【作者】 程兰玲；

【导师】 曹贵方；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2012， 博士

【摘要】 防御素是一种富含精氨酸的阳离子低分子短肽,对G+、G-菌,真菌、包膜病毒和螺旋体等都有广谱的杀伤作用。它不仅是新型高效的抗菌多肽,能直接杀菌,抵御入侵机体的病原微生物,而且能调节机体免疫应答、调节组织创伤修复、在介导获得性免疫反应过程中起着重要作用。防御素是动物机体内防御系统中重要的成员,提示防御素可能在奶牛乳腺中抵抗微生物感染、维持乳腺健康方面起着至关重要的作用。在奶牛发生乳腺炎时,防御素应参与了乳腺的防御过程,但防御素产生了怎样的变化,在变化的过程中又是如何调控的仍不清楚。目前防御素在抗乳腺炎和其防御机制方面的研究报道非常少,而关于β-防御素抗乳腺炎的作用过程更是少之又少,也未见其在奶牛乳腺中的表达调控机制方面的研究。为此我们初步研究了β-防御素基因在奶牛乳腺的表达情况和LPS诱导时β-防御素基因的表达变化及其可能调控的信号通路。其结果如下：1、克隆了奶牛乳腺组织EBD、TAP、LAP、BNBD4、BNBD5和BNBD7基因。以奶牛乳腺组织为材料,提取乳腺组织总RNA,根据NCBI数据库中牛β-防御素cDNA的保守序列分别设计特异性引物,采用RT-PCR技术扩增基因片段,纯化后连接pMD19-T载体,转化入大肠杆菌后挑取阳性重组质粒测序,测序结果证实EBD、TAP、LAP、 BNBD4、BNBD5和BNBD7在奶牛乳腺组织有表达。2、建立稳定的奶牛乳腺上皮细胞培养体系,并确定乳腺上皮细胞6种β-防御素基因的表达情况。采用胶原酶消化法和胰蛋白酶选择性消化法对奶牛乳腺上皮细胞进行分离、培养和纯化。对奶牛乳腺上皮细胞进行原代培养以及传1代细胞的传代培养。经形态学、核型分析和运用RT-PCR方法检测α-酪蛋白基因的表达,绘制生长曲线等方法对所培养的奶牛乳腺上皮细胞进行鉴定和分析,结果证实所培养的细胞为上皮细胞,细胞生长状态良好,符合细胞生长规律。在成功培养奶牛乳腺上皮细胞的基础上,提取乳腺上皮细胞总RNA,运用RT-PCR方法扩增6种β-防御素(EBD、BNBD4、BNBD5、BNBD7、LAP、TAP),测序结果证明6种β-防御素基因在奶牛乳腺上皮细胞有表达。3、获得奶牛乳腺组织与乳腺上皮细胞6种β-防御素mRNA的基础表达水平。通过RT-qPCR方法对6种β-防御素进行扩增及数据统计分析。结果表明,乳腺组织中6种β-防御素基因的相对表达量有显著性差异(P<0.01),仅BNBD4与BNBD5之间的相对表达量没有显著性差异,其中LAP相对表达量最多,BNBD7、EBD、BNBD5、BNBD4次之,TAP的相对表达量最低。奶牛乳腺上皮细胞中6种β-防御素基因mRNA水平的相对表达量有显著性差异(P<0.01),仅EBD与BNBD4之间的相对表达量没有显著性差异,其中LAP相对表达量最多,BNBD7、BNBD5、BNBD4、EBD次之,TAP的相对表达量最低。4、为了研究乳腺上皮细胞中6种β-防御素mRNA表达水平,我们培养乳腺上皮细胞,添加代表大肠杆菌为主要侵染力的脂多糖(LPS)建立试验性乳腺炎的乳腺上皮细胞模型。首先,确定了最佳的传1代细胞来研究6种β-防御素的表达变化。然后在奶牛乳腺上皮细胞中添加不同剂量(50、100、200、400、800ng/ml) LPS处理不同时间(2、4、8、16、24、48、72h),采用实时RT-qPCR方法检测乳腺上皮细胞中6种β-防御素mRNA的表达水平。结果表明：①乳腺上皮细胞中6种β-防御素mRNA表达量与空白对照组相比存在一定的剂量和时间效应,有些随着浓度增加而表达量增高,如LAP,这可能与其基础表达量有关；在时间上总体趋势是当LPS诱导后主要在8h、48h与72h时相对表达量达到高值,在2h、4h、16h和24h表达量相对较低,推测与其信号通路基因的调控时间相关。②LAP表达量差异较大,增加幅度较高,在奶牛乳腺防御中起重要的防御作用。③乳腺上皮细胞中6种β-防御素mRNA的表达量都显著增加,表明6种β-防御素的表达都呈诱导型表达。5、确定奶牛乳腺上皮细胞中是否有TLR2、TLR4、NF-κB P65、CREB表达,根据已发表的基因各自序列设计特异性引物,经RT-PCR鉴定及测序证明4种基因在奶牛乳腺上皮细胞可表达。然后采用RT-qPCR技术检测4个基因mRNA的表达变化。结果表明：①LPS刺激后TLRs中TLR2与TLR4的介导作用中,TLR2主要在4h时起介导作用,在2h、8h和48h起部分调控作用；TLR4在除4h外的几个时间段起主要的介导作用。②TLR2与TLR4都介导了LPS刺激时的调控过程,在整个调控过程中以TLR4起主要介导作用。③CREB在与NF-κB并行的信号通路中,CREB和核因子NF-κB都参与了炎症的调控过程。④在调控过程中CREB主要在8h时起调节作用,NF-κB在各时段都有显著性差异,调控增幅较大。⑤脂多糖对于4种调控因子的表达量都有促进作用。6、为了进一步确定NF-κB途径是否为脂多糖诱导防御素表达的信号通路之一我们在传1代乳腺上皮细胞添加特异性抑制剂PDTC以阻断NF-κB信号通路,然后采用RT-qPCR技术检测BNBD5、BNBD7、TAP、LAP、EBD和BNBD4等基因mRNA表达水平。结果表明：BNBD5、BNBD7、TAP、LAP、EBD和BNBD4mRNA表达量均有下降,在荷斯坦奶牛乳腺上皮细胞中NF-κB为LPS诱导β-防御素基因表达的信号通路之更多还原

【Abstract】 Defensins are a group of cationic peptide, which contain cysteine-rich, broad-spectrum effect on killing G+, G-bacteria, fungi, enveloped viruses, and spirochetes. It is not only efficient antimicrobial peptides, a direct bactericidal effect against the invading organism pathogenic microorganisms, but also can regulate the immune responsing and tissue repairing, playing an important role in mediating acquired immune response in the process. Due to defensins is the largest members in animal defenses, which indicates that defensins may play crucial role in resisting microbe infection and maintaining breast health. Defensins should participate in mammary gland defense processes, but be unknown to defensins produced what have changed and how to carry out adjusting.Nowadays there is seldom research of defensins in resistance to mastitis and its defense mechanism. At the same time, the β-defensins resistance to mastitis in the process is very few. Thus, expression of beta defensin and its possible signal pathways by LPS stimulated were studied initially in this paper. The results are listed as follows:1. At first, to obtain the amplified six kinds of beta defensin (EBD、 BNBD4、 BNBD5、 BNBD7、 LAP、 TAP) gene from the cow mammary gland tissue. Total RNA was extracted from the mammary tissue of a cow and, cDNA encoding beta defensin was amplified by the reverse transcription-PCR (RT-PCR) with the pair of primers which were designed according to the cDNA of NCBI database conserve sequences of reported P-defensins. The objective fragments after purification were connected to the pMD19-T vector, recombinant plasmid positive selection by transformed into Escherichia coli, and samples sequencing. The sequencing results confirmed that the6β-defensins of cloning expressed in mammary gland tissue.2. To establish stable culture system of mammary epithelium cell in cow and confirm the six kinds of P-defensins expression in mammary epithelial cell. We used collagenase digestion and trypsin chosen digestion method to isolate and purify bovine mammary epithelial cells, and culture the original generation of mammary epithelium cells, at least the cells culture processing to the first filial generation. The consequence shows that it is confirmed that the cultured cells are epithelial cell through the verification from morphological and detecting the expression of a-casein by using RT-PCR method. Analyzing chromosomal karyotype and drawing auxodrome curve to the first filial generation cells are confirm cell growth in good condition and compliance with cell growth law. Total RNA was extracted from the mammary epithelial cells which cultured successfully above and cDNA encoding beta defensin was amplified by the reverse transcription-PCR (RT-PCR). The sequencing results confirmed that the6P-defensins of cloning expressed in mammary epithelial cells.3. We detected the basic expression of6kinds of (3-defensins in cow breast tissue and mammary epithelial cells by fluorescence quantitative PCR method in order to study the defense functions of defensins. The results showed that the expression of the relative volume of6β-defensins have significant differences (P<0.01), only the relative expression of BNBD4and BNBD5non-significant difference in breast tissue. The result indicated that relative expression quantity of β-defensins to LAP is the highest, to BNBD7、EBD、BNBD5、BNBD4is in the middle, to TAP is the lowest. The results showed that the expression of the relative volume of6β-defensins have significant differences (P<0.01), only the relative expression of BNBD4and EBD non-significant difference in bovine mammary epithelial cells. The relative expression level of LAP is the highest in six β-defensins, BNBD7、BNBD5、BNBD4、EBD is the middle, the TAP is the minimum.4. After that, establishing mammary epithelial cells model of experimental mastitis by adding LPS produced mainly by E.coli infection in order to studying the six kinds of β-defensins mRNA in mammary epithelial cells. Firstly, we choose the best passage1cells to study changes. The dairy cow mammary epithelial cells were stimulated with different concentration (50,100,200,400and800ng/ml) of LPS. Then the total RNA was extracted after stimulated in seven different times (2,4,8,16,24,48and72h), and the mRNA expression levels of six kinds of β-defensins were evaluated by real-time quantitative PCR. The results show that:①LPS significantly down-regulated a-casein mRNA expression in a time and dose dependent, and the mammary epithelial cells produced a certain inflammatory by adding the LPS. The expression of six kinds of beta-defensins mRNA has significant differences in a time and dose dependent. Some increased with the concentration. The time overall trend of the relative expression is amount high value in8h、48h and72h, and a lower expression level in2h、4h、16h and24h after LPS stimulated mammary epithelial cells.②The expression level differences of LAP gene mRNA is larger and the amplitude is higher than other beta-defensin.③The expression level of the6beta-defensins increased significantly indicated that they are all inducible expression and the increased extent related to the basic expression levels in cow mammary epithelial cells.5. Initial we determine whether TLR2mRNA、TLR4mRNA、 NF-κB P65mRNA、 and CREB mRNA are expressed in cow mammary epithelial cells. We designed specific primers based on the published TLR2、 TLR4、 NF-κB P65、 and CREB sequence. By RT-PCR analysis and sequencing revealed that these two genes are expressed in cow mammary epithelial cells. Then the expression changes were detected by fluorescence quantitative PCR. The results indicate that:①the regulation of TLR2plays a major role in4h, and partial regulation in2h、8h and48h, at the same time, TLR4play a main role in regulating in several time segment, except4h after LPS stimulated.②TLR2and TLR4of TLRs were involved in the whole regulated process, and TLR4as the main control function.③The results show that CREB and NF-κB were involved in the whole regulated process.④the regulation of CREB plays a major role in8h, and the expression of NF-κB have significant differences in each of the slots.⑤Four kinds of nuclear factors were involved in the expression regulation between LPS and P-defensins.6. In order to further investigate NF-κB involved mediating the effect of LPS on the expression of defensin (BNBD5、 BNBD7、TAP、 LAP、 EBD and BNBD4) in mammary epithelial cells. The changes in the mRNA expression of BNBD5、 BNBD7、 TAP、LAP、 EBD and BNBD4in mammary epithelial cells treated with estrogen nuclear receptor inhibitor were studied by real-time PCR. The results show:The expression of BNBD5、 BNBD7、 TAP、LAP、EBD and BNBD4mRNA were decreased. The signaling pathway of NF-κB is one of signaling pathways for LPS-induced beta-defensin gene expression in Holstein cow mammary epithelial cells.更多还原