呼吸道合胞病毒感染对大鼠脊髓背根神经节转录因子活性的影响
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摘要
目的:在研究RSV感染后气道神经源性炎症和气道神经可塑性致气道高反应性的基础上,研究呼吸道合胞病毒(RSV)感染后大鼠脊髓背根神经节神经元转录因子的表达变化,筛选可能与气道神经可塑性相关的转录因子,进一步从细胞内信号转导和转录调控机制深入探讨气道神经可塑性的细胞内调控机制,阐明气道神经可塑性导致气道高反应性和哮喘的机理,为哮喘的防治探索一条新的途径。
方法:RSV病毒株接种于铺满单层Hela细胞的细胞培养瓶中,待病变达100%时收获病毒。-70℃保存备用。收集无病毒的Hela单层细胞培养瓶中培养基上清液和细胞溶解产物,从而获得病毒基质作为阴性对照。进行感染性(50%组织细胞发生病变的感染剂量即TCID50/0.1ML)的测定(毒力测定)。1-2周龄SD大鼠(湖南省农业大学动物部)随机区组分为2组,每组30只。1组:空白对照组:2组:RSV感染组①对照组(30只),用2%戊巴比妥钠40mg/kg腹腔注射麻醉大鼠,每一个鼻孔内滴入0.4u/g无病毒培养基。②RSV感染组(30只),用2%戊巴比妥钠40mg/kg腹腔注射麻醉大鼠,在每一个鼻孔内滴入0.4ul/g滴度为5X10~4TCID50/0.1ML的RSV病毒悬液。以上操作每周重复一次。两组第8周按编号后抽签方法将其中5只大鼠进行气道反应性测定,气道反应性测定后,取左肺,HE染色观察病理改变,行免疫组化染色检测,在冰面上迅速剖取脊髓背根神经节C7-T5于冻存管中,保存于液氮中。原位杂交法检测肺组织RSV蛋白的存在,免疫组化检测气道、神经节SYN、NF的表达,用转录因子活性芯片初步筛选调控气道神经可塑性的信号转录因子,western blot检测EGR-1在肺组织和脊髓背根节的表达变化。
结果:(1)实验组大鼠渐出现卡他性鼻炎、呼吸气促、毛乱、活动度降低的临床表现,实验组大鼠与对照组大鼠在平均体重差异无显著性,体温差异有显著性(P＜0.05)。(2)实验组大鼠吸入梯度浓度组胺后气道阻力明显高于正常对照组,差异有显著性(均P＜0.05),说明存在气道高反应性。(3)光镜下实验组大鼠肺组织符合典型间质性肺炎的病理改变。(4)实验组气道壁、神经节中SYN、NF蛋白表达平均阳性系数显著高于对照组(P＜0.05)。(5)利用TranSignal蛋白/DNA组合芯片(345)检测筛选:RSV反复感染引起大鼠C7-T5背根节345个转录因子中的55个表达上调2倍以上,43个表达下调2倍以上。(6)western blot检测EGR-1在肺部和脊髓背根节细胞的表达变化,结果显示EGR-1在RSV感染组表达明显低于对照。
结论:(1)采用RSV滴鼻的方式建立SD大鼠气道高反应性模型,能成功复制出气道高反应性大鼠模型。(2)反复呼吸道合胞病毒感染引起幼鼠气道神经网络发育异常改变(神经可塑性改变)。(3)转录因子活性芯片初步筛选出了反复呼吸道合胞病毒感染后脊髓背根节神经元细胞内发生活性改变的转录因子98种,为进一步探索反复呼吸道合胞病毒感染后气道神经可塑性变化的关键转录因子提供了研究基础。
Objective: On the basis of studying on airway hyperreactivity induced by airway neurogenic inflammation and airway neural plasticity, This project aims to investigate the expression of intracellular transcription factor in the C7-T5 spinal adorsal root Ganglion cells of SD rats after recurrent respiratory syncytial virus (RSV) infection and to identify the key intracellular transcription factors of involving in airway neural plasticity. Thus, to elucidate the mechanism of airway neural plasticity leading to the airway hyperreactivity and bronchial asthma after recurrent respiratory syncytial virus infection.
Methods: RSV is inoculated on the Hela cell bespreaded on the culture flask,and harvested when all of cells were infected undermicro-scope.The suspension was kept in reserve at -70℃.The virus-free medium was obtained as a negative control. The virus stock was titrated and diluted as final titer of 5×10~4 50% tissue culture infective dose (TCID50)in 0.1ml (virulence determination). Sixty 2-week-old SD rats(Hu Nan Agriculture University ,china) were randomly assigned into two groups (n=30 in each group). A volume of 0.4ml/kg body wt of RSV suspension was inoculated in the nostril of RSV-infected group while the rat was under pentobarbital sodium anesthesia(40mg/kg),control group were administered the same volume of virus-free medium.The treatment . above is repeated once a week.The rats were tested airway responsiveness before sarcrificed at 8th week and the left lung was removed for hema-toxylin and eosin, immunohistochemistry staining. C7-T5 dorsal root ganglion are dissected on the ice surface and put into the cryovial and stored in liquid nitrogen. RSV protein was assayed by hybridization in situ. The expression of SYN and NF in the airway and dorsal root ganglion were assayed by immunohistochemistry and transcription factors in C7-T5 dorsal root ganglion are preliminarily screened by the TranSignal~(TM) Protein/DNA Combo Arrays.The expression of EGR-1 in lung tissue and C7-T5 dorsal root ganglion were detected by western blot.
Results: (1)RSV infected rats gradually showed these syndromes such as catarrhalic rhinitis, gasping, mussy hair and activity decreasing.There was no significant difference between the two groups in the average body weigh. The temperature between the two groups has significant difference (P<0.05). (2)Airway resistance were significantly increased in the model groups than in the normal control group (P<0.05) after inhaled escalating histamine, indicating the airway hyperreactivity existed in RSV infected group. (3) The RSV infected group showed typical interstitial pneumonia under light microscope. (4) Comparing with the rats of control group, the expression of SYN and NF increased significantly (P<0.05) in RSV infected rats. (5) Protein/DNA array analysis showed that the expression of 55 transcription factors increased at least 2 fold in the C7-T5 spinal ganglion cells of RSV infected group, and the expression of 43 transcription factors decreased at least 2 fold in the C7-T5 spinal ganglion cells of the RSV-inoculated group. (6)The expression of EGR-1 was decreased both in the lung and in the C7-T5 spinal ganglion cells after RSV infection.
Conclusion: (1)The airway hyperreactivity in rats can be established successfully by RSV nostril inoculation. (2)The airway showed neural plasticity by SYN and NF staining after recurreent RSV infection. (3) TranSignal? Protein/DNA Combo Arrays analysis show that RSV infection result in the expression alteration of 98 transcription factors and build a foundation for futher study. EGR-1 may not be the key intracellular transcription factors of regulating airway neural plasticity.
方法:RSV病毒株接种于铺满单层Hela细胞的细胞培养瓶中,待病变达100%时收获病毒。-70℃保存备用。收集无病毒的Hela单层细胞培养瓶中培养基上清液和细胞溶解产物,从而获得病毒基质作为阴性对照。进行感染性(50%组织细胞发生病变的感染剂量即TCID50/0.1ML)的测定(毒力测定)。1-2周龄SD大鼠(湖南省农业大学动物部)随机区组分为2组,每组30只。1组:空白对照组:2组:RSV感染组①对照组(30只),用2%戊巴比妥钠40mg/kg腹腔注射麻醉大鼠,每一个鼻孔内滴入0.4u/g无病毒培养基。②RSV感染组(30只),用2%戊巴比妥钠40mg/kg腹腔注射麻醉大鼠,在每一个鼻孔内滴入0.4ul/g滴度为5X10~4TCID50/0.1ML的RSV病毒悬液。以上操作每周重复一次。两组第8周按编号后抽签方法将其中5只大鼠进行气道反应性测定,气道反应性测定后,取左肺,HE染色观察病理改变,行免疫组化染色检测,在冰面上迅速剖取脊髓背根神经节C7-T5于冻存管中,保存于液氮中。原位杂交法检测肺组织RSV蛋白的存在,免疫组化检测气道、神经节SYN、NF的表达,用转录因子活性芯片初步筛选调控气道神经可塑性的信号转录因子,western blot检测EGR-1在肺组织和脊髓背根节的表达变化。
结果:(1)实验组大鼠渐出现卡他性鼻炎、呼吸气促、毛乱、活动度降低的临床表现,实验组大鼠与对照组大鼠在平均体重差异无显著性,体温差异有显著性(P＜0.05)。(2)实验组大鼠吸入梯度浓度组胺后气道阻力明显高于正常对照组,差异有显著性(均P＜0.05),说明存在气道高反应性。(3)光镜下实验组大鼠肺组织符合典型间质性肺炎的病理改变。(4)实验组气道壁、神经节中SYN、NF蛋白表达平均阳性系数显著高于对照组(P＜0.05)。(5)利用TranSignal蛋白/DNA组合芯片(345)检测筛选:RSV反复感染引起大鼠C7-T5背根节345个转录因子中的55个表达上调2倍以上,43个表达下调2倍以上。(6)western blot检测EGR-1在肺部和脊髓背根节细胞的表达变化,结果显示EGR-1在RSV感染组表达明显低于对照。
结论:(1)采用RSV滴鼻的方式建立SD大鼠气道高反应性模型,能成功复制出气道高反应性大鼠模型。(2)反复呼吸道合胞病毒感染引起幼鼠气道神经网络发育异常改变(神经可塑性改变)。(3)转录因子活性芯片初步筛选出了反复呼吸道合胞病毒感染后脊髓背根节神经元细胞内发生活性改变的转录因子98种,为进一步探索反复呼吸道合胞病毒感染后气道神经可塑性变化的关键转录因子提供了研究基础。
Objective: On the basis of studying on airway hyperreactivity induced by airway neurogenic inflammation and airway neural plasticity, This project aims to investigate the expression of intracellular transcription factor in the C7-T5 spinal adorsal root Ganglion cells of SD rats after recurrent respiratory syncytial virus (RSV) infection and to identify the key intracellular transcription factors of involving in airway neural plasticity. Thus, to elucidate the mechanism of airway neural plasticity leading to the airway hyperreactivity and bronchial asthma after recurrent respiratory syncytial virus infection.
Methods: RSV is inoculated on the Hela cell bespreaded on the culture flask,and harvested when all of cells were infected undermicro-scope.The suspension was kept in reserve at -70℃.The virus-free medium was obtained as a negative control. The virus stock was titrated and diluted as final titer of 5×10~4 50% tissue culture infective dose (TCID50)in 0.1ml (virulence determination). Sixty 2-week-old SD rats(Hu Nan Agriculture University ,china) were randomly assigned into two groups (n=30 in each group). A volume of 0.4ml/kg body wt of RSV suspension was inoculated in the nostril of RSV-infected group while the rat was under pentobarbital sodium anesthesia(40mg/kg),control group were administered the same volume of virus-free medium.The treatment . above is repeated once a week.The rats were tested airway responsiveness before sarcrificed at 8th week and the left lung was removed for hema-toxylin and eosin, immunohistochemistry staining. C7-T5 dorsal root ganglion are dissected on the ice surface and put into the cryovial and stored in liquid nitrogen. RSV protein was assayed by hybridization in situ. The expression of SYN and NF in the airway and dorsal root ganglion were assayed by immunohistochemistry and transcription factors in C7-T5 dorsal root ganglion are preliminarily screened by the TranSignal~(TM) Protein/DNA Combo Arrays.The expression of EGR-1 in lung tissue and C7-T5 dorsal root ganglion were detected by western blot.
Results: (1)RSV infected rats gradually showed these syndromes such as catarrhalic rhinitis, gasping, mussy hair and activity decreasing.There was no significant difference between the two groups in the average body weigh. The temperature between the two groups has significant difference (P<0.05). (2)Airway resistance were significantly increased in the model groups than in the normal control group (P<0.05) after inhaled escalating histamine, indicating the airway hyperreactivity existed in RSV infected group. (3) The RSV infected group showed typical interstitial pneumonia under light microscope. (4) Comparing with the rats of control group, the expression of SYN and NF increased significantly (P<0.05) in RSV infected rats. (5) Protein/DNA array analysis showed that the expression of 55 transcription factors increased at least 2 fold in the C7-T5 spinal ganglion cells of RSV infected group, and the expression of 43 transcription factors decreased at least 2 fold in the C7-T5 spinal ganglion cells of the RSV-inoculated group. (6)The expression of EGR-1 was decreased both in the lung and in the C7-T5 spinal ganglion cells after RSV infection.
Conclusion: (1)The airway hyperreactivity in rats can be established successfully by RSV nostril inoculation. (2)The airway showed neural plasticity by SYN and NF staining after recurreent RSV infection. (3) TranSignal? Protein/DNA Combo Arrays analysis show that RSV infection result in the expression alteration of 98 transcription factors and build a foundation for futher study. EGR-1 may not be the key intracellular transcription factors of regulating airway neural plasticity.
引文
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[50]Shibasaki F, Price ER, Milan D, et al. Role of kineses and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF-AT4. Nature, 1996, 382:370-373
[51]Jain J, McCaffrey PG, Valge Archer VE, et al. Nuclear factor of activated T cells contains Fos and Jun. Nature 1992; 356: 801-804
[52]Northrop JP, Ullman KS, Crabtree GR. Characterization of the nuclear and cytoplasmic components of the lymphoid-specific nuclear factor of activated T cells (NF-AT). J Biol Chem, 1993, 268: 2917-2923
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[54]Wright LC, Cammisuli S, Baboulene L, et al . Cyclosporin A and glucocorticoids interact synergistically in T lymphocytes: implications for asthma therapy. Am J Respir Crit Care Med, 1995, 151: A675.
[55]Lee HJ, Matsuda I, Naito Y, et al. Signals and nuclear factors that regulate the expression of interleukin-4 and interleukin-5 genes in helper T cells. J Allergy Clin Immunol ,1994, 94: 594-604
[56]Hodge MR, Rooney JW, Glimcher LH. The proximal promoter of the IL-4 gene is composed of multiple essential regulatory sites that bind at
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