| Background:Premature ovarian failure (POP) is defined as amenorrhoea, infertility, decreased estrogen level and elevated gonadotropin level before 40 years due to certain reasons. Patients with POF can suffer from a series of climacteric symptom, such as hot flashes, hyperhidrosis, irritability insomnia, fatigue, emotional instability, memory decay, hypomnesis, vaginal dryness, dyspareunia, urgent urination, osteoporosis and so on due to lacking of estrogen. And most women will suffer from infertility, which will lead to great pain both physically and metally. Laboratory examination indicate FSH and LH is persistently above 40IU/L,with E2 usually below 50-70pmol/L. Through transvaginal ultrasound, the uterus and ovaries of POF patients are smaller than normal women of periodofduration, and there are no follicle or few ones no more than 10mm in the ovaries.There are neither no follicle growth nor maturity during continuous monitoring.The etiology of POF is complicated, it may related to autoimmune dysfunction, chromosomal abnormalities, genetic factors, metabolism, infectious factors or iatrogenic factors. In recent years, the incidence of carcinoma has an increasing tendency, and there are more and more patients suffering from ovarian deficiency and infertility caused by chemotherapy which becomes one of the important etiological factors of POF. The mechanism of follicles damaged by chemotherapeutics is unclear, but recent researches have proved that ovarian damage is closely correlated to the apoptosis of ovarian granulosa cells and oocytes. The prevention and treatment of chemotherapy-induced ovarian damage perplex clinical doctors. There is not effective treatment for infertility due to chemotherapy up to now. Currently the hormone replacement is the main method of treatment, which can ameliorate the symptoms in some patients. But it may lead to serious side effect for long time usage. As the improvement of significant long-term survival for cancer patients, the increased tendency of cancer incidence in young persons, it is urgent to find out a new way method to cure chemotherapy-induced premature ovarian failure.The Foxo3a transcription factors belong to the subfamily of the Forkhead transcription factor "O"family. Foxo3a is inhibited by PKB-mediated phosphorylation and nuclear exclusion. It has been found that Foxo3a is a crucial regulator and suppressor of follicular activation. Foxo3a-/-mice show a massive activation of primordial follicles. As a result of the massive activation of primordial follicles, these mice are depleted of functional oocytes at a considerably earlier age than wild type mice, and become rapidly infertile. Foxo3a can also up-regulation cyclin G2 to make cells cease at G0/G1 phase. The fertility life of female dependents on the amount of primordial follicles at birth and the rate of follicular activate. The primary cause of POF is the excessive depleted primordial follicles. Therefore if we can up-regulate Foxo3a, primordial follicles activation will be suppressed, which means women's fertility life can be longer. It can also make granular cell cease at interphase, which can reduce the damage of chemomedic chemotherapy agents.As the development of molecular biology in recent years, gene therapy has become a new way for many incurable diseases. The key of gene therapy is to transduct the target gene into host cells safely and effectively, which requires a special vector. Adenovirus vector is of most use because of its advantages. Mesenchymal stem cells (MSCs) have advantages of convenience and auto-transplantation, and can avoid the immune rejection and ethics, moral problems. Therefore, the somatic stem cells have vast prospect of application. Our study intent to structure the defect type recombination adenovirus vector carring rat Foxo3a gene by using molecular biology methods, which can infect the rat mesenchymal stem cells, which provides a foundation for gene therapy for premature ovarian failure by Foxo3a gene.Objective:In order to go deep into study the therapeutic effect in studying chemotherapy induced ovarian failure, we intent to construct the recombinant adenovirus vector expressing the rat Foxo3a, and prepared adenovirus particle which have the infecting ability using corresponding package cell line. After that, we infected rat mesenchymal stem cells with recombination Foxo3a adenovirus, and observed the possible influence on MSCs. According to the results, we could deeply investigate the function and mechanism of Foxo3a, and would lay down basis for the research on Foxo3a's therapeutical effect on POF using animal model in future.Method:1. Using Overlap-PCR method, rat Foxo3a cDNA sequences was synthetized and cloned into pMD19-T vector. After plasmid extraction was done for the positive cloning, gene sequencing was performed. Homology analysis between sequencing result and GenebBank rat Foxo3a-cDNA sequence was performed by the means of the Internet software.2. The Foxo3a gene after purificated and reacted with restricted enzyme, which called XbaI and KpnI, then cloned into the pAdTrack-CMV which carring the green fluorecence protein (GFP).The new recombinant transfer vector plasmid is named pAdTrack-fox, after amplification, purification, restricted analysis and DNA sequencing, the new recombinant transfer vector plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E.coli strain BJ5183.3. After amplification and purification,the new recombinant adenoviral backbone plasmid then was transfected into HEK 293 cells by liposome 2000.The recombinant adenovirus Ad-fox was packaged and propagated in HEK293 cells with high titers, purification by cesium chloride gradient density centrifugalization. The titer was detected by tissue culture infective dose 50 (TCID50) method.RT-PCR was used to detect the Foxo3a mRNA in HEK293 cells. 4. MSCs cell line was infected with the integrated Foxo3a adenovirus. RT-PCR was performed to detect the expression of Foxo3a and cell growth curve assay was drew between infected cells and control cells.Results:1.Total RNA was extracted from rat spleen tissue and agarose gel electrophoresis showed three clear RNA band (28S,18S and 5S) and cDNA was obtained by reverse transcription, and then after PCR amplification, agarose gel electrophoresis showed about 2019bp DNA strip. The positive clones have been digested into 2019bp and 2700bp electrophoresis zone, and then the plasmid extracted were sequenced. Homology between the two sequencing results are 99.9% by homology analysis.2.The Foxo3a gene fragment was inserted into the same restrictive enzyme cutting points of a defect adenovirus plasmid vector. Through sequencing analysis, the Foxo3a gene of recombinant plasmid has two point mutation.To compare the Foxo3a gene data of GenBank, this mutation belongs to a silent mutation type completely and the coded amino acid is still arginine, which can not affect the protein expression of Foxo3a.3.The pAdEasy-fox was digested with Pac I to liberate linear adenoviral genomes, then transfected into HEK293 cells.To assess whether the packaged viral particles could be observed, the transfected cells were monitored daily for GFP expression by a fluorescence microscope.GFP expression was visible 24 hours after transfection. About 14 days post-transfection, most of the cells became pycnosis and floating.The cells were lysed with three consecutive freeze-thaw cycles, and the virus was collected from supernatant and proved by PCR. The titer of Ad-fox was 1.2×10-10 pfu/ml.4. MSCs cells were infected by the recombinant adenovirus with MOI of 10 and 100,10% and 90% of the cells expressing the GFP.The Foxo3a specificity 2019bp gene fragment was noted in the gene-transfer group,but not in the empty vector group,suggesting a successful tansfection of Ad-fox in MSCs cells and a specific expression of Foxo3a mRNA. There showed no significant difference in cell growth curve assay between infected cells and control cells. Conclusions:1. Successful cloning of rat Foxo3a gene in the experiment can lay the foundation for further study on the Foxo3a cDNA gene function and gene therapy.2. We have constructed successfully recombinant adenovirus vector carrying rat Foxo3a gene and green fluorescent protein gene and packaged high-titer, high-purity adenovirus.3. Foxo3a gene can be transducted into rat MSCs by recombinant adenovirus vector with continued expression. When MOI value is 100, infection efficiency is up to 90%, the cells in the best status of growth. Foxo3a gene of rats could effectively carry out the transcription. There showed no significant difference in cell growth curve assay between infected cells and control cells.The study lay down basis for the research on Foxo3a's therapeutical effect on POF using animal model in future. |
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