冷冻保存对猪耳成纤维细胞H19和IGF2R基因DNA甲基化和基因表达的影响
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摘要
为探索冷冻保存对猪体细胞H19和IGF2R基因DNA甲基化的影响,本研究以梅山猪耳成纤维细胞为材料,采用亚硫酸氢盐测序和RT-PCR技术,检测了新鲜和冷冻保存细胞中H19和IGF2R基因的DNA甲基化状态和表达量,并对甲基化相关基因的表达水平进行分析。结果表明:冷冻组中H19基因DMR1和DMR3的甲基化率极显著高于新鲜组(P<0.01),H19基因DMR2表现为去甲基化,极显著低于新鲜组中的甲基化率(P<0.01),且该基因表达量极显著高于新鲜组(P<0.01);冷冻组IGF2R基因DMR2表现为超甲基化,冷冻组极显著高于新鲜组中的甲基化率(P<0.01),但IGF2R基因表达量无显著差异(P>0.05);冷冻组中DNMT3A和DNMT1的基因表达量极显著高于新鲜组(P<0.01),DNMT3B基因表达量则无显著差异(P>0.05)。本实验条件下,冷冻保存影响了H19和IGF2R基因甲基化控制区的DNA甲基化状态,从而影响了该基因的表达水平。
In order to explore the effects of methylation status of H19 and IGF2 R genes in porcine somatic cells induced by cryopreservation, DNA methylation status and expression levels of H19, IGF2 R in Meishan pig ear fibroblast cells before and after cryopreservation were detected with bisulfite sequencing and RT-PCR technology. The expression levels of methylation related genes were also detected and analyzed. The results showed that the methylation rates of H19 DMR1 and DMR3 in cryopreserved cells were significantly higher than those in fresh cells(P <0.01). H19 DMR2 was demethylation, and the methylation rates of H19 DMR2 was significantly lower than that in fresh cells(P<0.01), and expression level of H19 gene was significantly higher than that in the fresh cells(P<0.01). The IGF2 R DMR2 was hypermethylation, and its methylation rates was significantly higher than that in the fresh cells(P<0.01), but there was no significant difference in the expression level of IGF2 R DMR2 between fresh and cryopreserved cells(P>0.05). The expression levels of DNMT3 A and DNMT1 in cryopreserved cells were significantly higher than those in fresh cells(P<0.01), and there was no significant difference in the expression level of DNMT3 B(P>0.05) between two groups. The results indicated that the cryopreservation could affect the methylation status of H19 and IGF2 R DMR, thus changing their gene expression levels
In order to explore the effects of methylation status of H19 and IGF2 R genes in porcine somatic cells induced by cryopreservation, DNA methylation status and expression levels of H19, IGF2 R in Meishan pig ear fibroblast cells before and after cryopreservation were detected with bisulfite sequencing and RT-PCR technology. The expression levels of methylation related genes were also detected and analyzed. The results showed that the methylation rates of H19 DMR1 and DMR3 in cryopreserved cells were significantly higher than those in fresh cells(P <0.01). H19 DMR2 was demethylation, and the methylation rates of H19 DMR2 was significantly lower than that in fresh cells(P<0.01), and expression level of H19 gene was significantly higher than that in the fresh cells(P<0.01). The IGF2 R DMR2 was hypermethylation, and its methylation rates was significantly higher than that in the fresh cells(P<0.01), but there was no significant difference in the expression level of IGF2 R DMR2 between fresh and cryopreserved cells(P>0.05). The expression levels of DNMT3 A and DNMT1 in cryopreserved cells were significantly higher than those in fresh cells(P<0.01), and there was no significant difference in the expression level of DNMT3 B(P>0.05) between two groups. The results indicated that the cryopreservation could affect the methylation status of H19 and IGF2 R DMR, thus changing their gene expression levels
引文
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[2]Riesco M F,Robles V.Cryopreservation causes genetic and epigenetic changes in zebrafish genital ridges[J].PLoS One,2013,8(6):1-9.
[3]Daniels R,Hall V,Trounson A O.Analysis of gene transcription in bovine nuclear transfer embryos reconstructed with granulosa cell nuclei1[J].Biol Reprod,2000,63(4):1034-1040.
[4]Goldberg A D,Allis C D,Bernstein E.Epigenetics:a landscape takes shape[J].Cell,2007,128(4):635-638.
[5]Chat ter jee A,Saha D,Niemann H,et al.Effects of cryopreservation on the epigenetic profile of cells[J].Cryobiology,2017,74:1-7.
[6]Merrifield M,Kovalchuk O.Epigenetics in radiation biology:a new research frontier[J].Front Genet,2013,4:1-16.
[7]Dimond J L,Roberts S B.Germline DNA methylation in reef corals:patterns and potential roles in response to environmental change[J].Mol Ecol,2015,25(8):1895-1904.
[8]Baust J G,Baust J M.Advances in biopreservation[J].Crc Press,2010,13(6):951-961.
[9]Zampolla T,Spikings E,Srirangarajah S,et al.Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish(Danio rerio)ovarian fragments[J].Cryo Letters,2011,32(6):525-536.
[10]Cheng K R,Fu X W,Zhang R N,et al.Effect of oocyte vitrification on deoxyribonucleic acid methylation of H19,Peg3and Snrpn differentially methylated regions in mouse blastocysts.[J].Fertil Steril,2014,102(4):1183-1190.
[11]Wang Z,Xu L,He F.Embryo vitrification affects the methylation of the H19/Igf2 differentially methylated domain and the expression of H19 and Igf2[J].Fertil Steril,2010,93(8):2729-2733.
[12]Urrego R,Rodriguezosorio N,Niemann H.Epigenetic disorders and altered gene expression after use of assisted reproductive technologies in domestic cattle[J].Epigenetics,2014,9(6):803-815.
[13]陈秀莉,马利兵.DNA甲基化与基因表达调节[J].生物技术通报,2010(5):7-10.
[14]刘律君.MicroRNA-375过表达转基因小鼠IGF2R基因的印记状态研究[D].重庆:中南大学,2010.
[15]Tlj K,Jm T.Developmental biology:frontiers for clinical genetics reproductive epigenetics[J].Clinical Genetics,2004,5:247-260.
[16]Chan D,Delbès G,Landry M,et al.Epigenetic alterations in sperm DNA associated with testicular cancer treatment[J].Toxicol Sci,2012,125(2):532-543.
[17]董文娟,潘庆杰,闵令江,等.印迹基因Igf2r的甲基化进程与兔卵母细胞直径关系的研究[J].青岛农业大学学报(自然科学版),2011,28(1):1-4.