusion testUsing cell counting plate,counted the received single cell suspension for living cells and dead cells respectively, calculated the rate of live cells.2.2.5 Detection of spermatogonium purity by flow cytometryFixed the received single cell suspension, rinsed by PBS, and then centrifuge to collect cells.added rabbit anti-mouse c-Kit polyclonal antibody,incubated in dark at 4℃overnight.After rinsed by PBS,added goat anti-rabbit IgG-FITC(fluorescence of second anti),incubated in dark at 4℃.rinsed by PBS twice, and then re-suspended the cells by PBS,detected by flow cytometry. At the same time, took PBS in place of the first anti in contrast,detected spermatogonium purity by flow cytometry before adjusting the corresponding negative control to zero in order to eliminate the background autofluorescence.2.2.6 The received single cell suspension were cultured and observed , immunohistochemistry identified,trypan blue exclusion and flow cytometry (FCM)tested.we compared and analysized the collected datas.3 Results3.1 The growth of cell suspension,received among Groupâ… ,Groupâ…¡and Groupâ…¢was in line with SSCs.3.2 Taking c-kit receptor as a specific marker of SSCs to immunohistochemistry identify,the result dispiays cell membrane and ambinuclear cytoplasmic have a strong expression of c-kit. Takingα₆-Integrin receptor as a specific marker of SSCs to immunohistochemistry identify,the result dispiays cell membrane and ambinuclear cytoplasmic have a strong expression ofα₆-Integrin. The two corresponding controls were negative.3.3 Trypan blue exclusion test displays the rate of live cells: Group I: (93.26±1.06)%,Group II: (93.50±0.85)%,Group III: (94.73±0.82)%; The results of the rate of live cells in three ways was no statistically difference (P> 0.05).3.4 Spermatogonium purities of single cell suspensions respectivly dispiays: Group I: (63.34±0.79)%; Group II: (74.98±0.26)%; Group III: (79.17±0.83)%. There is a statistically difference (P<0.05),the results shows that the purities have differences among the three ways:the purities of SSCs inâ‘¡way andâ‘¢way was higher than that inâ‘ way,â‘¢way higher thanâ‘¡way. the purity of SSCs inâ‘ ,â‘¡,â‘¢way has an increased trend(P<0.05).4 Conclusions4.1 The influence in the rate of live cells among mechanical method,two-step enzymatic method,percoll separation method,differential attachment method is little.4.2 The purity of SSCs inâ‘ way is relately high. Combined with Percoll separation ,it is higher.4.3 SSCs can be further enriched by differential attachment method afer mechanical method,two-step enzymatic method,percoll separation method.