科研動態(tài)

為研究無乳鏈球菌（Streptococcus agalactiae，S. agalactiae）作用奶牛乳腺成纖維細胞（BMFBs）后對MMPs/TIMPs與uPA系統(tǒng)（uPA/PAI-1）表達的影響，以進一步闡明MMPs/TIMPs與uPA系統(tǒng)在調(diào)控細胞外基質(zhì)（ECM）代謝的作用，將107 cfu/mL熱滅活S. agalactiae菌液作用于BMFBs，在作用后不同時間點（6、12、24、48 h）通過RT-qPCR和Western Blot檢測MMP-2、MMP-9、TIMP-1、TIMP-2、uPA、PAI-1 mRNA轉(zhuǎn)錄水平和相應(yīng)蛋白表達水平，并用明膠酶譜法檢測MMP-2、MMP-9的酶活性。結(jié)果顯示：熱滅活S. agalactiae作用后，MMP-9、TIMP-2、uPA、PAI-1 mRNA轉(zhuǎn)錄水平均有不同程度下降趨勢，但MMP-2 mRNA水平上調(diào)，TIMP-1 mRNA水平呈先上升后下降趨勢；明膠酶譜法檢測MMP-2的酶活性較MMP-9明顯，二者均在作用6 h、48 h活性最強；MMP-2和MMP-9蛋白表達水平與對照組相比主要呈先升高后下降趨勢，但在作用后期MMP-2蛋白表達又出現(xiàn)了回升趨勢；TIMP-1蛋白水平與對照組相比主要呈先下降后上升趨勢，在作用前期與MMP-9蛋白表達相反，TIMP-2蛋白表達與對照組相比呈上升趨勢；uPA和PAI-1蛋白水平與對照組相比均呈先降低后升高再降低趨勢，均在24 h達到峰值，隨后呈下降趨勢。研究表明，熱滅活S. agalactiae菌液可誘導(dǎo)BMFBs中MMPs/TIMPs表達，影響uPA系統(tǒng)參與調(diào)控MMPs的表達及活性。

The Effect of Inactivated S. agalactiae on Metalloproteinase and uPA System Expression in BMFBs

In order to study the effect of Streptococcus agalactiae（S. agalactiae）on bovine mammary fibroblasts（BMFBs）and subsequently on the expression of MMPs/TIMPs and uPA system（uPA/PAI-1），and to further clarify the role of MMPs/TIMPs and uPA system in regulating extracellular matrix（ECM）metabolism，107cfu/mL heat-inactivated S. agalactiae was acted on BMFBs，the levels of MMP-2，MMP-9，TIMP-1，TIMP-2，uPA，PAI-1 mRNA transcription and corresponding protein expression were detected by RT-qPCR and Western Blot at different time points（6，12，24 and 48 h），then the enzymatic activities of MMP-2 and MMP-9 were detected by gelatin zymography. The results showed that after the reaction of heat-inactivated S. agalactiae，the level of transcription of MMP-9，TIMP-2，uPA and PAI-1 mRNA trended to reduce to different extent，except that of MMP-2 mRNA，and that of TIMP-1 mRNA increased firstly and then decreased；the enzymatic activity of MMP-2 was more obvious than that of MMP-9 as detected by gelatin zymography，both of which were strongest in 6 and 8 h after reaction；as compared with the control group，the level of protein expression of MMP-2 and MMP-9 trended to increase prior to decrease，but the protein expression of MMP-2 rose again at later stage；as compared to the control group，the level of protein of TIMP-1 showed a trend of decreasing prior to rising，which was contrary to that of MMP-9 at early stage，and TIMP-2 trended to increase；the levels of protein of uPA and PAI-1 both decreased first then increased，and decreased finally，reaching the peak at 24 h after reaction. In conclusion，the expression of MMPs/TIMPs in BMFBs could be induced by heat-inactivated S. agalactiae，which could influence uPA system in regulating the expression and activity of MMPs.

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