| Spermatogonial stem cells (SSCs), the postnatal male germline stem cells, are not only capable of self - renewing and differentiating, but also the only adult stem cells in normal postnatal body that undergo self-renewal throughout life and transmit gene to offspring. SSCs located at the base of the seminiferous cord in the testis and close to seminiferous epithelium.They are round or oval-shaped; the diameter of it is adout 12μm; ribsome is the only well developed organelle in the cytoplasm. SSCs have some specific features which others adult stem cells lacked. SSCs is a cell group having high capability of self-renew and potentiality of differentiation. SSCs can differentiate to different stages of spermatogonial and produce spermatozoa at last.There are many investigations about sorting, cultivation, proliferation and differentiation in mice already. A new strategy that creates transgene animal using SSCs as vehicle cell has also been paid much attention by scientist.But we know little about the sorting ,cultivation and surface molecule character of the domestic animals, and the research is not enough about SSCs of pig. This research is the groundwork to make transgene animal.1.The SSCs are from the WZSP, and the histological characteristics of immune organs were observed by paraffin section and HE staining of the testis about 9-12 day,2 month old and 4 month old. We found that the morphology of germ cells were uniform in seminiferous tubules and did not enter the spermatogenesis. There are many SSCs, so we choose the testis from the pig of 9-12 day.2. In order to explore the different feeder layer on proliferation of SSCs, the experiment compared STO, PEF, MEF, Sertoli cells as feeder layer cultures, and ultimately chose the Sertoli cell feeder.3. Single cell suspension gained from testis after two steps enzymic digestion and then sorted according to Percoll density and different speed of adherence, most somatic cell was removed. The spermatogonium puirity to achieve above 82 % by percoll density and the spermatogonium purity by centrifugal elutriation were only 60 %, but the cells viability from the latter method was better.4. The study has optimised the culture system for long term maintenance spermatogonial stem cells by referring the research development in mouse stem cells. In view of higher serum concentration accelerated the proliferation of somatic cells, the 5 % of FBS was used. The growth factors, GDNF, LIF and bFGF, were added into the culture system. The cells proliferations were enhanced by growth factors,the shape and the size of colonies were also improved.5. Single cell suspension SSCs can proliferation form a lot of cell clone cluster. The SSCs can go on its proliferation after passage procedure and maintenance proliferation and living for two months, but SSCs lost its morphologic characteristics after the fifth passage. The Wuzhishan porcine SSCs express stem cell characteristic molecule such as THY-1 (CD90), CD9, intergrinβ-1 (CD29), Gfrα-1 and OCT4 according to the result of immune identification. SSCs total RNA was extracted and RT-PCR was performed to verification the immune result. The result of RT-PCR also revealed SSCs expressing some pluripotency gene of stem scell, and c-kit did not express in SSCs cultured in the research which can conclude the SSCs maintenance the state of undifferentiation.The pig SSCs can be proliferated in vitro culture at least 2 months in Sertoli feeder layer containing 10 ng/mLSCF,10 ng/mL LIF and 5 %FBS. The characterization of the surface molecule markers provided basic information for high effect sorting of SSCs using immune method.SSCs transgene technical is expected to be a hopeful way for creating transgene pigs.
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