人羊膜间充质干细胞修复卵巢功能的实验研究

发布时间：2018-05-17 01:12

本文选题：卵巢功能减退 + 卵巢早衰　；参考：《南京医科大学》2014年博士论文

【摘要】：干细胞治疗近年来发展迅速,已广泛应用于多个领域,为多种难治性疾病,例如糖尿病、帕金森、阿尔兹海默症等提供了新的治疗希望。卵巢功能减退是指卵巢产生卵子能力减弱,卵泡质量下降,导致女性生育能力下降及性激素缺乏。卵巢功能减退会进一步发展导致卵巢功能衰竭,其中卵巢早衰(premature ovarian failure, POF)是指妇女在40岁以前因某种原因引起的闭经、不育、雌激素缺乏以及促性腺激素水平升高为特征的一种疾病。目前为止,除年龄因素引起的生理性卵巢功能减退外,大多数病理性卵巢减退的病因仍不明确。临床上治疗效果不佳,辅助生殖治疗成功率也较低。研究发现恶性血液病患者行骨髓移植后部分病人能恢复生育能力,提示间充质干细胞可能具有修复卵巢功能的作用。因此,探讨间充质干细胞在卵巢功能减退中的作用效果及作用机制将为卵巢早衰治疗与研究开辟新的领域。本研究第一部分,通过培养和鉴定人类胎儿羊膜来源干细胞及成体脂肪来源间充质干细胞系,建立人类胚胎干细胞(human embryonic stem cell, hESCs)系,鉴定并比较它们的干细胞特性。在研究第二部分,构建年老导致生理性卵巢功能减退以及放疗导致病理性卵巢功能衰竭小鼠作为卵巢功能减退的动物模型；在研究第三部分,选择人羊膜间充质干细胞(human amniotic mesenchymal stem cells, hAMSCs)作为卵巢功能修复研究的种子细胞,通过小鼠尾静脉注射移植途径,分析hAMSCs在小鼠体内的归巢、存活能力,研究]hAMSCs对卵巢功能的修复作用并探讨其作用机制。本研究为hAMSCs在生殖医学应用领域积累了实验研究资料,揭示了hAMSCs治疗卵巢功能减退的可能性。一、人羊膜间充质干细胞的培养及与其它类型干细胞的比较研究 1.分离、培养、鉴定人类胎儿羊膜来源干细胞采用胰酶消化法获取人类羊膜上皮细胞(hAECs)以及后续胶原酶消化法分离羊膜间充质干细胞(hAMSCs),流式细胞分析hAECs、hAMSCs均不表达CD45,不表达免疫排斥标志MHC-Ⅱ分子,hAMSCs表达CD29, CD44, CD73,CD90和CD105等间充质干细胞标志,hAECs特异性表达上皮细胞特异性标志EpCAM。hAMSCs体外扩增能力强,可在体外诱导分化为中胚层成脂、成骨、成软骨细胞分化；hAECs具有上皮样细胞特性,部分表达胚胎干细胞多能性分子,体外扩增困难,上述细胞均可以在不同代次冷冻保存,在液氮中冻存6-12个月后复苏不影响细胞的生长/增殖,可用于进一步的研究。 2.分离成人脂肪来源间充质干细胞采用酶消化法和贴壁培养方法获得了脂肪间充质干细胞(ADSCs),发现腹部来源的ADSCs体外扩增5代以上易衰老,眼睑部来源的ADSCs具有更好的体外扩增能力。两种来源的hADSCs均不表达造血干细胞标志CD34和CD45,不表达免疫排斥标志MHC-Ⅱ分子,ADSCs表达CD29、CD44、CD73、CD90和CD105等间充质干细胞标志；均可在体外诱导成脂、成骨、成软骨细胞分化容易；可以在不同代次冷冻保存,在液氮中冻存6-12个月后复苏不影响细胞的生长增殖情况,可用于进一步的研究。 3.通过去除三原核废弃胚胎多余原核的方法建立人胚胎干细胞系本研究通过去除三原核废弃胚胎多余原核的方法建立了2株1hESC系,并命名为CCRM22、CCRM23。细胞系拥有胚胎干细胞的特性：克隆状生长,体外自我扩增,表达干细胞特征性标志物,在体内外分化为3个胚层来源组织。核型分析结合arrayCGH分析显示2株细胞系均为正常二倍体(46XY),说明可以利用临床上废弃的三原核胚胎通过去核技术构建胚胎干细胞系,降低伦理学障碍。本研究建立的hESC系可为后续研究hESCs体外向生殖细胞分化、胚胎发育风险、hESCs替代治疗等提供基础。 4.人羊膜间充质干细胞与其它类型干细胞的比较通过细胞材料来源、体外扩增能力、免疫原性、致瘤性、不同批次间细胞差异性等方面的比较,发现hAMSCs具有来源丰富、取材无创、细胞活力较好、扩增能力稳定、无免疫原性、无致瘤性等优点,因此比较适合用于移植治疗。然而,目前并无1hAMSCs用于卵巢功能减退修复的研究报道,本研究选择hAMSCs作为卵巢功能减退治疗实验的种子细胞,对于研究该细胞的功能以及卵巢功能减退的细胞治疗均具有重要意义。二、卵巢功能减退小鼠模型的建立1.建立年老导致生理性小鼠卵巢功能减退的实验模型分析不同年龄段雌性C57B6/L小鼠检测血清性激素(FSH、E2、AMH)水平、卵巢组织形态学,发现与正常8周性成熟小鼠相比,32周雌性C57B6/L小鼠血清E2浓度下降,FSH升高,AMH水平下降,各级卵泡数减少,可作为生理性卵巢功能减退模型。 2.放疗导致病理性卵巢功能减退模型选择8周雌性C57B6/L小鼠接受全身单次X射线照射,照射剂量4Gy,分别在照射后24小时、1周、2周、4周和8周不同时间点观察小鼠一般情况、动情周期、血清FSH、E2、AMH浓度、卵巢结构及卵泡计数。发现4Gy照射后小鼠动情周期紊乱,观察8周仍未恢复动情周期；FSH浓度升高,E2、AMH浓度降低；卵巢体积和卵巢质量系数降低。照射后1周原始卵泡数目显著减少,中大卵泡不受影响；3周后卵巢间质增生,皮质菲薄,部分残留初级卵泡样结构；Tunel实验揭示照射后1周卵巢颗粒细胞凋亡增加明显；透射电镜表现照射后1周卵巢内线粒体肿胀、空泡化,溶酶体增加,细胞间隙清晰,2周之后卵巢线粒体肿胀、线粒体空泡化,间质胶原纤维增生明显。除卵巢功能外,照射后小鼠体重减轻、毛色灰暗、脱毛明显,8周时毛色灰白、脱毛、脊柱弯曲等全身衰老症状明显。三、人羊膜间充质干细胞移植修复小鼠卵巢功能减退的实验研究移植实验分为4组,每组10只。用绿色荧光蛋白(GFP)标记iAMSCs,生理盐水悬浮成2×107/ml单细胞悬液,0.7ml经尾静脉注射移植。不同时间段分批处死小鼠,荧光显微镜下观察卵巢有无绿色荧光及绿色荧光的分布。发现移植后1月,卵巢内可见明亮的绿色荧光,且持续至移植后2月；绿色荧光分布在卵巢间质组织,卵泡结构内见不到绿色荧光；生理盐水对照组无绿色荧光表达。 1.小鼠生理性卵巢功能减退修复分为注射AMSCs组和生理盐水对照组,每组10只,分别接受尾静脉等体积的hAMSCs或生理盐水注射。注射后除观察小鼠一般情况外,在8周观察结束时行促排卵后取卵、体外授精以评估卵巢功能；并对取卵时血清性激素(E2、AMH、 INHb)浓度、卵巢结构、卵泡黄体计数、获卵数和胚胎发育率进行比较。结果发现老龄组小鼠在AMSCs移植移植前后排卵前血清E2分别为24.22±2.64ng/L和89.39±5.32ng/L, p0.05,有统计学差异；INHb水平分别为70.13±2.11ng/L和112.41±19.0ng/L, p0.05,有统计学差异,但不能上升到正常8周育龄期小鼠水平；血清AMH水平由58.9±3.12ng/L上升至70.0±10.30ng/L,但无统计学差异(p=0.051)；移植组促排卵治疗后获卵数及黄体数、受精率以及囊胚形成率较对照组分别由6.10±2.40个、65.00%+8.25%及13.20%±10.50%增加至13.90±6.13个、77.30%±8.66%和30.00%±9.50%,无统计学差异,且均不能达到正常8周育龄期小鼠水平。 2.小鼠病理性卵巢功能减退修复 8周雌性小鼠经过4Gy全身照射1周后,分为注射AMSCs组和生理盐水对照组,每组10只,分别接受尾静脉等体积的AMSCs或生理盐水注射,观察一般情况、动情周期、血清性激素水平,卵巢结构及超微结构改变情况。与生理盐水对照组相比,POF鼠hAMSCs后移植后2月基础E2水平由24.22±2.64ng/L上升至47.91±6.21ng/L(p=0.032),差异显著；FSH则由27.25+6.19IU/L下降至19.17±7.11IU/L (p=0.057),无统计学差异；AMH由8.50±4.14ng/L升高至13.06±3.05ng/L(p=0.050),但无统计学差异；hAMSCs移植鼠卵巢内可见未成熟卵泡,但促排卵后无成熟卵泡排出；电镜显示hAMSCs移植后小鼠卵巢胶原纤维增生明显减少。 3.芯片检测hAMSCs培养基上清液中细胞因子以无细胞培养的正常培养液为对照,利用细胞因子芯片检测hAMSCs培养基上清液中hAMSCs所分泌的细胞因子。结果显示,每1×106个hAMSCs48小时可分泌总蛋白达150mg；高分泌143种细胞因子,包括细胞营养与生长因子、趋化因子及受体、促炎症反应因子、抗炎症反应因子和血管生长因子等；进一步Elisa定量检测发现,hAMSCs分泌的IGF-1高达619.±50.6pg/ml, HGF则达490.5±22.3pg/ml。 hAMSCs所分泌的细胞因子改善了卵巢局部微环境,促进了细胞增殖、减少细胞凋亡,从而修复卵巢功能。结论：本研究成功分离、培养了hADSCs、hAECs和hAMSCs,并从3PN废弃胚胎中建立正常二倍体核型的hESCs,比较后发现hAMSCs比较适合作为细胞移植治疗的种子细胞。生理性卵巢功能减退小鼠、放疗导致病理性卵巢功能衰竭小鼠可分别作为卵巢功能部分减退、卵巢功能全面衰竭模型。hAMSCs静脉注射移植方法安全有效,移植后少量hAMSCs可迁移至损伤的小鼠卵巢组织中存活；单次hAMSCs移植后可显著改善年老导致鼠卵巢功能,能有效改善、但不能完全重建POF模型鼠的卵巢储备；移植的1hAMSCs分化为卵泡组分的可能性较小,但hAMSCs可分泌多种细胞因子,推测这些细胞因子可改善卵巢微环境、甚至是全身微环境,从而改善卵巢功能。
[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 卤 2.64ng / L and 89.39 卤 5.32ng / L , p0.01 respectively .
The levels of INHb were 70.13 卤 2.11ng / L and 112.41 卤 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 卤 3.12ng / L to 70.0 卤 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 卤 2.40 , 65.00 % + 8.25 % , 13.20 % 卤 10.50 % , 13.90 卤 6.13 , 77.30 % 卤 8.66 % and 30.00 % 卤 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 卤 2.64ng / L to 47.91 卤 6.21ng / L ( p = 0.032 ) , and the difference was significant .
FSH decreased from 27.25 + 6.19IU / L to 19.17 卤 7.11IU / L ( p = 0.057 ) .
AMH increased from 8.50 卤 4.14ng / L to 13.06 卤 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 . 卤 50.6 pg / ml , and HGF reached 490.5 卤 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 .
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.75

【共引文献】