| Spermatogonial stem cell(SSC) can both maintain themselves by self-renewal and generate differentiated germ cells by committed differentiation. They are the only adult stem cells in the normal postnatal body that undergo self-renewal throughout life and transmit gene to progeny. In vitro culture of spermatogonia stem cell is an important way of studying spermatogenesis and its influencing factors,optimize the culture conditions,regulate cell culture procedures, clarifying cell number, cell proliferation, apoptosis and other biological characteristics, is the essential foundation of further research..But there are some influencing factors on survive and proliferation of the SSCs, and it is unclear about the culture condition and some gene aspect.So it is necessary to research the culture codtion,proliferation and apoatosis related gene.Using the combination enzyme digestion,selective adhesion method, we gained high purity spermatogonial stem cells in mice. And we designed conditioned medium,GDNF treatment medium and control medium to cultivate the spermatogonial stem cells. Using CASY cell viability analyzer, laser confocal microscope, alkaline phosphatase staining to identification and detect number of spermatogonial, marker of spermatogonial stem cell c-kit receptor protein,the expression of PCNA and Bcl-2 families'proteins.The results:1. Using the combination enzyme digestion,selective adhesion method,the purity of sertoli cells reached 90% higher.2. Using the CASY cell viability analyzer to determine the number and viability of spermatogonial, which showed that during the period of cell culture, the peak of cell proliferation at the third day and the seventh day. The cell number of condtioned medium group is higher than the GDNF treatment group during the period; At the seventh day of culture, there is an insignificant difference between GDNF treatment group and control group.3. Using lkaline phosphatase staining and c-kit protein detection to identify spermatogonial stem cell.4. Confocal microscope detection experiments showed that: At the first day of cell culture, About the expression of Bax, Bcl-2 and PCNA, there is less differences between condition medium group and GDNF treatment group; The expression of Bax in the condition medium group and GDNF treatment group is weaker than control group;The expression of Bcl-2 and PCNA in the condition medium group and GDNF treatment group is stronger than control group, suggesting that at the initial stage of cell culture, condition medium group and GDNF treatment group is in the same condition, both are better than control group.5. At the third day of cell culture, the expression of Bax in the condition medium group and GDNF treatment group is weaker than control group, the condition medium group and GDNF treatment group are in the same level; About the expression of Bcl-2 and PCNA, the condition medium group is stronger than GDNF treatment group, GDNF treatment group is stronger than control group, suggesting that at the first peak of proliferation, the condition medium group is better than GDNF treatment group, GDNF treatment group is better than control group.6. At the fifth day of the cell culture, the expression of Bax in the three culture system has little difference, and maintain at a high level. About the expression of Bcl-2, the condition medium group is stronger than GDNF treatment group, GDNF treatment group is stronger than control group; About the expression of PCNA, the condition medium group is stronger than GDNF treatment group and control group, GDNF treatment group and control group have the same condition. Suggesting that three groups of cell are at a high apotosis rate, the cell proliferation of conditioned medium group is better than GDNF treatment group and control group.7. At the seventh day of the cell culture, the expression of Bax in the conditioned medium group is lower than GDNF treatment group and control group, GDNF treatment group and control group have little difference; The expression of Bcl-2 and PCNA in the conditioned medium group is higher than GDNF treatment group and control group, GDNF treatment group and control group are in the same level. It is suggesting that at the second peak of proliferation, the conditioned medium group remained at a high level of proliferation, and apoptosis rate is not high; The proliferation of GDNF treatment group and control group is in the same condition, and at a higher level of apoptosis.These results displayed that, at a certain period of culture spermatogonial stem cells, conditioned medium could significantly promote cell proliferation, inhibiting cell apoptosis; GDNF could promote cell proliferation at initial stage, but the effect of GDNF on proliferation and apoptosis at the end of cultivate is not obvious.
|
PDF Full Text Request