无乳链球菌对奶牛乳腺成纤维细胞几种生长因子表达的影响

发布时间：2018-05-19 23:28

  本文选题：奶牛乳腺成纤维细胞 + 无乳链球菌　； 参考：《内蒙古农业大学》2017年硕士论文

【摘要】：无乳链球菌(Streptococcus aagalactiae,S.agalactiae)通过乳头管侵入奶牛乳房内部,引起奶牛隐性或部分临床型乳腺炎。奶牛乳腺炎可引起乳腺损伤和机能障碍,最终会造成乳腺纤维化。奶牛乳腺成纤维细胞(BMFBs)是乳腺纤维化的主要参与细胞,在奶牛乳腺发生纤维化时可以产生细胞外基质(ECM)。本文探讨S.aagalactiae对BMFBs纤维化相关的细胞因子和受体的表达的影响,以便为乳腺纤维化的机制丰富数据。本研究以体外培养的BMFBs作为研究对象,在6h、12h、24h和48h后,分别用无血清培养液和105CFU/mL、106CFU/mL、108CFU/mL的不同浓度的热灭活S.agalactiae作用于BMFBs,采用RT-qPCR法检测转化生长因子-β1(TGF-β1)、碱性成纤维细胞生长因子(bFGF)、血小板源性生长因子(PDGF-BB)和对应的受体 TβRI、FGFR2、PDGFRβ 以及 Toll 样受体(TLR2、TLR4)的 mRNA 的表达,采用 Western-blot 法检测 TGF-β1、bFGF、PDGF-BB及对应的受体 TβRI、FGFR2、PDGFRβ、Toll 样受体(TLR2、TLR4)、NF-κB和AP-1的蛋白的表达。结果显示:(1)菌液处理组中的TGF-β1 mRNA相比对照组表达量明显升高,24h时表达量最大(P0.05);TGF-β1蛋白表达量于12h时达到最大值,然后蛋白表达量开始降低(P0.05)。TβRI mRNA的表达量在48h达到峰值(P0.05);TβRI蛋白在12h的表达量低于6h时,在24h时达到峰值,之后又下降(P0.05)。bFGF和FGFR2的mRNA的表达量均是108CFU/mL菌液浓度处理组的最大(P0.05);bFGF蛋白表达量在48h时的表达量最大;FGFR2蛋白表达量在6h时表达量最大(P0.05)。PDGF-BB mRNA表达量在48h的106CFU/mL浓度处理组达到最大值,PDGFRβmRNA表达量在12h的108CFU/mL菌液浓度处理组达到最大值(P0.05);PDGF-BB蛋白表达量在6h的106CFU/mL菌液浓度处理组的表达量最大,PDGFRβ蛋白表达量在24h时、105CFU/mL菌液浓度处理组的表达量最大(P0.05)。(2)与对照组比较,TLR2除了 48h时外,TLR4除了 6h时外,TLR2、TLR4的mRNA的表达量在108CFU/mL处理组最大,且差异性显著(P0.05)。在同一时间段,TLR2蛋白的表达量会随着处理组菌液浓度的加大而降低。随着作用时间的延长,菌液浓度增高,TLR4的蛋白的表达量会变低。(3)热灭活的无乳链球菌可促进NF-κB和AP-1蛋白的表达。在同一时间段,NF-κB和AP-1蛋白的表达量在105CFU/mL处理组最大,与对照组比较差异显著(P0.05)。研究表明,热灭活的无乳链球菌能够促进体外培养的BMFBs上述三种细胞因子及对应的受体和TLR2、TLR4的mRNA及蛋白的表达,热灭活的无乳链球菌也能够促进NF-κB和AP-1蛋白的表达,这些与纤维化有关的生长因子及其受体和TLR2、TLR4在奶牛乳腺纤维化中可能发挥着重要作用。
[Abstract]:Streptococcus aagalactiaeus S.agalactiae) invades the breast through the papillary tube and causes recessive or partial clinical mastitis. Cow mastitis can cause breast damage and dysfunction, eventually leading to breast fibrosis. Bovine breast fibroblasts (BMFBs) are the main cells involved in breast fibrosis, which can produce extracellular matrix (ECM) during the development of breast fibrosis in dairy cows. To investigate the effect of S.aagalactiae on the expression of cytokines and receptors related to BMFBs fibrosis in order to enrich the data on the mechanism of breast fibrosis. In this study, BMFBs cultured in vitro was used as the research object. BMFBswere exposed to different concentrations of heat-inactivated S.agalactiae in serum-free medium and 105CFU / mLX 106CFU / mL, respectively. Transforming growth factor- 尾 _ 1tGF- 尾 _ (1), basic fibroblast growth factor (bFGF-), platelet-derived growth factor (PDGF-BBB) and corresponding receptor T 尾 -RII-FGFR2PDGFR 尾 were detected by RT-qPCR assay. The expression of mRNA in Toll like receptor TLR2 and TLR4. Western-blot assay was used to detect the expression of NF- 魏 B and AP-1 protein in TGF- 尾 1, bFGFG, PDGF-BB and its corresponding receptor, T 尾 RII-FGFR2, PDGFR 尾 -Toll-like receptor, TLR2 and TLR4, respectively. The results showed that the expression of TGF- 尾 1 mRNA in the solution group was significantly higher than that in the control group. The maximum expression level of TGF- 尾 1 protein reached the maximum at 12 h after 24 h treatment. Then the protein expression level began to decrease, the expression of P0.05N 路T 尾 RI mRNA reached the peak at 48h and reached the peak at 12h when the expression level was lower than 6h, and reached the peak at 24h. Then the mRNA expression of P0.05, bFGF and FGFR2 were both the maximum expression of P0.05 and P0.05 bFGF protein in 108CFU/mL solution treatment group. The maximum expression amount of FGFR2 protein at 48h was the highest expression amount of P0.05FGFR2 protein at 6h. P0.05U. PDGF-BB mRNA expression at 48h was the largest in the 106CFU/mL group treated with 108CFU/mL solution concentration. The maximum expression of PDGFR 尾 mRNA was reached in the treatment group with 12h concentration of PDGFR 尾. The expression of PDGF-BB protein was the highest in the group treated with P0.05 and PDGFR 尾 at 6h. The expression of PDGFR 尾 protein was the highest in the group treated with the concentration of 105CFU / mL at 24 h. Compared with the control group, the mRNA expression of TLR2 was the highest in the 108CFU/mL treatment group except at 48 h, except at 6 h. The difference was significant (P 0.05). At the same time, the expression of TLR2 protein decreased with the increase of the concentration of the treated group. With the prolongation of the action time, the protein expression of TLR4 increased with the increase of the concentration of bacteria. The expression of NF-kappa B and AP-1 protein was enhanced by heat-inactivated streptococcus lactis. The expression of NF- 魏 B and AP-1 protein was the highest in the 105CFU/mL treatment group at the same time, and the difference was significant compared with the control group (P 0.05). The results showed that the heat-inactivated streptococcus actinomycetes could promote the expression of the above three cytokines and corresponding receptors of BMFBs in vitro and the mRNA and protein of TLR2TLR4, and the heat-inactivated streptococcus actinoides could also promote the expression of NF- 魏 B and AP-1 protein. These fibrogenic growth factors and their receptors and TLR2 TLR4 may play an important role in dairy breast fibrosis.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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