發(fā)布時(shí)間：2018-05-01 05:36

  本文選題：線粒體膜電位 + mtDNA拷貝數(shù)��； 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：哺乳動物胚胎體外培養(yǎng)體系影響著早期胚胎的發(fā)育潛能。在目前模擬體內(nèi)輸卵管及子宮的低氧環(huán)境的培養(yǎng)系統(tǒng)中,活性氧(reactive oxygen species,ROS)被認(rèn)為是影響胚胎發(fā)育的一個(gè)重要因素。胚胎合子基因組激活(zygotic gene activation,ZGA)是早期胚胎正常發(fā)育的關(guān)鍵,研究認(rèn)為ZGA的延遲或失敗與早期胚胎發(fā)育阻滯有關(guān)。為探究ROS對小鼠早期胚胎發(fā)育阻滯的影響,本研究比較了不同品種小鼠在不同培養(yǎng)液中發(fā)生阻滯的情況,檢測了 ZGA相關(guān)基因、胞質(zhì)ROS濃度及ROS相關(guān)基因的表達(dá);同時(shí)利用2,2-偶氮二(2-甲基丙基咪)二鹽酸鹽AAPH處理昆明白小鼠體外受精卵建立ROS引起阻滯的細(xì)胞模型,對線粒體機(jī)能相關(guān)的線粒體膜電位、線粒體ROS(mtROS)及線粒體分布狀態(tài)、mtDNA的拷貝數(shù)進(jìn)行了檢測;另外,檢測了線粒體相關(guān)基因Grm2、Drd2、Polg2、TE4M的表達(dá)變化及其調(diào)節(jié)區(qū)的DNA甲基化的調(diào)節(jié)。1、不同品系小鼠胚胎在不同培養(yǎng)液中合子基因組激活與ROS相關(guān)性研究本研究利用實(shí)時(shí)熒光定量PCR的方法檢測了小鼠2-細(xì)胞胚胎中ZGA相關(guān)基因(Zscan4、MuERV-L、Eif-1a、Hsp70.1)及ROS相關(guān)基因(Nox1、Gpx4、Gpu6、Prdx2)的表達(dá),并利用ROS特異性染料DCFH-DA對胚胎胞質(zhì)內(nèi)ROS水平進(jìn)行檢測。首先比較了阻滯品系小鼠KM與非阻滯品系小鼠BDF1受精卵在M16培養(yǎng)液中發(fā)生阻滯的情況,結(jié)果表明,在M16培養(yǎng)液中,KM小鼠受精卵的4-細(xì)胞發(fā)育率為44.2%,而BDF1小鼠受精卵的發(fā)育率為90.3%,KM小鼠2-細(xì)胞胚中ZGA相關(guān)基因MuERV-L、Eif-1a的表達(dá)量顯著低于BDF1小鼠2-細(xì)胞胚(P0.05)。KM小鼠2-細(xì)胞胚胎的胞質(zhì)內(nèi)ROS水平明顯低于BDF1胚胎,ROS相關(guān)基因Gp和Prdx2的表達(dá)顯著高于BDF1小鼠。以上研究表明,小鼠胚胎發(fā)育阻滯與ZGA相關(guān)基因表達(dá)有關(guān),ROS水平的差異反映出不同品系小鼠胚胎對ROS的耐受性不同。之后利用M16培養(yǎng)液和KSOM培養(yǎng)液比較了阻滯品系KM小鼠胚胎在不同培養(yǎng)液條件下的發(fā)育。結(jié)果顯示,在KSOM培養(yǎng)液中,KM小鼠受精卵的4-細(xì)胞率為90.8%,不發(fā)生2-細(xì)胞阻滯。KSOM培養(yǎng)液中培養(yǎng)的KM小鼠2-細(xì)胞胚胎Eif-1a、Hsp70.1表達(dá)量顯著高于M16培養(yǎng)液中培養(yǎng)的2-細(xì)胞胚胎。兩種培養(yǎng)液中的KM小鼠2-細(xì)胞胚胎的胞質(zhì)內(nèi)ROS水平和ROS相關(guān)基因表達(dá)無明顯差異。以上研究表明,小鼠胚胎體外培養(yǎng)液成分的不同影響ZGA相關(guān)基因表達(dá)的變化,對小鼠胚胎發(fā)育阻滯產(chǎn)生影響。2、ROS對發(fā)育阻滯胚胎中線粒體相關(guān)機(jī)能的影響本實(shí)驗(yàn)探究了 ROS在小鼠體外發(fā)育2細(xì)胞阻滯胚胎中對線粒體相關(guān)機(jī)能的影響。使用AAPH處理昆明白小鼠體外受精卵,通過熒光探針檢測處理組和對照組中線粒體膜電位、線粒體ROS(mtROS)及線粒體分布狀態(tài)的變化,并利用實(shí)時(shí)熒光定量PCR檢測ROS相關(guān)基因、線粒體相關(guān)基因(Grm2、Drd2、Polg2、TFAM)mRNA的表達(dá)量及mtDNA的拷貝數(shù),利用重亞硫酸鹽測序(BSP)檢測了線粒體四個(gè)相關(guān)基因的甲基化變化。研究結(jié)果顯示,使用1.0 mmol/L濃度的AAPH分別處理的胚胎,具有較高的阻滯率(66.16%)和較低的損傷率(24 h畸形率9.09%,48 h死亡率10.39%),ROS染色實(shí)驗(yàn)結(jié)果顯示,AAPH處理能夠使胚胎內(nèi)胞質(zhì)ROS水平明顯升高,所檢測的ROS相關(guān)基因mRNA表達(dá)量升高。1.0 mmol/L處理后二細(xì)胞期胚胎線粒體膜電位及mtROS水平明顯高于對照組,mtDNA的拷貝數(shù)增加,實(shí)時(shí)熒光定量PCR結(jié)果顯示,處理組與對照組的Grm2、Drd2、Polg2、TFAM基因表達(dá)量呈現(xiàn)顯著性差異,處理組Grm2、Drd2表達(dá)顯著低于對照組,處理組Polg2、TFAM表達(dá)顯著高于對照組。利用亞硫酸鹽測序(bisulfite genomic sequencing,BSP)方法檢測DNA甲基化的結(jié)果,AAPH處理之后的胚胎中基因調(diào)節(jié)區(qū)DNA甲基化水平升高;Polg2、TFAM甲基化水平降低。以上研究表明,AAPH處理引起的氧化應(yīng)激反應(yīng)及發(fā)育阻滯的胚胎中,線粒體活性升高,線粒體數(shù)目增加,線粒體相關(guān)基因表達(dá)變化,表明線粒體參與調(diào)節(jié)細(xì)胞內(nèi)ROS的動態(tài)平衡,ROS可能通過影響線粒體相關(guān)基因啟動子區(qū)DNA甲基化的變化調(diào)控線粒體相關(guān)基因的時(shí)序性表達(dá)。綜上所述,不同品系小鼠胚胎對ROS的耐受性不同,體外培養(yǎng)環(huán)境中小鼠胚胎發(fā)育阻滯與ZGA相關(guān)基因表達(dá)有關(guān),線粒體參與調(diào)節(jié)細(xì)胞內(nèi)ROS的動態(tài)平衡影響胚胎發(fā)育。本研究初步探究了 ROS與線粒體相關(guān)功能變化的關(guān)系,為優(yōu)化哺乳動物胚胎體外培養(yǎng)體系提供參考。
[Abstract]:In vitro culture system of mammalian embryos affects the developmental potential of early embryos. Reactive oxygen species (ROS) is considered as an important factor affecting embryo development in the culture system that simulates the hypoxic environment of the oviduct and uterus in the body at present. The genome activation of the embryo of the embryo (zygotic gene activation, ZGA) is early. The key to the normal development of the embryo is that the delay or failure of ZGA is related to the early embryonic development block. In order to explore the effect of ROS on the early embryonic development block of mice, this study compared the arrest of different mice in different cultures, and detected the ZGA phase gene, the concentration of cytoplasmic ROS and the table of ROS related genes. At the same time, using 2,2- azo two (2- methyl proprom) two hydrochloride AAPH to treat the cell model of ROS block in vitro fertilized eggs of Kunming white mice, the mitochondrial membrane potential related to mitochondrial function, mitochondrial ROS (mtROS) and mitochondrial distribution state, and the number of mtDNA copy scallop were detected, and the mitochondrial related gene Gr was detected. The expression changes of M2, Drd2, Polg2, TE4M and the regulatory.1 of DNA methylation in the regulatory region, the study on the correlation between the activation of zygote genomes in different strain mice embryos and the correlation of ROS in different cultures. This study used real time fluorescence quantitative PCR to detect ZGA phase Guan Jiyin in mouse 2- cell embryos. The expression of the gene (Nox1, Gpx4, Gpu6, Prdx2) and the ROS specific dye DCFH-DA were used to detect the ROS level in the cytoplasm of the embryo. First, the arrest of the BDF1 fertilized eggs of the block mouse KM and the non block strain mice in the M16 culture solution was compared. The results showed that the growth rate of the fertilized egg of the KM mice was 44. in the M16 culture medium. 2%, the growth rate of the fertilized eggs of BDF1 mice was 90.3%, and the ZGA related gene MuERV-L in the 2- cell embryos of the KM mice was significantly lower than that of the BDF1 mouse 2- cell embryos (P0.05).KM mice. The ROS level in the cytoplasm of the 2- cell embryos was significantly lower than that of the embryos. The embryonic development block was related to the expression of ZGA related genes. The difference of ROS level reflected the different tolerance of mouse embryos from different strain mice to ROS. Then the development of KM mouse embryos in the block line was compared with the M16 culture and KSOM culture. The results showed that the 4- cells of the fertilized eggs of KM mice were in the KSOM culture solution. The rate was 90.8%, the KM mouse 2- cell embryo Eif-1a was not cultured in the.KSOM culture medium with 2- cell block. The expression of Hsp70.1 was significantly higher than that of the 2- cell embryos cultured in the M16 culture solution. The ROS level and the ROS related gene expression in the cytoplasm of the KM mouse 2- cell embryos in the two cultures were not distinct. The above study showed that the mouse embryos were in vitro The effects of the different components of the culture medium on the expression of ZGA related genes, the effect of.2, and the effect of ROS on the mitochondrial related function in the developmental block embryos, the effect of ROS on the mitochondrial related function in the 2 cell block embryos developed in vitro in mice was investigated. The use of AAPH in the treatment of Kunming white mice in vitro was studied. Sperm egg, the mitochondrial membrane potential, mitochondrial ROS (mtROS) and mitochondrial distribution in the treatment group and the control group were detected by the fluorescence probe, and the ROS related genes were detected by real-time fluorescence quantitative PCR, the amount of mRNA and the copy number of mtDNA of the mitochondrial related genes (Grm2, Drd2, Polg2, TFAM) were detected by the heavy sulphite sequencing (BSP) detection. The change of methylation of four related genes of mitochondria was observed. The results showed that the embryos treated with 1 mmol/L concentration of AAPH had higher block rate (66.16%) and lower damage rate (24 h malformation rate 9.09%, 48 h mortality 10.39%). The results of ROS staining showed that AAPH treatment could significantly increase the ROS level in the cytoplasm of the embryo. The expression of ROS related gene mRNA increased by.1.0 mmol/L. The mitochondrial membrane potential and mtROS level of the two cell stage embryos were significantly higher than those of the control group. The copy number of mtDNA increased. The real-time fluorescence quantitative PCR results showed that the Grm2, Drd2, Polg2, and TFAM base of the treated group and the control group showed significant difference. The expression of Polg2 and TFAM in the treatment group was significantly higher than that in the control group. The results of DNA methylation were detected by the method of bisulfite genomic sequencing (BSP), and the level of DNA methylation in the gene regulatory region of the embryos after AAPH treatment was increased; Polg2, TFAM methylation level decreased. The above study showed that AAPH treatment caused it. In the oxidative stress reaction and the development block embryo, the mitochondrial activity is elevated, the number of mitochondria is increased, and the mitochondrial related gene expression changes, indicating that mitochondria are involved in regulating the dynamic balance of ROS in cell, and ROS may regulate the sequence of mitochondrial related genes by affecting the changes of DNA methylation in the promoter region of the mitochondrial related genes. To sum up, the tolerance of mouse embryos in different strains of ROS was different. The development of mouse embryo retardation was related to the expression of ZGA related genes in the culture environment. Mitochondria involved in regulating the dynamic balance of ROS in the cell. This study explored the relationship between ROS and mitochondrial related function, in order to optimize the mammal. It provides reference for the culture system of embryo in vitro.

【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q954.4

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本文編號：1827986