本文選題：奶牛乳腺成纖維細(xì)胞 + 無乳鏈球菌��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：無乳鏈球菌(Streptococcus aagalactiae,S.agalactiae)通過乳頭管侵入奶牛乳房?jī)?nèi)部,引起奶牛隱性或部分臨床型乳腺炎。奶牛乳腺炎可引起乳腺損傷和機(jī)能障礙,最終會(huì)造成乳腺纖維化。奶牛乳腺成纖維細(xì)胞(BMFBs)是乳腺纖維化的主要參與細(xì)胞,在奶牛乳腺發(fā)生纖維化時(shí)可以產(chǎn)生細(xì)胞外基質(zhì)(ECM)。本文探討S.aagalactiae對(duì)BMFBs纖維化相關(guān)的細(xì)胞因子和受體的表達(dá)的影響,以便為乳腺纖維化的機(jī)制豐富數(shù)據(jù)。本研究以體外培養(yǎng)的BMFBs作為研究對(duì)象,在6h、12h、24h和48h后,分別用無血清培養(yǎng)液和105CFU/mL、106CFU/mL、108CFU/mL的不同濃度的熱滅活S.agalactiae作用于BMFBs,采用RT-qPCR法檢測(cè)轉(zhuǎn)化生長(zhǎng)因子-β1(TGF-β1)、堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)、血小板源性生長(zhǎng)因子(PDGF-BB)和對(duì)應(yīng)的受體 TβRI、FGFR2、PDGFRβ 以及 Toll 樣受體(TLR2、TLR4)的 mRNA 的表達(dá),采用 Western-blot 法檢測(cè) TGF-β1、bFGF、PDGF-BB及對(duì)應(yīng)的受體 TβRI、FGFR2、PDGFRβ、Toll 樣受體(TLR2、TLR4)、NF-κB和AP-1的蛋白的表達(dá)。結(jié)果顯示:(1)菌液處理組中的TGF-β1 mRNA相比對(duì)照組表達(dá)量明顯升高,24h時(shí)表達(dá)量最大(P0.05);TGF-β1蛋白表達(dá)量于12h時(shí)達(dá)到最大值,然后蛋白表達(dá)量開始降低(P0.05)。TβRI mRNA的表達(dá)量在48h達(dá)到峰值(P0.05);TβRI蛋白在12h的表達(dá)量低于6h時(shí),在24h時(shí)達(dá)到峰值,之后又下降(P0.05)。bFGF和FGFR2的mRNA的表達(dá)量均是108CFU/mL菌液濃度處理組的最大(P0.05);bFGF蛋白表達(dá)量在48h時(shí)的表達(dá)量最大;FGFR2蛋白表達(dá)量在6h時(shí)表達(dá)量最大(P0.05)。PDGF-BB mRNA表達(dá)量在48h的106CFU/mL濃度處理組達(dá)到最大值,PDGFRβmRNA表達(dá)量在12h的108CFU/mL菌液濃度處理組達(dá)到最大值(P0.05);PDGF-BB蛋白表達(dá)量在6h的106CFU/mL菌液濃度處理組的表達(dá)量最大,PDGFRβ蛋白表達(dá)量在24h時(shí)、105CFU/mL菌液濃度處理組的表達(dá)量最大(P0.05)。(2)與對(duì)照組比較,TLR2除了 48h時(shí)外,TLR4除了 6h時(shí)外,TLR2、TLR4的mRNA的表達(dá)量在108CFU/mL處理組最大,且差異性顯著(P0.05)。在同一時(shí)間段,TLR2蛋白的表達(dá)量會(huì)隨著處理組菌液濃度的加大而降低。隨著作用時(shí)間的延長(zhǎng),菌液濃度增高,TLR4的蛋白的表達(dá)量會(huì)變低。(3)熱滅活的無乳鏈球菌可促進(jìn)NF-κB和AP-1蛋白的表達(dá)。在同一時(shí)間段,NF-κB和AP-1蛋白的表達(dá)量在105CFU/mL處理組最大,與對(duì)照組比較差異顯著(P0.05)。研究表明,熱滅活的無乳鏈球菌能夠促進(jìn)體外培養(yǎng)的BMFBs上述三種細(xì)胞因子及對(duì)應(yīng)的受體和TLR2、TLR4的mRNA及蛋白的表達(dá),熱滅活的無乳鏈球菌也能夠促進(jìn)NF-κB和AP-1蛋白的表達(dá),這些與纖維化有關(guān)的生長(zhǎng)因子及其受體和TLR2、TLR4在奶牛乳腺纖維化中可能發(fā)揮著重要作用。
[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.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.23

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