微囊化卵巢颗粒细胞长期体外培养及同种异体体内移植的研究
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摘要
绝经及绝经后期是妇女卵巢功能消失及其后的一段很长的时期。绝经所带来的一系列症状严重影响了妇女的自信心和生活质量,尤其是对那些POF和卵巢切除手术的年轻妇女。
对绝经期的更年期综合征,临床上通常采用的是激素替代治疗(HRT)或雌激素替代治疗(ERT),来弥补雌激素缺乏所带来的一系列症状。但HERS和WHI的两份报告对HRT的受益／风险比的重新进行界定使全球范围内的HRT在过去的几年的应用产生了停滞甚至是倒退。因此,探讨可行的内源性激素替代治疗方法对解决无生育要求的绝经妇女,特别是对POF的妇女意义重大。
细胞移植技术可以用于治疗人类某种蛋白或激素缺乏的疾病。细胞微囊化技术可以克服细胞移植治疗中的免疫排斥反应。以往对细胞微囊化移植的研究主要集中于胰岛、嗜铬细胞、肝细胞、甲状旁腺细胞、甲状腺细胞等治疗内分泌／代谢疾病和神经系统疾病,而对同样具有内分泌功能的卵巢颗粒细胞微囊化治疗绝经后的雌激素缺乏相关症状的研究目前国内外尚无报道。
为探讨微囊化卵巢颗粒细胞移植治疗绝经综合征的可行性,本研究主要从以下几个方面进行了研究：1.卵巢颗粒细胞的获取及体外活性和功能的研究；2.微囊化卵巢颗粒细胞的制备及体外活性和功能的研究；3.微囊化卵巢颗粒细胞异体移植对同种异体去势大鼠内分泌功能和生殖系统的影响；为临床上POF及绝经妇女微囊化卵巢颗粒细胞的同种异体移植提供理论基础。
第一部分卵巢颗粒细胞的获取及体外活性和功能的研究
目的：观察卵巢颗粒细胞长期在体外无血清培养体系中形态和内分泌功能的改变,探讨卵巢颗粒细胞在无血清体系中最长生存时间和生物学活性。
方法：1从26日龄未成年雌性SD大鼠获取原代卵巢颗粒细胞,采用FSHR对颗粒细胞进行免疫组化鉴定；
2 WST方法测定颗粒细胞在不同浓度FSH(0,2.5,25, 100ng/ml)刺激48小时后的OD值,判断颗粒细胞对FSH刺激的增殖反应；
3卵巢颗粒细胞长期体外培养45天,培养液中加添不同浓度的FSH(0,2.5,25ng／ml),显微镜下观察颗粒细胞的形态和数量的变化,ELISA法检测颗粒细胞培养液上清中E2和P的分泌。
结果：1免疫组化的结果提示卵巢颗粒细胞的FSHR位于细胞浆,获得的颗粒细胞的FSHR的阳性率>90%；
2 WST方法检测到,体外培养的卵巢颗粒细胞的增殖对不同浓度的FSH(0,2.5,25,50,100ng／ml)呈剂量依赖性；WST作用后颗粒细胞的OD值随FSH浓度的增大而增大,FSH 100ng/ml组OD值最高,与其他组相比差异有显著性(P<0.05)；
3原代卵巢颗粒细胞24-48小时贴壁,贴壁后的成短梭形或多角形。第7-9天,细胞伸展成长梭形并完全融合；培养的第9天到第20天左右,细胞连接成片状,状态良好。从体外培养的第21天开始,细胞开始萎缩,凋亡,逐渐变得如干枯状,数量减少；至培养的第30天,细胞数量减少明显,剩余的细胞簇所占面积大约占六孔板每个孔面积的1／6至1／8左右。培养的第31天至45天,细胞凋亡的进程加快,到培养的第45天时,颗粒细胞大部分已解体,细胞结构消失。FSH2.5ng/ml和25ng／ml组未见明显加快颗粒细胞贴壁和融合,减慢颗粒细胞凋亡的作用。
4颗粒细胞在体外培养的第3,9,15,21,27,33,39和45天E2的分泌水平不同,差异有显著性(P＜0.001),而不同浓度的FSH对颗粒细胞分泌E2的影响差异也有显著性(P=0.01)。FSHOng/ml组和FSH2.5ng/ml组培养的卵巢颗粒细胞在培养的第9天,E2的分泌达到最高峰(2244.6～2369.9) pg/ml,与培养的第3天相比差异有显著性(P<0.05)；培养的第15天到第39天内,E2水平保持较高水平,变化不大；到第45天时,颗粒细胞E2的分泌水平降至第3天水平(P>0.05)。FSH25ng/ml组,在体外培养的第3天E2水平即达到最高峰2244.6pg/ml,第9天到第39天E2的分泌维持高水平,与第3天比差异无显著性(P>0.05)。第45天E2水平降低至1869 pg／ml,显著低于第3天(P＜0.05)。颗粒细胞体外培养第3天时E2的分泌水平对FSH浓度成剂量依赖性的增加,在FSH25ng/ml组分泌水平最高达2244.6pg／ml,差异有显著性(P=0.016)。
5颗粒细胞在体外培养的第3,9,15,21,27,33,39和45天P的分泌水平不同,差异有显著性(P=0.039)；FSH浓度对颗粒细胞分泌P的影响差异也有显著性(P=0.035)。在FSHOng/ml组中,颗粒细胞在培养的第9天达到最高峰1.07ng/ml(P=0.006),第15天开始下降,一直维持在(7.6～8.1)ng／ml左右直到体外培养的第45天；而在FSH2.5ng/ml和FSH25ng/ml组在各时间点颗粒细胞分泌的P水平差异无显著性(P>0.05)。在体外培养的第9天,FSH2.5ng/ml和FSH25ng/ml组P的产生量显著低于FSHOng/ml组,差异有显著性(P=0.021),其余各时间点不同浓度FSH对颗粒细胞产生P无显著影响(P>0.05)
第二部分微囊化卵巢颗粒细胞的制备及体外活性和功能的研究
目的：制备微囊化的卵巢颗粒细胞,探讨微囊化卵巢颗粒细体外培养的生物学特性。
方法：1应用海藻酸钠和氯化钡采用“一步成囊法”制备微囊化卵巢颗粒细胞。
2 WST方法测定微囊化颗粒细胞在不同浓度FSH(0,2.5,25,50, 100ng/ml)刺激48小时的OD值,判断颗粒细胞微囊化后对FSH刺激后的增殖反应。
3对微囊化卵巢颗粒细胞进行长期体外培养60天,培养液中添加不同浓度的FSH(0,2.5,25ng/ml),显微镜下观察微囊及微囊内细胞的形态和数量的变化,ELISA方法检测微囊化细胞培养液上清中中E：和P的分泌情况。
结果：1.结果显示所制备的微囊直径750-800μm,呈规则球形或椭球形,颗粒细胞在微囊内分布均匀。体外培养60天,微囊形态保持良好,微囊内颗粒细胞无明显增多,FSH添加对微囊内颗粒细胞数量无显著影响。
2.通过WST方法检测到,微囊化卵巢颗粒细胞在体外培养48小时FSH各组间的OD值无显著性差异(P=0.876)。
3.微囊化的卵巢颗粒细胞在体外培养的第3,9,15,21,27,33,39,45,51,57和60天E2的分泌水平相似,在各时间点的E2水平差异无显著性(P=0.184)。不同浓度的FSH对微囊内颗粒细胞分泌E2的影响差异也无显著性(P=0.481)。但在体外培养的第3天,FSH2.5ng/ml组微囊内颗粒细胞分泌E2水平显著高于FSH0ng/ml和FSH25ng/ml组,差异有显著性(P=0.005)
4.微囊化的卵巢颗粒细胞在体外培养的第3,9,15,21,27,33,39,45,51,57和60天P的分泌水平不同,差异有显著性(P=0.002),而不同浓度的FSH对颗粒细胞分泌P的影响差异无显著性(P=0.069)。三组微囊化的卵巢颗粒细胞在培养的第15天时P的分泌增加显著(P=0.001),在体外培养的第21天达到分泌的高峰(P<0.001),培养的第45天时,P的分泌开始下降,但仍保持稳定到培养的第60天。
第三部分微囊化卵巢颗粒细胞同种异体腹腔内移植对去势大鼠生殖系统和内分泌功能的影响
目的：观察微囊化卵巢颗粒细胞异体移植对去势大鼠血清中E2和P的分泌及子宫内膜、阴道脱落细胞的影响,探讨微囊化卵巢颗粒细胞异体移植作为激素替代治疗的可行性。
方法：12周龄成年SD大鼠分为4组,正常大鼠组,单纯去势组,微囊化卵巢颗粒细胞移植组、空微囊移植组。SD大鼠去势4周后腹腔内移入微囊化颗粒细胞和空微囊。移植后对各研究组的大鼠每10天尾静脉采血一次,分离大鼠血清,ELISA法统一测定血清中E2和P的水平；每天取阴道脱落细胞,在光镜下观察雌激素的影响；移植后60天,大鼠处死,采用HE染色方法对移植后大鼠的子宫进行染色,观察子宫内膜的变化。
结果：1移植后60天,大鼠处死后发现所移植微囊大部分粘附、包裹在腹腔脏器表面；回收的微囊形态完整,无破裂,表面被成纤维细胞、巨噬细胞等包裹。
2移植后大鼠体内随移植时间变化,E2分泌水平有显著性差异(P<0.001),各分组间E2分泌水平也有显著性差异(P=0.001)。微囊化卵巢颗粒细胞移植组大鼠在移植后的第30天,E2的水平达最高峰,接近正常组大鼠的E2的水平(P>0.05)。微囊化卵巢颗粒细胞移植组大鼠移植后第30天和第40天的E2的水平显著高于移植前水平(P=0.001,P=0.021),而其他时间E2的水平与移植前无显著差异(P>0.05)。微囊化卵巢颗粒细胞移植对去势大鼠的P分泌无影响。
3去势大鼠子宫内膜变薄、腺体萎缩,微囊化卵巢颗粒细胞移植后子宫内膜增厚不明显,但内膜内出现腺体,腺腔较小
4微囊化卵巢颗粒细胞移植组的阴道脱落细胞与正常大鼠的发情前期或发情后期的表现相似,但无周期性变化。
结论
1.卵巢颗粒细胞微囊化前后均可在体外无血清培养体系中长期存活,并保持内分泌功能,可以作为产生内源性雌激素来源的细胞用于细胞移植。
2.颗粒细胞微囊化前后,E2分泌量差异显著,可能与微囊化过程和微囊材料的生物相容性有关。
3.FSH对颗粒细胞促进E2分泌作用显著,但对微囊化后的颗粒细胞促进E2分泌作用有限,可能与微囊限制了颗粒细胞的增殖有关。
4.微囊化卵巢颗粒细胞异体移植后可以使去势大鼠血清中E2阶段性升高,对去势大鼠的生殖系统有雌激素样作用,改善去势大鼠的“低雌状态”,有可能成为绝经后激素替代治疗一种新的治疗手段。
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 study
Objective: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 vitro
Methods: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 ELISA
Results:
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 study
Objective: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 transplantation
Objective:To explore the feasibility of the alternative of HRT with microencapsulated granulosa cells allogenetic transplantation in postmenopausal women
Methods: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
对绝经期的更年期综合征,临床上通常采用的是激素替代治疗(HRT)或雌激素替代治疗(ERT),来弥补雌激素缺乏所带来的一系列症状。但HERS和WHI的两份报告对HRT的受益／风险比的重新进行界定使全球范围内的HRT在过去的几年的应用产生了停滞甚至是倒退。因此,探讨可行的内源性激素替代治疗方法对解决无生育要求的绝经妇女,特别是对POF的妇女意义重大。
细胞移植技术可以用于治疗人类某种蛋白或激素缺乏的疾病。细胞微囊化技术可以克服细胞移植治疗中的免疫排斥反应。以往对细胞微囊化移植的研究主要集中于胰岛、嗜铬细胞、肝细胞、甲状旁腺细胞、甲状腺细胞等治疗内分泌／代谢疾病和神经系统疾病,而对同样具有内分泌功能的卵巢颗粒细胞微囊化治疗绝经后的雌激素缺乏相关症状的研究目前国内外尚无报道。
为探讨微囊化卵巢颗粒细胞移植治疗绝经综合征的可行性,本研究主要从以下几个方面进行了研究：1.卵巢颗粒细胞的获取及体外活性和功能的研究；2.微囊化卵巢颗粒细胞的制备及体外活性和功能的研究；3.微囊化卵巢颗粒细胞异体移植对同种异体去势大鼠内分泌功能和生殖系统的影响；为临床上POF及绝经妇女微囊化卵巢颗粒细胞的同种异体移植提供理论基础。
第一部分卵巢颗粒细胞的获取及体外活性和功能的研究
目的：观察卵巢颗粒细胞长期在体外无血清培养体系中形态和内分泌功能的改变,探讨卵巢颗粒细胞在无血清体系中最长生存时间和生物学活性。
方法：1从26日龄未成年雌性SD大鼠获取原代卵巢颗粒细胞,采用FSHR对颗粒细胞进行免疫组化鉴定；
2 WST方法测定颗粒细胞在不同浓度FSH(0,2.5,25, 100ng/ml)刺激48小时后的OD值,判断颗粒细胞对FSH刺激的增殖反应；
3卵巢颗粒细胞长期体外培养45天,培养液中加添不同浓度的FSH(0,2.5,25ng／ml),显微镜下观察颗粒细胞的形态和数量的变化,ELISA法检测颗粒细胞培养液上清中E2和P的分泌。
结果：1免疫组化的结果提示卵巢颗粒细胞的FSHR位于细胞浆,获得的颗粒细胞的FSHR的阳性率>90%；
2 WST方法检测到,体外培养的卵巢颗粒细胞的增殖对不同浓度的FSH(0,2.5,25,50,100ng／ml)呈剂量依赖性；WST作用后颗粒细胞的OD值随FSH浓度的增大而增大,FSH 100ng/ml组OD值最高,与其他组相比差异有显著性(P<0.05)；
3原代卵巢颗粒细胞24-48小时贴壁,贴壁后的成短梭形或多角形。第7-9天,细胞伸展成长梭形并完全融合；培养的第9天到第20天左右,细胞连接成片状,状态良好。从体外培养的第21天开始,细胞开始萎缩,凋亡,逐渐变得如干枯状,数量减少；至培养的第30天,细胞数量减少明显,剩余的细胞簇所占面积大约占六孔板每个孔面积的1／6至1／8左右。培养的第31天至45天,细胞凋亡的进程加快,到培养的第45天时,颗粒细胞大部分已解体,细胞结构消失。FSH2.5ng/ml和25ng／ml组未见明显加快颗粒细胞贴壁和融合,减慢颗粒细胞凋亡的作用。
4颗粒细胞在体外培养的第3,9,15,21,27,33,39和45天E2的分泌水平不同,差异有显著性(P＜0.001),而不同浓度的FSH对颗粒细胞分泌E2的影响差异也有显著性(P=0.01)。FSHOng/ml组和FSH2.5ng/ml组培养的卵巢颗粒细胞在培养的第9天,E2的分泌达到最高峰(2244.6～2369.9) pg/ml,与培养的第3天相比差异有显著性(P<0.05)；培养的第15天到第39天内,E2水平保持较高水平,变化不大；到第45天时,颗粒细胞E2的分泌水平降至第3天水平(P>0.05)。FSH25ng/ml组,在体外培养的第3天E2水平即达到最高峰2244.6pg/ml,第9天到第39天E2的分泌维持高水平,与第3天比差异无显著性(P>0.05)。第45天E2水平降低至1869 pg／ml,显著低于第3天(P＜0.05)。颗粒细胞体外培养第3天时E2的分泌水平对FSH浓度成剂量依赖性的增加,在FSH25ng/ml组分泌水平最高达2244.6pg／ml,差异有显著性(P=0.016)。
5颗粒细胞在体外培养的第3,9,15,21,27,33,39和45天P的分泌水平不同,差异有显著性(P=0.039)；FSH浓度对颗粒细胞分泌P的影响差异也有显著性(P=0.035)。在FSHOng/ml组中,颗粒细胞在培养的第9天达到最高峰1.07ng/ml(P=0.006),第15天开始下降,一直维持在(7.6～8.1)ng／ml左右直到体外培养的第45天；而在FSH2.5ng/ml和FSH25ng/ml组在各时间点颗粒细胞分泌的P水平差异无显著性(P>0.05)。在体外培养的第9天,FSH2.5ng/ml和FSH25ng/ml组P的产生量显著低于FSHOng/ml组,差异有显著性(P=0.021),其余各时间点不同浓度FSH对颗粒细胞产生P无显著影响(P>0.05)
第二部分微囊化卵巢颗粒细胞的制备及体外活性和功能的研究
目的：制备微囊化的卵巢颗粒细胞,探讨微囊化卵巢颗粒细体外培养的生物学特性。
方法：1应用海藻酸钠和氯化钡采用“一步成囊法”制备微囊化卵巢颗粒细胞。
2 WST方法测定微囊化颗粒细胞在不同浓度FSH(0,2.5,25,50, 100ng/ml)刺激48小时的OD值,判断颗粒细胞微囊化后对FSH刺激后的增殖反应。
3对微囊化卵巢颗粒细胞进行长期体外培养60天,培养液中添加不同浓度的FSH(0,2.5,25ng/ml),显微镜下观察微囊及微囊内细胞的形态和数量的变化,ELISA方法检测微囊化细胞培养液上清中中E：和P的分泌情况。
结果：1.结果显示所制备的微囊直径750-800μm,呈规则球形或椭球形,颗粒细胞在微囊内分布均匀。体外培养60天,微囊形态保持良好,微囊内颗粒细胞无明显增多,FSH添加对微囊内颗粒细胞数量无显著影响。
2.通过WST方法检测到,微囊化卵巢颗粒细胞在体外培养48小时FSH各组间的OD值无显著性差异(P=0.876)。
3.微囊化的卵巢颗粒细胞在体外培养的第3,9,15,21,27,33,39,45,51,57和60天E2的分泌水平相似,在各时间点的E2水平差异无显著性(P=0.184)。不同浓度的FSH对微囊内颗粒细胞分泌E2的影响差异也无显著性(P=0.481)。但在体外培养的第3天,FSH2.5ng/ml组微囊内颗粒细胞分泌E2水平显著高于FSH0ng/ml和FSH25ng/ml组,差异有显著性(P=0.005)
4.微囊化的卵巢颗粒细胞在体外培养的第3,9,15,21,27,33,39,45,51,57和60天P的分泌水平不同,差异有显著性(P=0.002),而不同浓度的FSH对颗粒细胞分泌P的影响差异无显著性(P=0.069)。三组微囊化的卵巢颗粒细胞在培养的第15天时P的分泌增加显著(P=0.001),在体外培养的第21天达到分泌的高峰(P<0.001),培养的第45天时,P的分泌开始下降,但仍保持稳定到培养的第60天。
第三部分微囊化卵巢颗粒细胞同种异体腹腔内移植对去势大鼠生殖系统和内分泌功能的影响
目的：观察微囊化卵巢颗粒细胞异体移植对去势大鼠血清中E2和P的分泌及子宫内膜、阴道脱落细胞的影响,探讨微囊化卵巢颗粒细胞异体移植作为激素替代治疗的可行性。
方法：12周龄成年SD大鼠分为4组,正常大鼠组,单纯去势组,微囊化卵巢颗粒细胞移植组、空微囊移植组。SD大鼠去势4周后腹腔内移入微囊化颗粒细胞和空微囊。移植后对各研究组的大鼠每10天尾静脉采血一次,分离大鼠血清,ELISA法统一测定血清中E2和P的水平；每天取阴道脱落细胞,在光镜下观察雌激素的影响；移植后60天,大鼠处死,采用HE染色方法对移植后大鼠的子宫进行染色,观察子宫内膜的变化。
结果：1移植后60天,大鼠处死后发现所移植微囊大部分粘附、包裹在腹腔脏器表面；回收的微囊形态完整,无破裂,表面被成纤维细胞、巨噬细胞等包裹。
2移植后大鼠体内随移植时间变化,E2分泌水平有显著性差异(P<0.001),各分组间E2分泌水平也有显著性差异(P=0.001)。微囊化卵巢颗粒细胞移植组大鼠在移植后的第30天,E2的水平达最高峰,接近正常组大鼠的E2的水平(P>0.05)。微囊化卵巢颗粒细胞移植组大鼠移植后第30天和第40天的E2的水平显著高于移植前水平(P=0.001,P=0.021),而其他时间E2的水平与移植前无显著差异(P>0.05)。微囊化卵巢颗粒细胞移植对去势大鼠的P分泌无影响。
3去势大鼠子宫内膜变薄、腺体萎缩,微囊化卵巢颗粒细胞移植后子宫内膜增厚不明显,但内膜内出现腺体,腺腔较小
4微囊化卵巢颗粒细胞移植组的阴道脱落细胞与正常大鼠的发情前期或发情后期的表现相似,但无周期性变化。
结论
1.卵巢颗粒细胞微囊化前后均可在体外无血清培养体系中长期存活,并保持内分泌功能,可以作为产生内源性雌激素来源的细胞用于细胞移植。
2.颗粒细胞微囊化前后,E2分泌量差异显著,可能与微囊化过程和微囊材料的生物相容性有关。
3.FSH对颗粒细胞促进E2分泌作用显著,但对微囊化后的颗粒细胞促进E2分泌作用有限,可能与微囊限制了颗粒细胞的增殖有关。
4.微囊化卵巢颗粒细胞异体移植后可以使去势大鼠血清中E2阶段性升高,对去势大鼠的生殖系统有雌激素样作用,改善去势大鼠的“低雌状态”,有可能成为绝经后激素替代治疗一种新的治疗手段。
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 study
Objective: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 vitro
Methods: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 ELISA
Results:
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 study
Objective: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 transplantation
Objective:To explore the feasibility of the alternative of HRT with microencapsulated granulosa cells allogenetic transplantation in postmenopausal women
Methods: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
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