| Stem cells could be extensively found in the adult mammalian organs, now they could bedetected in a variety of tissues and organs. However, just in terms of female mammalian ovary,before2004, the traditional biological view is that stem cells do not exist in ovarian andfollicular database update does not occur simultaneously. But in2004, due to statistical mouseovaries statistics by Johnson, the existence of follicular database could updates. Number ofatretic follicle and non atretic follicle are disaccord, after that many articles found FemaleGermline Stem Cells, FGSCs could be detected in mice and humans. At the same time, theovaries also found in other types of stem cells: mesenchymal stem cells, theca cells, and stemcell characteristics with granulosa cells. Recently, ovarian factor caused infertility accountsfor about15%to25%, including ovarian dysfunction or premature ovarian failure, and mostimportantly infertility diagnosis rate continues to increase. FGSCs could treat female ovariandysfunction and premature study provides a broad prospect. But now there exists lowseparation efficiency of FGSCs in mouse. Therefore, this research utilized porcine for thestudy, to explore a similar pattern as with the human animal, whether it also has FGSCs, anddetect these FGSCs specific positioning. And try to establish porcine FGSCs cell lines invitro. The results showed that: there may be a follicular capsular district female germline stemcells, in vitro serum-free culture system, energy-amplified with multiple features FGSCs. RFPlabeled lentivirus to transfect cultured FGSCs, busulfan transplanted to mice treated in vivo,two months after the labeled cells are still alive, but did not find it to differentiate into cells ofoocyte-like structure.1. Localization of germline stem cell in juvenile porcine ovaryPorcine ovaries after puberty processed into paraffin-embedded tissue sections, the use ofproven pluripotency and reproductive antibodies such as OCT4, SOX2, VASA, SSEA4, TERT,LIN28, CD49f, NANOG, C-KIT, C-MYC, SSEA1, CD133to find whether the pig ovariesalso has female germline stem cells. Mechanical isolated theca, ovarian granulosa cells andgerminal epithelium of three parts, by quantitative PCR and Western Blot to detect three partsand reproductive pluripotency gene and protein expression levels relative. And usingheterogeneous protein homologous OCT4OCT4A to be confirmed. The results show thatwith the female germline stem cell labeling FGSCs mainly located in the follicle capsularplace, such tag may make people rediscover follicle library update mechanism. 2. Isolation and detection of FGSCs in vitroIn this study, the use of mechanical separation method FGSCs, through in vitro serum-freemedium without feeder layer formed after cell clone, clone group formed afterimmunofluorescence staining found that the same expression in reproductive and pluripotencymarkers. Clones were picked by mechanical after collagenase digestion after stabilizationpassaged8times, while isolated from the clone individual cells stained by OCT4A can still bedetected. Finally, PCR validation results show that mechanical separation of FGSCs in vitroserum stability under the conditions without feeder layer passaged several times and relatedmarks are not lost.3.Inducement of porcine FGSCs in vivo and in vitroRFP fluorescence labeled using a band to infect lentivirus FGSCs. And separated from thecomplete removal of follicular granulosa cells into the empty capsule was prepared, theexpression of RFP FGSCs digested and transferred to the previously prepared an empty bag,and with transplanted into busulfan treated mice prepared under the kidney capsule. Twomonths later the mice were sacrificed grafts prepared as paraffin, after identification, cellsexpressing RFP found still exist, and those with the same cells expression of RFP andreproduction pluripotent markers, but did not detect these cells itself can differentiate intooocyte-like cells, and on mice ovaries did not find repair. |
PDF Full Text Request