修回日期: 2003-08-10
接受日期: 2003-10-12
在线出版日期: 2004-02-15
目的: 研究产毒幽门螺杆菌(H. pylori)的浓缩培养上清液(CCS)对小鼠胃黏膜的致病作用及胃黏膜保护剂硫糖铝和三九胃泰对CCS所致小鼠胃黏膜损伤的保护作用.
方法: 56只健康♂Balb/c小鼠随机分成7组: 生理盐水组(Ⅰ组)、单纯损伤组(ⅡA组、ⅡB组)、硫糖铝保护组(ⅢA组、ⅢB组)、三九胃泰保护组(ⅣA组、ⅣB组), 其中A组为小剂量毒素组, B组为大剂量毒素组. 分别用硫糖铝和三九胃泰提前给小鼠灌胃, 其后用不同剂量的产毒H. pylori菌株(NCTC11637)的CCS灌胃致急性胃黏膜损伤, 然后在显微镜及电镜下观察胃黏膜组织学改变, 分别测定各组胃黏膜损伤积分(EDS), 以评价胃黏膜损伤程度及药物的预防保护效果.
结果: 产毒H. pylori的CCS可以对小鼠胃黏膜产生明显的损害, 包括空泡变性、腺体排列紊乱以及糜烂、溃疡形成, 但炎症反应不明显. 在超微结构水平, H. pylori的CCS引起细胞间隙增宽、空泡变性、细胞质肿胀、线粒体及内质网扩张、微绒毛排列紊乱及脱落、吞噬溶酶体增多等改变. 小剂量毒素引起的损害不如大剂量毒素引起的损害严重. Ⅰ, ⅡA, ⅡB, ⅢA, ⅢB, ⅣA, ⅣB各组的胃黏膜损伤积分依次为1.13±0.35, 2.25±0.46, 3.63 ±0.52, 1.25±0.46, 1.75±0.71, 1.50±0.53, 1.63±0.74; 单纯损伤组(ⅡA, ⅡB)与生理盐水组(Ⅰ组)比较, 胃黏膜损伤明显(P<0.01); 硫糖铝保护组(ⅢA, ⅢB)、三九胃泰保护组(ⅣA, ⅣB)与单纯损伤组(ⅡA, ⅡB)比较, 不论小剂量毒素组, 还是大剂量毒素组, 胃黏膜损伤程度均减轻, 损伤积分均明显下降(P<0.05).
结论: H. pylori的细胞毒素对鼠胃黏膜上皮细胞损害起重要作用, 但并不引起明显的炎症反应; 胃黏膜保护剂硫糖铝和三九胃泰对不同浓度H. pylori培养上清液所致的小鼠胃黏膜损伤有明显的预防和保护作用.
引文著录: 崔梅花, 胡伏莲, 董欣红. 胃黏膜保护剂预防幽门螺杆菌培养上清液所致小鼠胃黏膜损伤. 世界华人消化杂志 2004; 12(2): 355-358
Revised: August 10, 2003
Accepted: October 12, 2003
Published online: February 15, 2004
AIM: To determine the roles of H. pylori concentrated culture supernatants (CCS) on the gastric mucosa of mouse and to investigate the protective effects of gastric mucosal protectives sucrafate and sanjiuweitai on CCS-induced gastric mucosal lesion in Balb/c rats.
METHODS: Fifty-six healthy male Balb/c rats were randomly divided into seven groups: normal saline control (Ⅰ), injured simply (ⅡA, ⅡB), sucrafate pretreatment (ⅡA, ⅡB), sanjiuweitai pretreatment (ⅡA, ⅡB). Group A was dealt with small amounts of CCS and group B with large amounts of CCS. CCS were drawn from cytotoxic H. pylori strain (NCTC11637). The four protective groups were pretreated with sucrafate and sanjiuweitai separately, and then infused orally with different amounts of CCS. The pathological changes on histological sections and ultrastructural sections of gastric mucosa were assessed under microscope or electron microscope. The epithelial damage scoring (EDS) of the gastric mucosa was measured.
RESULTS: The management with large amounts of CCS from cytotoxic strains induced various epithelial lesions, which included vacuolation, erosions, ulcers and loss of gastric gland architecture. Infiltration of inflammatory cells in the lamina propria was not significant. At ultrastructural level, there was the presence of intracytoplasmic vacuoles, dilation of endoplasmic reticulum and mitochondrion, increasing of phagolysosomes, loose connection between cells and degenerative changes of microvilli. Small amounts of CCS from cytotoxic strain induced epithelial lesions less seriousey than large amounts of CCS. The results of the EDS of the gastric mucosa in the groups Ⅰ, ⅡA, ⅡB, ⅢA, ⅢB, ⅣA and ⅣB arranged successively as follows, 1.13±0.35, 2.25±0.46, 3.63±0.52, 1.25±0.46, 1.75±0.71, 1.50±0.53 and 1.63±0.74 respectively. A remarkable protection was found in gastric mucosa pretreated with sucrafate and sanjiuweitai. In comparison with the purely injured group, the EDS of the gastric mucosa descended significantly (P<0.05).
CONCLUSION: Cytotoxin has an important role in the induction of gastric mucosa lesions, but not in eliciting obvious inflammation; The gastric mucosal protection of sucrafate and sanjiuweitai against CCS-induced gastric mucosal lesion in rats is significant.
- Citation: Cui MH, Hu FL, Dong XH. Preventive effects of gastric mucosal protective on H. pylori CCS-induced gastric mucosal lesion in rats. Shijie Huaren Xiaohua Zazhi 2004; 12(2): 355-358
- URL: https://www.wjgnet.com/1009-3079/full/v12/i2/355.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v12.i2.355
幽门螺杆菌(H. pylori)与慢性胃炎、消化性溃疡、胃癌及胃黏膜组织相关性(MALT)淋巴瘤等疾病关系密切[1-15], 其确切的致病机制还不十分清楚. 目前关于H. pylori毒素对胃黏膜的损伤作用已经得到许多学者的关注, 而H. pylori毒素对胃黏膜的病理损伤以及胃黏膜保护剂对H. pylori毒素所致的胃黏膜损伤是否有保护作用, 目前研究甚少. 我们研究胃黏膜保护剂硫糖铝和三九胃泰干预H. pylori毒素对Balb/c小鼠胃黏膜的损伤作用如下.
H. pylori培养及CCS制备参照Leunk (J Med Microbiol 1998; 26: 93-99)[16]的方法. 标准产毒菌NCTC11637接种于固体培养基上, 在37 ℃微需氧条件下培养3-7 d. 用无菌接种环挑取适量H. pylori菌落, 接种于液体培养基中, 松瓶盖, 在CO2培养箱(37℃, 50 mL/L CO2)放置2 h, 然后盖紧瓶口, 置于恒温振荡器上(37 ℃, 140-150 r/min)培养48-72 h. 液体培养基离心后加等量饱和硫酸铵溶液沉淀上清, 留集沉淀, 加入0.1 mol/L PBS稀释, 用0.22 μm的滤膜过滤除菌, 分装保存于-70 ℃冰箱备用. 取固体培养菌落及液体培养沉淀, 分别进行革兰染色、尿素酶试验、过氧化氢试验等, 证明无杂菌生长才留取CCS. 用考马斯亮兰法在紫外分光光度计上测定H. pylori培养上清液的蛋白含量. 硫糖铝(协和药业有限公司)配置成浓度为48 g/L, 相当于600 mg/kg; 三九胃泰颗粒(由三桠苦、九里香、白芍、生地、木香等组成, 深圳南方制药厂)配置成浓度为60 g/L, 相当于750 mg/kg.
健康♂Balb/c小鼠 (北京大学医学部动物实验室提供)56只, 质量20±2 g, 随机分为7组, 每组8只, 分别为生理盐水组(Ⅰ)、单纯损伤组(ⅡA, ⅡB)、硫糖铝保护组(ⅢA, ⅢB)、三九胃泰保护组(ⅣA, ⅣB), 其中A为小剂量毒素组, B为大剂量毒素组. 所有动物实验前禁食12 h, 可自由饮水, 然后分别予生理盐水0.25 mL, 硫糖铝(600 mg/kg)或三九胃泰(750 mg/kg)灌胃, 0.5 h后予生理盐水或不同浓度H. pylori的CCS 100 μg (0.25 mL), 1000 μg (0.25 mL)灌胃, 2次灌胃结束2 h后进食. 48 h后将小鼠断颈处死(处死前一晚禁食), 立即开腹取胃, 沿胃大弯侧剪开胃壁, 在胃窦小弯侧剪下1块组织放置于40 g/L甲醛溶液中固定, 石蜡包埋, 制成4-6 μm厚的切片, 采用HE染色, 光镜下观察胃黏膜的改变. 胃黏膜损伤组织形态学分级, 以黏膜损伤积分(EDS)计数[17-18] : 黏膜正常为1分, 黏膜表层细胞受损为2分, 损伤累及腺体细胞为3分, 黏膜糜烂、出血或溃疡形成为4分. Ⅰ, ⅡA, ⅡB, ⅣB四组各取1只在胃窦部剪下0.1 cm×0.1cm黏膜组织块置于20 g/L戊二醛中, 制成电镜标本, 观察超微结构改变.
统计学处理 采用方差分析, P<0.05表示差异有显著性.
I组黏膜层、固有层、肌层结构完整连续, 依次排列, 腺体排列紧密, 极少炎症细胞浸润. ⅡB组小鼠胃黏膜上皮细胞可见大量的空泡, 部分上皮细胞及腺体排列紊乱, 有的腺体结构被完全破坏. 5只发现糜烂, 但炎症细胞浸润不明显, 偶见几个单核细胞, 其中2只发现溃疡, 2只在胃黏膜固有层内见到灶性出血; ⅡA组可见部分上皮细胞里有空泡, 与大剂量毒素组比较, 空泡数量少, 有1只发现糜烂, 未发现溃疡, 上皮细胞及腺体排列凌乱不明显, 炎症细胞浸润不明显. 药物保护组各层组织排列完整连续, 损伤情况明显较单纯损伤组为轻. ⅣA组, ⅣB组各层排列整齐, 偶见表层黏膜上皮细胞损伤, 未见糜烂、溃疡、出血灶, 炎症细胞浸润不明显(图1).
I组小鼠的超微结构可见细胞排列紧密, 细胞器结构完整, 未见到变性, 微绒毛排列整齐, 无脱落, 吞噬溶酶体少见, 可见到正常的分泌颗粒及黏液颗粒. ⅡA组小鼠的超微结构见细胞排列比较紧密, 可见到线粒体及内质网肿胀变性, 微绒毛排列紊乱、肿胀、部分脱落, 溶酶体增加; ⅡB组小鼠的超微结构可见到细胞间隙明显增宽, 微绒毛稀疏、脱落, 线粒体和内质网高度肿胀扩张, 基本结构不清楚, 有的形成空泡, 吞噬溶酶体增多. ⅣB组小鼠其超微结构基本类似于生理盐水组(图2, 3).
ⅡA, ⅡB组损伤积分(2.25±0.46, 3.63 ±0.52)均明显高于Ⅰ组(1.13±0.35)(P<0.01); 硫糖铝和三九胃泰保护组的损伤积分, 不论小剂量毒素处理组(1.25±0.46, 1.50±0.53), 还是大剂量毒素处理组(1.75±0.71, 1.63±0.74), 均低于单纯损伤组(P<0.01, ⅣA vs ⅡA, P<0.05); 而硫糖铝与三九胃泰保护组比较, 不论小剂量毒素处理组, 还是大剂量毒素处理组, 胃黏膜损伤程度均无明显差异(P>0.05).
幽门螺杆菌(H. pylori )的细胞毒素引起了人们的普遍关注[19-22]. 我们探讨了H. pylori的毒力因子-细胞毒素对胃黏膜的损伤作用及胃黏膜保护剂硫糖铝和三九胃泰颗粒干预后的影响. 孙兆金et al[23]应用EDTA抑制尿素酶活性, 产毒株与非产毒株采用同样条件处理后, 产毒株的CCS引起明显的胃黏膜上皮损伤, 而非产毒株的CCS却没有引起相应的损伤, 说明胃黏膜上皮损伤是由H. pylori培养上清液里的空泡毒素导致的. 我们[24]曾证明H. pylori细胞毒素可引起胃黏膜细胞c-met, c-myc基因高表达, 从而造成细胞损伤. 我们采用产毒H. pylori的CCS灌服Balb/c小鼠, 观察到黏膜上皮细胞产生了大量的空泡、腺体结构排列紊乱及黏膜糜烂、溃疡形成. 超微结构发现细胞质肿胀变性, 微绒毛排列紊乱、脱落, 细胞间隙增宽, 吞噬溶酶体增多等改变, 这些损伤改变与文献[16,25-26]报道类似. 本结果表明H. pylori毒素可致胃黏膜病理损伤, 提示细胞毒素在导致胃部疾病方面起重要作用.
胃黏膜损伤的程度依赖于灌服CCS的次数, 给小鼠灌服一次即可引起胃黏膜损伤, 若48 h后再灌服一次可获得更加严重的损伤, 口服多次并不能加重胃黏膜损伤程度[24]. 胃黏膜损伤程度还依赖于灌服的剂量[25], 我们的研究亦发现大剂量毒素组所引起的鼠胃黏膜损伤远重于小剂量毒素组. 众所周知, 胃黏膜屏障的破坏、黏膜损伤及溃疡形成是由于损害因素与防御因素的失衡. 胃黏膜的防御因素通常包括黏膜 、黏液屏障、黏液及碳酸氢盐的分泌、黏膜血流、细胞更新、生长抑素、表皮生长因子、前列腺素、氨基己糖、NO、巯基等[27-29]. 损害因素则包括了H. pylori. 如前所述, H. pylori能引起胃黏膜损伤, 诱发胃十二指肠糜烂及溃疡. 如何预防和保护H. pylori所致的胃黏膜损伤就成为目前摆在大家面前需要研究和解决的问题.
硫糖铝是传统的胃黏膜保护剂, 长期以来作为主要胃黏膜保护剂广泛用于临床. 三九胃泰颗粒[30-31]是中药复方制剂, 具有消炎止痛, 理气健胃等功效. 药效学研究证明其有消炎、止血, 促进胃黏膜上皮修复, 纠正胃肠功能紊乱, 促进胃、脑等组织器官蛋白质合成, 抑制和黏附胃蛋白酶, 促进胸腺核蛋白、胸腺RNA、脾脏RNA的合成, 增强和调节机体免疫功能等作用, 因此三九胃泰也是治疗慢性胃炎的常用药物. 北京地区的一个多中心研究[32]还显示了"三九胃泰四联疗法"在治疗消化性溃疡以及根除幽门螺杆菌的作用. 国内作者报道[33]用三九胃泰能预防或减轻乙醇所致的小鼠急性胃黏膜损伤. 三九胃泰是否对H. pylori毒素所致的胃黏膜损伤有预防保护作用, 目前国内外尚无类似报道. 我们在此实验中利用三九胃泰对H. pylori 浓缩培养上清液造成的小鼠胃黏膜损伤的保护作用的小鼠模型作了初步探讨. 其结果发现三九胃泰保护组小鼠胃黏膜各层组织排列完整连续, 损伤情况较单纯损伤组明显减轻, 偶见表层黏膜上皮细胞损伤, 未见糜烂、溃疡、出血灶, 炎症细胞浸润不明显, 其超微结构也基本正常. 无论小剂量毒素处理组, 还是大剂量毒素处理组其胃黏膜损伤积分均低于单纯损伤组, 差异明显, 但与硫糖铝保护组比较则无明显差异. 结果提示胃黏膜保护剂硫糖铝和三九胃泰颗粒对H. pylori的CCS所致胃黏膜损伤都具有明显的预防保护作用.
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