Premilinary Research Of Expression Of β-defensins MRNA And Involved Signaling Pathway In Mammary Epithelial Cells From Holstein Cows

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.