导　　师： 王加启;卜登攀

学科专业： 090502

授予学位： 博士

作　　者： ;

机构地区： 中国农业科学院

摘　　要： 乳腺是动物成年后经历增殖、分化、凋亡的唯一器官。奶牛乳腺发育和泌乳机理的研究对提高奶牛的生产性能及乳品质非常重要。microRNAs是一类在转录后调节基因表达的短的非编码RNA。从microRNA的角度研究其对奶牛乳腺发育和泌乳机理的调控是一个较新的研究领域。本试验选取在奶牛分娩前后有差异表达的bta-miR-145为研究对象，通过生物信息学分析、文献报道比较、3’RACE测序、细胞转染、RT-qPCR、Western Blotting、双荧光报告分析等方法在牛乳腺上皮细胞中初步研究了bta-miR-145的功能。主要研究内容如下： 1.为了挖掘更多的与bta-miR-145相关的信息，对bta-miR-145进行了生物信息学分析。通过MEGA5.05软件对牛的bta-mir-145进行种系发育分析，发现牛与人和猪的microRNA145同源关系较近；通过miR TarBase数据对miR-145进行已验证靶标收集，发现在人和小鼠上miR-145靶向IRS1；将Target Scan6.2预测的bta-miR-145的648个靶标带入DAVID数据库进行KEGG信号通路分析，发现大部分靶基因位于MAPK信号通路，其中包括IRS1；PicTar、miRanda、miRBase和miRGen这几个预测软件都预测到miR-145靶向IRS1，Target Scan6.2预测bta-miR-145靶向CSN2；通过RNAhybrid数据库对IRS1和CSN2的bta-miR-145的预测结合位点进行最小自由能分析，发现自由能都小于-15kcal//mol。以上结果表明bta-miR-145可能靶向IRS1和CSN2，并调节MAPK信号通路。 2.为了进一步确定bta-miR-145对牛的IRS1和CSN2基因的靶向关系，通过NCBI和Ensemble数据库对这两个基因进行3’UTR序列分析，结果发现牛的IRS1基因为预测基因，于是通过3’RACE的方法对牛IRS1的3’端进行快速扩增，然后利用Sequencher软件对扩增片段进行拼接，获得牛IRS13’UTR序列，进一步分析发现其上仍然存在预测的bta-miR-145的结合位点；CSN2基因的3’UTR序列在两个数据库中也有一些差异，但都存在预测的bta-miR-145的结合位点。 3.为了获取本研究的试验模型，采用机械破碎法成功获得增殖旺盛的原代牛乳腺上皮细胞，并对细胞进行纯化，纯化后的细胞及纯化后传二十代的细胞都检测到角蛋白-18的表达，表明仍然具有上皮细胞的典型特征。采用不同激素和细胞因子诱导乳腺上皮细胞，发现100ng//mLIGF-1可显著提高乳腺上皮细胞中β-酪蛋白的基因表达，为后续试验提供了分泌功能的细胞模型。 4.为了探索最佳的转染条件，研究了细胞的接种密度、转染时RNAi与脂质体的添加量、bta-miR-145的过表达和抑制表达效果，结果表明：以5×104-1×105//mL的密度接种细胞可以满足转染时细胞汇合率50/%的要求；转染时RNAi与脂质体2000选择/(30/)/(1.5/)组合/(24孔板/)，可以达到最佳转染效率；150pmol//孔的mimic能够达到过表达bta-miR-145的效果，300pmol//孔的inhibitor能够达到抑制表达bta-miR-145的效果/(6孔板/)。 5.为了研究bta-miR-145对牛乳腺上皮细胞增殖的影响，将牛乳腺上皮细胞接种于96孔板，48小时后利用CCK-8试验盒进行检测，结果表明bta-miR-145对牛乳腺上皮细胞生长具有一定的调控作用。 6.为了研究bta-miR-145过表达和抑制表达是否会影响其它microRNA的表达，通过RT-qPCR的方法对bta-miR-214、bta-miR-181a、bta-miR-21进行检测，发现bta-miR-181a与bta-miR-145呈相同的表达趋势，bta-miR-214与bta-miR-145呈相反的表达趋势，bta-miR-21基本不受bta-miR-145过表达和抑制表达的影响。 7.为了研究bta-miR-145是否影响IRS1和CSN2的基因表达和蛋白表达，将bta-miR-145mimic和inhibitor转染入牛乳腺上皮细胞中，利用RT-qPCR和Western blotting方法分别检测细胞中IRS1和CSN2的mRNA及蛋白表达的变化。结果表明Bta-miR-145影响牛IRS1、CSN2的蛋白表达，但并不影响它们的mRNA表达。 8.为了研究bta-miR-145是否与IRS1和CSN2的3’UTR有相互作用，采用双荧光报告基因载体pmiR-RB-REPORTTM构建分别含有IRS1、CSN23’UTR的重组载体，将此重组载体与bta-miR145mimic一起转染入293T细胞。结果表明，以cel-miR-67作对照，bta-miR-145mimic没有引起IRS1和CSN2双荧光比值的明显下调，因此，bta-miR-145可能对IRS1和CSN2的3’UTR并没有明显的相互作用。 综上所述，bta-miR-145对奶牛乳腺发育和泌乳有一定的调控作用，它可以调节牛乳腺上皮细胞的增殖，调节IRS1和CSN2的蛋白表达，但并不调节它们的基因表达，一种microRNA的表达变化会引起其它一系列的microRNA发生变化，应以系统生物学的观点来解释相关的生物学现象。 Mammary gland is the only organ undergoes proliferation and differentiation, apoptosis in adultanimal. Research on bovine mammary development and lactation mechanism is very important forimproving production performance and milk quality of dairy cows. MicroRNAs are a class of shortnon-coding RNAs, which regulate gene expression at post-transcription level. Study on the regulation ofbovine mammary gland development and lactation mechanism from the perspective of microRNA is arelatively new field. This experiment selected bta-miR-145as research object, which was differentiallyexpressed before and after parturition of dairy cows. By bioinformatics analysis, references comparison,3'RACE sequencing, cells transfection, RT-qPCR, Western Blotting, and dual fluorescence analysis,preliminary study of bta-miR-145function was carried out in bovine mammary epithelial cells. Themain research contents are as follows: 1. In order to dig more information related to bta-miR-145, bioinformatics analysis of bta-miR-145was made. Phylogenetic analys is of bta-mir-145was performed by using MEGA5.05software.MicroRNA-145of cow had the closer homologous relationship with pig and human. Validated targets ofmiR-145were collected by miR TarBase database. It was found that miR-145targets IRS1in humanand mouse. Six hundred and forty eight targets of bta-miR-145predicted by Target Scan6.2were putinto DAVID database for analysis of KEGG signal pathway. And most of target genes were located inMAPK s ignaling pathway, including IRS1. Several softwares predicted miR-145targeting IRS1,including PicTar, miRanda, miRBase and miRGen. And Target Scan6.2predicted bta-miR-145targeting CSN2. For binding sites of IRS1and CSN2for bta-miR-145, the minimum free energy /(MFE/)was analyzed through RNAhybrid database. Values of MFE for biding sites were all less than-15kcal//mol. These results suggested that bta-miR-145might target IRS1and CSN2, and regulate MAPKsignaling pathway. 2. In order to further determine the target relationship between IRS1//CSN2and bta-miR-145,3'UTR sequence of two genes was analyzed in NCBI database and Ensemble database. Results showedthat bovine IRS1is a predicted gene. Thus,3' rapid amplification of cDNA end /(RACE/) of bovine IRS1was carried out and amplified fragments were spliced by Sequencher software. Further analysis showedthat the probable binding site for bta-miR-145remained on the3’UTR of IRS1. There were somedifferences in CSN23’UTR sequence in two databases. But the predicted binding site of CSN2forbta-miR-145all existed in both databases. 3. In order to obtain experimental model of this study, highly proliferative primary bovinemammary epithelial cells were successfully cultured by mechanical crushing method. Then, the cellswere purified. The expression of cytokeratin-18was detectable in the purified cells and the cells oftwenty passages after purification. Different hormones and cytokines were used to induce mammaryepithelial cells. It was found that100ng//mL IGF-1significantly increased gene expression of betacasein in mammary epithelial cells. These provided a cell model for subsequent experiment. 4. In order to explore the optimal transfection conditions, inoculation density of cells, adding doseof RNAi and liposome, and over-expression and down-expression of bta-miR-145were studied. Resultsshowed that5×104-1×105//mL of cell density could meet the requirement of50/%of confluent rate.Combination of RNAi and liposome2000/(30/)/(1.5/)/(24hole plate/) could achieve the optimaltransfection efficiency. And150p mol//hole mimic could achieve the effect of bta-miR-145over-expression, and300p mol//hole inhibitor could restrain the expression of bta-miR-145./(6holeplate/). 5. In order to study the effect of bta-miR-145on proliferation of bovine mammary epithelial cells,bovine mammary epithelial cells were cultured in96well plates, and were detected after48h by CCK-8test kit. Results showed that bta-miR-145had a regulatory effect on the growth of bovine mammaryepithelial cells. 6. In order to study whether over-expression or down-expression of bta-miR-145could influencethe expression of other microRNAs or not, bta-miR-214, bta-miR-181a and bta-miR-21was detected byRT-qPCR. It was found that bta-miR-181a had the same trend with bta-miR-145; bta-miR-214showedthe opposite trend with bta-miR-145; and bta-miR-21was not affected by bta-miR-145over-expressionor down-expression. 7. To investigate the effect of bta-miR-145on gene and protein expression of IRS1and CSN2,bta-miR-145mimic or inhibitor was transfected into bovine mammary epithelial cells. Expressionchanges of mRNA and protein of IRS1and CSN2were detected by RT-qPCR and Western blotting.Results showed that bta-miR-145affected protein expression of bovine IRS1and CSN2, but not theirmRNA expression. 8. To study whether there were interactions between bta-miR-145and IRS1//CSN23'UTR,recombinant vectors containing IRS13’UTR and CSN23' UTR, respectively, were constructed by dualfluorescence reporter vector pmiR-RB-REPORTTM. Recombinant plasmid was transfected into293Tcells with bta-miR145mimic. The results showed that bta-miR-145mimic did not lead to obviousreductions of dual fluorescence ratio of IRS1and CSN2, compared with cel-miR-67. Therefore,bta-miR-145had no obvious interactions with IRS1//CSN23'UTR. In conclusion, bta-miR-145had a certain regulation effect on bovine mammary development andlactation. It could adjust bovine mammary epithelial cell proliferation, and regulate IRS1and CSN2protein expression, but not their gene expression. Expression change of a kind of microRNA couldcause changes of a series of microRNAs. The view of systems biology should be adopted to explain therelated biological phenomenon.

关 键 词： 乳腺上皮细胞 增殖

分 类 号： [S823]

领　　域： [农业科学] [农业科学]