Investigation of the control mechanism of gene coding glucose metabolic protein in preimplantation embryo

着床前胚胎葡萄糖代谢蛋白基因调控机制研究

• 批准号: 12671632
• 负责人: AYABE Takuya
• 金额: $ 1.98万
• 依托单位: Teikyo University, School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671632/
• 关键词: in situ PCR; in situ RT-PCR; follicle growth; polycystic ovary syndrome

We intended, at first, to investigate the control mechanism of gene coding protein which play a role on glucose metabolism in preimplantation embryo. As a method of analysis, we utilized in situ hybridization. In order to analyze a sample for this method, an egg should be prepared with cryostat. This step was, however, difficult and could not achieved because an egg was so small. So, we changed our object from an egg to a follicle. In a menstrual cycle, many follicles are recruited at once but only one follicle matures, and remaining go into atresia by apoptosis.Up to now gene working on apoptosis of follicles were analyzed using a whole ovary. In this study, we investigated the gene in a follicle level. Mouse ovary was collected, then was frozen in OCT compound Cryostat sections of 4-10 micrometer were collected onto pre-coated slideglass. Samples were pre-treated with proteinase K in order to get penetration of the PCR primers into the cell and allow the target sequences to be accessed for amplification. The permeabilization that allows the primers to penetrate the cell must be carefully controlled so that tissue morphology is maintained and the larger products of amplification do not diffuse out Primers for Fas receptor gene were used and a target in 374 bp was amplified by in situ PCR. Two-step reaction program was used and 10 cycles achieved. Digoxygenin(DIG) was incorporated into dNTP, and the amplified product was detected using anti-DIG antibody. Fas receptor gene was detected, and it gathered in inner layer of a follicle. This result may be achieved by in situ hybridization method, but through in situ PCR method we may be able to achieve an investigation of messenger RNA by in situ RT-PCR method.In polycystic ovary syndrome, not one but many follicles grow up at once, so we suppose apoptosis mechanism is deteriorated in this syndrome, Further study may reveal the pathogenesis of this syndrome.

首先，我们打算研究基因编码蛋白对着床前胚胎葡萄糖代谢的调控机制。作为一种分析方法，我们采用原位杂交。为了用这种方法分析样品，应该用低温恒温器准备一个鸡蛋。然而，这一步是困难的，而且无法实现，因为鸡蛋太小了。所以，我们把我们的实验对象从卵子变成了卵泡。在月经周期中，许多卵泡同时被招募，但只有一个卵泡成熟，其余的卵泡通过凋亡进入闭锁状态。迄今为止，对卵泡凋亡相关基因的分析都是在整个卵巢中进行的。在这项研究中，我们在卵泡水平上研究了该基因。收集小鼠卵巢，在OCT复合冷冻中冷冻，收集4-10微米的Cryostat切片于预涂玻片上。样品用蛋白酶K预处理，以使PCR引物渗透到细胞中，并允许进入目标序列进行扩增。必须仔细控制引物渗透细胞的通透性，以保持组织形态，并且扩增的较大产物不会扩散出去，使用Fas受体基因的引物，并通过原位PCR扩增了374 bp的靶标。采用两步反应程序，完成了10个循环。将Digoxygenin（DIG）掺入dNTP中，用抗DIG抗体检测扩增产物。检测到Fas受体基因，该基因聚集在卵泡内层。该结果可以通过原位杂交方法获得，但通过原位PCR方法，我们可以通过原位RT-PCR方法实现对信使RNA的研究。在多囊卵巢综合征中，不是一个而是多个卵泡同时发育，因此我们认为该综合征的细胞凋亡机制恶化，进一步的研究可能揭示该综合征的发病机制。