| Background:Each year,millions of women world wide are diagnosed with cancer.About 10% of those women were in their reproductive years.Due to the recent advances in multimodality treatments and supportive care,almost 90% of young women with cancer can suvive when they are eraly-diagnosed,but risk long-term complications from their treatment.One of the most significant long-term sequelae of exposure to cytotoxic drug treatments is infertility,secondary to premature ovarian failure(POF)or primary ovarian insufficiency(POI).For young gynecologic oncology patients who have taken fertility preserving surgery,they may still suffer from fertility loss due to irreversible reproductive damage caused by aggressive chemotheropy.Losing the ability to have biological children has been seriously affect their quality of life of cancer survivors.As a result,the protection of ovarian reserve and prevention of infertility has become the primary quality of life issue facing patients and their physicians.Clinical studies and animal experiments show that in the process of chemotherapy of malignant tumor,chemotherapy drugs can damage ovarian structure damage,effect follicle initiation,growth,development and maturation.Different chemotherapy drugs are different in the way and severity of ovarian damage,but the specific mechanism is not clear.Current options for female fertility preservation in the face of cytotoxic treatments include gonadotropin releasing hormone therapy,hormone replacement therapy and fertility cryopreservation.However,these methods are limited by patient age and status,and can not be widely used in clinical treatment.With the development of regenerative medicine,stem cells have been used in the treatment of a variety of diseases,which play an important role in tissue repair and regeneration.At present,studies have shown that stem cell transplantation can repair the ovarian structure,improve ovarian function and improve fertility,but the mechanism is not clear.In recent years,the discovery and research of human amniotic fluid stem cells have opened up a new field of research for stem cells.Due to a wide source of amniotic fluid,small trauma of obtaining amniotic fluid,low immunogenicity,strong ability of proliferation and differentiation,and no ethical restrictions,h AFS cells have wide application prospect in the field of medicine.h AFS cells are expected to become a new method to protect and repair the damage caused by chemotherapy of ovarian,and give young female cancer patients hope for preserving fertility function.Objective:To study the biological characteristics and application safety of h AFS cells;To establish a stable and reliable rat model of cisplatin induced ovarian injury in vivo and cell model of cisplatin induced ovarian granulosa cell injury in vitro,and investigate the possible ways of cisplatin induced ovarian damage;To explore the feasibility and mechanism of h AFS cells' preventive protection and therapeutic repair on cisplatin induced ovarian damage in rats,and also to research the feasibility and possible approaches of h AFS cells in the repair of cisplatin damaged granulosa cells in vitro.This study will establish the theoretical basis to the prevention and treatments on POF caused by chemotherapy drugs,and provide new ideas and strategies to clinical protection of ovarian function.Methods:Part one: Isolation,culture,identification and biological characteristics of h AFSc1.Amniotic fluid specimens were collected by prenatal diagnosis or voluntary labor,and h AFS cells were collected and purified by differential adhesion and mechanical isolation.2.h AFS cells were identified by cell morphology observation,flow cytometry detection of stem cell surface markers and MTT test of cell proliferation ability.3.The biological characteristics of h AFS cells were analyzed by MTT test of cell proliferation ability before and after frozen,Western Blot detection on the expression of pluripotent stem cell markers Oct3/4,Nanog and Sox-2,and inducing the differentiation of h AFS cells into osteoblasts and adipocytes;4.The tumorigenicity of h AFS cells was detected by subcutaneous injection of h AFSc in nude mice to evaluate the safety.Part two: The study on the establishment of cisplatin induced ovarian damage model in rats and injury pathway8 weeks old SD rats with normal estrous cycle were randomly divided into 4 groups: control group(0.9% saline),low dose cisplatin treatment group(1.0mg/kg),middle dose cisplatin treatment group(1.5mg/kg),and high dose cisplatin treatment group(2.0mg/kg).Blood samples were collected in each group before the model was made.Cisplatin was injected intraperitoneally for 6 days.The rats were sacrificed at 7d,15 d,30d and 45 d to obtain blood samples,bilateral ovaries and uterus.1.During the experiment,the general state of rats was observed,the body weight was recorded and the survival curve was drawn;2.The effects of different concentrations of cisplatin on ovarian and uterine tissue injury were evaluated by gross observation,HE staining and follicle count;3.The levels of FSH and E2 in serum of rats were detected by Elisa method to analyzed the effects of cisplatin on ovarian endocrine function with different concentrations;4.TUNEL staining and Western Blot method were used to detect the apoptosis of ovarian tissue and the expression of FSHR protein in rats,and explore the possible ways of cisplatin on ovarian damage.Part three: The study on the prevention and treatment of cisplatin induced ovarian damage in rats by h AFSc transplantation in vivoThe third generation h AFS cells labeled with PHK26 fluorescent dye were used for the treatment and prevention experiment of h AFS cells transplantation.The 8 week old SD rats with normal estrous cycle were included in the experiment?In the h AFS cells treatment experiment of ovarian injury,rats were randomly divided into 3 groups: control group(0.9% saline),model group(1.5mg/kg cisplatin for 6 days),and h AFS cells treatment group(1.5mg/kg cisplatin for 6 days and h AFSc).For the first day after six days of cisplatin injection,h AFS cells were transplanted into the rats in treatment group by intravenous injection.Blood samples were collected from the tail tip at 1d,7d and 30 d,and the rats were sacrificed at 15 d and 45 d respectively to obtain blood samples,bilateral ovaries and uterus.1.During the experiment,the general state of rats was observed.The survival and localization of PKH26 labeled h AFS cells in ovary were observed by frozen section of ovary;2.The levels of FSH and E2 in serum of rats were detected by Elisa method to analyzed the effects of h AFS cells transplantation on ovarian endocrine function;3.The effect of h AFS cells on the prevention and repair of ovarian tissue in rats was evaluated by the gross of ovary,HE staining and follicle count;4.Western Blot method was used to detect the protein expression of PTEN,p-PTEN,Akt,p-Akt in PI3K/PTEN/Akt signaling pathway and downstream molecules FOXO3 a,p-FOXO3 a,GSK3?and p-GSK3?,to investigate the protective mechanism of h AFS cells transplantation preventively on primordial follicles during cisplatin treatment;5.The expressions of Bax and Bcl-2 were detected by Western Blot method o explore the effect of h AFS cells transplantation on cisplatin induced apoptosis in ovarian tissue.In the h AFS cells prevention experiment of ovarian injury,rats were randomly divided into 3 groups: control group(0.9% saline),model group(1.5mg/kg cisplatin for 6 days),and h AFS cells prevention group(h AFS cells and 1.5mg/kg cisplatin for 6days).For the second day before cisplatin injection,h AFS cells were transplanted into the rats in prevention group by intravenous injection.Blood samples were collected from the tail tip before h AFS cells transplantation and at 30 d,and the rats were sacrificed at 7d,15 d and 45 d respectively to obtain blood samples,bilateral ovaries and uterus.The methods were the same as those of h AFS cells treatment experiment of ovarian injury.Part four: The effect of h AFSc on cisplatin induced injury of rat granulosa cells in vitro1.The SD rat ovary granulosa cells were isolated under anatomical microscope and cultured primarily;2.Granulosa cells were identified by cell morphology observation and immunofluorescence staining detection of the expression of FSHR.The growth state of granulosa cells were analyzed by MTT test of cell proliferation ability;3.To select the appropriate cisplatin concentration and action time and establish an in vitro model of cisplatin induced rat ovarian granulosa cell injury,the effects of different concentrations(0mg/L,1.25mg/L,2.5mg/L,5mg/L,10mg/L,20mg/L)of cisplatin on the growth,apoptosis and specific protein expression of ovarian granulosa cells acting 24 hours or 48 hours were analyzed by the following methods: MTT method was used to detect the inhibition rate of granulosa cell proliferation,crystal violet staining was used to observe morphological changes of cells,and DAPI staining was used to detect the apoptosis of granulosa cells;4.The effect of cisplatin on the expression of FSHR protein in granulosa cells was detected using Western Blot method;5.h AFS cells were co-cultured with normal granulosa cells or cisplatin injured granulosa cells using Transwell chamber(8?m),and the ability of h AFS cells migrating to the damaged granulosa cells was analyzed by counting the number of h AFS cells migrating through the polycarbonate membrane;6.h AFS cells were co-cultured with normal granulosa cells or cisplatin injured granulosa cells using Transwell chamber(0.4?m).The ability and possible pathway of h AFS cells to repair injured granulosa cells were analyzed by TUNEL staining test of granulosa cell apoptosis and Western Blot detection on the expression of FSHR protein.Results:Part one:1.The purified h AFS cells were homogeneous and fibroblast like in morphology.The cells were arranged in a spiral or radial shape and grew colony-like;2.h AFS cells expressed pluripotent stem cell markers Oct3/4,CD117,SSEA-4,mesenchymal stem cell markers CD29,CD44,CD73,CD90,CD105 and MHCI class molecule HLA-ABC.h AFSc didn't express MHCII class molecule HLA-DR and hematopoietic stem cell markers CD34,CD45;3.The fifteenth generation of h AFS cells still maintained a high proliferative capacity,and there was no significant difference in the expression of pluripotent stem cell specific protein Oct3/4,Nanog and Sox-2 among different generation of h AFS cells;4.h AFS cells could differentiate into adipocytes and osteocytes;5.h AFS cells didn't have tumorigenicity.Part two:1.Cisplatin induced weight loss in rats,and high dose cisplatin group had a high mortality rate;2.Cisplatin could cause the increase of serum FSH and decrease of E2 in rats.On the forty-fifth day,FSH remained a high level and E2 remained a low level in the middle and high dose group,however,the FSH and E2 levels in low dose group had significant recovery already;3.Cisplatin could cause atrophy of ovary and uterus,and affected the number of ovarian follicles.On the seventh day,the primordial follicles and antral follicles in the ovary were reduced,while the number of preantral follicles and atresia follicles were increased;On the thirtieth day,the number of atresia follicles increased,while follicles at other stages were all reduced significantly.Besides,the ovarian stroma was lack of vessles and was interstitial fibrosis seriously;4.Cisplatin could induce the apoptosis of granulosa cells in growing follicles and decrease the expression of FSHR protein in ovary.Part three:1.PKH26 labeled h AFS cells were able to migrate and survive for a long time in cisplatin damaged rat ovaries;2.Preventive or therapeutic transplantation of h AFS cells could improve the level of FSH and E2 in rats,and the protective effect of preventive transplantation was better than that of therapeutic transplantation after cisplatin injury;3.Both of preventive and therapeutic transplantation of h AFS cells had protective and repairing effects on the ovarian structure.The number of primordial follicles and growing follicles in the preventive or therapeutic group significantly increased in comparison with the model group.Besides,the protective effect on primordial follicles is more obvious when transplant h AFS cells before cisplatin injury,which was better than the treatment effects of transplantation group;4.Compared with the control group,the relative value of phosphorylated form normalized by the amount of the respective total key proteins in PI3K/PTEN/Akt pathway was elevated significantly in cisplatin group,whereas this increase was significantly reduced by transplanting h AFS cells preventively;5.Compared with the control group,cisplatin could up regulate the expression of Bax and down regulate the expression of Bcl-2,whereas this change was significantly alleviated when transplanted h AFS cells.Part four:1.Primary ovarian granulosa cells of rats were polygonal,stellate or spindle,highly expressd FSHR antigen.After 1-2 days of incubation,the cells entered the logarithmic growth phase,and began to enter the plateau stage on the sixth day,and the cells began to degenerate on the eighth day;2.Cisplatin could inhibit the proliferation of granulosa cells and increase the apoptosis of granulosa cells.Cisplatin(2.5mg/L)could induce granulosa cells apoptosis in 48 hours with the detection of nuclear condensation,nuclear fragmentation,nuclear dissolution,and apoptotic bodies.After modeling,the cell death rate is low and the state is stable;3.Cisplatin could reduce the expression of FSHR protein in granulosa cells.Cisplatin(1.25mg/L)had no effect on the proliferation of granulosa cells,but could induce the decrease of FSHR expression in granulosa cells in 24 hours;4.h AFS cells can migrate to cisplatin damaged granulosa cells;5.h AFS cells could inhibit cisplatin induced apoptosis of granulosa cells,and up regulate the expression of FSHR protein in damaged granulosa cells.Conclusions:1.h AFS cells have strong and stable proliferation ability,low immunogenicity and no tumorigenicity.The application of h AFS cells is safe and there is wide application prospect in stem cell therapy.2.Through continuous intraperitoneal injection of cisplatin(1.5mg/kg)for 6 days,we can establish a stable animal model of cisplatin induced ovarian damage in rats.Cisplatin(2.5mg/L)acting for 48 h can be used to establish a stable model of cisplatin induced ovarian granulosa cell injury in vitro.The model is stable,reliable and repeatable3.Cisplatin has a damage effect on the ovary,which may be related to promoting the activation of primordial follicles,promoting the apoptosis of granulosa cells and decreasing the expression of FSHR protein.4.h AFS cell transplantation can preventive protect and therapeutic repair the damage of ovarian structure and function,and the effect of prophylactic transplantation is better than that of post transplantation,particularly in the significant protective effect on the primordial follicles.5.The protective effect of h AFS cells may be mediated by inhibiting the activation of the PI3K/PTEN/Akt signaling pathway,thereby reducing the activation of primordial follicle induced by cisplatin and maintaining ovarian reserve.6.The therapeutic effect of h AFS cells may be mediated by inhibiting the apoptosis of ovarian granulosa cells,increasing the expression of FSHR protein and maintaining the growth of follicles,thereby indirectly inhibiting the activation of primordial follicles. |
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