| BackgroundPremature ovarian failure is a kind of disease referring to women in the age of 40 with amenorrhea, infertility, estrogen deficiency and gonadotropins elevation. Currently known reasons are genetic, antoimmune disease,ldiopathic disease and iatrogenic injury such as chemotherapy and radiotherapy which is the most important factor. As everyone knows, chemotherapeutic drugs inhibit cancer cell proliferation by promoting cell apoptosis to prolong patients’survival time. However, the reproductive toxicity of those drugs affect ovarian function seriously. Researches have shown that there are 70-90% women undergoing chemotherapy have different degree of ovarian damage. Premature ovarian failure not only reduces fertility and result in perimenopausal syndrome but also increase the patients’ psychological burden and lower living quality. With the trend of yonger women getting malignancy, we should try to protect their ovarian function.At present, the treatment and preventive measures of CIPOF are embryo preservation, ovary transplantation, hormonotherapy and so on. GnRH-a can reduce the sensitivity for ovarian cells to chemotherapeutic drugs by inhibiting the gonad axis, so as to avoid premature follicle activation. Because GnRH-a can stimulate transient elevations in gonadotropin levels promoting primordial follicle split into active and might cause ovarian cells more sensitive to chemotherapeutic drugs, thus the one to two weeks before chemotherapy using is better, which violate the principles of "early found, early treatment" in tumors. As well as, the security of GnRHa for some hormone-dependent tumor such as breast cancer is still an open question. Recently, stem cell therapy have become the focus in the prevention of ovarian function because stem cells have proliferation and differentiation potential, but the safety of its use needs to be further explored as well. Ovary or oocyte cryopreservation and transplantation technology has good prospects for development, but it was still in the stage of experiment and may not be widely applied due to the high cost. Therefore, the key to prevent CIPOF is not only reducing the sensitivity of ovary for chemotherapeutic drugs to reduce cytotoxic effects, but also ensuring efficacy on tumors at the same time.The main cause of mammalian ovarian function failure is a decline in both number and quality of follicles. The size for primordium follicle pool and atresia for growing follicles affect the reproductive age of female mammals directly. Granulose cells, surrounding oocyte, are the most important cells to stabilize ovarian endocrine and constitute normal follicle structure. Some studies found that the growth of granulosa cells started ahead of oocytes, which show that proliferation and differentiation of GCs is the key of primordium follicle activation and development. Researches shows that granulose cells arresting in Go/Gi phase is not sensitive to chemotherapeutic drugs, so it is significant to research some genes involved in regulating these processes on the ovarian function prevention. It is comfirmed that chemotherapy for ovarian damage related to drug categories, dosage and time, and patient’s age. In the early chemotherapy, drugs induce oocytese and granulose cells spoptosis and activate static follicles leading to excessive follicles deplention; in the late period, ovarian microcirculatory disorder and partial endocrine environment change cause follicle dysmaturity. Cisplatin is a common chemotherapy drug, can excessive activate primordium follicles though PTEN/AKT pathway and accelerate oocytes apoptosis, leading to decreases ovarian reserve function in rats and cause POF further. So how to avoid the excessive apoptosis of granulose cells and primordial follicle depletion is one of the most important research direction for ovarian protection in advance.FOXO transcription factors family have a regulatory roles for biological development, proliferation, caducity and tumor growth. Foxo3a transcription factor is the downstream genes in PTEN/PI3K/PKB(AKT) pathway. PKB can make Foxo3a phosphorylation and combine with 14-3-3 protein transferring from nucleus to cytoplasm and losing transcriptional activity. In one hand, Foxo3a can regulate cell apoptosis though Fas/Fasl, Bim, and Caspase, in the other hand, it can arrest cell cycle in G0/G1 phase by p27 in kinds of cells. Studies have shown that Foxo3a may play an important role in mammalian follicular development, but its regulation and mechanism remains controversial. Some scholars consider foxo3a gene is good to ovarian protection:Castrillon, Schneider and Pelosi found that Foxo3a-/-mice have poor fertility due to the depletion of primordial follicles, oocytes atresia and follicular stagnation. However, genetically modified rats have better ovarian reserve function and fertility than wild types. Meng Chunhua found that Foxo3a gene can inhibit pig granulose cells apoptosis. But some researchers disagree above:Liu.H discovered that over-expressed Foxo3a inhibit granulose cells proliferating by reducing expression of BMP-15 and Smad protein. Liu.L considered that Foxo3a gene overexpression in the oocytes cause oocyte and follicular developmental delays. The studies confirmed that oocytes apoptosis cause primordial and primary follicles atresia in early development of follicles in mammals, but the mature follicle atresia is started by granulose cells apoptosis and there is different regulatory mechanism in various phases. Furthermore, there are many programmed death-ligand system regulating the development and apoptosis of granulose cells and follicles.Above all, the purpose of our experimental study is to promote granulose cell arresting in G0/G1 phase of cell cycle and make preantral follicles static by over-expressing Foxo3a gene and then reduce ovary sensitivity to chemotherapeutic drugs and prevent CIPOF. Our study will over-express Foxo3a in rat ovarian granulose cells and preantral follicles culturing in vitro by recombinant adenovirus, observe it’s regulation and mechanism for granulose cells and preantral follicles and whether it can reduce the ovarian toxicity by cisplatin, and look for a new method for prevention and treatment of premature ovarian failure.Objective:1. Establishing rat Foxo3a gene over-expression vector, packaging and purificating of recombinant adenovirus; rat primary ovarian granulose cells culturing in vitro and exploring the best MOI for recombinant adenovirus and observe Foxo3a expression in target cells.2. Discussing the regulation and mechanism in rat ovarian granulose cell developing in vitro by over-expressed Foxo3a gene and observing whether Foxo3a can prevent CIPOF.3. Establishing rat preantral follicle culturing in vitro and observing the toxicity by cisplatin. Exploring the feasibility of recombinant adenovirus transfection for preantral follicles cultures in vitro.Methods:1. To construct recombinant adenovirus:Querying Foxo3a gene sequences of rats in the Genebank,constructing pUC57 vector containing the purpose gene, connecting it with adenovirus plasmid vector pDC315 though enzyme digestion reaction and ligation, and then take pDC315-rFoxo3a plasmid though bacterial transformation, collecting the positive clone and identifying it by DNA sequencing. The recombinant adenovirus AD-rFoxo3a were obtained with pDC315-rFoxo3a plasmid and framework plasmid cotransfected into HEK293 cells and identified by DNA sequencing. The method for determination of virus titer is TCID50.2. Rat ovarian granulosa cell was released by mechanical method from ovaries and cultured in vitro. HematoxylinEosin(HE)and immunohistochemical staining of FSHR were used for ovarian granulose cell identification. Determining the optimal MOI for granulosa cells. Foxo3a was detected by RT-qPCR and western-blot. The experiment was divided into three groups:AD-Foxo3a group,rAD group and Control group. Observing each group cell proliferation and drawing cell growth curve. Detecting expression of protein cyclinD, p27, Bax and Bim by western-blot. The cell apoptosis and cell cycle were detected by Hoechst33342/PI staining and flow cytometer. Adding cisplatin 24h after, cell apoptosis was detected by flow cytometer.3. Rat ovarian preantral follicle separating and culturing in vitro. Observing the morphological changes and atresia of follicles in normal environment and adding cisplatin after. Observing the expression of green fluorescent protein EGFP with fluorescence microscope and dectecting the expression of Foxo3a by RT-PCR after recombinant adenovirus transfecting.Results:1. In our study, the plasmid carrying Foxo3a gene of rat was digested by enzyme digestion electrophoresis, and the length of fragment (about 2000bp) was consistent with the length of rat Foxo3a gene found in genebank(2019bp), which was identified by sequencing and homology analysis was 99%. The recombinant adenovirus plasmids pDC315-rFoxo3a was confirmed by restriction enzyme digestion and sequencing. We found the viral plaques after 15 days cotransfecting on 293T cells by pDC315-rFoxo3a plasmid and framework plasmid, then collecting P1 virus at 21 days. The recombinant adenovirus AD-rFoxo3a was successfully constructed by agarose gel ecectrophoresis and DNA sequencing. The titer of recombinant adenovirus was 1.106×1010PFU/mL.2. Over 90% of the cultured cells were survived which containing more than 95% ovarian granulose cells. The infection rate of recombinant adenovirus was the highest and there was no obvious toxicity to granulosa cells when MOI was 50. The expression of Foxo3a mRNA was detected by RT-PCR and the molecular weigh of 80KD was found by Western-Blot in experimental group, while no expression was detected in the control group. The Foxo3a mRNA and protein was found 36h and 48h after Ad-rFoxo3a infection. Thus the Foxo3a was successfully induced by recombinant adenovirus AD-rFoxo3a in rat ovarian granulosa cells.3. (1)Plotting the cell growth curve, we found that the growth of experimental group of ovarian granulosa cells was inhibited compared with the two control groups, while the growth of blank control group and negative control group had no significant difference, showing S type growth curve both. (2) Cell proliferation of granulose cells can be inhibited by Ad-rFoxo3a, but whose apoptotic rate can increase and cell cycle can arrest in G1 phrase. (3) The apoptosis rates in treatment group added cisplatin were significantly increased as compared with those in control group. (4)We found that the expression of Bim、p27、CyclinD1protein were overexpression in experimental group, while there were no significant difference in expression of Bax. (5) The apotosis rate of ovarian granulosa cells in the experimental group was higher than that in the control groups, while there was no significant difference between the blank control group and negative control group.4. The preantral follicle isolated by mechanical method combined with enzyme digestion method from rat ovaries againsted after 24h cultured in vitro, and the emergence of follicular atresia such as oocyte escape, basement membrane rupture was found in average 7 days. The follicular atresia rate was increased signigicantly after cisplatin administration. In addition, there was no expression of EGFP in fluorescence microscopy or Foxo3a protein detected by RT-PCR with different titer of recombinant adenovirus transfection on preantral follicles.Conclusion:1. We successfully constructed recombinant adenovirus AD-rFoxo3a which having high titer and efficiency transtecting of rat ovarian granulosa cells in virto, and laiding the foundation for further study of the research on the function of Foxo3a gene and the effect on chemotherapy-induced ovarian damage.2. Overexpressing Foxo3a gene can promote granulose cell apoptosis by increasing Bim expression, and make cells arrest in Gi phrase of cell cycle by increasing cyclinDi and p27 expression. We cannot found the repair effects of overexpressing Foxo3a on the cisplatin-induced rat ovarian granulosa cells damage cultured in virto.3. In the study,we consider that the transfection efficiency of recombinant adenvirus on rat preantral follicles was very low, which may be related to the virus that can not be passed though the basement membrane of the follicle surface.4. The effect of Foxo3a on the regulation of follicular development and atresia as well as ovarian function depends on further in vivo experiments. |
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