Screening The Functional Genes Of Ovarian Aging And Further Investigating The Function Of Selected Targets By The Technology Platform Of Tumor Molecular Biology

We have payed more attention to the organ aging with the occurrence of the aging society. Perimenopausal symptom and several senium disease resulted from ovarian aging have affected women's healthy seriously. A series of molecular biotechnology platform were established during the exploration of the c-FLIP function in cervical adenocarcinoma. To screen the functional geens in the process of ovarian aging and analyse the mechanisim of the target gene in the follicular development which help to further investigate the molecular mechanism of ovarian aging by using the molecular biotechnology platform had been established.MethodsLaser capture microdissection was used to obtain the single follicle from the ovary. The amplified RNA which was extracted from the LCM cells was hybridized to the Affymetrix GeneChip Mouse Genome 430 2.0 Array and the signal was scanned. Immunohistochemistry, real time-PCR and Weston blot were used to test the location and expression level of target gene. RNA interference technology and monoclony antibody were used to downregulate the expression level of target genes. MTT and flow cellsorting were taken to test the proliferation and apoptosis ratio of the cells respectively. The size and morphology and its ulturestructure of the cells was observed with confocal microscop and electron microscop. Enzyme Linked Fluorescence Analysis was used to test the hormone level.Results(1) The proliferation of cervical adenocarcinoma cell Hela was inhibited and its apoptosis was induced as the result of cFLIP downregulated with the change of caspase-8 protein level. cFLIP downregulation sensitized the Hela cell to radiotherapy and chemotherapy.(2) 334 functional genes of ovarian aging were abtained by combinging LCM with cDNA array.(3) One of the 334 genes 898-a specifically expressed in ovarian follicle and its expression level reduced with the mouse development. The diameter of follicles cultured was smaller and hormone levels were higher in the proup of 898-a down-regulated compared with that of the control, while the proliferation and hormone secretion level of the granulose cells both were no significantly different between the two groups. The morphology of oocyte was abnormal and its ultrastructure was destroyed and the density of zona pellucida was much thicker and its projections were disappearance with 898-a down-regulated, while there was no change in granulose cells. The expression levels of oocyte specific genes were increased with 898-a downregulated, while that of the steroidogenesis enzyme and LHR were reverse. The gene expression level of the gap junctions between oocyte and granulose cell was decreased while the gene expression level of gap junctions between granulose cells was reverse.(4) PRLR expression level was increased significantly with the mouse development. The expression level of PRLR was increased during the process of follicular recruitment and selection while it was a little lower at the time of ovulation but still much higher than the control, than it increased again during the formation of corpus luteum.Conclusion(1) cFLIP plays an important effection on the biology behaviour and it may become a new target for the cervical adenocarcinoma therapy.(2) It is successful to obtain the key genes of ovarian aging with the tumor molecular biotechnology platform.(3) It is an effectively and feasible technique that combining LCM with cDNA array applied in the screening functional genes.(4) Granulose cells were differentiation to mature abnormally as the result of inhibition of oocyte function with 898-a downregulated which may be associated with the accelerated exhaustion of follicles during the ovarian aging.(5) PRLR may be involved in folliculogenesis through regulating the proliferation of follicular cells and may be associated with the formation and maintenance of corpus luteum in the mouse ovary.