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[1]罗晓强,蒲 静,邱意开,等.卵泡液微环境对人羊水干细胞体外分化为雌性生殖细胞的影响[J].宁夏医科大学学报,2019,(06):561-567,576.[doi:10.16050/j.cnki.issn1674-6309.2019.06.005]
 LUO Xiaoqiang,PU Jing,QIU Yikai,et al.Effect of Follicular Microenvironment on Differentiation of Human Amniotic Fluid Stem Cells into Female Germ Cells in Vitro[J].Ningxia Medical University,2019,(06):561-567,576.[doi:10.16050/j.cnki.issn1674-6309.2019.06.005]
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卵泡液微环境对人羊水干细胞体外分化为雌性生殖细胞的影响(PDF)
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《宁夏医科大学学报》[ISSN:1005-8486/CN:64-1029/R]

卷:
期数:
2019年06期
页码:
561-567,576
栏目:
论著
出版日期:
2019-06-30

文章信息/Info

Title:
Effect of Follicular Microenvironment on Differentiation of Human Amniotic Fluid Stem Cells into Female Germ Cells in Vitro
文章编号:
1674-6309(2019)06-0561-07
作者:
罗晓强12 蒲 静1 邱意开1 刘心蕊1 王国平3 张樱馨1 田 媛1 裴秀英1 俞晓丽1
(1. 生育力保持教育部重点实验室,宁夏生殖与遗传重点实验室,银川 750004; 2. 宁夏医科大学基础医学院,银川 750004; 3. 宁夏银川市妇幼保健院,银川 750004)
Author(s):
LUO Xiaoqiang12 PU Jing1 QIU Yikai1 LIU Xinrui1 WANG Guoping3 ZHANG Yingxin1 TIAN Yuan1 PEI Xiuyin1 YU Xiaoli1
(1. Key Laboratory of Fertility Preservation and Maintenance,Key Laboratory of Reproduction and Genetics in Ningxia,Yinchuan 750004,China; 2.Ningxia Medical University,Basic Medical School,Yinchuan 750004,China; 3. Yinchuan Maternal and Child Health Hospital,Yinchuan 75004,China)
关键词:
人羊水干细胞卵泡液分化雌性生殖细胞
Keywords:
human amniotic fluid stem cellsfollicular fluiddifferentiation female germ cells
分类号:
R329
DOI:
10.16050/j.cnki.issn1674-6309.2019.06.005
文献标志码:
A
摘要:
目的 观察不同直径猪卵泡内卵泡液诱导人羊水干细胞(human amniotic fluid stem cells,hAFSCs)-070607株向雌性生殖细胞分化的效果。探讨卵泡内微环境对体外雌性生殖细胞形成的影响。方法 选取不同直径猪卵泡内的卵泡液(S组:直径<3 mm、M组:直径3~6 mm、L组:直径>6 mm),检测卵泡液内雌二醇(E2)和卵泡刺激素(FSH)的含量及骨形态发生蛋白15(BMP15)的表达。将不同分组卵泡液添加到雌性生殖细胞分化体系中,经过5 d的体外诱导培养,观察生殖细胞基因OCT4、FIGLα、GDF9、FSHR和甲基化基因DNMT3B的表达变化。继续用M组卵泡液诱导hAFSCs-070607,观察基因及蛋白表达的变化。结果 通过基因、蛋白和三胚层分化的检测,确定hAFSCs-070607具有良好的多能性及分化潜能。3组不同直径的卵泡液内都含有FSH,但不含E2;BMP15的未成熟蛋白在M组中的含量略高于S、L组。M组添加到诱导分化培养基后,E2的产量较高,生殖相关标记基因检测结果发现,OCT4、FIGLα、GDF9、FSHR和DNMT3B基因的表达依次按照大、中、小卵泡组上升。继续用M组卵泡液诱导hAFSCs-070607,诱导10d后,有卵母细胞样细胞(oocyte-like cells,OLCs)和卵泡样结构的出现,经EGFP-BMP15转染后,这些结构显示EGFP表达阳性,表明有OLCs的产生。荧光定量PCR结果证实,生殖细胞形成相关基因OCT4、FIGLα、GDF9、FSHR和表观学基因DNMT3B的表达逐渐呈上升的趋势。经细胞免疫荧光技术检测发现,生殖相关蛋白OCT4、FRAGILIS和SCP3呈阳性表达。结论 hAFSCs-070607株人羊水干细胞具有良好的多能性和体外分化潜能,经猪卵泡液体外诱导分化后,可分化为雌性生殖细胞,且M组卵泡液诱导后细胞分泌的E2高于S组和L组。长时间诱导后,生殖细胞相关基因和蛋白上调,并能形成卵泡样结构。
Abstract:
Objective To observe the effect of follicular fluid in different diameter porcine follicles on the differentiation of human amniotic fluid stem cells(hAFSCs)-070607 into female germ cells in vitro. And to investigate the effects of microenvironment in follicles on the formation of female germ cells in vitro. Methods The pluripotency and differentiation potential of hAFSCs-070607 were determined by detection of gene,protein and triple germ layer differentiation. The follicular fluids(diameter<3mm,diameter3~6mm,diameter>6mm)in follicles of different diameters were selected to detect the content of estradiol (E2) and follicle stimulating hormone (FSH) in follicular fluid,and the expression of bone morphogenetic protein15(BMP15). Meanwhile,different groups of follicular fluids were added to the female germ cell differentiation system,and the expression of germ cell genes OCT4, FIGLα, GDF9, FSHR and methylation gene DNMT3B were tested by Real time PCR after 5 days of differentiation. Secondary follicles were selected for further study. The hAFSCs were induced by M group follicular fluid, and the changes of gene and protein expression were observed. Results Through the detection of gene,protein and triple germ layer differentiation,hAFSCs-070607 had good pluripotency and differentiation potential. FSH was contained in all kinds of follicular fluids,but E2 was not. The content of BMP15 immature protein in medium-sized follicular fluid was higher than that of the other two groups. The medium follicular fluid was added to the induced differentiation medium,and the yield of E2 was high. The results of reproductive-related marker gene detection showed that the expression of OCT4,FIGLα,GDF9,FSHR and DNMT3B genes increased in accordance with the large,medium and small follicle groups. Continuing to induce hAFSCs with group M follicular fluid, oocyte-like cells(OLCs) and follicular-like structures were found after 10 days of induction, and these structures showed positive expression of EGFP after transfection with EGFP-BMP15, which indicated that there were oocyte in follicle-like structures. The results of Real time PCR confirmed that the expression of germ cell formation related genes OCT4,FIGLα,GDF9,FSHR and epigenetic gene DNMT3B gradually increased,and the reproductive related proteins OCT4,FRAGILIS and SCP3 were positive by cellular immunofluorescence assay. Conclusion hAFSCs-070607 strain human amniotic fluid stem cells have good pluripotency and differentiation potential in vitro. After induced by porcine follicular fluid,they can differentiate into female germ cells,and the E2 secreted by cells in group M is higher than that of S group. Group L. After prolonged induction,germ cell related genes and proteins are up-regulated and can form a follicular-like structure.

参考文献/References:

[1] Hübner K,Fuhrmann G,Christenson L K,et al. Derivation of oocytes from mouse embryonic stem cells [J]. Science,2003,300 (5623):1251-1256.
[2] Dyce PW,Wen L,Li J. In vitro germline potential of stem cells derived from fetal porcine skin[J]. Nature Cell Biology,2006,8 (4):384-390.
[3] Yu X,Wang N,Qiang R,et al. Human amniotic fluid stem cells possess the potential to differentiate into primordial follicle oocytes in vitro1[J]. Biology of Reproduction,2014,90 (4):73.
[4] Gattazzo F,Urciuolo A,Bonaldo P. Extracellular matrix: A dynamic microenvironment for stem cell niche [J]. Biochimica et Biophysica Acta (BBA) - General Subjects,2014,1840 (8):2506-2519.
[5] Munakata Y,Ueda M,Kawahara-Miki R,et al.Follicular factors determining granulosa cell number and developmental competence of porcine oocytes [J]. J Assist Reprod Genet,2018,35 (10):1809-1819.
[6] Ducolomb Y,Gonzalez-Marquez H,Fierro R,et al. Effect of porcine follicular fluid proteins and peptides on oocyte maturation and their subsequent effect on in vitro fertilization[J]. Theriogenology,2013,79 (6):896-904.
[7] Cheng X,Chen S, Yu X, et al. BMP15 gene is activated during human amniotic fluid stem cell differentiation into oocyte-like cells [J]. DNA Cell Biol, 2012, 31 (7):1198-1204.
[8] 俞晓丽,王宁,马洋洋,等. 人卵母细胞样细胞体内分化模型的建立[J]. 生物工程学报,2015,31(3):394-402.
[9] Zuccotti M,Merico V,Redi CA,et al. Role of Oct-4 during acquisition of developmental competence in mouse oocyte [J]. Reprod Biomed Online,2009,19 Suppl 3:57-62.
[10] Yamaguchi S,Kimura H,Tada M,et al. Nanog expression in mouse germ cell development [J]. Gene Expression Patterns,2005,5 (5):639-646.
[11] Ikenishi K,Tanaka TS. Spatio-temporal expression of Xenopus vasa homolog,XVLG1,in oocytes and embryos:the presence of XVLG1 RNA in somatic cells as well as germline cells [J]. Dev Growth Differ,2000,42 (2):95-103.
[12] Payer B,Saitou M,Barton SC,et al. Stella is a maternal effect gene required for normal early development in mice[J]. Current Biology,2003,13 (23):2110-2117.
[13] Xue W,Wang H,Wang Z,et al. Cloning and characterization of ifitm1 and ifitm3 expression during early zebrafish development[J]. Zygote,2016,24 (01):149-158.
[14] Tsuda M,Kiso M,Saga Y. Implication of nanos2-3′UTR in the expression and function of nanos2 [J]. Mechanisms of Development,2006,123(6):440-449.
[15] Tepekoy F,Ozturk S,Sozen B,et al. CD90 and CD105 expression in the mouse ovary and testis at different stages of postnatal development[J]. Reprod Biol, 2015,15 (4):195-204.
[16] Giebel J,Rune GM. Relationship between expression of integrins and granulosa cell apoptosis in ovarian follicles of the marmoset(Callithrix jacchus) [J]. Tissue Cell,1997,29 (5):525-531.
[17] Hsueh AJ. Fertility: the role of mTOR signaling and KIT ligand [J]. Curr Biol,2014,24(21): R1040-R1042.
[18] Hashimoto O,Moore RK,Shimasaki S. Posttranslational processing of mouse and human BMP-15: potential implication in the determination of ovulation quota[J]. Proc Natl Acad Sci U S A,2005,102 (15):5426-5431.
[19] Britt KL,Saunders PK,McPherson SJ,et al. Estrogen actions on follicle formation and early follicle development[J]. Biol Reprod,2004,71(5):1712-1723.
[20] Joshi S,Davies H,Sims LP,et al. Ovarian gene expression in the absence of FIGLA,an oocyte-specific transcription factor[J]. BMC Dev Biol,2007,7:67.
[21] Monestier O,Servin B,Auclair S,et al. Evolutionary origin of bone morphogenetic protein 15 and growth and differentiation factor 9 and differential selective pressure between mono- and polyovulating species[J]. Biol Reprod,2014,91 (4):83.
[22] Demeestere I,Streiff AK,Suzuki J,et al. Follicle-stimulating hormone accelerates mouse oocyte development In vivo[J]. Biology of Reproduction,2012,87(1):3.
[23] Petrussa L,Van de Velde H,De Rycke M. Dynamic regulation of DNA methyltransferases in human oocytes and preimplantation embryos after assisted reproductive technologies[J]. Mol Hum Reprod,2014,20 (9):861-874.

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备注/Memo

备注/Memo:
收稿日期:2019-01-17
基金项目:国家自然科学基金(31560329);宁夏自然科学基金(NZ17070)
作者简介:罗晓强(1992-),男,汉族,在读硕士研究生。
通信作者:俞晓丽(1984-),女,讲师,从事干细胞与生殖生物学研究。E-mail: yuxiaoli1221@126.com
更新日期/Last Update: 2019-06-30