| BackgroundPremature ovarian failure (POF) is defined as women have decreasing ovarian function from puberty to 40 years old. POF is diagnosed by serum follicle stimulating hormone (FSH)> 40IU/L, estradiol (E2)<25pg/ml and immature follicles atresia. It is charactrized by amenorrhea, infertility, and a series of low estrogen symptoms as like hectic fever, sweating, facial flushing, vaginal dryness, low libido and so on.With the developing the mechanism of ovarian follicles, the cause of POF can be dived into genetics, enzyme deficiencies, autoimmune and iatrogeny. Increasing survival rate of patients with tumor, clinicians pay close attentions to chemotherapy-induced POF.Chemotherapy is a common modality in some groups of patients with cancer and autoimmune diseases. It is widely used in clinic because of its great efficacy.Platinum currently used clinically in common, cisplatin (CDDP) is a preferred agent. The mechanism of CDDP damages the function of DNA in order to inhibit the copy of cells. With the increasing dose of CDDP, the side effects become a limited place in the management of clinical treatment.At present, it is known cisplatin's side effects is nephrotoxicity, the reproductive toxicity is increasing popular.Oocytes had been identified in the fetal periods, the average of 400 is mature. Follicle consists of oocyte, granulosa cells and theca cells, no longer increase after birth. The fate cycle of follicles:primordial follicles,primary follicles, secondary follicles develop into preantral follicles, antral follicles and eventually develop into the Graafian follicle. The preantral follicle is a crucial stage in follicle,with the active of replication in DNA. It is reported rat preantral follicles turn to be atresia significantly in intraperitoneal injection of CDDP in animal model. However, the preantral follicles are influenced on autocrine and paracrine. With complicated mechanisms. It is benefit for us to observe the process of preantral follicle in physiology and pathology.In recent years stem cells research behavior type understanding paroxysm occurrence development and expand new of treatment the method provided a brand-new of path. Stem cells is one type to have ego replication with many to divided potential of earlier period do not divide cell, according to source dissimilarity can is divided into embryo stem cells and become body stem cells. MSCs is a preferred agent of body stem cells, with the advantage that can not neglect and get material convenience, without ethics, law, morals problems. As a result, MSCs have vast of application foreground. Reccent research manifestation, MSCs can synthesize with secrete nourishment factor, cell factor and nerve protection factor, repair hurt of nervous tissue, treat extand myocardial disease and myocardial infaraction and divide into cardiomyocytes and vasular endothelial cells ect. It is lately reported MSCs and proximal tubule epthelial cells(PTECs) co-culture can be reduced the nephrotoxicity of CDDP. Well, MSCs and rat preantral follicles co-cultured and whether there is a protective effect on the injury of CDDP? The morphology of preantral follicles and the level of E2 how to change? We want to further in depth study.Objective1.In order to study the side effect of CDDP on rat preantral follicles, first of all,they are cultured in vitro, which survive up to 7 days. Rat preantral follicles are treated with cisplatin in culture medium. After 24 hours, the preantral follicles were observed under inverted microscope and fluorescent double staining Hoechst33342/PI, with chemiluminescence detection in culture medium of E2. Therefore, we can establish the cisplatin-induced rat preantral follicles model.2. Rat preantral follicles are cultured in a medium with the third generation of MSCs. The preantral follicles also are observed under inverted microscope and chemiluminescence detection in culture medium of E2 from CDDP treated 3 to 7days. All above are for further study of the mechanism of CDDP induced rat preantral follicles atresia.Methods1.14 days old female SD rats intraperitoneal injected with pregnant mare serum(PMSG:gonadotropin extract,30IU/only) were killed off after cervical spine, obtained under sterile conditions, bilateral ovaries, employing a-MEM+10% fetal calf serum in Petri dishes, with lml syringe connected to 29G needles 40 times in the body as mechanical separation of follicles under the microscope. The criteria of rat preantral follicles is a diameter of 120-150um, the oocyte in core, the granulosa cells clearly, the theca cells completely. The isolated of preantral follicles cultured in 24-well adding 1ml by a-MEM+10% FCS+ITS (5μg/ml insulin,5μg/ml transferrine, 5ng/ml selenium)+0.15mIU/ml hMG+1.5U/ml rhCG composition of culture medium,37℃,5% CO2,95% saturated humidity cultured for 24 hours.60 preantral follicles were divided into 5 groups:blank control,0.25μg/ml CDDP,0.5μg/ml CDDP, 1μg/ml CDDP,2μg/ml CDDP. After different concentrations CDDP have impacted on rat preantral follicle 24 hours, the morphological changes in microscope and fluorescent double staining Hoechst33342/PI and chemiluminescence detection of preantral follicles in culture medium of E2.2. After anesthesia, the tibia and femur of rat separated in sterile conditions, prepared into single cell suspension,1000 rev/separation 8 minutes, discard supernatant, add 10% FBS inα-MEM culture medium re-suspended cells, according purified adherent cells,with 5×105/ml of cells in wells. Examed surface CD29,CD34,CD44,CD45 of the adoption flow type cell instrument of expression of the third generations MSCs. And then, rat preantral follicles under sterile conditions were co-cultured with MSCs.30 rat preantral follicles were divided equally in two groups. (1) Experimental group:MSCs cultured in 24-well plates and washed twice with PBS, then rat preantral follicles were co-cultured adding 1ml medium consisted of a-MEM+10% FCS+ITS (5μg/ml insulin,5μg/ml transferrine,5ng/ml selenium) +0.15 mIU/ml hMG+1.5 U/ml rhCG. (2) Control group:Rat preantral follicles were cultured in the same medium without MSCs.37℃,5% CO2,95% saturated humidity.We observed the morphological changes preantral follicles in microscope; chemiluminescence detection of preantral follicles in culture medium of E2.3.90 rat preantral follicles were divided equally in three group. (1) experimental group:Take the third generation MSCs was inoculated into 24-hole culture plate. To be adherent growth of MSCs, will be under sterile conditions, isolated rat preantral follicles moved to culture plate. After 24 hours co-cultured, added 1μg/ml CDDP in the culture medium; (2) experimental control group:rat preantral follicles cultured lonely for 24 hours also added 1μg/ml CDDP; (3) The blank control group:rat preantral follicles were cultured without CDDP and MSCs. We also observed the morphological changes in microscope; chemiluminescence detection of preantral follicles in culture medium of E2.Results1.MSCs cultured for 72 hours with high majority the cell stick wall, shaped into a spindle, showed colony-like growth, became to be refraction poorly.Development of the third generation cell above 90% expression CD29 and CD44, but CD34 and CD45 present fminine gender, confirmtion is Chong quality stem cell.2.When CDDP concentration<1μg/ml, there is no significant morphological changes of rat preantral follicles about microscope and fluorescent double staining Hoechst33342/PI and the concentration of E2. When CDDP concentration 1μg/ml, the morphological changes of rat preantral follicles became larger in diameter, the theca cells were not clear, the oocytes also turned to be vague, PI also went through membrane and combined together the nucleus DNA.When CDDP concentration 2μg/ml, the rat preantral follicles became atresia clearly, they were observed loss of the normal structure, cell membranes were rupture, granulosa cells and oocytes escaped in microscope and fluorescent double staining Hoechst33342/PI.The concentrations of E2 in medium decreased significantly.3. Rat preantral follicles were survived up to 7 days in virto alone. When MSCs and rat preantral follicles were co-cultured for 24 hours, there were significant morphological changes in microscope, oocytes were clearly visible, located in the center of follicles. Rat preantral follicles with MSCs indicated that apparent stability in follicles, turned to be transparent, and 53.3% oocytes of the rat preantral follicles developed to germinal vesicle(GV). By contrast,60% rat preantral follicles cultured alone degenerated in the first five days, turned darken,losed structure, became vague, occurred rupture in membranes and only 26.7% of oocytes developed GV there. The concentrations of E2 in co-culture medium were significantly higher than control medium.4. Rat preantral follicles were survived up to 7 days in vitro in the experiment. Rat preantral follicles with MSCs were cultured for 24 hours after added CDDP concentration of 1μg/ml in culture medium. Also 24 hours later, we observed the morphological changes of rat preantral follicles and the concentrations of E2.The experimental group of rat preantral follicles, we observed the follicle walls clearly visible, oocytes located in the central follicles compared with the experimental control group and blank control group, the first 7 days preantral follicles still slightly dark, most of the follicle profiles were relatively clear and inside the oocytes were also clearly visible.The rat preantral follicles in experimental control group,they began to be darken, membrane of follicles turned unclearly in the third days, the most of rat preantral follicles were atresia in the first 7 days.Rat preantral follicles of the control group were slightly dark, but the follicles membrane with a clear outline,oocytes clearly visible in the center in the first 3 days; the majority of preantral follicles became larger in diameter and oocytes were not clear,but membrane of follicles were rupture in the first 7 days.The concentrations of E2 experimental group were statistically higher compared with other groups.Conclusions1.The results showed that with a concentration (1μg/ml),CDDP caused the rat preantral follicles to be early atresia in 24 hours. The model preantral follicles morphology and endocrine function in a noticeable change in human cisplatin-induced disease process are similar to premature ovarian failure.2. When MSCs and preantral follicles were cultured, it can enhance the rat preantral follicles survived in vitro, oocytes can develop to GV. It shows preantral follicles in co-cultured can enter the nucleus rapid proliferation phase, in order to prepare for meiosis finally to be the Graafian follicle.3. MSCs have a protective effect on cisplatin-induced injury of rat preantral follicles, promote preantral follicles of endocrine function, delay the early atresia of rat preantral follicles.
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