宣肺定喘汤对大鼠气道平滑肌细胞增殖的影响

【作者】 余常乐；

【导师】 李家庚；

【作者基本信息】 湖北中医学院 ， 中医临床基础， 2007， 硕士

【摘要】 支气管哮喘（bronchial asthma，BA，简称哮喘）是由多种细胞（如嗜酸性粒细胞、肥大细胞、T淋巴细胞、中性粒细胞、气道上皮细胞等）和细胞组分参与的气道慢性炎症性疾病。近年来，关于哮喘气道炎症、气道重塑（airway remodeling，ARM）的研究层出不穷，其中一个重大的突破是明确哮喘的实质是气道的慢性炎症。但由于单纯用气道炎症不能完全解释支气管哮喘，人们再次把气道重塑作为哮喘研究领域的重点。大量尸体解剖资料显示，哮喘患者气道平滑肌层较正常人增厚50％以上，它的增厚并不仅是细胞代偿、肥大的结果，而更主要是气道平滑肌细胞（airway smooth muscle cell，ASMC）本身增殖所致。ASMC增殖是产生ARM的重要原因，并与气道高反应性（Airway hyper-responsiveness，AHR）密切相关。因此，在常规抗炎治疗的基础上研发抑制ASMC增殖的药物，有可能为BA的治疗提供新的更为有效的途径。目的本文通过观察宣肺定喘汤（XFDCT）含药血清对体外培养的大鼠ASMC增殖的影响，并初步探讨其作用机理，以了解XFDCT在哮喘气道重塑的一个重要方面——ASMC增殖的直接作用。方法1．清洁级雄性SD大鼠一只，脊椎脱臼法处死，于无菌条件下，取左肺下叶、右中及右下肺叶，组织液中分离各肺叶支气管。将剥离好的支气管制成1mm³大小的组织块，用贴块法原代培养ASMC。2．用组胺（Histamine，His）诱导体外培养的气道平滑肌细胞建立增殖细胞模型。3．清洁级Wistar大鼠20只，随机分为5组：XFDCT高、中、低剂量组，西药组及空白组，每组4只。XFDCT高、中、低剂量组大鼠分别给予该方水煎液按40g／（kg·d）、20g／（kg·d）、10g／（kg·d）灌胃，20ml／kg；西药组予地塞米松2.54×10^-4g／（kg·d）等容积溶液灌胃；空白组予等容积生理盐水灌胃，连续5天。于末次给药前禁食12h，但不禁水，末次灌胃1h后，10％水合氯醛（1ml／kg）麻醉，腹主动脉取血，离心管收集血液4℃过夜，待其血清充分析出，3000rpms／15min离心，收集血清，56℃／30min灭活补体，0.22μm微孔滤膜过滤分装，-20℃保存备用。分别作用于各组ASMC。4．倒置相差显微镜下观察细胞形态及其生长状况并摄片，四唑盐比色法（MTT法）检测细胞增殖活力及免疫细胞化学染色（SABC法）检测增殖细胞核抗原（proliferation cell nuclear antigen，PCNA）的表达。结果1．通过免疫细胞化学染色，抗平滑肌a-肌动蛋白抗体阳性染色，细胞纯度达96％。2．不同浓度His作用于ASMC后，在不同的时间点上A_570nm值均高于对照组，其中浓度10^-4mol／L的His对ASMC的作用最明显，其在36h的A_570nm值是对照组的1.19倍（为0.152±0.002比0.128±0.003，P＜0.05，n=5）。表明His对ASMC有明显的促增殖作用（P＜0.05，n=5），且呈时间但非浓度依赖性。3．XFDCT高、中剂量组含药血清能显著降低His促大鼠ASMC增殖模型MTT法OD值，与对照组有显著性差异（P＜0.05），与地塞米松组无显著差异（P＞0.05）；而XFDCT低剂量组与对照组无显著差异（P＞0.05）。且存在一定的时间、剂量依赖关系。4．PCNA定位于细胞核，阳性细胞核内出现棕黄色的颗粒，MIAS2000图象分析显示XFDCT高、中剂量组PCNA表达的平均光密度明显高于对照组（P＜0.05），存在显著差异；与地塞米松组比较无显著性差异（P＞0.05）。而XFDCT低剂量组与对照组无显著差异（P＞0.05）。结论1．His在一定浓度范围内能以时间但非浓度依赖的方式促进ASMC的增殖，且10^-4mol／L时His对ASMC的促增殖作用最强。2．XFDCT具有良好的抑制由组胺诱导ASMC增殖的作用，且存在一定的时间、剂量依赖关系。3．XFDCT可以通过抑制ASMC增殖，参与气道重塑的治疗。更多还原

【Abstract】 Bronchial asthma is a combination of cells （such as eosinophils, mast cells, Tlymphocytes, neutrophils, airway epithelial cells, etc.） and cellular componentsinvolved in the chronic airway inflammatory diseases. In recent years, on airwayinflammation, airway remodeling in endless, a major breakthrough is clear asthma is,in essence, the chronic airway inflammation. However, simply using the airwayinflammation can not fully explain bronchial asthma, People again as airwayremodeling in asthma research focus. Large autopsy information shows that patientswith asthma airway smooth muscle layer thickness than 50% above normal, Itsthickness is not just compensatory cells, mast results More is the main airway smoothmuscle cells due to the proliferation itself. ASMC proliferation of the ARM haveimportant reasons, with airway hyperresponsiveness are closely related. Therefore, theconventional anti-inflammatory treatment on the basis of research and developmentASMC inhibit the proliferation of drugs, BA likely to treat new and more effectiveapproaches.ObjectiveBy observation Xuanfei Dingchuantang-containing serum of the rats in vitroproliferation of ASMC. and to explore the mechanism of its action, to understandXFDCT in airway remodeling—ASMC an important aspect of the proliferation ofdirect role.Methods1. Clean a male Sprague-Dawley rats, spinal dislocation law were killed in asepticconditions, from the middle left lung, the right middle and lower lobes of the lungtissue fluid separated the bronchial. Will be separated into good beating bronchialtissue block size, with the original explant culture ASMC.2. Histamine （histamine. His） induced apoptosis in cultured airway smooth musclecells proliferation of cells.3. Clean 20 Wistar rats were divided into five groups: XFDCT high, medium and low dose group, Western and control groups, each group four. XFDCT high, mediumand low dose rats were given to the parties by Decoctions 40g/（kg·d）, 20g/（kg·d）,10g（kg·d）, respectively, 20ml/kg. WM group to dexamethasone 2.54X10^-4g （kg·d） volume suspension gavage; blank group to the same dose of saline wasadministered for five consecutive days. In the last administration before fasting for 12h, but could not help but water, the last hour after gavage. 10% chloral hydrate（1ml/kg） anesthesia, abdominal aortic blood, blood collection tube 4℃overnight.Serum full until precipitation 3000rpms/15min centrifugation, the collection of serum,56℃/30min inactivated complement,0.22um filter membrane-packing,-20℃standby.Respectively role in the group ASMC.4. Inverted microscope observation of cell morphology and growth conditions andradiography. MTT assay and cell proliferation immunocytochemical staining （SABC）in the detection of proliferating cell nuclear antigen expression.Resuls1. Through anti-α-actin irnmunohistochemistry staining,all of the cells’staining areMasculine,the purity coefficient 95%.2. His role in different concentrations in ASMC, in different time points werehigher than A570nm value, 10^-4mol/L in which the concentration of ASMC His mostvisible role, 36h in the A570nm value of the control group was 1.19 times （0.152±0.002 compared with 0.1 28±0.003, P＜0.05,n=5）. His right ASMC showedsignificant role in promoting proliferation （P＜0.05,n=5）. Time-dependent relationshipexists but not a concentration-dependent manner.3. Serum containing high or middle dose XFDCT could decrease the OD value ofASMC tested by MTT method,and there was singificant difference （P＜0.05）comparedwith the control group,but there was not singificant difference（P＞0.05）compared withthe dexamethasone group. XFDCT low-dose group and the control group were notsignificantly different （P＞0.05）. And there is a certain time and dose dependentmanner.4. PCNA located in the nucleus, the positive nuclei within the brown granules,MIAS2000 image analysis showed high-dose and middle-dose group PCNA expression in the the average optical density was significantly higher than the controlgroup （P＜0.05）, there are significant differences; and dexamethasone group was nosignificant difference （P＞0.05）. XFDCT low-dose group and the control group werenot significantly different （P＞0.05）.Conclusions1. His(10^-3～10^-6mol/L)could stimulate the cell proliferation in a time-dependentbut not dose-dependent manner and reached the maximal effect at 10^-7 mol/L.2. XFDCT good inhibited histamine-induced by the proliferation of ASMC, andthere is a certain time and dose dependent manner.3. XFDCT could treat asthma by inhibiting the proliferation of ASMC.更多还原