摘要
目的:观察以养阴润燥、清热解毒为基本治法组方的中药复方制剂地黄管食通口服液对放射疗法治疗后食管癌细胞凋亡的影响,分析该口服液作用机制。方法:实验于2004-11/2005-08在郑州大学动物实验中心SPF级实验室(动物实验)和郑州大学分子医学重点实验室(其他实验)完成。①选用清洁级Wistar大鼠125只,6～8周龄,雌雄不拘。取20只大鼠作为空白对照组;其余大鼠均皮下注射甲基戊基亚硝胺溶液5mg/kg造成食管癌模型,每周1次,连续16周,每周称体质量1次,根据体质量增长调节注射给药量。末次大鼠注射给予甲基戊基亚硝胺溶液后,除20只大鼠(模型组)外,将其余造模大鼠用氯胺酮腹腔注射麻醉穴0.lg/kg,2mL/kg雪,应用60Co放射治疗机进行大鼠食管局部60Co照射,剂量为2Gy/d,连续5d。②末次照射24h后,分别给予相应受试药物,动物分组及给药情况如下:空白对照组(n=20):灌胃蒸馏水10mL/穴kg·d雪;模型组穴n=20雪:灌胃蒸馏水10mL/(kg·d);放疗组穴n=17雪:灌胃蒸馏水10mL/(kg·d)鸦六味地黄丸组穴n=17雪:灌胃六味地黄丸(成分为熟地黄、山药、山茱萸、泽泻、牡丹皮、茯苓;河南宛西制药股份有限公司生产,批号:20010691;9g/粒)生药含量4.5g/(kg·d)鸦地黄管食通口服液低、中、高剂量组穴n=17雪:灌胃地黄管食通口服液(地黄管食通口服液:成分为熟地黄、山药、山茱萸、泽泻、牡丹皮、茯苓、山豆根、冬凌草;由河南中医学院第一临床医学院制剂中心提供,批号:020117;10mL/支)生药含量0.5,1.0,1.5g/(kg·d)。每组均干预5周。③采用原位末端标记法检测细胞凋亡情况,计数细胞凋亡比例的均数为凋亡指数。④采用免疫组化方法检测P53,Bcl-2蛋白表达:P53和Bcl-2蛋白阳性反应均呈棕黄色颗粒,按显色有无及阳性细胞多少可分为4个等级:阴性穴-雪:整个切片未见阳性染色;弱阳性穴+雪:阳性细胞数<25%;中度阳性穴荻雪:阳性细胞数25%～50%;强阳性穴莘雪:阳性细胞数>50%。⑤两组计量和计数资料差异比较采用t检验和χ2检验。结果:纳入125只大鼠,各组取10只用于结果分析。①细胞凋亡指数:地黄管食通各剂量组明显高于模型组(P<0.05～0.01),六味地黄丸组和地黄管食通各剂量组明显高于放疗组(P<0.05～0.01)。②P53蛋白表达阳性率:模型组、放疗组、六味地黄丸组均明显高于空白对照组(P<0.05～0.01)。地黄管食通口服液各剂量组明显低于放疗组(P<0.05～0.01),以地黄管食通口服液高剂量组与放疗组差异最明显穴P<0.01雪。③Bcl-2蛋白表达阳性率:模型组、放疗组、六味地黄丸组均明显高于空白对照组(P<0.05～0.01)。地黄管食通口服液各剂量组明显低于放疗组(P<0.05～0.01),以地黄管食通口服液高剂量组与放疗组差异最明显穴P<0.01雪。结论:地黄管食通口服液可促进放射疗法治疗后食管癌细胞凋亡,该作用发生可能与抑制P53蛋白表达,下调Bcl-2蛋白表达有关。
AIM： To observe the effect of compound Chinese herb oral liquid dihuang guangshi tong, which was characterized by nourishing yin to moisten dryness and clearing heat and removing toxic substance, on apoptosis of esophageal carcinoma after radiotherapy and analyze its mechanism.
METHODS： The experiment was completed at the SPF Laboratory of Animal Experimental Center Affiliated to Zhengzhou University （animal experiment） and Key Laboratory of Molecular Medicine Affiliated to Zhengzhou University （other experiments） from November 2004 to August 2005. ①A total of 125 Wistar rats of clean grade and either gender aged 6-8 weeks were selected in this study. Among them, 20 rats were regarded as blank control group, and other rats were injected subcutaneously with 5 mg/kg methyl-amyl-nitrosamines to establish models of esophageal carcinoma once a week for successive 16 weeks. Body mass was weighted once a week, and the dosage was accordance to the increase of body mass. After the last injection, except 20 rats in model group, other rats were anesthetized intravenously with 0.1 g/kg ketamine （2 mL/kg）. ^60 Co radiation therapeutic machine was used to exposure local esophagus with the dosage of 2 Gy/d for 5 days. ② 24 hours after the last irradiation, rats in each group were treated with relevant medicines as follow： 20 rats in blank control group were perfused with 10 mL/（kg·d） distilled water; 20 rats in model group were perfused with 10 mL/（kg·d） distilled water; 17 rats in radiotherapy group were perfused with 10 mL/（kg·d） distilled water; 17 rats in liuwei dihuang wan group were perfused with liuwei dihuang wan [components： shudihuang, shanyao, shanzhuyu, zexie, mudanpi and fuling, Henan Wanxi Pharmaceutical Company Limited, batch number： 20010691; 9 g/pill; 4.5 g/（kg ·d） raw meterials]; 17 rats in low, middle and higher dosage groups of oral liquid dihuang guangshi tong were perfused with dihuang guangshi tong [components： shudihuang, shanyao, shanzhuyu, zexie, mudanpi, fuling, shandougen and donglingcao; Agent Center of the First Clinical Medical College Affiliated to Henan College of Traditional Chinese Medicine, batch number：. 020117; 10 mL/ampoule; 0.5, 1.0 and 1.5 g/（kg·d） raw materials]. Rats in each group were interfered for 5 weeks. ③ Apoptosis was assayed with in situ end-labeling （ISEL）, and average amount was regarded as apoptosis index. ④ Expressions of P53 and Bcl-2 protein were detected with immunohistochemical method. Positive reaction of P53 and Bcl-2 protein was buffy, and could be classified into 4 grades according to number and color of positive cells： negative （-）： Positive cells were not observed in the whole section; mild positive （＋）： Amount of positive cell was less than 25%; moderate positive （＋＋）： Amount was from 25% to 50%; great positive （＋＋＋）： Amount was more than 50%. ⑤ Measurement data and enumeration data were compared with t test and X^2 test.
RESULTS： Among 125 rats, 10 from each group were involved in the final analysis. ① Apoptosis index： Index in dosage groups of dihuang guangshi tong was higher than that in model group （P 〈 0.05-0.01）, and index in liuwei dihuang wan group and dosage groups of dihuang guangshi tong was higher than that in radiotherapy group （P 〈 0.05-0.01）, ② Positively expressive rate of P53 protein： Rate in model group, radiotherapy group and liuwei dihuang wan group was higher than that in blank control group （P 〈 0.05-0.01）, and rate in dosage groups of dihuang guangshi tong was lower than that in radiotherapy group （P 〈 0.05-0.01）. There was significant difference between high-dosage group of dihuang guangshi tong and radiotherapy group （P 〈 0.01）. ③ Positively expressive rate of Bcl-2 protein： Rate in model group, radiotherapy group and liuwei dihuang wan group was higher than that in blank control group （P 〈 0.05-0.01）, and rate in dosage groups of dihuang guangshi tong was lower than that in radiotherapy group （P 〈 0.05 -0.01）. There was significant difference between high-dosage group of dihuang guangshi tong and radiotherapy group （P 〈 0.01）.
CONCLUSION： Oral liquid dihuang guangshi tong can promote apoptosis of esophageal carcinoma after radiotherapy, and the effect is possibly related with inhibiting expression of P53 protein and decreasing expression of Bcl-2 protein.
出处
《中国临床康复》
CAS
CSCD
北大核心
2006年第19期111-114,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家中医药管理局资助项目(02-03JP25)~~
