【作者】 丁鹏；

【导师】 王世鑫； 高宏生；

【作者基本信息】 河北医科大学 ， 劳动卫生与环境卫生学， 2008， 硕士

【摘要】 目的:本研究以大鼠矽肺动物模型为研究对象,通过测定矽肺相关生物活性介质并确定相关介质致纤维化的节点。初步探讨肿瘤坏死因子α(tumor necrosisαfactor,TNF-α)可溶性受体对二氧化硅(silicon dioxide,SiO2)粉尘致大鼠肺纤维化的防治作用以及相关细胞因子表达的影响。方法:根据随机化原则将大鼠分为3个实验组,即对照组(control group,CL)、染矽尘组(silica-instilled group,SA)、干扰组(intervention group,IN),对照组、染矽尘组每组48只,干扰组10只。对照组大鼠气管内注入生理盐水,染矽尘组大鼠气管内注入矽尘混悬液1ml(50mg/ml),干扰组大鼠气管内注入矽尘混悬液1ml(50mg/ml)后于1、5、8d给予500ug TNF-α可溶性受体皮下注射。气管暴露法建立大鼠矽肺模型,模型建立后,对照组、染矽尘组于1d、3d、7d、14d、21d、28d 6个时间点分别取8只大鼠将其处死。干扰组于7d、14d 2个时间点取5只大鼠处死。HE染色观察大鼠肺组织形态学的变化。血细胞计数仪测定支气管肺泡灌洗液细胞总数。天狼猩红染色法结合偏振光显微镜观察肺组织Ⅰ、Ⅲ型胶原合成。用Image-Pro Plus Version 4.5 for windows TM图象分析系统对图像进行分析。一氧化氮(nitrogen monoxidum,NO)测定采用南京建成生物工程研究所NO试剂盒。酶联免疫吸附法( enzyme-labeledimmunosorbent assay,ELISA)检测对照组、染矽尘组和干扰组各时间点外周血核因子κB(nuclear factorκB,NF-κB)、转化生长因子β1(transforming growth factorβ1,TGF-β1)、白介素(interleukin-1,IL-1β)、TNF-α等4种生物活性介质表达水平。所有数值均用x±s表示,两组间采用t检验,不同组间的比较用单因素方差分析的LSD法,全部数据的统计学处理均采用SPSS15.0 for windows分析软件。结果: 1 HE染色法证实气管暴露注入SiO2粉尘悬液成功建立矽肺模型。2 TNF-α可溶性受体对染矽尘大鼠肺脏器系数的影响。与对照组比较,染矽尘组大鼠7d以后各时间点肺脏器系数高于对照组(P<0.05);与染矽尘组比较,干扰组大鼠各时间点肺脏器系数低于染矽尘组,但高于对照组,差异有显著性(P<0.05)。3 TNF-α可溶性受体对染矽尘大鼠肺泡灌洗液(broncho alveolar lavage fluid,BALF)白细胞总数的影响。与对照组比较,染矽尘组相应时间点BALF白细胞总数均不同程度增高,在14d、21d、28d具有显著性差异(P<0.01);与染矽尘组比较,干扰组各相应时间点BALF白细胞总数均小于染矽尘组,在14d差异有显著性(P<0.05)。4 TNF-α可溶性受体对染矽尘大鼠Ⅰ、Ⅲ型胶原合成的影响。与对照组比较,染矽尘组7d、14d、21d、28dⅠ型胶原均高于对照组,差异有显著性(P<0.01);与染矽尘组比较,干扰组各时间点Ⅰ胶原低于染矽尘组组,在14d差异有显著性(P<0.05)。与对照组比较,染矽尘组各时间点Ⅲ胶原在21d时上升陡然趋缓,仅在7d、14d、21d时有显著性差异(P<0.01);与染矽尘组比较,干扰组各时间点Ⅲ胶原均低于染矽尘组,但仅在14d差异有显著性(P<0.05)。5 TNF-α可溶性受体对染矽尘大鼠外周血清NO表达的影响。与对照组比较,染矽尘组各时间点外周血清NO的浓度均高于对照组(P<0.01),在7d、14d、21d、28d具有显著性差异(P<0.05);与染矽尘组比较,干扰组各时间点外周血清NO浓度低于染矽尘组,在14d时差异有显著性(P<0.05)。6 TNF-α可溶性受体对染矽尘大鼠外周血清IL-1β表达的影响。与对照组比较,染矽尘组各时间点外周血清IL-1β的浓度均高于对照组(P<0.01),但仅在3d、7d时有显著性差异(P<0.05)。与染矽尘组比较,干扰组各时间点外周血清IL-1β浓度低于染矽尘组,在7d时差异有显著性(P<0.01)。7 TNF-α可溶性受体对染矽尘大鼠外周血清TGF-β表达的影响。与对照组比较,染矽尘组在14d、21d、28d时外周血清TGF-β的浓度高于对照组(P<0.01,P<0.05),其表达高峰在染矽尘后的第21d;与染矽尘组比较,干扰组各时间点外周血清TGF-β浓度低于染矽尘组,在14d时差异有显著性(P<0.05)。8 TNF-α可溶性受体对染矽尘大鼠外周血清NF-κB表达的影响。与对照组比较,染矽尘组各时间点外周血清NF-κB的浓度均高于对照组(P<0.01,P<0.05),其表达高峰在染矽尘后的第21d;与染矽尘组比较,干扰组各时间点外周血清NF-κB浓度低于染矽尘组,差异有显著性(P<0.05)。9 TNF-α可溶性受体对染矽尘大鼠外周血清TNF-α表达的影响。染矽尘组与干扰组各时间点外周血清TNF-α的浓度比较无统计学意义;与染矽尘组比较,干扰组各时间点外周血清TNF-α浓度低于染矽尘组,但差异无显著性。结论:本研究认为:1大鼠支气管内滴入矽尘可引起大鼠肺组织炎性和纤维化性病理变化,并有矽结节形成。2 TNF-α可溶性受体可以缓解矽尘诱导的大鼠肺脏器系数的增加。3 TNF-α可溶性受体对矽尘诱导的大鼠BALF中细胞总数的显著增加有抑制作用。4 TNF-α可溶性受体能够抑制矽尘诱导的大鼠肺组织Ⅰ、Ⅲ型胶原的增加。5 TNF-α可溶性受体能够抑制矽尘诱导的大鼠肺外周血清中NO、IL-1β、TGF-β1、NF-κB、TNF-α的表达水平。更多还原

【Abstract】 Objective: This study was designed to calculate 5 kind of biological activity medium in the blood serum of rats exposed to silica, and speculate the key profibrotic points. Primarily investigate the preventive and therapeutic effect of soluble TNF-αreceptor and the expression of other cytokinesMethod: Wistar rats were randomly divided into three experimental groups: control group(n=48), silica group(n=48), and intervention group(n=20). silica-instilled groups were received lightly anesthetized with pentobarbital and received a single intratracheal instillation of silicon suspension of 1ml at the concentration of 50mg/ml. The control group was treated in the same way but injection of 1 ml sterilized saline instead. Rats in drug-treated groups were given soluble TNF-αreceptor (500ug) in the 1, 5, 8 day after operation. At 1d, 3d, 7d, 14d, 21d, 28d after establishment of the animal model, eight rats in control and silica-instilled group were sacrificed. At 7d, 14d after establishment of the animal model, five rats in intervention group were sacrificed. The changes of lung histomorphology of rats were observed by HE staining of histological section. Total cell count and differential cell counts in bronchoalveolar lavage fluid (BALF) were estimated by cytometer. Sirius red polarization microscopy detectsⅠandⅢcollagen. The NO activity was also assayed by spectrophotometer, using the kits purchased from Nanjing Jiancheng Bioengineering Institute. The expression of NF-κB, TGF-β1, IL-1β, TNF-αwas assayed by ELISA kit.Result: 1 Histochemistry (HE staining) proved that the rat model of silicosis was successfully established by intratracheal infusion of silica dust suspension.2 Exposure to silica can increased the coefficient of organ to body of rats at each time point, and at 7d, 14d, 21d, 28d this difference was significant(P<0.05). soluble TNF-αreceptor can ameliorated the silica-induced increase in the coefficient of organ to body of rats, this difference was significant at 7d, 14d (P<0.05).3 Effect of soluble TNF-αreceptor on total white cell count in bronchoalveolar lavage fluid(BALF) of silica-treated rats. Compared with that of control group, total white cell count in BALF of silica goup, silica group at every time points increased, this difference was significant at 14d, 21d, 28d (P<0.01). Compared with that of silica group, total white cell count in BALF of Intervention group at 14d decreased significantly (P<0.05).4 Effect of soluble TNF-αreceptor on synthesis ofⅠⅢcollogen of silica-treated rats. In comparison to control group,Ⅰcollagen positive area percent of lung in silica-induced group exhibited a sustained significant increase at 7d, 14d, 21d, 28d (P<0.01) , but the increase ofⅢcollagen positive area percent of lung in silica-induced groups began to be small at 21d, this increase was just significant at 7d, 14d, 21d (P<0.01). ForⅠcollagen, such increases were reduced significantly by TNF-αmonoclonal antibody treatment at 14d (P<0.05), and forⅢcollagen significant at 14d too (P<0.05).5 Effect of soluble TNF-αreceptor on NO content in circumference blood serum of rats exposed to silica. Compared to the control, a relatively increase in the NO content in the serum was observed from silica-exposed rats, this increase was significant at 7d, 14d, 21d, 28d (P<0.05). soluble TNF-αreceptor treatment had obvious effect on the silica-induced increase in this parameter, this reduction was significant at 14d (P<0.05) .6 Effect of soluble TNF-αreceptor on IL-1βprotein expression in circumference blood serum of rats exposed to silica. Compared to the control, a relatively smaller increase in the IL-1βcontent in the serum was observed from silica-exposed rats, this increase was just significant at 3d, 7d (P<0.05) . soluble TNF-αreceptor treatment had marked effect on the silica-induced increase in this parameter, this reduction was significant at 7d (P<0.05) .7 Effect of soluble TNF-αreceptor on TGF-β1 protein expression in circumference blood serum of rats exposed to silica. In comparison to the control, there was a significant increase in TGF-β1 content in the serum from silica-exposed group rats at 14d, 21d, 28d (P<0.01, P<0.05), and the highest level of TGF-β1 was found at 21d. Compared with that of silica group, the level of TGF-β1 was significantly decreased at 14d by treatment of soluble TNF-αreceptor.8 Effect of soluble TNF-αreceptor on NF-κB protein expression in circumference blood serum of rats exposed to silica. Compared to the control, the level of NF-κB increased significantly at at all time points(P<0.01, P<0.05). and it peaked at 3d after rats exposed to silica. such increases were reduced significantly by treatment of soluble TNF-αreceptor at 7d, 14d (P<0.05).9 Effect of soluble TNF-αreceptor on TNF-αprotein expression in Circumference blood serum of rats exposed to silica. No significant differences were found in serum levels of TNF-αbetween rats received different treatment.Conclusion: 1 The bronch of the rat instilled silica dust suspension can cause phlegmonosis and fibrosis pathological change in the lung tissues of the rat and form silicotic nodule. 2 soluble TNF-αreceptor can ameliorated the silica-induced increase in the coefficient of organ to body of rats. 3 The increases of the numbers of total cells in BALF in silica-treated rats were significantly suppressed by soluble TNF-αreceptor. 4 The formation ofⅠandⅢcollagen in lung tissues in silica-treated rats can be suppressed by soluble TNF-αreceptor. 5 soluble TNF-αreceptor may plays roles of anti-fiborosis through intervention the key cytokine such as TNF-α、NF-κB、TGF-β1、IL-1βin cytokine network in silicotic process.更多还原