【作者】 张伟；

【导师】 方朝义；

【作者基本信息】 河北医科大学 ， 中医诊断学， 2009， 硕士

【摘要】 目的:观察补气活血法对慢性支气管炎（CB）肺气虚证大鼠细胞凋亡及相关基因蛋白bcl-2、fas的影响,探讨其治疗作用机理,进一步揭示CB肺气虚证形成的病理机制,为临床防治该病提供实验依据。方法:选用健康Sprague Dawley（SD）大鼠50只（雌雄各半）,实验前一周置于实验环境中喂养。采用改良烟熏法复制CB肺气虚证动物模型。将造模各组大鼠置于特制的1m×1m×1m的烟室中点燃熏烟,每日2次,每次30分钟,持续40天。正常对照大鼠则置于正常无烟环境中饲养。模型复制成功后,将CB肺气虚证大鼠随机分为分为模型对照组（模型组,model group）、补气活血高剂量组（高剂组,high-dose group）、补气活血低剂量组（低剂组,low-dose group）、补肺汤组（tonify lung recipe group）,并设不加任何处理的正常对照组（normal group）。每组10只,雌雄各半。5组均于造模结束后第2天起连续灌胃生理盐水或药物21天,每日1次。正常组与模型组灌服生理盐水,均为10ml/kg;高剂组每日用药剂量为25.6g/kg（相当于临床成人用药量的14倍）,以0.18g/ml灌胃;低剂组每日用药剂量为12.8g/kg（相当于临床成人用药量的7倍）,以0.09g/ml灌胃;补肺汤组每日用药剂量为8.63g/kg（相当于临床成人用药量的7倍）,以0.09g/ml灌胃。于灌胃结束后第2天,各组大鼠分别麻醉后处死,开胸取右侧肺脏中部组织0.5cm×0.5cm×0.5cm大小组织块,用4%多聚甲醛溶液（PH7.4磷酸盐缓冲液配制）固定24h,常规石蜡包埋,切片,用于普通HE染色及TUNEL检测、免疫组织化学染色。取数块组织,用于流式细胞术检测。结果:1一般状态观察:造模组大鼠在烟熏12天后开始出现呼吸困难,咳嗽并兼有痰鸣音,蜷卧倦怠,饮食量及活动量减少,毛发光泽渐失,鼻腔可见分泌物,质地清稀,消瘦便溏,腹部有硬块,舌色紫暗。正常组未见异常。与正常组相比较,造模组体重增长缓慢,与造模40天时体重增幅比较,造模组增加（94.48±14.16）g,正常组增加（104.69±23.65）g,两组体重变化有明显差异（P<0.05）。2肺组织形态学观察2.1肉眼观察:正常组大鼠双肺外观无异常,肺叶轮廓清晰,表面光滑柔软,粉红色,弹性良好。模型组大鼠双肺可见灰黑色斑点,肺表面略显不平,表面无光泽,边缘有点状出血。2.2光镜观察:正常组可见细支气管黏膜上皮完整,细胞排列整齐,细支气管壁及肺泡无明显病理形态学改变。模型组可见细支气管管腔增大,管壁明显增厚,部分上皮细胞坏死脱落,伴有大量慢性炎细胞浸润,小静脉有淤血现象。各治疗组与模型组相比均有明显改善。3各组大鼠支气管及肺组织细胞凋亡3.1TUNEL和免疫组化检测各组大鼠肺炎性细胞凋亡3.1.1 TUNEL检测肺炎性细胞凋亡模型组大鼠支气管及肺组织炎细胞凋亡百分数明显低于正常组,有统计学意义（P<0.05）。治疗组与模型组炎细胞凋亡数比较明显增加,组间比较有统计学意义（P<0.01）,其中高剂组与低剂组相比,凋亡数目明显增加（P<0.01）;高剂组与补肺汤组相比明显增加,有统计学意义（P<0.01）;低剂组与补肺汤组比较无统计学意义（P>0.05）。3.1.2免疫组化法检测凋亡基因蛋白bcl-2模型组平均灰度值明显低于正常组,有统计学意义（P<0.01）。与模型组相比,各治疗组的平均灰度值明显升高,组间比较有统计学意义（P<0.01）,其中高剂组与低剂组相比平均灰度值升高,有统计学意义（P<0.01）;高剂组与补肺汤组比较平均灰度值升高,有统计学意义（P<0.01）;低剂组与补肺汤组相比较,平均灰度值升高,有统计学意义（P<0.05）。3.1.3免疫组化法测凋亡基因蛋白fas模型组平均灰度值明显高于正常组,有统计学意义（P<0.01）。与模型组相比,各治疗组的平均灰度值明显降低,组间比较有统计学意义（P<0.01）,其中高剂组与低剂组相比平均灰度值降低,有统计学意义（P<0.01）;高剂组与补肺汤组比较,平均灰度值降低,有统计学意义（P<0.01）;低剂组与补肺汤组相比较,平均灰度值降低,有统计学意义（P<0.05）。3.2流式细胞术检测各组大鼠肺实质细胞凋亡3.2.1流式细胞术检测各组大鼠肺实质细胞凋亡率模型组显著高于正常组,有统计学意义（P<0.01）。各治疗组大鼠细胞凋亡率与模型组相比明显降低,组间比较有统计学意义（P<0.01）,其中高剂组与低剂组相比较,高剂组细胞凋亡率有降低趋势,但经统计学处理无统计学意义（P>0.05）;高剂组与补肺汤组相比凋亡率明显降低,有统计学意义（P<0.01）;低剂组与补肺汤组相比凋亡率降低,有统计学意义（P<0.05）。3.2.2流式细胞术检测各组大鼠肺实质细胞周期G₀/G₁期（细胞静止期/合成前期）:模型组细胞百分数较正常组明显升高,有统计学意义（P<0.01）。各治疗组与模型组相比,细胞百分数均明显降低,有统计学意义（P< 0.01 ）,其中高剂组与低剂组相比,高剂组有降低趋势,但经统计学处理无统计学意义（P>0.05）;高剂组与补肺汤组相比明显降低,有统计学意义（P<0.05）;低剂组与补肺汤组相比有降低趋势,但经统计学处理无统计学意义（P>0.05）。S期（细胞合成期）:模型组细胞百分数较正常组明显降低,有统计学意义（P<0.01）。各治疗组与模型组相比,均明显升高,有统计学意义（P<0.01）,其中高剂组细胞百分数与低剂组相比有升高趋势,但经统计学处理无统计学意义（P>0.05）;高剂组与补肺汤组相比较明显升高,有统计学意义（P<0.05）;低剂组与补肺汤组比较有升高趋势,但经统计学处理无统计学意义（P>0.05）。G₂/M期（细胞合成后期/有丝分裂期）:模型组细胞百分数较正常组明显降低,有统计学意义（P<0.05）。高剂组与模型组相比较,细胞百分数明显增加,有统计学意义（P<0 .01）;低剂组与模型组比较,有升高趋势,但经统计学处理无统计学意义（P>0.05）;补肺汤组与模型组比较,细胞百分数明显增加,有统计学意义（P<0.05）。各治疗组之间比较未见统计学意义（P>0.05）。PI:模型组较正常组明显降低,有统计学意义（P<0.01）。各治疗组与模型组相比,均明显升高,组间比较有统计学意义（P<0.01）,其中高剂组与低剂组相比有升高趋势,但经统计学处理无统计学意义（P>0.05）;高剂组与补肺汤组相比较明显升高,有统计学意义（P<0.05）;低剂组与补肺汤组相比有升高趋势,但统计学处理无统计学意义（P>0.05）。结论:1 CB肺气虚状态下,支气管及肺组织炎性细胞存在凋亡延迟,凋亡相关基因蛋白bcl-2表达上调和fas表达下调。支气管及肺组织实质细胞存在凋亡过度导致支气管及肺组织结构的破坏,成为CB肺气虚证发生发展的机制之一。2补气活血方能够通过抑制炎性细胞凋亡相关基因蛋白bcl-2的表达并上调fas蛋白在肺炎症细胞的表达,增加CB肺气虚证支气管及肺组织内炎细胞的凋亡数目,促进炎症细胞以凋亡的形式清除,对于限制炎症及消散炎症起到作用。3补气活血方能够通过抑制肺实质细胞的凋亡并促进肺细胞的增殖来提高肺实质细胞的合成速度,恢复肺实质细胞的数量和功能,加强肺组织的自我修复能力,为中医药治疗CB肺气虚证的作用机制赋予了新的涵义。更多还原

【Abstract】 Objective:To observe the effect of the tonifying vital energy and promoting blood flow recipe on pulmonary apoptosis and related genic proteinum of the rats,insufficiency of lung-qi of chronic bronchitis（CB）,approach its functional mechanism,and explore the possible mechanisms,which will provide practical and theoretical evidence for developing new method of treating and preventing the insufficiency of lung-qi of CB.Methods:Take 50 healthy SD rats（paroecious about half）to raise in the experimental environment about one week.Adopt the improved smoking-fumigated to duplicate the model of the insufficiency of lung-qi of CB.The normal rats raise in the common environment without smoking-fumigated.After the model duplicate to succeed , divided into model group ,high-dose group of the tonifying vital energy and promoting blood flow,low-dose group of the tonifying vital energy and promoting blood flow, tonifying lung recipe group,and normal group without enay treatment（10 per-group and paroecious about half）. Give the isotonic Na chloride or drugs to the five groups retain twenty-one days after the model duplicate,one time everyday.Normal and model group all drench the isotonic Na chloride,volume-10ml/kg;The volume of the high-dose group is 25.6g/kg（amount to the 14 multiples of the adult）,0.18g/ml.The volume of the low-dose group is 12.8g/kg（amount to the 7 multiples of the adult）,0.09g/ml.The volume of the tonifying lung recipe group is 8.63g/kg（amount to the 7 multiples of the adult）,0.09g/ml.The rats in each group are put to be anaesthesia to death after two days of drench.Open the breast and take the lung tissue（0.5cm×0.5cm×0.5cm）,which were fix with 4% formal -dehyde,embedded with paraffin,maken sections by HE and immunohistochemical method stain.Meanwhile take some lung tissues by FCM.Results:1Observation of the gemeral condition12 days after smoking-fumigated the rats in model group showed the symptoms,such as dyspnea,cough,wheezy phlegm losing brightness of hair,lassitud and so on.While the rats in normal group were without any symptoms.The weight in model group increased much slowly than normal group,at the last day of smoking-fumigated,weight addition in model group was（94.48±14.16）g,the normal group was（104.69±23.65）g,there were significant difference between the two groups（P<0.05）. 2Observation of the lung’s histomorphology2.1Macroscopic observation:The lung’s appearance of normal group was common with color pink,surface smooth,elasticity good and lobes of lung edge sharpness.The lung’s appearance of model group was abnormal with color gray-black,surface concavity,without burnish and ecchymosis were seen.2.2Obeservation under light microscope:The result of phthomorphology manifested that in normal group mucous membrane of bronchiole epithelia was integrity、cells lined up orderly、bronchiole dissepiment and alveoli had no distinctive pathomorphologic alteration under light microscope.In model group,bronchioles cavity was enlarged、vessel wall was thicked significantly、part of epithelial cellular necrosis and abscission、there was a lot of chronic inflammatory cells infiltration and venule with stagnant blood.3Bronchi and lung tissues’apoptosis of every group rats3.1Inflammatory cells’apoptosis of bronchi and lung tissues of every group rats by TUNEL and immunohistochemical method3.1.1Inflammatory cells’apoptosis of bronchi and lung tissues by TUNELCompare with normal group,the percentage of inflammato -ry cells’apoptosis of model group was decreased obviously,has statistics significance（P<0.05）.Compare with model group,the percentage of every group to treatment was increased obviously,has statistics significance（P<0.01）,the percentage of high-dose group compare with low-dose group was increased （P<0.01）;the percentage of high-dose group compare with tonifying lung recipe group was increased（P<0.01）;the percentage of low-dose group compare with tonifying lung recipe group wasn’t have statistics significance（P>0.05）.3.1.2Detect apoptosis gene protein bcl-2 with immunohistoche -mistyThe average grey level of the model group is obviously lower than the normal group , has statistics significance（P<0.01）.Compaired with model group,the average grey level of each treatment group is obviously elevates,it has statistics significance between each group（P<0.01）,the average grey level of the high-dose group is higher than the low-dose group,has statistics significance（P<0.01）;Between the high-dose group and tonifying lung recipe group,the average grey level is elevates, it has statistics significance（P<0.01）;Between the low preparation group and tonifying lung recipe group,it has statistics significance（P<0.05）.3.1.3Detect apoptosis gene protein fas with immunohistochemis -tyThe average grey level of the model group is obviously higer than the normal group , has statistics significance （P<0.01）.Compaired with model group,the average grey level of each treatment group is obviously reduses,it has statistics significance between each group（P<0.01）,the average grey level of the high-dose group is lower than the low-dose group,has statistics significance（P<0.01）;Between the high-dose group and tonifying lung recipe group,the average grey level is reduses,it has statistics significance（P<0.01）;Between the low-dose group and tonifying lung recipe group,the average grey level is reduses,it has statistics significance（P<0.05）.3.2Detect each group of the rat’s lung parenchyma with flow cytometry3.2.1Detect the rate of apoptosis through each group of the rat’s lung parenchyma with flow cytometryThe model group is obviously higher than the normal group,has statistics significance（P<0.01）.Compaired with model group,the rate of apoptosis of each treatment group is obviously reduses,it has statistics significance between each group（P<0.01）,the rate of apoptosis of the high-dose group is lower than the low-dose group, but does not have statistics significance（P>0.05）;Between the high-dose group and tonifying lung recipe group,the a rate of apoptosis is reduses, it has statistics significance（P<0.01）;Between the low-dose group and tonifying lung recipe group,the rate of apoptosis is reduses ,it has statistics significance（P<0.05）.3.2.2Detect cell cycle of each group of the rat’s lung parenchym -a with flow cytometryG₀/G₁ period（silent period of cell/presynthetic phase）:The cell percentage of the model group is obviously higher than the nomal group,has statistics significance （P<0.01）.Com -paired with model group,the cell percentage of each treatment group is obviously reduses,it has statistics significance between each group（P<0.01）,the cell percentage of the high-dose group is lower than the low-dose group,but does not have statistics significance（P>0.05）;Between the high-dose group and tonifying lung recipe group,the a rate of apoptosis is reduses, it has statistics significance（P<0.05）;Between the low-dose group and tonifying lung recipe group,the rate of apoptosis is reduses,but does not have statistics significance（P>0.05）.S period（synthesis period of cell）:The cell percentage of the model group is obviously lower than the nomal group,has statistics significance（P<0.01）.Compaired with model group,the cell percentage of each treatment group is obviously elevates,it has statistics significance between each group（P<0.01）,the cell percentage of the high-dose group is higher than the low-dose group, but does not have statistics significance（P>0.05）;Between the high-dose group and tonifying lung recipe group , the a rate of apoptosis is obviously elevates,it has statistics significance（P<0.05）;Between the low-dose group and tonifying lung recipe group,the rate of apoptosis is elevates , but does not have statistics significance（P>0.05）.G₂/M period（post-synthetic phase of cell/mitotic period）:The cell percentage of the model group is obviously lower than the nomal group, has statistics significance（P<0.05）.The cell percentage of the high-dose group is obviously higher than the model group,has statistics significance（P<0.01）;Between the low-dose group and the model group,the rate of apoptosis is elevates , but does not have statistics significance（P>0.05）;Between the tonifying lung recipe group and the model group,the rate of apoptosis is obviously elevates, has statistics significanc（eP<0.05）.Between each treatment group,there is no statistics significance（P>0.05）.PI:The model group is obviously lower than the normal group,has statistics significance（P<0.01）.Compared with the model group,eash treatment group is obviously elevates,there is statistics significance during the group comparison（P<0.01）,the high-dose group is higher than the low-dose group, but does not have statistics significance（P>0.05）;Between the high-dose group and tonifying lung recipe group,the a rate of apoptosis is obviously elevates,it has statistics significance（P<0.05）;Between the low-dose group and tonifying lung recipe group,the rate of apoptosis is elevates , but does not have statistics significance（P>0.05）.Conclusion:1 Under the condition around insufficiency of CB lung-qi,inflammatory cells’apoptosis of bronchi and lung tissues existed apoptosis to be delaved,The express was up-regulation that apoptosis related genic proteinum bcl-2 and down-regulation that genic proteinum fas . The bronchi and lung tissue’parenchyma cells existed excessus apoptosis that lead to the breakdown of bronchi and lung tissues’architectonic,that is one of the evolutionary mechanism about insufficiency of CB lung-qi.2 The tonifying vital energy and promoting blood flow recipe can increase percentage about the inflammatory cell’apoptosis of bronchi and lung tissues by abaissement the express of genic protein bcl-2 and up-regulation the express of fas, promote to discard the inflammatory cells to depend on apoptosis,which is very important to disappear the inflammatory reaction.3 The tonifying vital energy and promoting blood flow recipe can speed up the synthetical rate of lung parenchymary cells by hold-back its apoptosis , then can anastate the lung parenchymary numbers and function,strengthen its recovery capability by itself,which assign new connotation to cure insufficiency of lung-qi of CB.更多还原