α-黑素細胞刺激素及其類似物對組織因子和組織因子途徑抑制物的調節(jié)作用
發(fā)布時間：2018-10-30 13:40
【摘要】： 目的:通過進行小鼠腦微血管內皮細胞的分離與原代培養(yǎng),并將其用于本課題的體外實驗研究細胞模型;建立內毒素血癥小鼠模型,研究及評價α-黑素細胞刺激素(α-melanocyte stimulating hormone,α-MSH)及其類似物對小鼠內毒素血癥模型小鼠和腦微血管內皮細胞在炎癥因子LPS刺激下,產生組織因子(Tissue factor,TF)和組織因子途徑抑制物(tissue factor pathway inhibitor,TFPI)的調節(jié)作用。 方法:進行小鼠腦微血管內皮細胞的分離與原代培養(yǎng),建立本課題的細胞模型進行體外實驗研究,比較和探討濃度為10-7mol/L的α MSH、NDP-MSH、多肽39在不同時間段處理經LPS刺激的腦微血管內皮細胞,培養(yǎng)6h、8h分別收集上清和細胞,ELISA、RT-PCR檢測TF和TFPI的表達;每只小鼠腹腔注射1μgLPS和20mgD-Gal,建立內毒素血癥小鼠模型,進行體內實驗研究,內毒素血癥小鼠模型在不同時間段分別給予2.5mg/kgα MSH、NDP- MSH、多肽39,分別于LPS刺激后6h、8h取小鼠眼眶血,小鼠采血后頸椎脫臼處死小鼠,取肝、肺、脾、腎等臟器,ELISA檢測小鼠血清中TF和TFPI的含量,RT-PCR檢測小鼠組織中TF mRNA、TFPI mRNA表達。 結果:體外實驗研究結果顯示,濃度為10-7mol/Lα MSH、NDP-MSH、多肽39,均能抑制原代培養(yǎng)的小鼠腦微血管內皮細胞產生TF(P0.01),且多肽39抑制TF產生的作用優(yōu)于α MSH、MSH-NDP;但α MSH、NDP-MSH、多肽39三種多肽沒有顯示出上調TFPI產生的作用。體內實驗結果顯示,內毒素血癥小鼠模型在不同時間段給予三種多肽,均能顯著的抑制TF的表達(P0.01),6h和8h采血組統(tǒng)計學處理無顯著性差異;三種多肽均有上調TFPI的表達作用(P0.05),在6h采血時,在LPS刺激后2h給予三種多肽,多肽39的作用要優(yōu)于α MSH、NDP-MSH;8h采血時,與LPS同時或者之后1h給予三種多肽,上調TFPI表達作用顯著(P0.05),多肽39優(yōu)于α MSH、NDP-MSH。 結論:本研究結果表明α-MSH、NDP-MSH、多肽39對TF和TFPI表達具有調節(jié)作用,迄今未見相同研究報道,為進一步闡明α-MSH、NDP-MSH、多肽39的抗炎作用機制,以及將來的應用研究提供了新的、可信的實驗依據。
[Abstract]:Objective: to isolate and culture mouse brain microvascular endothelial cells (MECs) and to study the cell model in vitro. To establish the model of endotoxemia in mice, to study and evaluate the effects of 偽-melanocyte stimulating hormone, 偽-MSH and its analogues on mice with endotoxemia and cerebral microvascular endothelial cells stimulated by inflammatory factor LPS. Production of tissue factor (Tissue factor,TF) and tissue factor pathway inhibitor (tissue factor pathway inhibitor,TFPI). Methods: the mouse brain microvascular endothelial cells were isolated and primary cultured, and the cell model was established in this paper. The experimental study was carried out in vitro, and the concentration of 10-7mol/L 偽 MSH,NDP-MSH, was compared and discussed. The supernatants and cells were collected at 6 h and 8 h after LPS stimulated brain microvascular endothelial cells were treated with polypeptide 39 at different time points. The expression of TF and TFPI were detected by ELISA,RT-PCR. Each mouse was injected intraperitoneally with 1 渭 gLPS and 20 mg D-Gal. to establish the model of endotoxemia and to carry out in vivo experimental study. The model of endotoxemia was treated with 2.5mg/kg 偽 MSH,NDP- MSH, polypeptide 39 at different time points. Mouse orbital blood was taken at 6h after LPS stimulation. The mice were killed by cervical dislocations after blood collection. The liver, lung, spleen, kidney and other organs were taken. The contents of TF and TFPI in serum were detected by ELISA, and the expression of TF mRNA,TFPI mRNA in the tissues of mice was detected by RT-PCR. Results: in vitro, the concentration of 10-7mol/L 偽 MSH,NDP-MSH, peptide 39 could inhibit the production of TF in primary cultured rat brain microvascular endothelial cells (P0.01), and the inhibitory effect of polypeptide 39 on TF production was superior to that of 偽 MSH,. MSH-NDP; However, three kinds of 偽-MSH,NDP-MSH, peptide 39 did not show the effect of up-regulating TFPI production. The results of in vivo experiment showed that three kinds of polypeptides could significantly inhibit the expression of TF in endotoxemia mice at different time periods (P0.01), but there was no significant difference between the 6 h and 8 h blood collection groups. The expression of TFPI was upregulated by all three peptides (P0.05). When blood was collected at 6 h, three kinds of peptides were given at 2 h after stimulation of LPS. The effect of polypeptide 39 was better than that of 偽 MSH,NDP-MSH;. When blood was collected at 8 h, three kinds of polypeptides were given at the same time or 1 hour after LPS, and the expression of TFPI was up-regulated significantly (P0.05), and the peptide 39 was superior to 偽 MSH,NDP-MSH.. Conclusion: the results of this study indicate that 偽-MSH,NDP-MSH, polypeptide 39 can regulate the expression of TF and TFPI. So far, no similar studies have been reported, in order to further elucidate the anti-inflammatory mechanism of 偽-MSH,NDP-MSH, polypeptide 39. And the future applied research provides a new and credible experimental basis.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363
本文編號：2300156
[Abstract]:Objective: to isolate and culture mouse brain microvascular endothelial cells (MECs) and to study the cell model in vitro. To establish the model of endotoxemia in mice, to study and evaluate the effects of 偽-melanocyte stimulating hormone, 偽-MSH and its analogues on mice with endotoxemia and cerebral microvascular endothelial cells stimulated by inflammatory factor LPS. Production of tissue factor (Tissue factor,TF) and tissue factor pathway inhibitor (tissue factor pathway inhibitor,TFPI). Methods: the mouse brain microvascular endothelial cells were isolated and primary cultured, and the cell model was established in this paper. The experimental study was carried out in vitro, and the concentration of 10-7mol/L 偽 MSH,NDP-MSH, was compared and discussed. The supernatants and cells were collected at 6 h and 8 h after LPS stimulated brain microvascular endothelial cells were treated with polypeptide 39 at different time points. The expression of TF and TFPI were detected by ELISA,RT-PCR. Each mouse was injected intraperitoneally with 1 渭 gLPS and 20 mg D-Gal. to establish the model of endotoxemia and to carry out in vivo experimental study. The model of endotoxemia was treated with 2.5mg/kg 偽 MSH,NDP- MSH, polypeptide 39 at different time points. Mouse orbital blood was taken at 6h after LPS stimulation. The mice were killed by cervical dislocations after blood collection. The liver, lung, spleen, kidney and other organs were taken. The contents of TF and TFPI in serum were detected by ELISA, and the expression of TF mRNA,TFPI mRNA in the tissues of mice was detected by RT-PCR. Results: in vitro, the concentration of 10-7mol/L 偽 MSH,NDP-MSH, peptide 39 could inhibit the production of TF in primary cultured rat brain microvascular endothelial cells (P0.01), and the inhibitory effect of polypeptide 39 on TF production was superior to that of 偽 MSH,. MSH-NDP; However, three kinds of 偽-MSH,NDP-MSH, peptide 39 did not show the effect of up-regulating TFPI production. The results of in vivo experiment showed that three kinds of polypeptides could significantly inhibit the expression of TF in endotoxemia mice at different time periods (P0.01), but there was no significant difference between the 6 h and 8 h blood collection groups. The expression of TFPI was upregulated by all three peptides (P0.05). When blood was collected at 6 h, three kinds of peptides were given at 2 h after stimulation of LPS. The effect of polypeptide 39 was better than that of 偽 MSH,NDP-MSH;. When blood was collected at 8 h, three kinds of polypeptides were given at the same time or 1 hour after LPS, and the expression of TFPI was up-regulated significantly (P0.05), and the peptide 39 was superior to 偽 MSH,NDP-MSH.. Conclusion: the results of this study indicate that 偽-MSH,NDP-MSH, polypeptide 39 can regulate the expression of TF and TFPI. So far, no similar studies have been reported, in order to further elucidate the anti-inflammatory mechanism of 偽-MSH,NDP-MSH, polypeptide 39. And the future applied research provides a new and credible experimental basis.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363
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