健脾清热化瘀药对胃溃疡大鼠表皮生长因子及其受体表达的影响

1、健脾清热化瘀药对胃溃疡大鼠表皮生长因子及其受体表达的影响         11-05-02 15:52:00     编辑：studa20                    作者：包永欣,吕冠华,赵金婷,李海泉【摘要】  目的 通过观察健脾清热化瘀药对血清和胃黏膜组织表皮生长

2、因子(EGF)含量及其受体(EGFR)表达的影响,探讨不同中药治法对实验性胃溃疡愈合质量的影响及其作用机制。方法 采用冰乙酸注射法制备大鼠胃溃疡模型,随机分为模型组、雷尼替丁组(西药组)、健脾中药组(中药A组)、清热中药组(中药B组)、化瘀中药组(中药C组)和健脾清热化瘀中药组(中药D组),并设立正常对照组(空白组)。在光镜下观察溃疡指数(UI)、再生黏膜厚度及黏膜肌层缺损宽度,应用ELISA法检测血清及胃黏膜EGF含量,免疫组化法检测胃黏膜EGFR的表达。结果 健脾、清热、化瘀中药及其合方都能使再生黏膜厚度增加,溃疡指数及黏膜肌层缺损宽度减少,与模型组比较差异有统计学意义(P0.01),中药

3、D组疗效明显优于西药组(P0.05或P0.01)。中药各组及西药组血清及胃黏膜EGF含量较模型组及空白组明显升高(P0.01),且可明显增强胃溃疡黏膜EGFR的表达(P0.05或P0.01),尤以中药D组疗效最为显著(P0.01)。结论 健脾、清热、化瘀中药可通过增加EGF和EGFR水平,促进胃黏膜上皮细胞增生及组织黏膜修复,提高溃疡愈合质量。 【关键词】  健脾清热化瘀药；表皮生长因子；表皮生长因子受体；溃疡愈合质量；大鼠    Abstract：Objective To study the effect and mechanism of invigo

4、rating spleen, clearing heat and eliminating stasis drugs on QOUH of gastric mucosa and expression of EGF and EGFR in rat model of gastric ulcer. Methods The rat model of gastric ulcer was constructed by injection of acetic acid. The rats were randomly divided into model group, ranitidine group, inv

5、igorating spleen group, clearing heat group, eliminating stasis group, complex prescription group and normal control group. The ulcer index (UI), regenerate mucosal thickness, lamina muscularis mucosal width were observed by HE, and the level of EGF was detected by ELISA. The expression of EGFR in t

6、he mucosa around the ulcer was detected by SABC kit immunohistochemical method. Results Compared with model group, the regenerate mucosal thickness, lamina muscularis mucosae defect width and UI in invigoraing spleen, clearing heat, elimianting stasis and complex prescription groups were different s

7、ignificantly (P0.01). And the effect of complex prescription group was better than ranitidine group (P0.05, or P0.01). Compared with model group and normal control group, the levels of EGF in serum and gastric mucosa in treating groups were increased significantly (P0.01). Positive expression of EGF

8、R around ulcerative tissues in treating groups were strengthened obviously (P0.05, or P0.01), and the effect of complex prescription group was the best (P0.01). Conclusion Invigorating spleen, clearing heat and eliminating stasis drugs can increase the level of EGF and EGFR, and promote gastric muco

9、sa epithelial hyperplasia and tissue mucous renovation and improve QOUH.    Key words：invigorating spleen, eliminating stasis drugs；EGF；EGFR；QOUH；rat     临床资料显示,胃溃疡的主要病机为脾胃虚弱、热毒内结、气滞血瘀。应用健脾清热化瘀中药不仅能消除溃疡病的损害因素,增强胃黏膜保护因素,而且在治愈溃疡病的同时,还能对紊乱的消化功能进行调整,提高溃疡的愈合质量,从而降低其复发率1

10、。本研究通过检测胃溃疡大鼠血清及再生黏膜表皮生长因子(EGF)及表皮生长因子受体(EGFR)表达的变化,探讨健脾清热化瘀中药提高溃疡愈合质量(QOUH)、减少溃疡复发的机制。1  实验材料1.1  动物    清洁级SD雄性大鼠,体重200220 g,共70只,沈阳军区总医院实验动物中心提供,许可证号：SCXK-(军)2007-001。1.2  药物    健脾中药：黄芪20 g,党参15 g,甘草5 g；清热中药：蒲公英15 g,紫花地丁15 g,黄连5 g,半枝莲15 g；化瘀中药：当归10 g,丹参

11、15 g,莪术15 g,延胡索10 g。上述药材单味药配方颗粒购自广东一方制药有限公司。雷尼替丁胶囊由杭州赛诺菲圣德拉堡民生制药有限公司生产,批号1059,150 mg/粒。1.3  仪器与试剂    EGF酶联免疫吸附法(ELISA)试剂盒由深圳晶美生物工程有限公司提供；EGFR多克隆抗体及即用型SABC免疫组化试剂盒由北京中杉生物工程有限公司提供。图像分析采用MPIAS多媒体彩色病理图纹分析系统。2  实验方法2.1  造模    应用冰乙酸浆膜下注射法制备大鼠胃溃疡模型2,造模前禁食不禁水24 h,

12、2%速麻安2号注射液10 mL/kg腹腔麻醉后,将大鼠固定,在上腹部皮肤正中切口,沿腹白线剪开腹壁组织,打开腹腔,以1 mL注射器在胃窦前壁胃壁肌层与浆膜层之间注入100%冰乙酸0.03 mL,然后覆盖网膜,间断缝合腹膜、腹壁各层组织和皮肤。2.2  分组及给药    70只大鼠随机分为7组,即空白组、模型组、雷尼替丁组(西药组)、健脾中药组(中药A组)、清热中药组(中药B组)、化瘀中药组(中药C组)和健脾清热化瘀中药组(中药D组),每组10只。空白组大鼠不做任何处理,其余6组造模3 d后开始给药。治疗组用药剂量按人与大鼠体表面积换算,200 g大鼠分别

13、给予5.4 mg的雷尼替丁、0.72 g的健脾中药、0.9 g的清热中药,0.72 g的化瘀中药、2.34 g的健脾清热化瘀中药,空白组与模型组给予0.9%氯化钠溶液。各组均按2 mL/200 g大鼠体重的剂量灌胃,每日1次,共14 d。2.3  标本采集与处理    实验结束后,将大鼠禁食24 h后麻醉,于腹主动脉采血4 mL,在室温下静置后,2 500 r/min离心10 min,取血清,-20 保存备用。打开腹腔,分别结扎贲门、幽门后取出胃,沿胃大弯剪开,冰生理盐水冲洗,在放大镜下用游标卡尺测量溃疡长径和短径,以溃疡瘢痕平行于胃长轴方向的最长径为中心取材,放入4%多聚甲醛中固定,24 h后石蜡包埋切片。留取溃疡边缘组织约100 mg,用0.01 mol/L PBS溶液制成10%的组织匀浆,离心后取上清液,-20 保存以备检测EGF。2.4  观察指标与检测方法    溃疡指数(UI)：采用游标卡尺(精确度0.02 mm)测量溃疡面的最大长径和垂直于最大长径的最大宽径,以二者的乘积作为溃疡指数。再生黏膜厚度与黏膜肌层缺损宽度：参照杨氏方法3,在HE染色的切片上,用低倍镜(