PLOD2反义寡核苷酸对TNBS诱导小鼠慢性肠纤维化的影响及疗效观察

发布时间：2018-06-14 23:37

  本文选题：炎症性肠病 + 肠壁纤维化　； 参考：《南昌大学》2014年硕士论文

【摘要】：研究背景： 炎症性肠病（inflammatory bowel disease,IBD）包括溃疡性结肠炎(ulcerativecolitis,UC)及克罗恩病(Crohn disease,CD)，为肠道的慢性非特异性炎性疾病，其病因和发病机制尚不确切，近年来，其发病率持续增高，引起了人们高度的重视。长期的慢性炎症刺激能导致肠壁纤维化，且克罗恩病的病变累及范围广，为肠壁全层性炎症，导致大量细胞外基质（extracellular matrix,ECM）异常沉淀引起肠壁各解剖层发生纤维化，易产生瘢痕性狭窄从而导致肠梗阻。目前，对于慢性肠壁纤维化的治疗，传统的治疗方法及外科手术治疗效果均不理想，预后不佳，尚缺乏十分有效的治疗手段。国内外研究发现，ECM的沉淀以胶原蛋白的沉积为主，赖氨酸羟化酶（lysyl hydroxylase,LH）在胶原蛋白的形成及稳定中起着重要作用，其中尤以LH2作用最为关键，而PLOD(procollage-lysine,2-oxiglutarate,5-dioxygerase)基因为LH的编码基因，，因此，PLOD2有望成为预防或治疗慢性肠纤维化的研究新方向。 目的： 前期研究表明，应用三硝基苯磺酸（trinitro-benzene-sulfonic acid,TNBS）灌肠可成功诱导BALB/C小鼠慢性肠纤维化模型的建立，在此基础上，我们合成PLOD2反义寡核苷酸，以此干预灌肠来探讨PLOD2反义寡核苷酸对BALB/C小鼠慢性肠壁纤维化的影响及作用机制。通过比较实验各组间小鼠的疾病活动指数（DAI），观察结肠组织的病理变化和胶原纤维的增生程度，比较分析TNF-α、PLOD2、Col-ⅢmRNA的表达水平及Col-Ⅲ蛋白的表达水平，进而分析PLOD2反义寡核苷酸在TNBS诱导小鼠慢性肠壁纤维化中的作用，探讨其作用机制，并为PLOD2反义寡核苷酸治疗慢性肠壁纤维化及开发相关基因治疗药物提供一定的实验及理论依据。 实验方法： 随机将40只体重为20-25g、6-8周龄的雌性BALB/C小鼠分为4组，每组10只，分别为生理盐水空白对照组（空白对照组），TNBS模型组（TNBS组），PLOD2错义寡核苷酸阴性对照组（MSODN组）及PLOD2反义寡核苷酸治疗组（ASODN组）。对BALB/C小鼠采用灌肠的方式建立动物模型，空白对照组每周给予生理盐水100ul灌肠后24小时再次给予生理盐水100ul灌肠；TNBS模型组每周先给予2mg TNBS/50%乙醇溶液100u1灌肠，24小时后给予生理盐水100ul灌肠；PLOD2错义寡核苷酸阴性对照组每周先给予2mg TNBS/50%乙醇溶液100u1灌肠，24小时后给予PLOD2错义寡核苷酸100ul灌肠；PLOD2反义寡核苷酸治疗组每周先给予2mg TNBS/50%乙醇溶液100u1灌肠，24小时后给予PLOD2反义寡核苷酸100ul灌肠；每组均连续灌肠6周，于最后1次灌肠后1周采取颈椎脱臼法处死小鼠并取结肠组织。采集的结肠组织分别进行如下处理：即HE染色评估结肠组织炎症程度；VG染色评估结肠组织纤维化程度；RT-PCR检测结肠组织中TNF-α、PLOD2、COL-III的mRNA的表达；免疫组化检测结肠组织中COL-III蛋白的表达水平。 结果： 1.TNBS组、MSODN组及ASODN组小鼠每次灌肠后出现不同程度的症状，如少食，少动，身体蜷缩，稀便，大便潜血阳性甚至肉眼血便，皮毛光泽度下降等，第3-4天后上述症状逐渐减轻。小鼠的体重大部分在第2-4天有不同程度的下降，部分第4天后体重较前有所增加，甚至超过灌肠前体重。整个实验期间，症状以前3周最重，第4周后症状相对减轻。三组小鼠均有个别小鼠死亡，死亡时间主要在前3周。空白对照组灌肠后进食及活动情况无明显改变，无小鼠死亡。TNBS组、MSODN组及ASODN组小鼠DAI评分均高于空白对照组（P＜0.05）；TNBS组、MSODN组及ASODN组组间相比较无统计学意义（P0.05）。 2.肉眼观察各组小鼠结肠组织的大体表现，可见TNBS组、MSODN组及ASODN组有不同程度的充血、水肿、粘连等，剖开后肠壁有不同程度的糜烂、溃疡，有的部位出现管壁增厚、狭窄、僵硬变形等表现，空白对照组小鼠结肠组织则无上述改变。HE染色镜下观察空白组小鼠结肠组织无明显炎症表现，TNBS组、MSODN组及ASODN组都可见不同程度的上皮细胞、腺体及隐窝的破坏，杯状细胞减少，以淋巴细胞及单核细胞为主的炎症浸润，可有淋巴滤泡增生，部分结肠组织可出现粘膜层溃疡，部分甚至达粘膜肌层。TNBS组、MSODN组及ASODN组小鼠炎症评分均高于空白对照组（P＜0.05）；而这三组间相比较无统计学意义（P0.05）。VG染色镜下可见空白对照组小鼠结肠组织无明显纤维化表现，而TNBS组及MSODN组中小鼠结肠组织中可见大量胶原蛋白沉积，固有肌层可有不同程度的增厚，部分甚至可见纤维分隔。ASODN组中小鼠结肠组织的胶原蛋白沉积及固有肌层增厚程度较TNBS组及MSODN组轻。TNBS组、MSODN组及ASODN组小鼠纤维化评分均高于空白对照组（P＜0.05）；TNBS组及MSODN组组间相比较无统计学意义（P0.05）；而ASODN组评分低于TNBS组及MSODN组（P＜0.05）。 3.TNBS组、MSODN组中PLOD2mRNA的表达高于空白对照组及ASODN组（P＜0.05），ASODN组高于空白组（P＜0.05），TNBS组及MSODN组间比较无统计学意义（P＞0.05）。对于TNF-αmRNA及Col-IIImRNA的表达，TNBS组、MSODN组及ASODN组的表达均高于空白对照组（P＜0.05），但这三组间比较无统计学意义（P＞0.05）。 4.TNBS组、MSODN组及ASODN组Col-III蛋白的表达较空白对照组高（P＜0.05），但ASODN组比TNBS组、MSODN组表达低（P＜0.05），而TNBS组及MSODN组间比较无统计学意义（P＞0.05）。 结论： 应用PLOD2ASODN治疗TNBS/50%EtOH诱导的小鼠慢性肠纤维化，其通过抑制PLOD2的表达，降低LH2的活性，从而减少III型胶原蛋白的合成，降低肠壁纤维化的程度。
[Abstract]:Research background:
Inflammatory bowel disease (IBD), which includes ulcerative colitis (ulcerativecolitis, UC) and Crohn's disease (Crohn disease, CD), is a chronic nonspecific inflammatory disease of the intestines. Its etiology and pathogenesis are still uncertain. In recent years, the incidence of the disease has been increasing, and people have paid great attention to the chronic inflammatory disease. Irritable energy leads to intestinal wall fibrosis, and the lesions of Crohn's disease are involved in a wide range, as a whole layer of intestinal inflammation, resulting in a large number of extracellular matrix (extracellular matrix, ECM) abnormal precipitations that cause fibrosis in the anatomic layers of the intestinal wall, causing scar stricture and causing intestinal obstruction. The results of the treatment and the surgical treatment are not ideal, and the prognosis is not good. There is still a lack of effective treatment. It is found that the precipitation of ECM is mainly deposited by collagen, and lysyl hydroxylase (LH) plays an important role in the formation and stability of collagen, especially the role of LH2 is the most important. The gene of PLOD (procollage-lysine, 2-oxiglutarate, 5-dioxygerase) is the encoding gene of LH. Therefore, PLOD2 is expected to be a new research direction for the prevention or treatment of chronic intestinal fibrosis.
Objective:
Previous studies have shown that Trinitro-benzene-sulfonic acid (Trinitro-benzene-sulfonic acid, TNBS) enema can successfully induce chronic intestinal fibrosis in BALB/C mice. On this basis, we synthesize PLOD2 antisense oligodeoxynucleotides to investigate the effect of PLOD2 antisense oligodeoxynucleotides on the chronic intestinal fibrosis in BALB/C mice and the effect of PLOD2 antisense oligodeoxynucleotides on the chronic intestinal fibrosis in BALB/C mice. By comparing the disease activity index (DAI) of mice in the experimental groups, the pathological changes of colonic tissue and the degree of proliferation of collagen fibers were observed. The expression level of TNF- alpha, PLOD2, Col- III mRNA and the expression level of Col- III protein were compared and analyzed, and then the PLOD2 antisense oligodeoxynucleotides were analyzed in the chronic intestinal fibrosis induced by TNBS in mice. The role of PLOD2 antisense oligodeoxynucleotides for the treatment of chronic intestinal fibrosis and the development of related gene therapy drugs for the development of antisense oligodeoxynucleotides provide some experimental and theoretical basis.
Experimental methods:
The female BALB/C mice of 40 20-25g and 6-8 weeks old were randomly divided into 4 groups, 10 in each group, respectively, the blank control group (blank control group), the TNBS model group (group TNBS), the PLOD2 missense oligonucleotide negative control group (group MSODN) and the PLOD2 antisense oligoside acid treatment group (ASODN group). The enema method was established in the BALB/C mice. In the blank control group, the blank control group was given the saline 100ul enema again after 24 hours of saline 100ul enema every week; the TNBS model group gave 2mg TNBS/50% ethanol solution 100u1 enema first, and then given the saline 100ul enema after 24 hours, and the PLOD2 missense oligonucleotide negative control group gave 2mg TNBS/50% ethanol solution 100u1 every week. The enema was given after 24 hours of PLOD2 missense oligonucleotide 100ul enema; PLOD2 antisense oligodeoxynucleotides were given 100u1 enema by 2mg TNBS/50% ethanol solution and PLOD2 antisense oligodeoxynucleotides 100ul enema 24 hours later; each group was consecutively enema for 6 weeks, and the cervical dislocations were executed and colonic group was taken 1 weeks after the last 1 enema. The colonic tissues collected were treated as following: HE staining was used to assess the degree of inflammation in colon tissue; VG staining was used to assess the degree of fibrosis in colon tissue; RT-PCR was used to detect the expression of mRNA in the colon of TNF- a, PLOD2, COL-III; and immunohistochemistry was used to detect the expression level of COL-III egg white in colon tissue.
Result锛

本文编号：2019480