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间充质干细胞条件培养基调节凋亡和应激相关基因表达,改善成熟情况并允许人工激活后未成熟人类卵母细胞的发育。

Mesenchymal Stem Cell-Conditioned Medium Modulates Apoptotic and Stress-Related Gene Expression, Ameliorates Maturation and Allows for the Development of Immature Human Oocytes after Artificial Activation.

作者信息

Akbari Hakimeh, Eftekhar Vaghefi Seyed Hassan, Shahedi Abbas, Habibzadeh Victoria, Mirshekari Tooraj Reza, Ganjizadegan Aboozar, Mollaei Hamidreza, Ahmadi Meysam, Nematollahi-Mahani Seyed Noureddin

机构信息

Department of Anatomy, Afzalipour Faculty of Medicine, Kerman University of Medical Sciences, 7616913555 Kerman, Iran.

Cellular and Molecular Research Center, Gerash University of Medical Science, 7441758666 Gerash, Iran.

出版信息

Genes (Basel). 2017 Dec 8;8(12):371. doi: 10.3390/genes8120371.

Abstract

The aim of the present study was to determine whether mesenchymal stem cell-conditioned medium (MSC-CM) modulates apoptotic and stress-related gene expression, and ameliorates maturation and developmental potential of immature human oocytes after artificial activation. A total of 247 surplus immature germinal vesicle (GV) oocytes obtained from infertile women were allocated into two in vitro maturation (IVM) groups: 1: GV oocytes ( = 116) matured in vitro (fIVM), and 2: GV oocytes ( = 131) that were vitrified, then in vitro matured (vIVM). Also, two maturation media were used: Alpha-minimum essential medium (α-MEM) and human umbilical cord-derived MSCs (hUCM). After 36 h of incubation, the IVM oocytes were examined for nuclear maturation. In IVM-matured oocytes, cytoplasmic maturation was evaluated after artificial activation through Ionomycin. Moreover, the quantitative expressions of B-cell CLL/lymphoma 2 (), BCL2-associated X protein (), superoxide dismutase (), and Heat shock proteins () in matured oocytes were assessed by quantitative Real-time polymerase chain reaction (qRT-PCR) and compared with fresh and vitrified in vivo matured oocytes, which were used as fIVM and vIVM controls, respectively. The highest maturation rate was found in hUCM in fIVM, and the lowest maturation rate was found using α-MEM in vIVM (85.18% and 71.42%, respectively). The cleavage rate in fIVM was higher than that in vIVM (83.4% vs. 72.0%). In addition, the cleavage rate in α-MEM was lower than that in the hUCM (66.0% vs. 89.4%). Furthermore, the difference between parthenote embryo arrested in 4-8 cells ( < 0.04) and the quality of embryo arrested in 8-cell ( < 0.007) were significant. The developmental stages of parthenote embryos in hUCM versus α-MEM were as follows: 2-4 cell (89.45% vs. 66.00%, respectively), 4-8 cell (44.31% vs. 29.11%, respectively), morula (12.27% vs. 2.63%, respectively), and blastocysts (2.5% vs. 0%, respectively). The messenger RNA (mRNA) expression levels of BCL2, BAX and SOD were significantly different ( < 0.05) between the matured IVM oocytes. Overall, hUCM showed potential efficacy in terms of ameliorating oocyte maturation and in promoting the development and mRNA expression of BAX, BCL2, and SOD.

摘要

本研究的目的是确定间充质干细胞条件培养基(MSC-CM)是否能调节凋亡和应激相关基因的表达,并改善人工激活后未成熟人类卵母细胞的成熟和发育潜能。从不孕妇女获得的总共247个多余的未成熟生发泡(GV)卵母细胞被分为两个体外成熟(IVM)组:1:在体外成熟的GV卵母细胞(n = 116)(fIVM),以及2:经玻璃化处理后再体外成熟的GV卵母细胞(n = 131)(vIVM)。此外,使用了两种成熟培养基:α-最低必需培养基(α-MEM)和人脐带间充质干细胞(hUCM)。孵育36小时后检查IVM卵母细胞的核成熟情况。在IVM成熟的卵母细胞中,通过离子霉素人工激活后评估细胞质成熟情况。此外,通过定量实时聚合酶链反应(qRT-PCR)评估成熟卵母细胞中B细胞淋巴瘤/白血病-2(BCL2)、BCL2相关X蛋白(BAX)、超氧化物歧化酶(SOD)和热休克蛋白(HSP)的定量表达,并与分别用作fIVM和vIVM对照的新鲜和玻璃化体内成熟卵母细胞进行比较。在fIVM中,hUCM组的成熟率最高,而在vIVM中,使用α-MEM组的成熟率最低(分别为85.18%和71.42%)。fIVM中的卵裂率高于vIVM(83.4%对72.0%)。此外,α-MEM中的卵裂率低于hUCM(66.0%对89.4%)。此外,4-8细胞期孤雌胚胎之间的差异(P < 0.04)和8细胞期胚胎质量之间的差异(P < 0.007)具有统计学意义。hUCM与α-MEM中孤雌胚胎的发育阶段如下:2-4细胞期(分别为89.45%对66.00%)、4-8细胞期(分别为44.31%对29.11%)、桑葚胚期(分别为12.27%对2.63%)和囊胚期(分别为2.5%对0%)。成熟的IVM卵母细胞中BCL2、BAX和SOD的信使核糖核酸(mRNA)表达水平存在显著差异(P < 0.05)。总体而言,hUCM在改善卵母细胞成熟以及促进BAX、BCL2和SOD的发育和mRNA表达方面显示出潜在疗效。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d545/5748689/d05ce70f4126/genes-08-00371-g001.jpg

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