先天性巨结肠小肠结肠炎模型的建立及鉴定

Identification and establishment of Sprague-Dawlay rat model with experimental Hirschsprung' sassociated enterocolitis

目的建立先天性巨结肠小肠结肠炎(Hirschsprung,s-associated enterocolitis,HAEC)大鼠模型,为进一步研究HAEC的发病机制及分子生物学机制提供可靠模型。方法将120只6~8周龄SD大鼠按随机数字表法平均分为对照组、先天性巨结肠(Hirschsprung's disease,HSCR)组和HAEC组。将HACE组的大鼠在造模前一周用大肠杆菌JM83按照1x10^9CFU/d的剂量灌胃。对照组和HSCR组用相同剂量的生理盐水灌胃。1周后将3组大鼠使用1%的苯巴比妥钠经腔注射麻醉后剖腹,提出结肠并显露直肠末段,HSCR组和HAEC组用0.1%苯扎澳镀(benzalkonium chloride,BAC)纱布紧贴包绕直肠末段作用45min以制作动物模型,对照组使用生理盐水。在造模后的第一、三、五、七周对模型进行大体观察,用髓过氧化物酶(MPO)活性检测评估经BAC或生理盐水处理段直肠的近端结肠组织以评估肠道炎症情况,用RTPCR检测乙酰胆碱酯酶(AchE)、胶质细胞源性神经营养因子(GDNF)的表达情况。在造模后第五周取直肠末段行病理学检查以检测建模是否成功,并用16SrRNA技术检测各组中小肠、横结肠、直肠中菌群结构的变化。结果术后第三周,HSCR组和HAEC组的大鼠逐渐出现腹胀、进食及排便减少。处理段肠道狭窄、近端结肠扩张、大便堆积。术后第五周HAEC组大鼠腹胀程度明显比HSCR组的大鼠明显。病理学检查发现BAC处理段在5周后神经节细胞数量明显减少,于7周后完全消失。MPO活性检测发现在第五周近端结肠组织的MPO活性:HAEC组(10.4±0.43)明显高于HSCR组(7.6±0.35)及对照组(2.8±0.16)(P<0.05),在第七周近端结肠组织的MPO活性:HAEC组(13.2±0.56)与HSCR组(11.2±0,35)的差异无统计学意义(P>0.05)。用RT-PCR检测AchE、GDNF的mRNA表达,结果表明AchEmRNA和GDNFmRNA在HSCR组及HAEC组的表达明显下调,第五周HSCR组和HAEC组的AchEmRNA表达量分别为(0.74±0.09,0.55±0.11);GDNFmRNA的表达量分别为(0.47±0.12,0.40±0.22);第七周HSCR组和HAEC组的AchEmRNA表达量分别为(0.67±0.03,0.46±0.13);GDNFmRNA的表达量分别为(0.51±0.06,0.31±0.09),第五周和第七周AchEmRNA和GDNFmRNA的表达量均较对照组降低,差异具有统计学意义(P<0.05),HSCR组和HAEC组之间的差异在各时间点均无统计学意义(P>0.05)。造模后第五周对HAEC组大鼠的近端结肠切片行HE及免疫荧光染色可见结肠黏膜在镜下呈炎性改变。用16SrRNA技术检测大鼠的小肠、横结肠和直肠三处肠道菌群的分布情况,共发现27种细菌种属。小肠、横结肠和直肠中肠道菌群占比情况:HAEC组以肠杆菌科占比最高,分别为17.3%、22.8%和34.7%,其次为梭菌目,占比分别为8.7%、12.4%和10.4%,粪杆菌属占比分别为6.9%、7.9%、8.6%,梭杆菌属占比分别为7.6%、3.8%和5.6%;在HSCR组中乳杆菌目占比最高,分别为14.5%、9.4%和5.7%,其次为拟杆菌类,占比分别为12.6%、9.4%和6.3%,肠杆菌科占比分别为4.3%、6.3%和7.1%,梭菌属占比分别为6.4%、5.2%和4.6%。结论采用0.1%BAC剖腹处理直肠末段结合灌胃大肠杆菌JM83的方法成功建立了先天性巨结肠小肠结肠炎的SD大鼠动物模型,为深入研究先天性巨结肠小肠结肠炎的发病机制及预防提供一个可靠的模型基础。

Objective To establish a Sprague-Dawlay (SD) rat model of experimental Hirschsprung's-associated enterocolitis (HAEC) for providing experimental rationales for elucidating the pathogenesis of HAEC. Methods According to a random number table, a total of 120 SD rats aged 6-8 weeks were divided into control, Hirschsprung's disease (HSCR) and HAEC groups. Subjects in HAEC group had an intragastric administration of Escherichia coli (E. coli) JM83 (109 CFU/day) for 1 week before modeling. All animal were operated under an anesthesia of 1 % phenobarbital sodium. A gauze soaked with 0. 1 % benzalkonium chloride (BAC) was wrapped around rectal serosa for 45 mins in both HSCR and HAEC groups. And 0. 9% saline was wrapped to rectum similarly in control group. The models were examined through gross observations. Myeloperoxidase (MPO) assay, hematoxylin & eosin (HE) staining and the expressions of AchE, glial cell line-derived neurotrophic factor (GDNF) were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) at the end of 1, 3, 5, 7 weeks post-modeling. Colonic wall was harvested for HE staining for identifying successful models in 5 weeks. HE staining, immunofluorescent staining and 16S rRNA were used for detecting the changes of intestinal flora structure at 5 weeks post-modeling. Results After 3-week BAC treatment, abdominal distension and loss of appetite appeared in both groups. Pathological biopsy revealed a narrow segment and marked dilation of proximal segment Abdominal distension was significantly higher in HAEC group than that in HSCR group after 5-week BAC treatment. Histological examination showed ganglion cells gradually decreased after 5-week BAC treatment and disappeared completely at 7 weeks after BAC treatment MPO assay showed that the activity in HAEC group (10. 4±0. 43) was higher than that of HSCR (7. 6 ± 0. 35) and control (2. 8 ± 0. 16) groups (P<0. 05) after 5-week BAC treatment However, no difference existed between HAEC and HSCR groups at 7 weeks [(13. 2 ± 0. 56) vs.(11. 2 ± 0. 35), P>0. 05). The mRNA expression of AchE, GDNF decreased dramatically in both HSCR and HAEC groups after BAC treatment The mRNA expression of AchE (0. 74 ± 0. 09, 0. 55 ± 0. 11 vs. 0. 67 ± 0. 03 , 0. 46 ± 0. 13) and GDNF (0. 47 ±0.12, 0. 40 ± 0. 22 vs 0. 51 + 0. 06, 0. 31 ± 0. 09) between HSCR and HAEC groups was lower than those of control group at 5, 7 weeks after BAC treatment (P<0. 05). No difference existed in HSCR group compared with HAEC group at 1, 3, 5, 7 weeks after BAC treatment. Colonic mucosa showed typical changes of enterocolitis in HAEC group after modeling at 5 weeks. Twenty-seven bacterial genera were found in rectum, transverse colon and ileum by 16S rRNA gene analysis. Genus Enterobacteriaceae (34. 7%, 22. 8%, 17. 3%) was the most prevalent in HAEC group, following Clostridium (10. 4%, 12.4%, 8.7%), Faecium (8.6%, 7. 9%, 6. 9%) and Fusobacterium (5. 6%, 3. 8%, 7. 6%);genus Lactobacillus (5. 7%, 9. 4%, 14. 5%) was the most prevalent in HSCR group, following Bacteroides (6.3%, 9. 4%, 12. 6%), Enterobacteriaceae (7. 1%, 6. 3%, 4. 3%) and Clostridium (4. 6%, 5. 2%, 6. 4%). Conclusions A rat experimental model for HAEC is successfully established by applying 0. 1 % BAC onto rectal serosa plus an intragastric administration of E. coli JM83. This mcxdel provides the basis for future studies of exploring the pathophysiology of HAEC.