血小板活化因子对大鼠肺微血管内皮细胞Src抑制的蛋白激酶C底物基因表达的影响
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摘要
目的:研究血小板活化因子(platelet-activating factor,PAF)对培养的大鼠肺微血管内皮细胞(rat pulmonary microvascular endothelial cells,RPMVEC)中Src抑制的蛋白激酶C底物(Src-suppressed C kinase substrate,SSeCKS)mRNA表达的影响,观察信号通路抑制剂对炎症介导SSeCKS mRNA表达的干预作用,结合我们既往的工作进一步探讨PAF参与急性肺损伤(acute lung injury,ALI)发生的机制,并初步开展SSeCKS在炎症诱导RPMVEC损伤过程中基因表达的信号转导途径研究。
方法:体外分离培养RPMVEC;建立RPMVEC中SSeCKS mRNA表达的原位杂交检测方法;根据PAF刺激时间和浓度的差异将RPMVEC随机分为PAF量效组和时效组:量效组分别以10-10、10-9、10-8、10-7 mol/L PAF与RPMVEC作用1.5 h,时效组以10-7 mol/L PAF与RPMVEC分别作用0.5、1.5、3、6、12、24 h;信号通路抑制剂干预:RPMVEC与10μmol/L核转录因子-κB(nuclear factor-κB,NF-κB)抑制剂吡咯烷二巯基氨甲酸(pyrrolidine dithiocarbamate,PDTC)预孵育1 h或10μmol/L蛋白激酶C(protein kinase C,PKC)抑制剂双吲哚基顺丁烯二酰亚胺(bis-indolylmaleimide,BIM)预孵育0.5 h,再与10-7 mol/L PAF或10 mg/L脂多糖(lipopolysaccharide,LPS)作用1.5 h,以上均设正常、阴性和阳性对照组;原位杂交技术(in situ hybridization,ISH)与计算机图像分析系统软件结合运用检测不同条件下RPMVEC中SSeCKS mRNA的表达变化。
结果: 1.体外成功进行RPMVEC的分离培养,并经形态学及FITC-BSI结合实验证实。2.成功建立经地高辛标记的寡核苷酸探针原位杂交检测RPMVEC中SSeCKS mRNA的实验方法。3.正常RPMVEC有SSeCKS mRNA少量表达,为棕黄色细颗粒状杂交信号,分布于胞质。10-10、10-9、10-8、10-7 mol/L PAF分别孵育RPMVEC 1.5 h,SSeCKS mRNA表达量随其浓度增加而逐渐升高,与正常对照组比较差异均有统计学意义。10-7 mol/L PAF刺激RPMVEC 0.5 h,SSeCKS mRNA表达水平即显著升高,1.5 h达峰值,之后逐渐下降,至24 h仍高于正常对照组。4. PDTC可显著下调PAF或LPS对RPMVEC中SSeCKS mRNA的诱导效应,而BIM对此效应无干预作用。
结论: 1.成功进行RPMVEC的体外原代培养及鉴定。2.通过ISH与计算机图像分析系统软件的结合运用,成功检测不同条件下RPMVEC中SSeCKS基因的表达变化。3. PAF呈浓度和时间依赖的方式上调RPMVEC中SSeCKS基因的转录水平。4. NF-κB信号通路而非PKC参与了PAF、LPS诱导SSeCKS基因表达的信号转导机制。
Objective: To investigate the effect of platelet-activating factor(PAF) on the production of Src-suppressed C kinase substrate(SSeCKS) mRNA in rat pulmonary microvascular endothelial cells(RPMVEC) and to observe the effect of signal inhibitors on inflammation-induced SSeCKS gene expression. On the basis of our previous work, to explore the mechanism of PAF involved in ALI and initially study the signal transduction pathways of SSeCKS gene expression in the inflammation-induced RPMVEC injury process.
Methods: RPMVEC were isolated and cultured in vitro, then according to the different culture dosage or time of PAF randomly grouped into two sections. One was using 10-10, 10-9, 10-8, 10-7 mol/L PAF incubated cells for 1.5 h, the other was using 10-7 mol/L PAF challenge for 0.5, 1.5, 3, 6, 12, 24 h. Pretreatment of 10μmol/L nuclear factor-kappa B(NF-κB) inhibitor pyrrolidine dithiocarbamate(PDTC) or protein kinase C(PKC) inhibitor bis-indolylmaleimide(BIM) was used to interfere PAF and LPS stimulation. The normal, negative and positive control groups were set concurrently. In situ hybridization(ISH) and computer image analysis software was jointly performed to reveal the change of SSeCKS mRNA expression in the cultured RPMVECs under each condition.
Results: 1. We have isolated and cultured the primary RPMVEC in vitro.These cells were further confirmed by typical morphology and FITC-BSI binding assay. 2. We have established a method for the detection of SSeCKS mRNA in RPMVEC by ISH approach using digoxin-labelled oligonucleotide probes. 3. Normal RPMVEC expressed SSeCKS mRNA at a low level, which appeared throughout the cytoplasm with specific hybridization signals. 1.5 h of 10-10, 10-9, 10-8, 10-7 mol/L PAF incubation induced a progressive increase in SSeCKS mRNA expression. When compared SSeCKS mRNA’s mean optical density with the normal control group, difference in each group had statistically significance. Within 0.5, 1.5, 3, 6, 12, 24 h of 10-7 mol/L PAF challenge, the level of SSeCKS mRNA expression markedly raised at 0.5 h, peaked at 1.5 h, then began to decline gradually, and still persisted at a higher level than the normal control group until 24 h. 4. Pre-incubation of 10μmol/L pyrrolidine dithiocarbamate(PDTC) that inhibits activity of nuclear factor-kappa B(NF-κB) in RPMVECs caused a conspicuous attenuation of PAF- or LPS-induced SSeCKS mRNA expression, whereas no change was found by pretreatment of protein kinase C(PKC) inhibitor bis-indolylmaleimide(BIM).
Conclusion: 1. We have successfully cultured and demonstrated the primary RPMVEC in vitro. 2. Through the combination of ISH and computer image analysis software ,We have successfully detected the change of SSeCKS gene expression under different conditions in RPMVEC. 3. PAF can up regulate expression of SSeCKS mRNA in a dose- and time-dependent manner in RPMVECs. 4. It is NF-κB rather than PKC signal pathway that involve in modulation of the intracellular signaling process that induce SSeCKS mRNA expression.
方法:体外分离培养RPMVEC;建立RPMVEC中SSeCKS mRNA表达的原位杂交检测方法;根据PAF刺激时间和浓度的差异将RPMVEC随机分为PAF量效组和时效组:量效组分别以10-10、10-9、10-8、10-7 mol/L PAF与RPMVEC作用1.5 h,时效组以10-7 mol/L PAF与RPMVEC分别作用0.5、1.5、3、6、12、24 h;信号通路抑制剂干预:RPMVEC与10μmol/L核转录因子-κB(nuclear factor-κB,NF-κB)抑制剂吡咯烷二巯基氨甲酸(pyrrolidine dithiocarbamate,PDTC)预孵育1 h或10μmol/L蛋白激酶C(protein kinase C,PKC)抑制剂双吲哚基顺丁烯二酰亚胺(bis-indolylmaleimide,BIM)预孵育0.5 h,再与10-7 mol/L PAF或10 mg/L脂多糖(lipopolysaccharide,LPS)作用1.5 h,以上均设正常、阴性和阳性对照组;原位杂交技术(in situ hybridization,ISH)与计算机图像分析系统软件结合运用检测不同条件下RPMVEC中SSeCKS mRNA的表达变化。
结果: 1.体外成功进行RPMVEC的分离培养,并经形态学及FITC-BSI结合实验证实。2.成功建立经地高辛标记的寡核苷酸探针原位杂交检测RPMVEC中SSeCKS mRNA的实验方法。3.正常RPMVEC有SSeCKS mRNA少量表达,为棕黄色细颗粒状杂交信号,分布于胞质。10-10、10-9、10-8、10-7 mol/L PAF分别孵育RPMVEC 1.5 h,SSeCKS mRNA表达量随其浓度增加而逐渐升高,与正常对照组比较差异均有统计学意义。10-7 mol/L PAF刺激RPMVEC 0.5 h,SSeCKS mRNA表达水平即显著升高,1.5 h达峰值,之后逐渐下降,至24 h仍高于正常对照组。4. PDTC可显著下调PAF或LPS对RPMVEC中SSeCKS mRNA的诱导效应,而BIM对此效应无干预作用。
结论: 1.成功进行RPMVEC的体外原代培养及鉴定。2.通过ISH与计算机图像分析系统软件的结合运用,成功检测不同条件下RPMVEC中SSeCKS基因的表达变化。3. PAF呈浓度和时间依赖的方式上调RPMVEC中SSeCKS基因的转录水平。4. NF-κB信号通路而非PKC参与了PAF、LPS诱导SSeCKS基因表达的信号转导机制。
Objective: To investigate the effect of platelet-activating factor(PAF) on the production of Src-suppressed C kinase substrate(SSeCKS) mRNA in rat pulmonary microvascular endothelial cells(RPMVEC) and to observe the effect of signal inhibitors on inflammation-induced SSeCKS gene expression. On the basis of our previous work, to explore the mechanism of PAF involved in ALI and initially study the signal transduction pathways of SSeCKS gene expression in the inflammation-induced RPMVEC injury process.
Methods: RPMVEC were isolated and cultured in vitro, then according to the different culture dosage or time of PAF randomly grouped into two sections. One was using 10-10, 10-9, 10-8, 10-7 mol/L PAF incubated cells for 1.5 h, the other was using 10-7 mol/L PAF challenge for 0.5, 1.5, 3, 6, 12, 24 h. Pretreatment of 10μmol/L nuclear factor-kappa B(NF-κB) inhibitor pyrrolidine dithiocarbamate(PDTC) or protein kinase C(PKC) inhibitor bis-indolylmaleimide(BIM) was used to interfere PAF and LPS stimulation. The normal, negative and positive control groups were set concurrently. In situ hybridization(ISH) and computer image analysis software was jointly performed to reveal the change of SSeCKS mRNA expression in the cultured RPMVECs under each condition.
Results: 1. We have isolated and cultured the primary RPMVEC in vitro.These cells were further confirmed by typical morphology and FITC-BSI binding assay. 2. We have established a method for the detection of SSeCKS mRNA in RPMVEC by ISH approach using digoxin-labelled oligonucleotide probes. 3. Normal RPMVEC expressed SSeCKS mRNA at a low level, which appeared throughout the cytoplasm with specific hybridization signals. 1.5 h of 10-10, 10-9, 10-8, 10-7 mol/L PAF incubation induced a progressive increase in SSeCKS mRNA expression. When compared SSeCKS mRNA’s mean optical density with the normal control group, difference in each group had statistically significance. Within 0.5, 1.5, 3, 6, 12, 24 h of 10-7 mol/L PAF challenge, the level of SSeCKS mRNA expression markedly raised at 0.5 h, peaked at 1.5 h, then began to decline gradually, and still persisted at a higher level than the normal control group until 24 h. 4. Pre-incubation of 10μmol/L pyrrolidine dithiocarbamate(PDTC) that inhibits activity of nuclear factor-kappa B(NF-κB) in RPMVECs caused a conspicuous attenuation of PAF- or LPS-induced SSeCKS mRNA expression, whereas no change was found by pretreatment of protein kinase C(PKC) inhibitor bis-indolylmaleimide(BIM).
Conclusion: 1. We have successfully cultured and demonstrated the primary RPMVEC in vitro. 2. Through the combination of ISH and computer image analysis software ,We have successfully detected the change of SSeCKS gene expression under different conditions in RPMVEC. 3. PAF can up regulate expression of SSeCKS mRNA in a dose- and time-dependent manner in RPMVECs. 4. It is NF-κB rather than PKC signal pathway that involve in modulation of the intracellular signaling process that induce SSeCKS mRNA expression.
引文
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[2] Kuebler WM, Parthasarthi K, Wang PM, et al. A novel signaling mechanism between gas and blood compartments of the lung. J Clin Invest, 2000, 105: 905-913.
[3] G?ggel R, Uhlig S. The inositol trisphosphate pathway mediates platelet-activating factor-induced pulmonary oedema. Eur Respir J, 2005, 25: 849-857.
[4] Miotla J, Jeffery P, Hellewell P. Platelet-activating factor plays a pivotal role in the induction of experimental lung injury. Am J Respir Cell Mol Biol, 1998, 18:197-204.
[5]王应灯,孙耕耘.山莨菪碱对血小板活化因子致肺微血管内皮细胞形态和骨架变化的影响.安徽医科大学学报, 2004, 39(1): 29-33.
[6] Gelman IH, Lee K, Tombler E, et al. Control of cytoskeletal architecture by the src-suppressed C kinase substrate, SSeCKS. Cell Motil Cytoskeleton, 1998, 41(1): 1-17.
[7] Kitamura H, Okita K, Fujikura D, et al. Induction of Src suppressed C Kinase substrate(SSeCKS) in vascular endothelial cells by bacterial lipopolysaccharide. J Histochem Cytochem, 2002, 50(2): 245-255.
[8] Nelson PJ, Moissoglu K, Vargas J, et al. Involvement of the protein kinase C substrate, SSeCKS, in the actin-based stellate morphology of mesangial cells. J Cell Sci, 1999, 112 (Pt 3): 361-70.
[9]张泓,孙耕耘.血管紧张素Ⅱ及其受体拮抗剂对大鼠肺微血管内皮炎性损伤效应的影响[J].中国危重病急救医学, 2004, 16(10): 608-610.
[10]王楠,张泓,孙耕耘.内毒素对体外培养大鼠肺微血管内皮细胞血管紧张素转换酶2表达的影响.中国危重病急救医学, 2008, 20(10): 582-584.
[11] Malik A, Si?inger-Birnboim A. Vascular endothelial barrier function and its regulation. New York, 1993: Plenum.
[12] Blum MS, Toninelli E, Anderson JM, et al. Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines. Am J Physiol Heart Circ Physiol, 1997, 273: H286–H294.
[13] Parker JC ,Yoshikawa S. Vascular segmental permeabilities at high peak in?ation pressure in isolated rat lungs. Am J Physiol Lung Cell Mol Physiol, 2003, 283: L1203–L1209.
[14] Khimenko PL and Taylor AE. Segmental microvascular permeability in ischemia-reperfusion injury in rat lung. Am J Physiol Lung Cell Mol Physiol, 1999, 276: L958–L960.
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