The Study Of Isolation, Culture And Transgene Of Porcine Spermatogonial Stem Cells

In male mammals, spermatogonial stem cells (SSCs) located at the base of the seminiferous cord in the testis and close to seminiferous epithelium. SSCs are round or oval-shaped; the diameter of it is adout 12μm; ribsome is the only well developed organelle in the cytoplasm. There are two kinds of spermatogonial described as type A and type B. The term undifferentiated spermatogonia refers to As, Apr, and Aal cell types. As spermatogonia are stem cells that may divide into two identical daughter As spermatogonia as stem cells or into two Apr (paired) daughter cells,the Apr cells will differentiate to type B spermatogonial. The primary spermatocyte comes from the type B spermatogonial, and then become spermatozoa after maiosises. SSCs have some specific features that 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 stage of spermatogonial and produce spermatozoa at least, it can transfer genetic information to posterity. SSCs is the only diplontic"immortalization"cell that can copy it self in Adult male mammal. There already many investigation about sorting, cultivation, proliferation and differentiation. A new strategy that create transgene animal using SSCs as vehivle cell is payed much attention by scientist.We know little about the surface molecule character the mechanism controlling cell proliferation/differentiate because the research is not enough about SSCs, especially the research about SSCs of large mammal as livestock.This research planed to establish a technical foundation for using SSCs as vehivle cell of transgene, containing the isolation and culture of SSCs, identification of the surface molecule character et al. SSC was isolated from the pubescence Wuzhishan pig. Sertoli cell of testis tissue was used as feeder cell. Single cell suspension gained from testis after two steps enzymic digestion and then sorted according to different speed of adherence, most somatic cell was removed. SSCs can proliferation form a lot of cell clone cluster;the SSCs can go on its proliferation after passage procedure, it can maintenance proliferation and living for two monthes, but SSCs lost its morphologic characteristics after the fifth passage; We found that stem cell factors (SCF) can improve the proliferation obviously. The Wuzhishan porcine SSCs express stem cell characteristic molecule such as THY-1 (CD90), CD9, integrinβ-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 SOX2 which described as a pluripotency gene of stem scell, and c-kit did not express in SSCs cultured in the research. So it can concluded that the SSCs maintenance the state of undifferentiation. EGFP gene was successful transfered into SSCs using lipoid methord, and then green fluorescent was observed using microscope.The SSCs cell line had not establishmented in this research, but the two monthes cultivation and proliferation of SSCs is enough for performing some transgene research. The identification of the surface molecule character provided reference for high effect sorting of SSCs using immune methord. SSCs transgene technical is hoped to be a new way to create transgene animal and do some research in clone animal.