本文選題：卵巢功能減退 + 卵巢早衰�。� 參考：《南京醫(yī)科大學(xué)》2014年博士論文

【摘要】：干細(xì)胞治療近年來(lái)發(fā)展迅速,已廣泛應(yīng)用于多個(gè)領(lǐng)域,為多種難治性疾病,例如糖尿病、帕金森、阿爾茲海默癥等提供了新的治療希望。卵巢功能減退是指卵巢產(chǎn)生卵子能力減弱,卵泡質(zhì)量下降,導(dǎo)致女性生育能力下降及性激素缺乏。卵巢功能減退會(huì)進(jìn)一步發(fā)展導(dǎo)致卵巢功能衰竭,其中卵巢早衰(premature ovarian failure, POF)是指婦女在40歲以前因某種原因引起的閉經(jīng)、不育、雌激素缺乏以及促性腺激素水平升高為特征的一種疾病。目前為止,除年齡因素引起的生理性卵巢功能減退外,大多數(shù)病理性卵巢減退的病因仍不明確。臨床上治療效果不佳,輔助生殖治療成功率也較低。研究發(fā)現(xiàn)惡性血液病患者行骨髓移植后部分病人能恢復(fù)生育能力,提示間充質(zhì)干細(xì)胞可能具有修復(fù)卵巢功能的作用。因此,探討間充質(zhì)干細(xì)胞在卵巢功能減退中的作用效果及作用機(jī)制將為卵巢早衰治療與研究開(kāi)辟新的領(lǐng)域。本研究第一部分,通過(guò)培養(yǎng)和鑒定人類胎兒羊膜來(lái)源干細(xì)胞及成體脂肪來(lái)源間充質(zhì)干細(xì)胞系,建立人類胚胎干細(xì)胞(human embryonic stem cell, hESCs)系,鑒定并比較它們的干細(xì)胞特性。在研究第二部分,構(gòu)建年老導(dǎo)致生理性卵巢功能減退以及放療導(dǎo)致病理性卵巢功能衰竭小鼠作為卵巢功能減退的動(dòng)物模型；在研究第三部分,選擇人羊膜間充質(zhì)干細(xì)胞(human amniotic mesenchymal stem cells, hAMSCs)作為卵巢功能修復(fù)研究的種子細(xì)胞,通過(guò)小鼠尾靜脈注射移植途徑,分析hAMSCs在小鼠體內(nèi)的歸巢、存活能力,研究]hAMSCs對(duì)卵巢功能的修復(fù)作用并探討其作用機(jī)制。本研究為hAMSCs在生殖醫(yī)學(xué)應(yīng)用領(lǐng)域積累了實(shí)驗(yàn)研究資料,揭示了hAMSCs治療卵巢功能減退的可能性。 一、人羊膜間充質(zhì)干細(xì)胞的培養(yǎng)及與其它類型干細(xì)胞的比較研究 1.分離、培養(yǎng)、鑒定人類胎兒羊膜來(lái)源干細(xì)胞 采用胰酶消化法獲取人類羊膜上皮細(xì)胞(hAECs)以及后續(xù)膠原酶消化法分離羊膜間充質(zhì)干細(xì)胞(hAMSCs),流式細(xì)胞分析hAECs、hAMSCs均不表達(dá)CD45,不表達(dá)免疫排斥標(biāo)志MHC-Ⅱ分子,hAMSCs表達(dá)CD29, CD44, CD73,CD90和CD105等間充質(zhì)干細(xì)胞標(biāo)志,hAECs特異性表達(dá)上皮細(xì)胞特異性標(biāo)志EpCAM。hAMSCs體外擴(kuò)增能力強(qiáng),可在體外誘導(dǎo)分化為中胚層成脂、成骨、成軟骨細(xì)胞分化；hAECs具有上皮樣細(xì)胞特性,部分表達(dá)胚胎干細(xì)胞多能性分子,體外擴(kuò)增困難,上述細(xì)胞均可以在不同代次冷凍保存,在液氮中凍存6-12個(gè)月后復(fù)蘇不影響細(xì)胞的生長(zhǎng)/增殖,可用于進(jìn)一步的研究。 2.分離成人脂肪來(lái)源間充質(zhì)干細(xì)胞 采用酶消化法和貼壁培養(yǎng)方法獲得了脂肪間充質(zhì)干細(xì)胞(ADSCs),發(fā)現(xiàn)腹部來(lái)源的ADSCs體外擴(kuò)增5代以上易衰老,眼瞼部來(lái)源的ADSCs具有更好的體外擴(kuò)增能力。兩種來(lái)源的hADSCs均不表達(dá)造血干細(xì)胞標(biāo)志CD34和CD45,不表達(dá)免疫排斥標(biāo)志MHC-Ⅱ分子,ADSCs表達(dá)CD29、CD44、CD73、CD90和CD105等間充質(zhì)干細(xì)胞標(biāo)志；均可在體外誘導(dǎo)成脂、成骨、成軟骨細(xì)胞分化容易；可以在不同代次冷凍保存,在液氮中凍存6-12個(gè)月后復(fù)蘇不影響細(xì)胞的生長(zhǎng)增殖情況,可用于進(jìn)一步的研究。 3.通過(guò)去除三原核廢棄胚胎多余原核的方法建立人胚胎干細(xì)胞系 本研究通過(guò)去除三原核廢棄胚胎多余原核的方法建立了2株1hESC系,并命名為CCRM22、CCRM23。細(xì)胞系擁有胚胎干細(xì)胞的特性：克隆狀生長(zhǎng),體外自我擴(kuò)增,表達(dá)干細(xì)胞特征性標(biāo)志物,在體內(nèi)外分化為3個(gè)胚層來(lái)源組織。核型分析結(jié)合arrayCGH分析顯示2株細(xì)胞系均為正常二倍體(46XY),說(shuō)明可以利用臨床上廢棄的三原核胚胎通過(guò)去核技術(shù)構(gòu)建胚胎干細(xì)胞系,降低倫理學(xué)障礙。本研究建立的hESC系可為后續(xù)研究hESCs體外向生殖細(xì)胞分化、胚胎發(fā)育風(fēng)險(xiǎn)、hESCs替代治療等提供基礎(chǔ)。 4.人羊膜間充質(zhì)干細(xì)胞與其它類型干細(xì)胞的比較 通過(guò)細(xì)胞材料來(lái)源、體外擴(kuò)增能力、免疫原性、致瘤性、不同批次間細(xì)胞差異性等方面的比較,發(fā)現(xiàn)hAMSCs具有來(lái)源豐富、取材無(wú)創(chuàng)、細(xì)胞活力較好、擴(kuò)增能力穩(wěn)定、無(wú)免疫原性、無(wú)致瘤性等優(yōu)點(diǎn),因此比較適合用于移植治療。然而,目前并無(wú)1hAMSCs用于卵巢功能減退修復(fù)的研究報(bào)道,本研究選擇hAMSCs作為卵巢功能減退治療實(shí)驗(yàn)的種子細(xì)胞,對(duì)于研究該細(xì)胞的功能以及卵巢功能減退的細(xì)胞治療均具有重要意義。二、卵巢功能減退小鼠模型的建立1.建立年老導(dǎo)致生理性小鼠卵巢功能減退的實(shí)驗(yàn)?zāi)Ｐ?分析不同年齡段雌性C57B6/L小鼠檢測(cè)血清性激素(FSH、E2、AMH)水平、卵巢組織形態(tài)學(xué),發(fā)現(xiàn)與正常8周性成熟小鼠相比,32周雌性C57B6/L小鼠血清E2濃度下降,FSH升高,AMH水平下降,各級(jí)卵泡數(shù)減少,可作為生理性卵巢功能減退模型。 2.放療導(dǎo)致病理性卵巢功能減退模型 選擇8周雌性C57B6/L小鼠接受全身單次X射線照射,照射劑量4Gy,分別在照射后24小時(shí)、1周、2周、4周和8周不同時(shí)間點(diǎn)觀察小鼠一般情況、動(dòng)情周期、血清FSH、E2、AMH濃度、卵巢結(jié)構(gòu)及卵泡計(jì)數(shù)。發(fā)現(xiàn)4Gy照射后小鼠動(dòng)情周期紊亂,觀察8周仍未恢復(fù)動(dòng)情周期；FSH濃度升高,E2、AMH濃度降低；卵巢體積和卵巢質(zhì)量系數(shù)降低。照射后1周原始卵泡數(shù)目顯著減少,中大卵泡不受影響；3周后卵巢間質(zhì)增生,皮質(zhì)菲薄,部分殘留初級(jí)卵泡樣結(jié)構(gòu)；Tunel實(shí)驗(yàn)揭示照射后1周卵巢顆粒細(xì)胞凋亡增加明顯；透射電鏡表現(xiàn)照射后1周卵巢內(nèi)線粒體腫脹、空泡化,溶酶體增加,細(xì)胞間隙清晰,2周之后卵巢線粒體腫脹、線粒體空泡化,間質(zhì)膠原纖維增生明顯。除卵巢功能外,照射后小鼠體重減輕、毛色灰暗、脫毛明顯,8周時(shí)毛色灰白、脫毛、脊柱彎曲等全身衰老癥狀明顯。 三、人羊膜間充質(zhì)干細(xì)胞移植修復(fù)小鼠卵巢功能減退的實(shí)驗(yàn)研究 移植實(shí)驗(yàn)分為4組,每組10只。用綠色熒光蛋白(GFP)標(biāo)記iAMSCs,生理鹽水懸浮成2×107/ml單細(xì)胞懸液,0.7ml經(jīng)尾靜脈注射移植。不同時(shí)間段分批處死小鼠,熒光顯微鏡下觀察卵巢有無(wú)綠色熒光及綠色熒光的分布。發(fā)現(xiàn)移植后1月,卵巢內(nèi)可見(jiàn)明亮的綠色熒光,且持續(xù)至移植后2月；綠色熒光分布在卵巢間質(zhì)組織,卵泡結(jié)構(gòu)內(nèi)見(jiàn)不到綠色熒光；生理鹽水對(duì)照組無(wú)綠色熒光表達(dá)。 1.小鼠生理性卵巢功能減退修復(fù) 分為注射AMSCs組和生理鹽水對(duì)照組,每組10只,分別接受尾靜脈等體積的hAMSCs或生理鹽水注射。注射后除觀察小鼠一般情況外,在8周觀察結(jié)束時(shí)行促排卵后取卵、體外授精以評(píng)估卵巢功能；并對(duì)取卵時(shí)血清性激素(E2、AMH、 INHb)濃度、卵巢結(jié)構(gòu)、卵泡黃體計(jì)數(shù)、獲卵數(shù)和胚胎發(fā)育率進(jìn)行比較。結(jié)果發(fā)現(xiàn)老齡組小鼠在AMSCs移植移植前后排卵前血清E2分別為24.22±2.64ng/L和89.39±5.32ng/L, p0.05,有統(tǒng)計(jì)學(xué)差異；INHb水平分別為70.13±2.11ng/L和112.41±19.0ng/L, p0.05,有統(tǒng)計(jì)學(xué)差異,但不能上升到正常8周育齡期小鼠水平；血清AMH水平由58.9±3.12ng/L上升至70.0±10.30ng/L,但無(wú)統(tǒng)計(jì)學(xué)差異(p=0.051)；移植組促排卵治療后獲卵數(shù)及黃體數(shù)、受精率以及囊胚形成率較對(duì)照組分別由6.10±2.40個(gè)、65.00%+8.25%及13.20%±10.50%增加至13.90±6.13個(gè)、77.30%±8.66%和30.00%±9.50%,無(wú)統(tǒng)計(jì)學(xué)差異,且均不能達(dá)到正常8周育齡期小鼠水平。 2.小鼠病理性卵巢功能減退修復(fù) 8周雌性小鼠經(jīng)過(guò)4Gy全身照射1周后,分為注射AMSCs組和生理鹽水對(duì)照組,每組10只,分別接受尾靜脈等體積的AMSCs或生理鹽水注射,觀察一般情況、動(dòng)情周期、血清性激素水平,卵巢結(jié)構(gòu)及超微結(jié)構(gòu)改變情況。與生理鹽水對(duì)照組相比,POF鼠hAMSCs后移植后2月基礎(chǔ)E2水平由24.22±2.64ng/L上升至47.91±6.21ng/L(p=0.032),差異顯著；FSH則由27.25+6.19IU/L下降至19.17±7.11IU/L (p=0.057),無(wú)統(tǒng)計(jì)學(xué)差異；AMH由8.50±4.14ng/L升高至13.06±3.05ng/L(p=0.050),但無(wú)統(tǒng)計(jì)學(xué)差異；hAMSCs移植鼠卵巢內(nèi)可見(jiàn)未成熟卵泡,但促排卵后無(wú)成熟卵泡排出；電鏡顯示hAMSCs移植后小鼠卵巢膠原纖維增生明顯減少。 3.芯片檢測(cè)hAMSCs培養(yǎng)基上清液中細(xì)胞因子 以無(wú)細(xì)胞培養(yǎng)的正常培養(yǎng)液為對(duì)照,利用細(xì)胞因子芯片檢測(cè)hAMSCs培養(yǎng)基上清液中hAMSCs所分泌的細(xì)胞因子。結(jié)果顯示,每1×106個(gè)hAMSCs48小時(shí)可分泌總蛋白達(dá)150mg；高分泌143種細(xì)胞因子,包括細(xì)胞營(yíng)養(yǎng)與生長(zhǎng)因子、趨化因子及受體、促炎癥反應(yīng)因子、抗炎癥反應(yīng)因子和血管生長(zhǎng)因子等；進(jìn)一步Elisa定量檢測(cè)發(fā)現(xiàn),hAMSCs分泌的IGF-1高達(dá)619.±50.6pg/ml, HGF則達(dá)490.5±22.3pg/ml。 hAMSCs所分泌的細(xì)胞因子改善了卵巢局部微環(huán)境,促進(jìn)了細(xì)胞增殖、減少細(xì)胞凋亡,從而修復(fù)卵巢功能。 結(jié)論： 本研究成功分離、培養(yǎng)了hADSCs、hAECs和hAMSCs,并從3PN廢棄胚胎中建立正常二倍體核型的hESCs,比較后發(fā)現(xiàn)hAMSCs比較適合作為細(xì)胞移植治療的種子細(xì)胞。生理性卵巢功能減退小鼠、放療導(dǎo)致病理性卵巢功能衰竭小鼠可分別作為卵巢功能部分減退、卵巢功能全面衰竭模型。hAMSCs靜脈注射移植方法安全有效,移植后少量hAMSCs可遷移至損傷的小鼠卵巢組織中存活；單次hAMSCs移植后可顯著改善年老導(dǎo)致鼠卵巢功能,能有效改善、但不能完全重建POF模型鼠的卵巢儲(chǔ)備；移植的1hAMSCs分化為卵泡組分的可能性較小,但hAMSCs可分泌多種細(xì)胞因子,推測(cè)這些細(xì)胞因子可改善卵巢微環(huán)境、甚至是全身微環(huán)境,從而改善卵巢功能。
[Abstract]:In this study , the effect and mechanism of stem cell therapy on ovarian function decreased .
In the third part , human amniotic mesenchymal stem cells ( hAMSCs ) were selected as the seed cells for ovarian function repair . The homing and survival abilities of hAMSCs in mice were analyzed by intravenous injection of mouse tail vein , and the mechanism of action of hAMSCs on ovarian function was studied .

Culture of human amniotic mesenchymal stem cells and comparison with other types of stem cells

1 . Isolation , culture and identification of human fetal amniotic - derived stem cells

hAMSCs express CD29 , CD44 , CD73 , CD90 and CD105 . hAMSCs express CD29 , CD44 , CD73 , CD90 and CD105 . hAMSCs express CD29 , CD44 , CD73 , CD90 and CD105 .
hAECs have the characteristics of epithelioid cells , partially express the pluripotent molecules of embryonic stem cells , and in vitro amplification is difficult , and the cells can be preserved in different generations , and the recovery does not affect the growth / proliferation of the cells after freezing for 6 - 12 months in liquid nitrogen , and can be used for further research .

2 . Isolation of adult adipose - derived mesenchymal stem cells

Adipose - derived mesenchymal stem cells ( ADSCs ) were obtained by enzymatic digestion and adherent culture . ADSCs were found to be able to amplify more than 5 generations of ADSCs in vitro . Both of the two derived hADSCs did not express hematopoietic stem cell markers CD34 and CD45 , which did not express immune rejection markers MHC - II molecules , and ADSCs express CD29 , CD44 , CD73 , CD90 and CD105 .
can be induced into fat in vitro , and can be formed into bone , so that the chondrogenic cell is easy to differentiate ;
can be stored in different generations , and the recovery does not affect the growth and proliferation of the cells after freezing for 6 - 12 months in liquid nitrogen , and can be used for further research .

3 . The human embryonic stem cell line is established by removing the extra prokaryotic expression of the three prokaryotic waste embryos

Two strains of 1hESC were established by removing the extra prokaryotic expression of three prokaryotic cells . The cell lines were named CCRM22 and CCR23 . The cell lines had the characteristics of embryonic stem cells : clonal growth , self - amplification in vitro , expression of characteristic markers of stem cells , and differentiation into three embryonic stem cells in vitro .

4 . Comparison of human amniotic mesenchymal stem cells with other types of stem cells

It is found that hAMSCs have the advantages of abundant sources , non - invasive materials , good cell viability , stable amplification ability , no immunogenicity , no oncogenicity , etc .

The levels of serum sex hormone ( FSH , E2 , AMH ) and the histomorphology of the ovarian tissues were analyzed in different age groups . Compared with the normal 8 - week mature mice , the serum E2 concentration decreased , the levels of FSH increased , the level of AMH decreased , and the number of follicles at all levels was decreased , which could be used as a model for the decline of physiological ovarian function .

2 . Model of pathological ovarian function caused by radiotherapy

Eight weeks after irradiation , the mice were treated with single X - ray irradiation , irradiation dose of 4Gy , respectively at 24 hours , 1 week , 2 weeks , 4 weeks and 8 weeks after irradiation . The normal conditions , dynamic cycle , serum FSH , E2 , AMH concentration , ovary structure and follicle count were observed .
The concentration of FSH increased , and the concentration of E2 and AMH decreased .
The ovarian volume and the ovarian mass index decreased . The number of primordial follicles decreased significantly in 1 week after irradiation , and the number of follicles in the middle and large follicles was not affected ;
After 3 weeks of ovarian stromal hyperplasia , the cortex was thin , partially residual primary follicle - like structure ;
Tunel experiment revealed that the apoptosis of ovary granular cells increased significantly in 1 week after irradiation .
After irradiation , mitochondria swelling , vacuolation , lysosome increase , cell gap were clear in 1 week after irradiation . After 2 weeks , the mitochondria swelling , mitochondria vacuolation , interstitial collagen fiber hyperplasia were obvious . Except for the function of ovarian function , the body weight of the mice was reduced , the hair color was gray and dark , the hair loss was obvious , and the whole body aging symptoms such as gray hair , hair loss , and spinal curvature were obvious at 8 weeks .

Experimental Study on the Effect of Human Amniotic Membrane Mesenchymal Stem Cells Transplantation on Ovarian Function in Mice

The experiment was divided into 4 groups , 10 in each group . iAMSCs were labeled with green fluorescent protein ( GFP ) , physiological saline was suspended in 2 脳 107 / ml single cell suspension , 0.7 ml was injected intravenously . The distribution of green fluorescence and green fluorescence was observed under fluorescence microscope .
Green fluorescence was found in the ovarian stromal tissue , and green fluorescence was found in the follicular structure .
There was no green fluorescence in normal saline control group .

1 . Repair of physiological ovarian function in mice

The rats were divided into two groups : AMSCs group and normal saline control group . 10 rats in each group were injected with hAMSCs or physiological saline , such as tail vein and other volumes . After injection , the eggs were collected after ovulation induction at the end of 8 weeks , and the ovarian function was evaluated in vitro .
The serum E2 , AMH , INHb concentration , ovary structure , follicle corpus luteum count , egg count and embryo development rate were compared . The results showed that the serum levels of E2 before and after the transplantation of AMSCs were 24.22 鹵 2.64ng / L and 89.39 鹵 5.32ng / L , p0.01 respectively .
The levels of INHb were 70.13 鹵 2.11ng / L and 112.41 鹵 19.0ng / L , p0.05 , respectively , but the level of INHb could not be increased to normal 8 weeks .
Serum AMH levels increased from 58.9 鹵 3.12ng / L to 70.0 鹵 10.30ng / L , but there was no statistical difference ( p = 0.051 ) .
The number of oocytes and the number of corpus luteum , fertilization rate and blastocyst formation rate were 6.10 鹵 2.40 , 65.00 % + 8.25 % , 13.20 % 鹵 10.50 % , 13.90 鹵 6.13 , 77.30 % 鹵 8.66 % and 30.00 % 鹵 9.50 % , respectively .

2 . Repair of pathological ovarian function in mice

After 4 Gy whole body irradiation for 1 week , 8 - week female mice were divided into four groups : AMSCs and normal saline control group . Ten rats in each group received AMSCs or physiological saline injections , such as tail vein , and observed general , dynamic , serum sex hormone levels , ovarian structure and ultrastructure changes . After transplantation , the level of basal E2 increased from 24.22 鹵 2.64ng / L to 47.91 鹵 6.21ng / L ( p = 0.032 ) , and the difference was significant .
FSH decreased from 27.25 + 6.19IU / L to 19.17 鹵 7.11IU / L ( p = 0.057 ) .
AMH increased from 8.50 鹵 4.14ng / L to 13.06 鹵 3.05ng / L ( p = 0.050 ) , but there was no statistical difference .
There were no mature follicles in the ovary of hAMSCs , but there were no mature follicles after ovulation .
Electron microscope showed that the proliferation of collagen fibers decreased significantly in mice after transplantation of hAMSCs .

3 . Detection of cytokines in supernatant of hAMSCs culture medium by chip

The cytokines secreted by hAMSCs in culture supernatant of hAMSCs were detected by cytokines . The results showed that the total protein reached 150 mg / 1 脳 106 hAMSCs48 hours .
high secretion of 143 cytokines , including cell nutrition and growth factor , chemokine and receptor , pro - inflammatory response factor , anti - inflammatory response factor and vascular growth factor , etc . ;
The results showed that the IGF - 1 secreted by hAMSCs reached 619 . 鹵 50.6 pg / ml , and HGF reached 490.5 鹵 22.3 pg / ml .

The cytokines secreted by hAMSCs improve local microenvironment of ovary , promote cell proliferation , reduce cell apoptosis , and thus repair ovarian function .

Conclusion :

hADSCs , hAECs and hAMSCs were isolated , hADSCs , hAECs and hAMSCs were cultured and normal diploid karyotype hESCs were established from 3PN discarded embryos .
After single hAMSCs transplantation , the ovarian function of rat can be improved significantly , which can improve the ovarian function effectively , but it can not completely rebuild the ovarian reserve of the rat ' s ovarian function ;
The migration of 1hAMSCs into follicular components is less likely , but hAMSCs can secrete a variety of cytokines , suggesting that these cytokines can improve ovarian microenvironment , and even systemic microenvironment , thereby improving ovarian function .
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R711.75

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