慢性阻塞性肺疾病模型大鼠肺组织差异基因的表达:正常组、模型对照组及中药治疗组的比较(英文)

Expression of different genes in the lung tissue of model rats with chronic obstructive pulmonary disease:A comparison among normal, model control and Chinese herb groups

背景:许多疾病都伴有基因表达的改变,能够为认识疾病的发病机制及治疗提供有价值的线索。目的:采用基因芯片技术筛查慢性阻塞性肺疾病模型大鼠肺组织中差异基因的表达情况。设计:开放性实验。单位:山东中医药大学附属医院,解放军军事医学科学院放射与辐射医学研究所,山东中医药大学。材料:实验于2005-04/10在山东中医药大学附属医院中心实验室进行。①选取SPF级健康雄性50d龄Wistar大鼠15只,随机数字表法分为正常组、模型对照组、中药治疗组,5只/组。②基因芯片类型为大鼠BiostarR-40S(上海博星基因芯片有限公司,批号G050510010052)。方法:①模型对照组、中药治疗组大鼠采用改良烟熏+气管滴加脂多糖的方法建立慢性阻塞性肺疾病模型。光学显微镜下观察大鼠气道病理改变,若出现气道黏膜充血水肿,上皮细胞变性坏死,肺内支气管的管腔及周围慢性炎性细胞浸润,管壁周围平滑肌和纤维细胞增生;间隔变薄、断裂,肺泡扩大融合;小动脉血管壁增厚,管腔变小,周围炎性细胞浸润,说明造模成功。②造模成功后第30天,中药治疗组灌服浓度为0.65g/mL的自制中药合剂(麻黄,杏仁,黄芪等),2mL/次,1次/d,连续给药14d。模型对照组每天给予2mL生理盐水灌胃。正常组不做任何干预,相同室内环境下自由饮食饮水。③给药完毕后处死各组大鼠,进行总RNA提取、探针的标记与杂交、洗片,采用芯片图像分析软件分析芯片灰度扫描图,得到芯片上每个基因点的原始信号值(包括前景信号值和背景信号值),从而进行差异表达基因的判定。检验水准是Ratio值大于2.0为上调基因,小于0.5为下调基因。为了提高检验的可靠性,本实验将检验水准设为Ratio值>2.5为上调基因,Ratio值<0.375为下调基因。主要观察指标:①与正常组比较模型对照组的差异表达基因。②与正常组比较中药治疗组的差异表达基因。③与模型对照组比较中药治疗组的差异表达基因。结果:15只大鼠全部进入结果分析。①模型对照组的基因表达情况较正常组有明显变化,差异表达基因达57个,涉及免疫、代谢、信号转导、基因表达调控、细胞周期、细胞转运、细胞迁移及纤维化等。②中药治疗组的基因表达大多恢复到正常组水平,与正常组比较差异表达基因减少到11个,涉及应激反应、信号转导及细胞骨架等。③与模型对照组比较,中药治疗组的差异表达基因有7个,涉及免疫及神经递质的分泌。结论:大量的基因表达改变可能是慢性阻塞性肺疾病发病机制的原因之一,而经药物治疗后,大多数差异表达基因能够恢复到正常水平,有利于纠正慢性阻塞性肺疾病的病理改变。

BACKGROUND： Many diseases accompany with changes of gene expression which can provide a valuable clue for pathogenesis and therapy of cognitive diseases. OBJECTIVE： To screen expression of different genes in lung tissue of model rats with chronic obstructive pulmonary disease （COPD） by gene expression profiling technique. DESIGN： Open study. SETTING： Affiliated Hospital of Shandong University of Traditional Chinese Medicine; Radiation Medical Institute of Academy of Military Medical Sciences of Chinese PLA; Shangdong University of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Central Laboratory of Affiliated Hospital, Shandong University of Traditional Chinese Medicine from April to October 2005. （1） Fifteen healthy male Wistar rats of SPF grade and aged 50 days were randomly divided into normal group, model control group and Chinese herb group with 5 in each group. （2） Type of gene chip was BiostarR-40S and provided by Shanghai Boxing Gene Chip Company Limited （batch number： G050510010052）. METHODS： （1） COPD models in model control group and Chinese herb group were established with modified smoking-fumigated method plus adding lipopolysaecharide in windpipe, Pathological changes of airway were observed under optic microscope. Models were established successfully based on the following phenomenon： hyperemia and edema in mucous membrane of airway; degeneration and necrosis of epithelial cells; infiltration of bronchial cavity in lung tissue and surrounding chronic inflammatory cells; proliferation of smooth muscle and fibrocyte surrounding dissepiment; thin and broken interval; amplifying confluence of pulmonary alveolus; thickness of vascular wall of arteriole; decrease of vaso-cavity; infiltration of surrounding inflammatory cells. （2） At 30 days after modeling, 0.65 g/mL self-made Chinese herb （mahuang, xingren, huangqi, etc.） was perfused into rats in Chinese herb group with 2 mL each time, once a day, for successive 14 days. Rats in model control group were peffused with 2 mL saline; however, rats in normal group were untouched. All rats drank freely under the same internal environment. （3） After administration, rats in each group were sacrificed to extract total RNA, label and hybridizated with probe, wash pieces, analyze gray value of clip with clip imaging software, obtain original signal of each gene point （signal values of foreground and background） and determine differently expressed genes. If size of test was more than 2.0, genes were regarded as up-regulation genes; otherwise, if ratio Was less than 0.5, they were regarded as down-regulation genes. In this study, based on reliability, if size of test was more than 2.5, they were regarded as up-relation genes; however, if ratio was less than 0.375, they were regarded as down-regulation genes. MAIN OUTCOME MEASURES： （1） Comparisons of differently expressed genes in normal and model control groups; （2） Comparisons of differently expressed genes in normal and Chinese herb groups; （3）Comparisons of differently expressed genes in model control and Chinese herb groups. RESULTS： Fifteen rats were all involved in the final analysis. （1） As compared with those in normal group, there were 57 differently expressed genes in model control group, involving in immunity, metabolism, signal transduction, adjusting of gene expression, cell cycle, cell transport, cell migration, fibrosis, etc. （2） Gene expression in Chinese herb group reached normal level and there were 11 differently expressed genes as compared with those in normal group, involving in stress, signal transduction, cytoskeleton, etc. （3） As compared with those in model control group, there were 7 differently expressed genes in Chinese herb group, involving in immunity and excretion of neurotransmitter. CONCLUSION： Changes of gene expression may be one of reasons of COPD pathogeneses; moreover, most differently expressed genes can recover the normal level after drug therapy which is beneficial to correct pathological changes of COPD.