SET和MYND结构域蛋白3（SET and MYND domain-containing protein 3,SMYD3）是一种组蛋白H3赖氨酸残基4（Lysine 4,K4）三甲基化转移酶（H3K4 trimethyltransferase）,其在多种癌细胞中高表达,能够促进癌细胞增殖、迁移和侵袭。为研究SMYD3在牛胎儿成纤维细胞（bovine embryonic fibroblast cells,b EFs）中的作用,以牛（Bos taurus）c DNA为模板,PCR扩增牛SMYD3基因CDS,并将其连接到巨细胞病毒（Cytomegalovirus,CMV）启动子表达载体p IRES2-Zsgreen1上,构建高表达SMYD3基因载体p SMYD3-IRES2-Zsgreen1。表达载体转染b EFs后,q RT-PCR和Western blot检测其表达,噻唑蓝（3-（4,5）-dimethylthiahiazo（-z-y1）-3,5-di-phenytetrazoliumromide,MTT）比色法监测其对细胞生长的影响。用鼠（Mus musculus）源POU5f1转录因子（octamer-binding transcription factor 4,Oct4）、性别决定基因相关转录因子2（SRY（sex determining region Y）-box 2,Sox2）、鸟类骨髓细胞瘤元癌基因同源基因（avian myelocytomatosis viral oncogene homolog,c-Myc）和Kruppel样因子4（Kruppel-like factor 4,Klf4）诱导高表达SMYD3基因的b EFs,研究SMYD3对b EFs诱导效率的影响。酶切鉴定结果表明,表达载体p SMYD3-IRES2-Zsgreen1构建成功;q RT-PCR和Western blot结果显示,SMYD3能够在b EFs正常表达,通过荧光筛选得到了稳定表达SMYD3基因的b EFs p SMYD3。MTT结果显示,SMYD3能够促进b EFs的生长;转录因子诱导p SMYD3细胞后,与对照组相比,细胞克隆形成率明显提高,且克隆均为OCT4、NANOG同源框（nanong homeobox,NANOG）和SOX2染色阳性,说明SMYD3能够促进诱导性多能干细胞（induced pluripotent stem cells,i PSCs）诱导效率。本研究表明SMYD3对b EFs的生长有促进作用,并且能够提高b EFs向i PSCs的诱导效率,为牛诱导干细胞制备及机理研究提供新的思路。

This experiment compared the effect of mature liquid that containing three different sources serum: high-quality fetal bovine serum,estrus goat serum,PG treated estrus goat serum,maturation rate were 65.95%,49.2%,76.47%(P0.01)respectively,the difference was extremely significant;compared SOFaa,CR1aa+ oviduct epithelial cells,and improved DMEM/F12 development system and found that,the blastocyst rate of SOFaa,CR1aa+ oviduct epithelial cells were 29.62%,24.73%,difference was not significant(P0.05),the DMEM/F12 system had higher blastocyst development rates 48.42%,the difference was very significant(P0.01).

In this study,we cultured and identified of primordial germ cells(PGCs) which were isolated from genital ridge of the Inner Mongolia cashmere goat fetus.Three groups(A,B,C) culture medium were detected establish culture system for culture of primordial germ cells from Inner Mongolia cashmere goat.After identified by RT-PCR,AKP staining and immunofluorescence,the results showed that the RT-PCR detection of β-actin,hTERT,GAPDH,Nanog and Oct3/4,AKP identified was positive,immunofluorescence detection of Oct3/4,SSEA-3,SSEA-4 were positive.Group A culture medium without any factors could only be passaged 1 generation,group B culture medium supplemented with LIF and insulin could be passaged 4 generations,group C culture medium supplemented with LIF,insulin and high-quality fetal bovine serum culture system could be passaged 9 generations.

ZAR1(Zygote Arrest 1)是母性效应基因,该基因在胚胎发育中起重要作用。本研究对成年牛组织中ZAR1基因表达情况及DNA甲基化状况进行了检测。利用RT-PCR检测成年牛心脏、肾脏、肝脏及睾丸中ZAR1基因mRNA的表达,结果ZAR1在睾丸中表达量较高,肾脏、心脏中较低,而在肝脏中不表达。选定该基因5′调控区中的11个CpG位点,利用亚硫酸盐测序PCR(bisulfite-sequencingPCR,BSP)检测其DNA甲基化状态,结果心脏中的甲基化程度明显高于其他3种组织。结果表明,DNA甲基化与牛ZAR1基因的组织特异性表达相关。