本文選題：細(xì)胞微囊化　切入點(diǎn)：顆粒細(xì)胞　出處：《浙江大學(xué)》2014年博士論文

【摘要】：婦女的卵巢功能在進(jìn)入更年期后逐漸衰退,雌二醇(E2)和孕酮(P4)分泌水平下降,導(dǎo)致更年期綜合癥。臨床上常用激素替代療法(HRT)來緩解患者的癥狀,但是,該療法頻繁地用藥給患者帶來了經(jīng)濟(jì)負(fù)擔(dān),長期的HRT治療還可能引起嚴(yán)重的副作用。本文研究卵巢細(xì)胞的微囊化與異體移植后體內(nèi)E2和P4的釋放,主要包括四部分： (1)以海藻酸鈉和殼聚糖為材料,使用靜電液滴發(fā)生法制備海藻酸-殼聚糖-海藻酸(ACA)微囊。通過改變靜電液滴發(fā)生裝置的針頭內(nèi)徑、電極距離、電壓和注射流速,摸索控制海藻酸鈣凝膠微球直徑的方法；以微囊膨脹率(Sw)作為機(jī)械強(qiáng)度的評價指標(biāo),研究殼聚糖濃度和交聯(lián)反應(yīng)時間對ACA微囊機(jī)械強(qiáng)度的影響；比較不同殼聚糖濃度制備的ACA囊膜的牛血清白蛋白(BSA)和γ-球蛋白的透過性能。結(jié)果顯示,使用較小的針頭內(nèi)徑,縮短電極距離,提高電壓和降低注射流速,可以制備直徑較小的海藻酸鈣凝膠微球；ACA微囊的Sw在10分鐘內(nèi)隨交聯(lián)反應(yīng)時間延長而降低,之后不再發(fā)生顯著變化；：1.2%(w/v)殼聚糖溶液制備的ACA微囊具有較高的機(jī)械強(qiáng)度和較弱的BSA滲透性能；0.6%和0.3%(w/v)殼聚糖溶液制備的ACA微囊的機(jī)械強(qiáng)度和BSA滲透性能均無顯著差異；三種濃度殼聚糖溶液制備的ACA囊膜對Y-球蛋白的阻隔能力無顯著差異。 (2)以非洲爪蟾作動物模型,研究ACA微囊化卵巢細(xì)胞異體移植后的E2和P4分泌能力。用免疫組化法檢測原代培養(yǎng)非洲爪蟾卵巢組織細(xì)胞的卵泡刺激素受體(FSHR)和黃體生成素受體(LHR)；用孕馬血清激素(PMSG)處理原代培養(yǎng)細(xì)胞48小時,酶聯(lián)免疫吸附法(ELISA)檢測培養(yǎng)液E2和P4濃度；用1.2%,0.6%和0.3%(w/v)殼聚糖溶液分別制備3組卵巢細(xì)胞ACA微囊,在培養(yǎng)28天內(nèi)用CFDA-SE/PI處理并測算細(xì)胞成活率,檢測培養(yǎng)60天內(nèi)0.3%(w/v)殼聚糖組的培養(yǎng)液E2和P4濃度；將0.3%(w/v)殼聚糖溶液制備的卵巢細(xì)胞ACA微囊移植入去勢雌性非洲爪蟾腹腔,ELISA法檢測60天內(nèi)血清的E2和P4水平。結(jié)果顯示,原代非洲爪蟾卵巢細(xì)胞的FSHR和LHR免疫染色呈陽性,在20IU/mL的PMSG處理時,培養(yǎng)細(xì)胞的E2和P4分泌水平最高；制備ACA微囊的最適殼聚糖濃度為0.3%(w/v)；在體外培養(yǎng)條件下,ACA微囊化卵巢細(xì)胞保持60天的E2和P4分泌,并在移植后60天內(nèi)顯著提高了去勢雌性非洲爪蟾血清E2和P4水平；移植后回收的微囊約90%保持完整和光滑。 (3)大鼠卵泡顆粒細(xì)胞(GCs)和卵泡膜細(xì)胞(TCs)的共微囊化。用免疫組化法分別檢測原代培養(yǎng)大鼠GCs和TCs的FSHR、LHR和CYP17A1;將GCs和TCs依不同數(shù)量比例混合并ACA微囊化,培養(yǎng)48小時后,檢測培養(yǎng)液E2和P4濃度；將GCs和TCs數(shù)量比例為1：2的GCs/TCs微囊移植入去勢雌性大鼠腹腔,檢測60天內(nèi)血清E2和P4水平。結(jié)果顯示,GCs/TCs共微囊化顯著提高了E2分泌水平,GCs/TCs數(shù)量比例為1：2時E2分泌水平最高,在移植后60天內(nèi)將去勢大鼠血清E2和P4維持在正常水平；移植后回收的微囊約90%保持完整和光滑。 (4)建立模擬卵泡自然結(jié)構(gòu)的卵巢細(xì)胞微囊化模式。微載體培養(yǎng)大鼠GCs (GCsMs),分別制備包有GCs、GCs+TCs、GCsMs和GCsMs+TCs的ACA微囊并體外培養(yǎng),WST-1法檢測微囊化GCs和GCsMs在培養(yǎng)24小時內(nèi)的生理活性；培養(yǎng)48小時后,檢測培養(yǎng)液E2和P4濃度；將模擬卵泡(GCsMs+TCs微囊)移植入去勢雌性大鼠腹腔,檢測60天內(nèi)血清E2和P4水平。結(jié)果顯示,微囊化GCsMs的生理活性顯著高于微囊化GCs；與其它三種微囊相比,模擬卵泡的E2分泌水平最高,移植后60天內(nèi)將去勢大鼠血清E2和P4維持在正常水平；移植后回收的微囊約90%保持完整和光滑。 綜上所述,本文以海藻酸鈉和殼聚糖為囊材,用靜電液滴發(fā)生法制備了卵巢細(xì)胞的ACA微囊,并分別在非洲爪蟾和大鼠上進(jìn)行了同種異體移植實(shí)驗,發(fā)現(xiàn)非洲爪蟾卵巢細(xì)胞微囊、大鼠GCs/TCs微囊,以及模擬卵泡在體外培養(yǎng)條件下都具有雌性激素分泌功能,移植后60天內(nèi)能夠持續(xù)釋放E2和P4,為婦女更年期綜合癥的治療開拓了新思路。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R943

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 劉袖洞;于煒婷;王為;雄鷹;馬小軍;袁權(quán);;海藻酸鈉和殼聚糖聚電解質(zhì)微膠囊及其生物醫(yī)學(xué)應(yīng)用[J];化學(xué)進(jìn)展;2008年01期

2 羅賢明;廖堅;王維;;脈絡(luò)叢細(xì)胞微囊腦內(nèi)移植前后的物理及生化性能研究[J];生命科學(xué)研究;2012年01期

3 李巖;田密;劉們巖;秦占芬;徐曉白;;非洲爪蟾肝細(xì)胞原代培養(yǎng)方法[J];生態(tài)毒理學(xué)報;2008年06期

4 王雅光;馬云勝;穆長征;蔡紅玉;王征;;海藻酸鈉-多聚賴氨酸-海藻酸鈉微囊內(nèi)細(xì)胞濃度與胰島素和C肽的釋放[J];中國組織工程研究與臨床康復(fù);2011年03期

5 潘月龍 ,鄭樹 ,彭佳萍,董琦;微囊化Maspin基因修飾細(xì)胞對乳腺癌細(xì)胞Bcap37運(yùn)動及黏附功能的影響[J];中華腫瘤雜志;2005年06期

，

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