摘要
肝X受体α(Liver X Receptorα, LXRα)是核受体超家族中非甾体类成员,LXRα特异表达于脂质代谢旺盛的组织,如肝、肠、肾、乳腺、脾和肌肉等。大量研究证明LXRα是机体保持胆固醇相对稳定的关键感受器。可通过调控胆固醇的输出、胆汁酸的产生、脂肪酸的合成及几种脂质转运蛋白从而保持机体脂质的动态平衡。在山羊上,目前还没有完整的LXRα基因序列。本研究克隆西农萨能奶山羊乳腺LXRα基因的cDNA序列并对其进行生物信息学分析;研究西农萨能奶山羊LXRα基因组织表达差异;研究LXRα激动剂TO901317对乳腺上皮细胞中LXRα基因、脂肪酸合酶(Fatty acid synthase, FASN)基因、脂蛋白脂酶(Lipoprotein Lipase, LPL)基因的表达及对细胞脂肪酸组成的影响。试验结果如下:
本研究采用RT-PCR和RACE技术,分离并克隆了西农萨能奶山羊LXRα基因的cDNA序列,其全长1654bp(GenBank收录号为:GU332719),包括5’UTR150bp,CDS 1344bp和3’UTR 160bp,共编码447个氨基酸。经序列比对发现山羊LXRα基因与其他物种相似性较高,其CDS与GeneBank中牛(BC103207)、小鼠(NM_013839)、大鼠(NM_031627)、猪(NM_001101814)和人(NM_005693)的LXRα基因相比较,CDS核苷酸相似性分别为98%、87%、87%、93%和90%,氨基酸的相似性分别为98%、91%、91%、96%和92%。其UTR核苷酸与牛、小鼠、大鼠、猪和人的相应序列比较,5’UTR相似性为93%、27%、82%、23%和74%,3’UTR相似性为98%、63%、65%、78%和79%。经蛋白质结构分析表明,推测山羊LXRα蛋白质的分子量为50.35kD,等电点为6.30,整个序列中不含信号肽与跨膜结构。该蛋白N端到C端均存在较强的亲水区域,按分值大小划分其疏水最大值为3.233,最小值为-3.089,分别位于第84和361氨基酸序列处。LXRα二级结构主要由49.66%不规则卷曲、49.00%α-螺旋和1.34%延伸链组成。其二级结构主要包括三个功能结构域:配体结合区域(LBD, Ligand-binding Domain)、DNA结合区域(DBD, DNA-binding Domain)和锌指蛋白C4型区域(zf-C4, Zinc finger C4 type)。
应用ABI PRISM?7300实时荧光定量PCR仪采用SYBR-Green法对西农萨能奶山羊11个组织LXRα基因表达量检测,以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)基因为内参。试验结果显示LXRα基因在垂体、肺脏、肝脏、瘤胃、卵巢、脾脏、皮下脂肪、乳腺、肾脏、心脏及小肠组织中均有表达。LXRα基因在小肠组织中的表达量最高,肝脏、脾脏、皮脂、乳腺次之,分析表明小肠表达量相对于其他组织差异显著(P<0.05),肝脏、脾脏、皮脂、乳腺基因表达量间差异不显著(P>0.05),心脏中表达量最低。
以西农萨能奶山羊乳腺上皮细胞为试验材料,应用激动剂TO901317(0、0.1、0.01、1、10)μmol/L浓度的培养基处理细胞(0μmol/L为空白对照),在相同处理时间下乳腺上皮细胞中LXRα、FASN、LPL基因mRNA表达量均上调,其中10μmol/L的LXRα激动剂表达量上调最大;同种浓度激动剂处理条件下,随着培养时间的延长(12、24、48、72)h,乳腺上皮细胞中LXRα、FASN、LPL基因mRNA表达量呈先升高后降低的趋势,培养48h达到最大值。相关分析表明,不同浓度(0.1、0.01、1、10)μmol/L激动剂对西农萨能奶山羊乳腺上皮细胞LXRα、FASN、LPL基因mRNA表达量呈正相关。对细胞总脂肪酸检测结果显示,细胞内总脂肪酸含量增加,不饱和脂肪酸的比例升高,表明LXRα基因对乳腺上皮细胞不饱和脂肪酸具有重要的调控作用。
LXRα(Liver x-activate receptorα)is an non-steroidal receptor in the nuclear receptor superfamily. The gene is expressed in specific lipid metabolism tissues, such as liver, intestine, kidney, breast, spleen and muscle. Numerous studies have demonstrated that LXRαis a key senser in the maintaining of cholesterol level in body. LXRαcontrols the cholesterol output and bile acid production, regulates fatty acid synthesis and several lipid-transfer proteins, and therefore contributes to the course of lipid homeostasis. So far in goats there is still no complete sequence of the LXRαgene. In this study, the mammary gland tissue of Xinong Saanen dairy goat was collected in the Northwest A & F University Saanen goats farm. The cDNA sequence of LXRαgene was cloned from total RNA of the tissue and analyzed using bioinformatics methods. The present study examined the LXRαgene expression in the different tissues of Saanen goats. Also, we analized the effects of TO901317, an LXRαagonist, in goat mammary gland epithelial cells by detecting the expression of the fatty acid synthase (FASN) gene, lipoprotein lipase (LPL) gene, and LXRαgene, as well as fatty acid composition in GMEC. The results are as follows:
The cDNA sequence of LXRαgene in Xinong Saanen dairy goat mammary gland was cloned by RT-PCR and RACE from total RNA of the mammary gland tissue. The cDNA of wild type LXRαgene was cloned successfully and registered in GenBank (GenBank accession No. GU332719). The cDNA sequence is 1654 bp in length, including 1344bp of open reading frame, which encodes a polypeptide of 447 amino acids. The UTR contains 160bp of 3’UTR and 150bp of 5’UTR.The nucleotide homology between the cloned goat LXRαgene and its counterparts in Bos taurus (BC103207), Mouse (NM_013839), Rattus norvegicus (NM_031627), Sus scrofa (NM_001101814) and Human (NM_005693) are 98%, 87%, 87%, 93%, and 90%; the homology of amino acid sequences are 98%, 91%, 91%, 96%, and 92%. The nucleotide homology of 5’UTR nucleotide sequence of LXRαgene with the counterparts of Bos taurus (BC103207), Mouse (NM_013839), Rattus norvegicus (NM_031627), Sus scrofa (NM_001101814) Human(NM_005693) are 93%, 27%, 82%, 23%, and 74%; The nucleotide homology of 3’UTR nucleotide sequence are 98%, 63%, 65%, 78%, 79% respectively. Protein structure analysis suggested that the relative molecular weight of LXRαreceptor is 50.35kD, its isoelectric point is 6.30. It most likely does not have a signal peptide structure and transmembrane domain. There are strong hydrophilic regions located at amino acid sequence 84 and 361. The secondary structure of the protein containsα-helices by 49.00%, an extension of the main chain(extended strand)by 1.34%, and random coil by 49.66%. LXRαprotein is mainly consists of three functional domains: a Ligand-binding Domain, a DNA-binding Domain, and a Zinc fingerr C4 region.
Taking the GAPDH gene as internal reference, use the RT-qPCR technology of ABI PRISM?7300 Real-Time PCR System with the SYBR-Green method to test 11 tissues about the LXRαgene expression on Xinong Saanen dairy goat. The results showed that LXRαgene were expressed in the pituitary, lung, liver, rumen, ovary, spleen, sebum, mammary gland, kidney, heart and intestinal tissues. The highest expression happened in the small intestine, followed by liver, spleen, sebum and mammary gland. Analysis of expression variance showed a significant difference in the small intestine compared to other tissues(P<0.05), no significant difference among liver, spleen, sebum, mammary gland(P>0.05).The lowest expression happened in heart.
Using the Saanen dairy goat mammary epithelial cells as experimental material, the mRNA expressions of LXRα, FASN, LPL genes had a trend to increase, with LXRαagonist TO901317 concentration’s increasing from 0 to 10μmol/L,When the dose of LXRαagonist was 10μmol/L, the mRNA expressions of genes above reached the maximum. With the extension of culture time, the mRNA expressions of LXRα, FASN, LPL genes in mammary epithelial cells had a trend to increase. When the culture time was less than 48 hours, however, it reduced after 48 hours. The maximum achieved at the 48th hour. The correlation analysis showed that the mRNA expressions in LXRα, FASN, LPL genes had a significant positive correlation with the doses of LXRαagonis(t0.01、0.1、1、10)μmol/L. Test of the total cellular fatty acids showed that the total content of fatty acid in cell increased, the proportion of unsaturated fatty acids increased, indicating that LXRαgene played an important role in regulation of unsaturated fatty acids in breast epithelial cell.
引文
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