α-黑素细胞刺激素及其类似物对组织因子和组织因子途径抑制物的调节作用
发布时间：2018-10-30 13:40
【摘要】： 目的:通过进行小鼠脑微血管内皮细胞的分离与原代培养,并将其用于本课题的体外实验研究细胞模型;建立内毒素血症小鼠模型,研究及评价α-黑素细胞刺激素(α-melanocyte stimulating hormone,α-MSH)及其类似物对小鼠内毒素血症模型小鼠和脑微血管内皮细胞在炎症因子LPS刺激下,产生组织因子(Tissue factor,TF)和组织因子途径抑制物(tissue factor pathway inhibitor,TFPI)的调节作用。 方法:进行小鼠脑微血管内皮细胞的分离与原代培养,建立本课题的细胞模型进行体外实验研究,比较和探讨浓度为10-7mol/L的α MSH、NDP-MSH、多肽39在不同时间段处理经LPS刺激的脑微血管内皮细胞,培养6h、8h分别收集上清和细胞,ELISA、RT-PCR检测TF和TFPI的表达;每只小鼠腹腔注射1μgLPS和20mgD-Gal,建立内毒素血症小鼠模型,进行体内实验研究,内毒素血症小鼠模型在不同时间段分别给予2.5mg/kgα MSH、NDP- MSH、多肽39,分别于LPS刺激后6h、8h取小鼠眼眶血,小鼠采血后颈椎脱臼处死小鼠,取肝、肺、脾、肾等脏器,ELISA检测小鼠血清中TF和TFPI的含量,RT-PCR检测小鼠组织中TF mRNA、TFPI mRNA表达。 结果:体外实验研究结果显示,浓度为10-7mol/Lα MSH、NDP-MSH、多肽39,均能抑制原代培养的小鼠脑微血管内皮细胞产生TF(P0.01),且多肽39抑制TF产生的作用优于α MSH、MSH-NDP;但α MSH、NDP-MSH、多肽39三种多肽没有显示出上调TFPI产生的作用。体内实验结果显示,内毒素血症小鼠模型在不同时间段给予三种多肽,均能显著的抑制TF的表达(P0.01),6h和8h采血组统计学处理无显著性差异;三种多肽均有上调TFPI的表达作用(P0.05),在6h采血时,在LPS刺激后2h给予三种多肽,多肽39的作用要优于α MSH、NDP-MSH;8h采血时,与LPS同时或者之后1h给予三种多肽,上调TFPI表达作用显著(P0.05),多肽39优于α MSH、NDP-MSH。 结论:本研究结果表明α-MSH、NDP-MSH、多肽39对TF和TFPI表达具有调节作用,迄今未见相同研究报道,为进一步阐明α-MSH、NDP-MSH、多肽39的抗炎作用机制,以及将来的应用研究提供了新的、可信的实验依据。
[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 伪-melanocyte stimulating hormone, 伪-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 伪 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 伪 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 伪 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 伪 MSH,. MSH-NDP; However, three kinds of 伪-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 伪 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 伪 MSH,NDP-MSH.. Conclusion: the results of this study indicate that 伪-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 伪-MSH,NDP-MSH, polypeptide 39. And the future applied research provides a new and credible experimental basis.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
本文编号：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 伪-melanocyte stimulating hormone, 伪-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 伪 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 伪 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 伪 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 伪 MSH,. MSH-NDP; However, three kinds of 伪-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 伪 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 伪 MSH,NDP-MSH.. Conclusion: the results of this study indicate that 伪-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 伪-MSH,NDP-MSH, polypeptide 39. And the future applied research provides a new and credible experimental basis.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
【参考文献】
相关期刊论文 前7条
1 徐剑文,王玮,康仲涵,张更,周琳瑛;小鼠脑微血管内皮细胞的体外培养[J];福建医科大学学报;2000年03期
2 顾江涛;急性炎症在急性冠状动脉事件发生机制中的作用[J];国外医学.心血管疾病分册;1999年04期
3 韩德平,田野苹;黑皮质素受体的研究进展[J];国外医学(生理、病理科学与临床分册);2000年05期
4 钱志远,黄强,周丽英,孙志方;鼠脑微血管内皮细胞的分离与长期培养[J];细胞生物学杂志;1999年01期
5 许熊飞,李润平,李泉,刘文武,练庆林,刘昀,孙学军,蒋春雷;大鼠脑微血管内皮细胞的分离与原代培养[J];细胞生物学杂志;2005年01期
6 董春霞,胡豫;内皮细胞表达组织因子的信号转导[J];医学综述;2005年05期
7 许彦钢,刘勇;大鼠脑皮质微血管内皮细胞培养和形态学观察[J];微循环技术杂志;1997年02期
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