【摘要】： 目的:通過進(jìn)行小鼠腦微血管內(nèi)皮細(xì)胞的分離與原代培養(yǎng),并將其用于本課題的體外實(shí)驗(yàn)研究細(xì)胞模型;建立內(nèi)毒素血癥小鼠模型,研究及評價(jià)α-黑素細(xì)胞刺激素(α-melanocyte stimulating hormone,α-MSH)及其類似物對小鼠內(nèi)毒素血癥模型小鼠和腦微血管內(nèi)皮細(xì)胞在炎癥因子LPS刺激下,產(chǎn)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(diào)節(jié)作用。 方法:進(jìn)行小鼠腦微血管內(nèi)皮細(xì)胞的分離與原代培養(yǎng),建立本課題的細(xì)胞模型進(jìn)行體外實(shí)驗(yàn)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(shí)間段處理經(jīng)LPS刺激的腦微血管內(nèi)皮細(xì)胞,培養(yǎng)6h、8h分別收集上清和細(xì)胞,ELISA、RT-PCR檢測TF和TFPI的表達(dá);每只小鼠腹腔注射1μgLPS和20mgD-Gal,建立內(nèi)毒素血癥小鼠模型,進(jìn)行體內(nèi)實(shí)驗(yàn)研究,內(nèi)毒素血癥小鼠模型在不同時(shí)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(dá)。 結(jié)果:體外實(shí)驗(yàn)研究結(jié)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(nèi)皮細(xì)胞產(chǎn)生TF(P0.01),且多肽39抑制TF產(chǎn)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(diào)TFPI產(chǎn)生的作用。體內(nèi)實(shí)驗(yàn)結(jié)果顯示,內(nèi)毒素血癥小鼠模型在不同時(shí)間段給予三種多肽,均能顯著的抑制TF的表達(dá)(P0.01),6h和8h采血組統(tǒng)計(jì)學(xué)處理無顯著性差異;三種多肽均有上調(diào)TFPI的表達(dá)作用(P0.05),在6h采血時(shí),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(shí),與LPS同時(shí)或者之后1h給予三種多肽,上調(diào)TFPI表達(dá)作用顯著(P0.05),多肽39優(yōu)于α MSH、NDP-MSH。 結(jié)論:本研究結(jié)果表明α-MSH、NDP-MSH、多肽39對TF和TFPI表達(dá)具有調(diào)節(jié)作用,迄今未見相同研究報(bào)道,為進(jìn)一步闡明α-MSH、NDP-MSH、多肽39的抗炎作用機(jī)制,以及將來的應(yīng)用研究提供了新的、可信的實(shí)驗(yàn)依據(jù)。
[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(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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