RSV感染大鼠气道神经营养因子水平与气道神经可塑性研究
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摘要
目的:建立呼吸道合胞病毒(RSV)感染SD大鼠动物模型。研究RSV感染后SD大鼠肺内神经营养因子(NGF、BDNF、NT3及NT4)浓度的变化与气道神经可塑性改变间的关系,为婴幼儿哮喘的预防治疗提供新的思路。
方法:1-2周龄SD大鼠共60只,随机分为2组,A组:正常对照组;B组RSV感染组。每组30只,采用每周1次呼吸道合胞病毒滴鼻法制作模型。在第8周进行气道阻力测定,留取左肺行HE染色观察气道炎症改变及免疫组化明确气道突触囊泡素和神经丝的表达变化,原位杂交法检测肺组织RSV RNA的表达,取右肺行组织匀浆后,以酶联免疫吸附(ELISA)法检测肺组织匀浆上清液神经营养因子(NGF、BDNF、NT3及NT4)浓度的变化。
结果:(1)一般情况: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。(2)体温:滴鼻后24小时内实验组动物体温上升幅度较基础体温有统计学差异(P＜0.05),而对照组滴鼻前后体温变化没有统计学差异。(3)体重:干预8周后,两组实验动物体重没有明显差别。(4)原位杂交检测肺组织RSV RNA的表达:对照组大鼠肺组织中未见RSV RNA阳性信号,而实验组大鼠肺间质细胞中可见明显的RSV RNA阳性信号。(5)气道反应性:干预8周后,各组间大鼠的气道阻力基础值差异无显著性,在RSV感染组随着盐酸组胺浓度的升高,气道阻力明显增高。(6)HE染色: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。(7)免疫组化:8周RSV组大鼠气道壁突触囊泡素及神经丝阳性分布部位及形态与对照组相似,但分布密度较对照组明显上调(P＜0.05),阳性产物为棕红色颗粒,颗粒与对照组相比较粗大。(8)神经营养因子表达水平:RSV感染组NGF及BDNF表达水平较对照组明显增高(P＜0.05);而NT_3及NT_4表达水平则较对照组没有明显差别。(9)相关性分析:NGF及BDNF浓度表达均与气道突触囊泡素表达呈正相关关系(r_1=0.892,r_2=0.995,P＜0.05),与神经丝表达呈正相关关系(r_1=0.949,r_2=0.936,P＜0.05),与气道高反应性也呈正相关关系(r_1=0.929,r_2=0.910,P＜0.05)。
结论:(1)本实验发现RSV感染可引起大鼠的气道神经可塑性的改变,表现为气道神经内SYN、NF的表达增加,此二者的密度可反映气道神经的密度分布;(2)RSV感染组大鼠肺组织匀浆中NGF及BDNF浓度较对照组明显增高;(3)NGF及BDNF可能参与了RSV感染所致大鼠气道神经可塑性改变的调节过程,从而导致气道高反应性的发生。
Objective: To develop an animal model of recurrent respiratory syncytial virus(RSV) infected SD rats and to study the relationship between the concentration of the NT(including NGF,BDNF,NT3 and NT4) in the lung and airway neural plasticity.
Methods: 60 SD rats were randomly divided into 2 groups with 30 rats in each group: control group(group A),RSV-infected group (group B,rats were infected with 5×10~4TCID50/0.1ML RSV each week),after 8 weeks, the bronchial responsiveness of the SD rats (RSV-inoculated group and control group) was assessed. The bronchial inflammation was assessed by light microscope. SYN and NF expression in the airway of SD rats were assayed by the immunohistochemistry stanining. Hybridization in situ to evaluate whether the RSV have planted in the lung. The concen -tration of NT(including NGF,BDNF,NT3 and NT_4) in the lung of SD rats were detected by ELISA.
Results: 1.general state of health: Model rats gradually emerged catarrhalic rhinitis、gasping、mussy hair and activity decreasing; 2.Body temperature:The body temperature was significantly increased compared with the control group(P<0.05) during 24 hours After RSV-inoculated; 3.Weight: There was no significant difference between the two groups; 4.Hybridization in situ to evaluate effects of butylph thalide on RSV RNA expression in rat's lung: Positive signal of RSV RNA can be found in the RSV-infected rat's lung compared with control group; 5. Bronchial responsiveness:There was no significant difference in all groups of the basal airway resistance before inhaled Histamine. The airway resistance kept rising with the escalating concentration of Histamine inhalation,the airway resistance was significantly increased compared with the control group(P<0.05); 6.The pathohistology in SD rats: The RSV infected group showed typical interstitial pneumonia under light microscope. 7.Immunohistochemistry : RSV-inoculated SD rats has a neural plasticity of the airways characterized by the increased expression of SYN and NF. The distribution density was significantly increased compared with the control group(P<0.05). Positive staining was brownish red color granule. Granule was much bulkier compared with the control group ; 8.Supernatant neurotrophins level of lung tissue homogenization: NGF and BDNF were significantly increased compared with the control group(P<0.05); The NT3 and NT4 level have no significant difference in all groups; 9.Relative analysis:The expression of NGF and BDNF are positively related to the expression of SYN (r1=0.892, r2=0.995, P<0.05 ) ,and are positively related to the expression of NF (r1=0.949,r2=0.936, P<0.05) ,and are positively related to the airway hyperresponsiveness (r1=0.929,r2=0.910, P<0.05) .
Conclusion: 1 .RSV-inoculated SD rats showed neural plasticity in the airway characterized by the increased expression of NF and SYN; 2.The expression of NGF and BDNF in the lung were increa sed after recurrent RSV infection; 3.NGF and BDNF may participat e in the development of airway neural plasticity and airway hyperre sponsiveness after recurrent RSV infection.
方法:1-2周龄SD大鼠共60只,随机分为2组,A组:正常对照组;B组RSV感染组。每组30只,采用每周1次呼吸道合胞病毒滴鼻法制作模型。在第8周进行气道阻力测定,留取左肺行HE染色观察气道炎症改变及免疫组化明确气道突触囊泡素和神经丝的表达变化,原位杂交法检测肺组织RSV RNA的表达,取右肺行组织匀浆后,以酶联免疫吸附(ELISA)法检测肺组织匀浆上清液神经营养因子(NGF、BDNF、NT3及NT4)浓度的变化。
结果:(1)一般情况: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。(2)体温:滴鼻后24小时内实验组动物体温上升幅度较基础体温有统计学差异(P＜0.05),而对照组滴鼻前后体温变化没有统计学差异。(3)体重:干预8周后,两组实验动物体重没有明显差别。(4)原位杂交检测肺组织RSV RNA的表达:对照组大鼠肺组织中未见RSV RNA阳性信号,而实验组大鼠肺间质细胞中可见明显的RSV RNA阳性信号。(5)气道反应性:干预8周后,各组间大鼠的气道阻力基础值差异无显著性,在RSV感染组随着盐酸组胺浓度的升高,气道阻力明显增高。(6)HE染色: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。(7)免疫组化:8周RSV组大鼠气道壁突触囊泡素及神经丝阳性分布部位及形态与对照组相似,但分布密度较对照组明显上调(P＜0.05),阳性产物为棕红色颗粒,颗粒与对照组相比较粗大。(8)神经营养因子表达水平:RSV感染组NGF及BDNF表达水平较对照组明显增高(P＜0.05);而NT_3及NT_4表达水平则较对照组没有明显差别。(9)相关性分析:NGF及BDNF浓度表达均与气道突触囊泡素表达呈正相关关系(r_1=0.892,r_2=0.995,P＜0.05),与神经丝表达呈正相关关系(r_1=0.949,r_2=0.936,P＜0.05),与气道高反应性也呈正相关关系(r_1=0.929,r_2=0.910,P＜0.05)。
结论:(1)本实验发现RSV感染可引起大鼠的气道神经可塑性的改变,表现为气道神经内SYN、NF的表达增加,此二者的密度可反映气道神经的密度分布;(2)RSV感染组大鼠肺组织匀浆中NGF及BDNF浓度较对照组明显增高;(3)NGF及BDNF可能参与了RSV感染所致大鼠气道神经可塑性改变的调节过程,从而导致气道高反应性的发生。
Objective: To develop an animal model of recurrent respiratory syncytial virus(RSV) infected SD rats and to study the relationship between the concentration of the NT(including NGF,BDNF,NT3 and NT4) in the lung and airway neural plasticity.
Methods: 60 SD rats were randomly divided into 2 groups with 30 rats in each group: control group(group A),RSV-infected group (group B,rats were infected with 5×10~4TCID50/0.1ML RSV each week),after 8 weeks, the bronchial responsiveness of the SD rats (RSV-inoculated group and control group) was assessed. The bronchial inflammation was assessed by light microscope. SYN and NF expression in the airway of SD rats were assayed by the immunohistochemistry stanining. Hybridization in situ to evaluate whether the RSV have planted in the lung. The concen -tration of NT(including NGF,BDNF,NT3 and NT_4) in the lung of SD rats were detected by ELISA.
Results: 1.general state of health: Model rats gradually emerged catarrhalic rhinitis、gasping、mussy hair and activity decreasing; 2.Body temperature:The body temperature was significantly increased compared with the control group(P<0.05) during 24 hours After RSV-inoculated; 3.Weight: There was no significant difference between the two groups; 4.Hybridization in situ to evaluate effects of butylph thalide on RSV RNA expression in rat's lung: Positive signal of RSV RNA can be found in the RSV-infected rat's lung compared with control group; 5. Bronchial responsiveness:There was no significant difference in all groups of the basal airway resistance before inhaled Histamine. The airway resistance kept rising with the escalating concentration of Histamine inhalation,the airway resistance was significantly increased compared with the control group(P<0.05); 6.The pathohistology in SD rats: The RSV infected group showed typical interstitial pneumonia under light microscope. 7.Immunohistochemistry : RSV-inoculated SD rats has a neural plasticity of the airways characterized by the increased expression of SYN and NF. The distribution density was significantly increased compared with the control group(P<0.05). Positive staining was brownish red color granule. Granule was much bulkier compared with the control group ; 8.Supernatant neurotrophins level of lung tissue homogenization: NGF and BDNF were significantly increased compared with the control group(P<0.05); The NT3 and NT4 level have no significant difference in all groups; 9.Relative analysis:The expression of NGF and BDNF are positively related to the expression of SYN (r1=0.892, r2=0.995, P<0.05 ) ,and are positively related to the expression of NF (r1=0.949,r2=0.936, P<0.05) ,and are positively related to the airway hyperresponsiveness (r1=0.929,r2=0.910, P<0.05) .
Conclusion: 1 .RSV-inoculated SD rats showed neural plasticity in the airway characterized by the increased expression of NF and SYN; 2.The expression of NGF and BDNF in the lung were increa sed after recurrent RSV infection; 3.NGF and BDNF may participat e in the development of airway neural plasticity and airway hyperre sponsiveness after recurrent RSV infection.
引文
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