| Menopausal symptoms have a strong impact on self-esteem and quality of postmenopausal women life, especially for young premature ovarian failure (POF) women. Hormone replacement therapy (HRT) is used clinically, but its risks and benefits need to be carefully weighed. Organ and cells transplantations are accepted treatment of end-stage organ failure with endogenous hormone, but immunological rejection is the most important issues those faced. Microencapsulation protects transplanted grafts from immune attack, preserving grafts physical function and it seems to be a promising method for organ or cells transplantations alternative. Growing evidence have shown that endocrine glands, such as, pancreatic islets, parathyroid, and other functional cells are appropriate to be microencapsulated to treat the relative diseases. Especial noticeably, it has been used in clinic to treat diabetes and patients with symptomatic persisting postoperative hypoparathyroidism in clinic with microencapsulated human islets and microencapsulated parathyroid recently.The successful microencapsulated human islets and parathyroid allotransplantation opened opportunities for other endocrine disorders with similar pathogenesis.To explore the feasibility of treatment with microencapsulated ovarian granulosa cells in postmenopausal women, The present study planned to be carried out from three aspects as follows:1.investigate the morphologic and functional changes of ovarian granulosa cells in long-term culture in vitro; 2.evaluate the morphology and function of microencapsulated ovarian granulosa cells in vitro study; 3.determine the effect of microencapsulated ovarian granulosa cells on endocrine and reproductive system in ovarectomized rats after allogenetic transplantation.Part I The activity and function of ovarian granulosa cells in vitro studyObjective:To observe the morphology and the endocrine function of ovarian granulosa cells in serum-free system, and to find out the longest survival time and biological activity of ovarian granulosa cells in serum-free culture system in vitroMethods:primary ovarian granulosa cells were obtained from 26 days SD rats and identified by immunohistochemistry with FSHR. Proliferations of granulosa cells were assessed by WST with different concentrations of FSH (0,2.5,25,100 ng/ml) stimulated for 48 hours.Ovarian granulosa cells were cultured in vitro for 45 days, with different concentrations of FSH(0,2.5,25 ng/ml) added.The morphology and the volume of granulose cells were observed under the microscope. The E2 and P secretion of the granulosa cells were detected by ELISAResults:1. FSHR localizes in cytoplasm of ovarian granulosa cells, the positive rate of FSHR was more than 90% ovarian granulose cells obtained.2. Proliferations of granulosa cells response to FSH were in a dose-dependent style and OD value of FSH 100ng/ml group is the highest (P<0.05). 3. The time for attachment of primary ovarian granulosa cells was about 24 to 48 hours after harvested. Cellular fusion rate was about 100% on culture day 7-9.No visible changes occurred during the culture days 9-20. From day 21, cellular atrophy and apoptosis began to appear.Compared to the cells on day 9,only about 1/6 to 1/8 cells left on day 30, and most of cells disintegrated and cell structure disappear on the day 45. FSH had no significant effect on speeding up the adherence, integration or the role on cell apoptosis.4. Significant differences of E2 level were found between the culture day 3,9,15,21,27,33,39,45 (P＜0.001) and among FSH groups(P= 0.01). In FSHOng/ml and FSH2.5ng/ml groups, E2 secretion reached the peak (2244.6～2369.9) pg/ml on day 9 (P<0.05). On day 45, E2 level reduced to the level of day 3 (P> 0.05).On the day 3, E2 secretion were dose-dependent to FSH,and reached maximum in FSH 25ng/ml group (P = 0.016).5. Significant differences of P level were found among FSH groups (P= 0.035). In FSHOng/ml group, P secretion reached peak 1.07ng/m on day 9 (P= 0.006), and began to decrease on day 15, but maintained at (7.6～8.1) ng/ml level until day 45. Little difference was found during culture in FSH2.5ng/ml and FSH25ng/ml groups (P> 0.05).But on day 9, P level in FSH2.5ng/ml and FSH25ng/ml group were significantly lower than that of FSHOng/ml group (P= 0.021).PartⅡThe activity and the function of microencapsulated granulosa cells in vitro studyObjective:To observe the morphology and the endocrine function of microencapsulated granulosa cells in vitro study. Methods:Granulosa cells were microencapsulated in alginate-barium microcapsules and culutured in vitro. Proliferations of granulosa cells in microcapsules were assessed by WST with different concentrations of FSH (0,2.5,25,100 ng/ml) stimulated for 48 hours. Microencapsulated granulosa cells cultured in vitro for 60 days, with different concentrations of FSH (0,2.5,25 ng/ml) added. Observation of Morphology and the volume of granulose cells in microcapsules were done under the microscope. The E2 and P secretion of the microencapsulated granulosa cells were detected by ELISA method.Results:1. Microcapsules obtained appeared like a sphere, with diameter of 750-800μm and smooth appearance.The microencapsulated granulosa cells survived over 60 days in microcapsules and responded little to FSH in vitro.2. No significant difference of OD value was found among FSH groups in WST test (P=0.876).3.No significant difference of E2 level was found among the culture days 3,9,15,21,27,33,39,45,51,57and 60 (P=0.184) and among FSH groups (p=0.481). But on day 3, E2 secretion in FSH 2.5ng/ml group was significantly higher than those of FSH0 ng/ml and FSH 25 ng/ml group (P=0.005).4. Significant differences of P level were found among the culture days 3,9,15,21,27, 33,39,45,51,57,60 (P=0.002),but no similar difference was found among FSH groups (P=0.069). P level significantly increased on day 15(P=0.001), reached to the peak on day 21(P<0.001), reduced on day 45 in all groups. From day 45, P level kept stable until day 60. PartⅢThe effect of microencapsulated granulosa cells on reproductive system and endocrine function of ovarectomized rats in vivo after allogeneic transplantationObjective:To explore the feasibility of the alternative of HRT with microencapsulated granulosa cells allogenetic transplantation in postmenopausal womenMethods:Adult SD rats of 12 weeks were divided into 4 groups:normal group (N),ovarectomized only group (OVX),ovarectomized+microencapsulated granulosa cells transplantation group (OVX+T), ovarectomized+hollow microcapsule transplantation group (OVX+HT). Transplantation was carried out 4 weeks after rats ovarectomized.Observation period was 60 days. Blood sampling from tail vein was used to determine the level of E2 and P every 10 days after transplantation.Vaginal exfoliate cells were observed under light microscope every day after transplantation.The uterus of rats were collected for HE staining 60 days after transplantation, to evaluate the changes of endometrium and glands.Results:1.60 days after transplantation, the microcapsules remained intact structurally, and most of the microcapsules adhered and wrapped on the surface of peritonea viscera.2. E2 levels of OVX+T group reached the peak on day 30 after transplantion, nearing to the level of normal group (P>0.05). The levels of E2 on day 30 and 40 were significantly higher than the level before transplantation (P= 0.001, P= 0.021 respectively). Microencapsulated granulosa cells transplanted had no effect on P secretion in ovariectomized rats.3. Endometrium of OVX rats was thin and the gland disappeared after OVX. In OVX+T group, no significant endometrial thickened was found. However, glands with small glandular cavity in endometrium occurred again.4. Vaginal exfoliated cells of rats in OVX+T group were similar to those of late proestrus and estrus in normal group, but without cyclical changes.Conclusions:1 The survival time of ovarian granulosa cells in vitro is up to 45 days in serum-free culture. FSH stimulates E2 secretion of granulosa cells, but had no effect on P secretion.2 Granulosa cells microencapsulated survive and maintain endocrine function in the microcapsules at least 60days.The effect of FSH on E2 stimulation of microencapsulated ovarian granulosa cells is limited, and FSH has no effect on P secretion of granulosa cells in microcapsules.3 Microencapsulated granulosa cells survive in vivo at least 60 days. Microencapsulated ovarian granulosa cells increase E2 level of OVX rats transiently, improve the the state of estrogen deficiency on reproductive system in OVX rats... |
