干扰素α-1b雾化吸入治疗呼吸道合胞病毒毒感感染的实验研究
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摘要
目的
呼吸道合胞病毒(RSV)是全球婴幼儿下呼吸道感染的首位病毒病原体，儿童2岁前都至少感染过一次RSV[ 1 ]，多数会发生再感染，与日后发生喘息密切相关，它是严重危害婴幼儿健康的一种病毒。重组人干扰素α-1b是我国第一个自主开发的基因工程新药，据研究报告称，与国内外同类产品相比，重组人干扰素α-1b在抗病毒治疗中不仅疗效高，而且不良反应轻，中和抗体产生率低，是适合中国人使用的基因工程药物。已有实验表明重组人干扰素α-1b作为一种较为安全和不产生耐药性的广谱抗病毒制剂，在RSV感染治疗中有巨大的优势。但到目前为止，国内应用重组人干扰素α-1b治疗RSV感染，多选肌肉注射给药，儿童依从性较差，且会引起发热、肌肉酸痛等不良反应，也有关于雾化吸入此药治疗呼吸道病毒感染的临床报告，但在雾化吸入干扰素α-1b治疗RSV感染方面缺乏相关实验室依据。为此，我们建立了RSV感染的小鼠模型，观察改变给药途径后（即经雾化吸入干扰素α-1b）其在小鼠体内对RSV的抑制作用，通过与雾化吸入利巴韦林的疗效比较，探讨雾化吸入干扰素α-1b治疗呼吸道合胞病毒感染的有效性，为今后的临床试验提供理论依据。
方法
将40只BALB/c小鼠滴鼻感染RSV，制备成RSV感染模型后随机分为4组：生理盐水对照组；干扰素α-1b治疗组；利巴韦林治疗组；干扰素α-1b+利巴韦林治疗组。分别雾化吸入生理盐水、干扰素α-1b、利巴韦林、干扰素α-1b+利巴韦林。第5天各组中随机抽取5只小鼠处死，取左肺组织处理后用于光镜观察肺组织病理改变，并计算病理积分(PS)，将右肺组织处理后用于透射电镜观察；各组中其它5只小鼠处死后无菌分离气管，用生理盐水进行支气管肺泡灌洗，收集支气管肺泡灌洗液（BALF），使用血球计数仪计数白细胞总数，离心处理沉淀物涂片后，按淋巴细胞、单核细胞、中性粒细胞及嗜酸性粒细胞形态学特点分类计数，上清液用于RSV实时荧光定量逆转录PCR的测定。
结果
1.病理学检查光镜形态学观察：三个治疗组小鼠肺病理变化较对照组明显减轻,且干扰素α-1b治疗组较利巴韦林治疗组病变更轻；经统计学处理光镜PS结果：三个治疗组较对照组显著减少，治疗组间无差异；电镜下观察：三个治疗组病理变化较对照组明显减轻。
2.细胞学检查：三个治疗组BALF白细胞计数及淋巴细胞比例显著低于对照组（P＜0.05)，干扰素α-1b治疗组和干扰素α-1b+利巴韦林治疗组无显著差异，两组均显著低于利巴韦林治疗组；各组BALF白细胞分类都以淋巴细胞为主。
3.支气管肺泡灌洗液RSV实时荧光定量逆转录PCR的测定结果：三个治疗组显著低于生理盐水对照组（P＜0.05)，干扰素α-1b治疗组及干扰素α-1b+利巴韦林治疗组无显著差异，两组均显著低于利巴韦林治疗组。
结论
本实验光镜形态学显示单用雾化吸入干扰素α-1b能明显减轻小鼠RSV肺炎的病理改变，效果同干扰素α-1b+利巴韦林，但优于单用利巴韦林；光镜病理积分显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林及雾化吸入利巴韦林能显著减轻肺部病理改变，三种方法效果相同；电镜显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林及雾化吸入利巴韦林能减轻小鼠肺病理改变，三种方法效果相同；BALF细胞学检查显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林均可明显减少气道炎症细胞，两种方法效果相同，但显著优于单用利巴韦林；支气管肺泡灌洗液RSV实时荧光定量逆转录PCR测定结果也显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林均能有效降低病毒的拷贝数，两种方法效果相同，但明显优于单独雾化吸入利巴韦林。因此单独雾化吸入干扰素α-1b与干扰素α-1b+利巴韦林合用治疗呼吸道合胞病毒感染的效果相同，但明显优于单独雾化吸入利巴韦林。故单独雾化吸入干扰素α-1b是治疗RSV感染的有效方法。
Objective
Human respiratory syncytial virus is the first viral pathogens of the lowerrespiratory tract infection in infants and young children around world. Childrenbefore the age of two infected with RSV at least once. Most of them will be reinfectedand closely related to asthma in the future. The health of the childrenwill be serious damaged. Recombinant human IFNα-1b is the China's first selfdevelopedgenetic engineering new drug. Compared to similar products at homeand abroad, recombinant human IFNα-1b has not only high efficacy, but alsohas light adverse effects and low rate of neutralize antibody production, which issuitable for Chinese people to use. As a safe and no resistant broad -spectrumanti-viral preparation. Recombinant human IFNα-1b has a huge advantage inthe treatment of pediatric viral diseases. However so far as, in the domestictreatment of RSV infection, most of them use intravenous or intr- amuscularadministration, and the children's compliance is poor. To this point, we haveestablished a RSV infection mouse model, in order to observe the inhibition of RSV in mice after the change in administration route (that is, through thenebulization of IFNα-1b). We discussed the effectiveness and safety of thetreatment of respiratory syncytial virus pneumonia through the nebulization ofIFNα-1b. Through animal experiments, it can provide a theore -tical basis forthe clinical trials in the future.
MethMethod
40 BALB / c mice which were intranasal instillation infected with RSVwere prepared to RSV infection model, and then were randomly divided intofour groups: blank control group; IFNα-1b group; ribavirin group; IFNα-1b +ribavirin group. And each group nebulization normal saline; IFNα-1b; ribavirin;IFNα-1b + ribavirin respectively. On the fifth day five mice were selected ineach group randomly and were killed. After the mice left lung organization weretreated, they were used for light microscopic observing the pathological score(PS) of lung tissue. And treat the right lung for the observation of the trans -mission electron microscopy; In each group we kill the other five mice, separatetrachea in sterile, bronchial lavage with normal saline , collect bronch -oalveolarlavage fluid, use blood cell counter to count the total number of white blood cells,centrifuge sediment smears, count the number by the classification of thelymphocytes, monocytes, neutrophils, and eosinophils morphologic features, andat last supernatant was used for the determination of RSV real-time fluorescencequantitative PCR.
Result
1.Pathological examination: PS of the three treatment group was signi -ficantly lower than the control group (P <0.05) by the light microscopyobservation, namely, pulmonary changes in mice treated group were significant -ly lighter than the saline nebulization group; pathological lesions in the IFNα- 1b group were significantly lighter than that in the control group under theelectron microscope. It did not find the RSV and the RSV particle inclusions.
2.Cytological examination: According to the bronchoalveolar lavage fluidwhite blood cells and lymphocyte count , the number of the treatment group islower than that of the control group (P <0.05), and the IFNα-1b group was lowerthan ribavirin group, namely, the airway inflammatory cells were reduced.. Ineach group, the classification of the BALF white blood cells are mainlylymphocytes.
3.The results of the Bronchoalveolar lavage fluid RSV real-time fluore -scence quantitative PCR determination: the levers of three treatment group weresignificantly lower than that of the control group; IFNα-1b group wassignificantly lower than the ribavirin group.ConclusiConclusion
The results of this study have shown that nebulization of IFNα-1b couldsignificantly reduce RSV pneumonia lesions in mice; nebulization of IFNα-1band ribavirin may reduce the airway inflammatory cells, but interferon issignificantly better than ribavirin; Similarly, the results of bronchoalveolarlavage fluid RSV real-time fluorescence quantitative PCR determination alsoshowed that nebulization of IFNα-1b can more effectively reduce the virus copynumber. So the anti-viral effects of nebulization of IFNα-1b are superior toribavirin. Therefore, nebulization of IFNα-1b is an effective method for thetreatment of RSV infection.
呼吸道合胞病毒(RSV)是全球婴幼儿下呼吸道感染的首位病毒病原体，儿童2岁前都至少感染过一次RSV[ 1 ]，多数会发生再感染，与日后发生喘息密切相关，它是严重危害婴幼儿健康的一种病毒。重组人干扰素α-1b是我国第一个自主开发的基因工程新药，据研究报告称，与国内外同类产品相比，重组人干扰素α-1b在抗病毒治疗中不仅疗效高，而且不良反应轻，中和抗体产生率低，是适合中国人使用的基因工程药物。已有实验表明重组人干扰素α-1b作为一种较为安全和不产生耐药性的广谱抗病毒制剂，在RSV感染治疗中有巨大的优势。但到目前为止，国内应用重组人干扰素α-1b治疗RSV感染，多选肌肉注射给药，儿童依从性较差，且会引起发热、肌肉酸痛等不良反应，也有关于雾化吸入此药治疗呼吸道病毒感染的临床报告，但在雾化吸入干扰素α-1b治疗RSV感染方面缺乏相关实验室依据。为此，我们建立了RSV感染的小鼠模型，观察改变给药途径后（即经雾化吸入干扰素α-1b）其在小鼠体内对RSV的抑制作用，通过与雾化吸入利巴韦林的疗效比较，探讨雾化吸入干扰素α-1b治疗呼吸道合胞病毒感染的有效性，为今后的临床试验提供理论依据。
方法
将40只BALB/c小鼠滴鼻感染RSV，制备成RSV感染模型后随机分为4组：生理盐水对照组；干扰素α-1b治疗组；利巴韦林治疗组；干扰素α-1b+利巴韦林治疗组。分别雾化吸入生理盐水、干扰素α-1b、利巴韦林、干扰素α-1b+利巴韦林。第5天各组中随机抽取5只小鼠处死，取左肺组织处理后用于光镜观察肺组织病理改变，并计算病理积分(PS)，将右肺组织处理后用于透射电镜观察；各组中其它5只小鼠处死后无菌分离气管，用生理盐水进行支气管肺泡灌洗，收集支气管肺泡灌洗液（BALF），使用血球计数仪计数白细胞总数，离心处理沉淀物涂片后，按淋巴细胞、单核细胞、中性粒细胞及嗜酸性粒细胞形态学特点分类计数，上清液用于RSV实时荧光定量逆转录PCR的测定。
结果
1.病理学检查光镜形态学观察：三个治疗组小鼠肺病理变化较对照组明显减轻,且干扰素α-1b治疗组较利巴韦林治疗组病变更轻；经统计学处理光镜PS结果：三个治疗组较对照组显著减少，治疗组间无差异；电镜下观察：三个治疗组病理变化较对照组明显减轻。
2.细胞学检查：三个治疗组BALF白细胞计数及淋巴细胞比例显著低于对照组（P＜0.05)，干扰素α-1b治疗组和干扰素α-1b+利巴韦林治疗组无显著差异，两组均显著低于利巴韦林治疗组；各组BALF白细胞分类都以淋巴细胞为主。
3.支气管肺泡灌洗液RSV实时荧光定量逆转录PCR的测定结果：三个治疗组显著低于生理盐水对照组（P＜0.05)，干扰素α-1b治疗组及干扰素α-1b+利巴韦林治疗组无显著差异，两组均显著低于利巴韦林治疗组。
结论
本实验光镜形态学显示单用雾化吸入干扰素α-1b能明显减轻小鼠RSV肺炎的病理改变，效果同干扰素α-1b+利巴韦林，但优于单用利巴韦林；光镜病理积分显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林及雾化吸入利巴韦林能显著减轻肺部病理改变，三种方法效果相同；电镜显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林及雾化吸入利巴韦林能减轻小鼠肺病理改变，三种方法效果相同；BALF细胞学检查显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林均可明显减少气道炎症细胞，两种方法效果相同，但显著优于单用利巴韦林；支气管肺泡灌洗液RSV实时荧光定量逆转录PCR测定结果也显示雾化吸入干扰素α-1b、雾化吸入干扰素α-1b+利巴韦林均能有效降低病毒的拷贝数，两种方法效果相同，但明显优于单独雾化吸入利巴韦林。因此单独雾化吸入干扰素α-1b与干扰素α-1b+利巴韦林合用治疗呼吸道合胞病毒感染的效果相同，但明显优于单独雾化吸入利巴韦林。故单独雾化吸入干扰素α-1b是治疗RSV感染的有效方法。
Objective
Human respiratory syncytial virus is the first viral pathogens of the lowerrespiratory tract infection in infants and young children around world. Childrenbefore the age of two infected with RSV at least once. Most of them will be reinfectedand closely related to asthma in the future. The health of the childrenwill be serious damaged. Recombinant human IFNα-1b is the China's first selfdevelopedgenetic engineering new drug. Compared to similar products at homeand abroad, recombinant human IFNα-1b has not only high efficacy, but alsohas light adverse effects and low rate of neutralize antibody production, which issuitable for Chinese people to use. As a safe and no resistant broad -spectrumanti-viral preparation. Recombinant human IFNα-1b has a huge advantage inthe treatment of pediatric viral diseases. However so far as, in the domestictreatment of RSV infection, most of them use intravenous or intr- amuscularadministration, and the children's compliance is poor. To this point, we haveestablished a RSV infection mouse model, in order to observe the inhibition of RSV in mice after the change in administration route (that is, through thenebulization of IFNα-1b). We discussed the effectiveness and safety of thetreatment of respiratory syncytial virus pneumonia through the nebulization ofIFNα-1b. Through animal experiments, it can provide a theore -tical basis forthe clinical trials in the future.
MethMethod
40 BALB / c mice which were intranasal instillation infected with RSVwere prepared to RSV infection model, and then were randomly divided intofour groups: blank control group; IFNα-1b group; ribavirin group; IFNα-1b +ribavirin group. And each group nebulization normal saline; IFNα-1b; ribavirin;IFNα-1b + ribavirin respectively. On the fifth day five mice were selected ineach group randomly and were killed. After the mice left lung organization weretreated, they were used for light microscopic observing the pathological score(PS) of lung tissue. And treat the right lung for the observation of the trans -mission electron microscopy; In each group we kill the other five mice, separatetrachea in sterile, bronchial lavage with normal saline , collect bronch -oalveolarlavage fluid, use blood cell counter to count the total number of white blood cells,centrifuge sediment smears, count the number by the classification of thelymphocytes, monocytes, neutrophils, and eosinophils morphologic features, andat last supernatant was used for the determination of RSV real-time fluorescencequantitative PCR.
Result
1.Pathological examination: PS of the three treatment group was signi -ficantly lower than the control group (P <0.05) by the light microscopyobservation, namely, pulmonary changes in mice treated group were significant -ly lighter than the saline nebulization group; pathological lesions in the IFNα- 1b group were significantly lighter than that in the control group under theelectron microscope. It did not find the RSV and the RSV particle inclusions.
2.Cytological examination: According to the bronchoalveolar lavage fluidwhite blood cells and lymphocyte count , the number of the treatment group islower than that of the control group (P <0.05), and the IFNα-1b group was lowerthan ribavirin group, namely, the airway inflammatory cells were reduced.. Ineach group, the classification of the BALF white blood cells are mainlylymphocytes.
3.The results of the Bronchoalveolar lavage fluid RSV real-time fluore -scence quantitative PCR determination: the levers of three treatment group weresignificantly lower than that of the control group; IFNα-1b group wassignificantly lower than the ribavirin group.ConclusiConclusion
The results of this study have shown that nebulization of IFNα-1b couldsignificantly reduce RSV pneumonia lesions in mice; nebulization of IFNα-1band ribavirin may reduce the airway inflammatory cells, but interferon issignificantly better than ribavirin; Similarly, the results of bronchoalveolarlavage fluid RSV real-time fluorescence quantitative PCR determination alsoshowed that nebulization of IFNα-1b can more effectively reduce the virus copynumber. So the anti-viral effects of nebulization of IFNα-1b are superior toribavirin. Therefore, nebulization of IFNα-1b is an effective method for thetreatment of RSV infection.
引文
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[40]. Hashmi NA, Cosgrove JF, MacMahon P. Prophylaxis in RSV in Fection(Palivizu -mab) is it worthwhile[J]. 1r Med J 2000,93(9) :284.
[41]. Wu H, Pfarr DS, Johnson S. Development of motavlzumab，an ultra-potentanti- body for the prevention of respiratory syncytial virus infection in theupper and lower respiratory tract[J]. J Mol Biol 2007, 368(3) :652-665.
[42]. Kalina WV, Woolums AR, Gershwin LJ. Formalin-inactivated bovineRSV vacc- ine influences antibody levels in bronchoalveolar lavage fluidand disease outcome in experimentally infected calves. Vaccine, 2005,23(37) :4625-4630.
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[46].赵宇红,申昆玲,刘亚谊,等.鱼腥草注射液治疗呼吸道合胞病毒感染的体外实验研究[J].首都医科大学学报,2005,26(5) :571-573.
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[50].钟琼,杨占秋,赵玲敏,等.穿心莲提取物体外抗呼吸道合胞病毒作用的研究[J].湖北中医学院学报,2005,(2):23-25.
[51].夏泉,黄赵刚,李绍平,等.莪术油抗流感病毒和呼吸道合胞病毒作用的实验研究[J].中国药理学通报,2004,20(3):357-358.
[52].董艳梅,李洪源,姚振江,等.甘草体外抑制呼吸道合胞病毒作用研究[J].中药材, 2004,27(6):425-427.
[53].杨桦．人参多糖抗呼吸道合胞病毒感染的作用机制研究[J]．中国药理学通报，1997，13(4)：382．
[54].邹毅,黄海鹭,徐军,等.小鼠的原发性呼吸道合胞病毒感染[J].中华结核和呼吸杂志,2001,24(8):484-486.
[55].黄艳,陈吉泉.雾化吸入干扰素γ阻断GATA23表达防治哮喘的实验研究[J].免疫学杂志, 2006,22(4):406-415.
[56].秦大莲主编.药理学实验指导[M].泸州医学院药理学教研室，2006：10
[57].李燕晖,张国成,许东亮.静脉注射用丙种球蛋白对呼吸道合胞病毒治疗的作用[J].细胞与分子免疫学杂志,2005, 21 (5):640-642.
[58]. Ng PC，Li G，Chui KM，el a1．Neutmphil CD64 is a sensitivediagnostic marker for early onset neonatalinfection[J]Pediatr Res，2004，56(5)：796—803．
[59].马壮,钱桂生,黄桂君,等.雾化吸入γ2干扰素对免疫低下大鼠肺部抗感染能力和细胞免疫的影响[J].中华结核和呼吸杂志,2001.24(2):112-113.
[60].田曼,葛传生.呼吸道合胞病毒感染的动物模型[J].中国抗感染化疗杂志,2001. 1(4):244-246.