微囊化卵巢细胞的雌二醇和孕酮释放研究

发布时间：2018-03-26 10:30

本文选题：细胞微囊化　切入点：颗粒细胞　出处：《浙江大学》2014年博士论文

【摘要】：妇女的卵巢功能在进入更年期后逐渐衰退,雌二醇(E2)和孕酮(P4)分泌水平下降,导致更年期综合症。临床上常用激素替代疗法(HRT)来缓解患者的症状,但是,该疗法频繁地用药给患者带来了经济负担,长期的HRT治疗还可能引起严重的副作用。本文研究卵巢细胞的微囊化与异体移植后体内E2和P4的释放,主要包括四部分： (1)以海藻酸钠和壳聚糖为材料,使用静电液滴发生法制备海藻酸-壳聚糖-海藻酸(ACA)微囊。通过改变静电液滴发生装置的针头内径、电极距离、电压和注射流速,摸索控制海藻酸钙凝胶微球直径的方法；以微囊膨胀率(Sw)作为机械强度的评价指标,研究壳聚糖浓度和交联反应时间对ACA微囊机械强度的影响；比较不同壳聚糖浓度制备的ACA囊膜的牛血清白蛋白(BSA)和γ-球蛋白的透过性能。结果显示,使用较小的针头内径,缩短电极距离,提高电压和降低注射流速,可以制备直径较小的海藻酸钙凝胶微球；ACA微囊的Sw在10分钟内随交联反应时间延长而降低,之后不再发生显著变化；：1.2%(w/v)壳聚糖溶液制备的ACA微囊具有较高的机械强度和较弱的BSA渗透性能；0.6%和0.3%(w/v)壳聚糖溶液制备的ACA微囊的机械强度和BSA渗透性能均无显著差异；三种浓度壳聚糖溶液制备的ACA囊膜对Y-球蛋白的阻隔能力无显著差异。 (2)以非洲爪蟾作动物模型,研究ACA微囊化卵巢细胞异体移植后的E2和P4分泌能力。用免疫组化法检测原代培养非洲爪蟾卵巢组织细胞的卵泡刺激素受体(FSHR)和黄体生成素受体(LHR)；用孕马血清激素(PMSG)处理原代培养细胞48小时,酶联免疫吸附法(ELISA)检测培养液E2和P4浓度；用1.2%,0.6%和0.3%(w/v)壳聚糖溶液分别制备3组卵巢细胞ACA微囊,在培养28天内用CFDA-SE/PI处理并测算细胞成活率,检测培养60天内0.3%(w/v)壳聚糖组的培养液E2和P4浓度；将0.3%(w/v)壳聚糖溶液制备的卵巢细胞ACA微囊移植入去势雌性非洲爪蟾腹腔,ELISA法检测60天内血清的E2和P4水平。结果显示,原代非洲爪蟾卵巢细胞的FSHR和LHR免疫染色呈阳性,在20IU/mL的PMSG处理时,培养细胞的E2和P4分泌水平最高；制备ACA微囊的最适壳聚糖浓度为0.3%(w/v)；在体外培养条件下,ACA微囊化卵巢细胞保持60天的E2和P4分泌,并在移植后60天内显著提高了去势雌性非洲爪蟾血清E2和P4水平；移植后回收的微囊约90%保持完整和光滑。 (3)大鼠卵泡颗粒细胞(GCs)和卵泡膜细胞(TCs)的共微囊化。用免疫组化法分别检测原代培养大鼠GCs和TCs的FSHR、LHR和CYP17A1;将GCs和TCs依不同数量比例混合并ACA微囊化,培养48小时后,检测培养液E2和P4浓度；将GCs和TCs数量比例为1：2的GCs/TCs微囊移植入去势雌性大鼠腹腔,检测60天内血清E2和P4水平。结果显示,GCs/TCs共微囊化显著提高了E2分泌水平,GCs/TCs数量比例为1：2时E2分泌水平最高,在移植后60天内将去势大鼠血清E2和P4维持在正常水平；移植后回收的微囊约90%保持完整和光滑。 (4)建立模拟卵泡自然结构的卵巢细胞微囊化模式。微载体培养大鼠GCs (GCsMs),分别制备包有GCs、GCs+TCs、GCsMs和GCsMs+TCs的ACA微囊并体外培养,WST-1法检测微囊化GCs和GCsMs在培养24小时内的生理活性；培养48小时后,检测培养液E2和P4浓度；将模拟卵泡(GCsMs+TCs微囊)移植入去势雌性大鼠腹腔,检测60天内血清E2和P4水平。结果显示,微囊化GCsMs的生理活性显著高于微囊化GCs；与其它三种微囊相比,模拟卵泡的E2分泌水平最高,移植后60天内将去势大鼠血清E2和P4维持在正常水平；移植后回收的微囊约90%保持完整和光滑。综上所述,本文以海藻酸钠和壳聚糖为囊材,用静电液滴发生法制备了卵巢细胞的ACA微囊,并分别在非洲爪蟾和大鼠上进行了同种异体移植实验,发现非洲爪蟾卵巢细胞微囊、大鼠GCs/TCs微囊,以及模拟卵泡在体外培养条件下都具有雌性激素分泌功能,移植后60天内能够持续释放E2和P4,为妇女更年期综合症的治疗开拓了新思路。
[Abstract]:Ovarian function gradually decline in women after menopause, estradiol (E2) and progesterone (P4) levels decreased, lead to climacteric syndrome. The common clinical hormone replacement therapy (HRT) to alleviate the symptoms, but the drug therapy frequently bring patients economic burden, long-term HRT treatment may also cause serious side effects. This paper studies the microencapsulated ovarian cells and transplantation of E2 in vivo and the release of P4, mainly includes four parts:
(1) using sodium alginate and chitosan as materials, the use of electrostatic droplet preparation of alginate chitosan alginate (ACA) microcapsules were occurred by changing the electrostatic droplet generator needle diameter, electrode distance, voltage and injection velocity, error control method of calcium alginate microspheres diameter; the micro expansion rate (Sw) as the evaluation index of mechanical strength. The effect of chitosan concentration and crosslinking time on the mechanical strength of ACA microcapsules; comparison of different concentrations of chitosan prepared ACA capsule of bovine serum albumin (BSA) and gamma globulin through performance. The results show that the needle diameter smaller, shortening the distance between electrodes to improve the voltage, and reduce the injection velocity, can calcium alginate gel microspheres with smaller diameter; ACA microcapsules Sw in 10 minutes with the crosslinking reaction time decreased, no significant change occurred after 1.2% (w/v); The preparation of chitosan solution ACA microcapsule has high mechanical strength and weak permeability for BSA; 0.6% and 0.3% (w/v) chitosan solution prepared ACA microcapsules mechanical strength and permeability of BSA had no significant difference; there was no significant difference between the three concentrations of chitosan solution preparation of ACA Capsule on Y- ball egg white blocking ability.
(2) in Xenopus animal model of ACA microencapsulated ovarian cells after allogeneic transplantation of E2 and P4 secretion ability. Detection of primary cultured Xenopus ovary cells by immunohistochemical method of follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR); with pregnant mare serum hormone (PMSG) treatment of primary cultured cells 48 hours, enzyme-linked immunosorbent assay (ELISA) detection of medium E2 and P4 concentration; 1.2%, 0.6% and 0.3% (w/v) of chitosan solution were prepared by 3 groups of ovarian cells in culture ACA microcapsules, within 28 days of treatment with CFDA-SE/PI and calculate the cell survival rate, cell culture 60 days 0.3% (w/v) E2 and P4 concentration in culture solution of chitosan group; 0.3% (w/v) chitosan solution prepared ACA microcapsules transplanted ovarian cells of ovariectomized female Xenopus was detected by ELISA within 60 days, serum E2 and P4 levels. The results showed that primary cultured Xenopus ovary cells FSHR And LHR showed positive immunostaining for PMSG, in 20IU/mL, the highest level of secretion of cultured cells of E2 and P4; preparation of ACA microcapsules the optimum chitosan concentration is 0.3% (w/v); in vitro, ACA microencapsulated ovarian cells maintained 60 days of E2 and the secretion of P4, and in 60 days after transplantation in ovariectomized female Xenopus significantly increased serum E2 and P4 levels were recovered after transplantation; about 90% remain intact and smooth.
(3) rat granulosa cells (GCs) and theca cells (TCs) were microencapsulated. Were detected by immunohistochemistry and cultured in GCs and TCs rats FSHR, LHR and CYP17A1; GCs and TCs according to different proportion of mixed and ACA microcapsulized, after cultured for 48 hours, the medium was detected E2 and P4 concentration of GCs and TCs; the ratio of the number of 1:2 GCs/TCs were transplanted into the abdominal cavity of ovariectomized female rats, detected within 60 days of serum E2 and P4 levels. The results showed that GCs/TCs co microencapsulated significantly increased the secretion of E2, GCs/TCs and the ratio of the number of 1:2 when the highest rate of E2 secretion in 60. Days after transplantation the serum E2 and P4 of castrated rats maintained at the normal level; the implantation recovered after about 90% to keep complete and smooth.
(4) establish ovarian cell microencapsulation model to simulate the natural structure of follicles. The microcarrier culture of rat GCs (GCsMs), were prepared by packets are GCs, GCs+TCs, GCsMs and GCsMs+TCs ACA were cultured in vitro and WST-1 assay, microencapsulated GCs and GCsMs in physiological training within 24 hours of activity; training 48 hours later, the medium was detected E2 and P4 concentration; the simulated follicles (GCsMs+TCs microcapsules) transplanted into the abdominal cavity of ovariectomized female rats, detected within 60 days of serum E2 and P4 levels. The results showed that the physiological activity of microencapsulated GCsMs was significantly higher than that of microencapsulated GCs; compared with the other three kinds of microcapsules, simulated follicular secretion of E2 the highest serum E2 and P4 of castrated rats maintained at normal levels 60 days after transplantation; transplantation of microcapsules recovered after about 90% to keep complete and smooth.
In summary, this paper using sodium alginate and chitosan as wall materials, ovarian cells of ACA microcapsules were prepared by electrostatic droplet generation, and respectively in Xenopus and rat by allogeneic transplantation experiments found that Xenopus microencapsulated ovarian cells, rat GCs/TCs microcapsules, and simulated follicles in culture conditions are female hormone secretion in vitro, sustained release of E2 and P4 within 60 days after transplantation, opens up a new way for the treatment of menopausal syndrome.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R943

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