【作者】 麻继臣；

【导师】 张晓岚；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 博士

【摘要】 溃疡性结肠炎(Ulcerative Colitis, UC)是一种病因不明的肠道慢性炎症性疾病,近年来在我国近年呈高发趋势,高复发率是其突出特征。然而,由于基本机制不明,目前UC治疗的根本目标仍然是控制症状,诱导缓解。如何更有效的防治UC复发是临床亟待解决的问题。近年UC发病机制的研究进展,给治疗学提供了新的启示。目前关于UC发病机制的基本共识是:易感个体在环境因素作用下,肠黏膜屏障削弱,肠道致病菌群及其有害成分穿过黏膜屏障,和有缺陷的免疫系统相接触,引发局部炎症反应,细菌不能被及时清除,使病情迁延反复。人体肠道处于复杂的带菌环境,肠黏膜表面积约有300m2,肠道内大约有500种共生菌,每克结肠内容物含有多达1012株共生菌,而易感个体肠黏膜屏障通透性显著升高,使细菌通过肠黏膜屏障的机会增加,由此可见,对于已经有遗传缺陷的个体,及时清除黏附于肠壁表面和偶然突破肠黏膜屏障的致病菌是防止疾病发生和复发的关键。抗菌肽在维持肠道黏膜屏障不被细菌入侵中起决定作用。它是迄今发现的最强的杀菌剂,对革兰氏阴性菌、革兰氏阳性菌、真菌、病毒和原虫均具有杀菌作用。防御素是人体肠道最主要的抗菌肽,结肠细胞主要表达防御素-1(humanβ-defensin 1, HBD-1)和防御素-2(humanβ-defensin 2, HBD-2)。研究表明,维生素D在调控防御素表达中起关键作用,流行病学调查发现UC患者普遍存在维生素D缺乏,动物实验证实维生素D受体基因敲除的小鼠更容易诱导出结肠炎,且结肠炎症状因补充活性维生素D获得缓解。只有活化的维生素D才具有生物学效能,然而活性维生素D即1,25-(OH)2D3循环浓度仅为0.03~0.06ng/ml,而其发挥抗菌作用所需生物作用浓度高达循环浓度数十甚至上百倍,提示维生素D可能存在一种局部活化形式。2001年Zehnder D证实结肠上皮细胞存在维生素活化的关键限速酶—25-羟维生素D 1-α-羟化酶(25-Hydroxyvitamin D3 1-alpha-hydroxylase, CYP27B1),因而我们推测CYP27B1可能是维生素D对防御素局部调控的关键。为此我们进行了如下研究:①收集UC患者和正常人肠黏膜组织标本,检测CYP27B1、防御素-2(HBD-2)在肠黏膜中的表达及其相关性;②采用过表达策略,构建重组腺病毒载体Ad-CYP27B1并感染Caco-2细胞,观察高表达CYP27B1对Caco-2细胞HBD-2和HBD-2表达的影响;③对比观察维生素D联合5-氨基水杨酸(5-ASA)与单独应用5-ASA对缓解期UC病人累积缓解率的影响,评价活性维生素D作为缓解期维持治疗药物的效能。实验内容主要包括以下三部分:第一部分CYP27B1在溃疡性结肠炎肠上皮组织中的表达及其与防御素-1、防御素-2的关系目的:检测人类溃疡性结肠炎患者结肠黏膜中CYP27B1和HBD-1、HBD-2在mRNA和蛋白水平的表达,探讨CYP27B1与防御素表达之间的关系,以及其在UC发病中的作用。方法:①依据临床资料、血液生化指标及内镜表现,选择在河北医科大学第二医院消化内科诊断明确的活动期UC患者。②免疫组织化学方法检测UC和正常对照结肠组织中的表达。③real-time Q-PCR方法测定UC患者和正常对照结肠上皮CYP27B1、HBD-1和HBD-2在转录水平的表达。④Western blot检测UC患者和正常对照结肠上皮CYP27B1、HBD-1和HBD-2蛋白的表达。实验分组如下:(1)con组:正常结肠黏膜;(2)uninfl组:UC患者非炎症结肠黏膜;(3)infl组:炎症结肠黏膜。结果:①UC患者35例,男19例,女16例。UC组轻度8例,中度18例,重度9例。②CYP27B1炎症黏膜组明显高于对照组(8.62±1.19 vs 5.14±0.86,P <0.01)及非炎症黏膜组(8.62±1.19 vs 5.86±0.74,P<0.05)后两者之间无统计学差异(5.86±0.74 vs 5.14±0.86,P >0.05);HBD-1在对照组及UC患者结肠上皮均有表达,对照组显著高于UC患者非炎症部位和炎症部位(7.28±0.93 vs 5.29±1.03,P <0.05;7.28±0.93 vs 4.47±1.24, P <0.01 );HBD-2在炎症部位显著高表达,与对照组比较(7.41±1.32 vs 3.46±0.81,P <0.01)及非炎症部位比较(7.41±1.32 vs 3.55±1.15,P <0.01)均具有统计学意义;Pearson相关性检验表明,炎症黏膜组织中CYP27B1的表达与HBD-2的表达呈高度正相关(r=0.710,P<0.01);③应用real-time Q-PCR技术检测各组CYP27B1、HBD-1和HBD-2的mRNA表达,并采用相对定量2-△△Ct法比较上述指标在各组结肠黏膜组织中的表达。以con组为对照组(其CYP27B1、HBD-1和HBD-2的mRNA表达量均为1),则uninfl组与infl组中CYP27B1 mRNA的表达量分别为1.355、5.559,HBD-1 mRNA的表达量分别为0.716、0.595,HBD-2的mRNA表达量分别为1.810、10.286。统计结果显示:infl组中CYP27B1、HBD2的mRNA的表达明显高于Con组(P<0.01,P<0.01),而HBD1的mRNA表达较con组显著降低(P< 0.01);④Western blot分析CYP27B1蛋白在各组中的表达,结果显示:infl组(1.22±0.10)显著高于con组(0.84±0.06)及uninfl组(0.99±0.19),P<0.05;而HBD-1蛋白在infl组(0.77±0.07)的表达显著低于con组(1.66±0.10)及uninfl组(1.39±0.10),P<0.05,P<0.01;HBD-2蛋白在infl组(0.87±0.04)的表达显著高于con组(0.11±0.04)及uninfl组(0.20±0.07),P<0.01,P<0.01。结论:①CYP27B1,HBD-1和HBD-2在正常对照组织和非炎症黏膜组织及炎症黏膜组织中的表达有显著差异,其中CYP27B1和HBD-2在UC病变部位显著高表达,而HBD-1在UC患者正常及病变黏膜均低表达。提示三者可能参与了UC发病的病理过程;②UC患者病变部位HBD-2表达和CYP27B1的表达呈显著正相关,而HBD-1和CYP27B1的关联有待进一步证实。第二部分CYP27B1腺病毒表达载体的构建及其体外促进防御素表达的效应观察目的:探讨CYP27B1过表达对体外培养的Caco-2细胞HBD-1及HBD-2的调控作用。方法:①重组腺病毒Ad-CYP27B1的构建:从Caco-2细胞提取RNA,经RT-PCR扩增得到目的基因CYP27B1片段,克隆至pTA2克隆载体,将测序正确的CYP27B1亚克隆至pAdTrack-CMV中后转化含pAdEasy-1的感受态BJ5183,通过同源重组获得腺病毒载体质粒,转染293包装病毒颗粒;②采用real-time Q-PCR技术和Western blot方法检测被感染的Caco-2细胞中CYP27B1的mRNA和蛋白表达以证实重组腺病毒Ad-CYP27B1的成功构建;③采用过表达策略,以腺病毒为载体,将Ad-CYP27B1及对照空病毒Ad-GFP转染体外培养的Caco-2细胞,分别采用real-time Q-PCR技术及Western blot检测Caco-2细胞中HBD-1及HBD-2在mRNA、蛋白表达水平的变化。实验分组如下:(1)con组,以含10%胎牛血清的DMEM细胞培养液培养细胞,在感染步骤加入无血清无抗生素DMEM代替病毒液;(2)Ad-GFP组,感染表达绿色荧光蛋白(green fluorescent protein, GFP)的空病毒Ad-GFP;(3)Ad-CYP27B1组,感染携带CYP27B1基因并表达GFP的重组腺病毒Ad-CYP27B1。结果:①重组腺病毒CYP27B1的构建:成功扩增出1527bp大小的CYP27B1片段,经测序与预期序列一致,将测序正确的CYP27B1成功克隆至pAdTrack-CYP27B1后,与骨架质粒pAdEasy-1在BJ5183细菌内同源重组,通过筛选、293细胞包装后获得了重组腺病毒Ad-CYP27B1,病毒滴度约为1011U/ mL②腺病毒感染Caco-2细胞后48 h,应用Real-time PCR检测Caco-2细胞CYP27B1 mRNA表达,并采用相对定量2-△△Ct法比较CYP27B1 mRNA在各组Caco-2细胞中的表达。以con组为对照组(其CYP27B1 mRNA表达量为1),Ad-CYP27B1组CYP27B1 mRNA的相对表达量为2.135,显著高于Con组(P<0.01),而Ad-GFP组的CYP27B1 mRNA相对表达量为0.886,与Con组无显著差异(P>0.05);进一步应用Western blot分析各组Caco-2细胞的CYP27B1蛋白表达,Ad-CYP27B1组(2.09±0.05)显著高于con组(1.52±0.09)及Ad-GFP组(1.35±0.13),P<0.01。Ad-GFP组与con组间CYP27B1蛋白表达均无显著差异(P>0.05),证明重组腺病毒Ad-CYP27B1成功感染体外培养的Caco-2细胞。③病毒感染Caco-2细胞后细胞状态良好,贴壁生长,无变圆、缩小或脱落等病理迹象,经过荧光倒置显微镜观察发现Ad-CYP27B1的转染效率在第24 h约48%,48 h约80%。④为评价CYP27B1对HBD1和HBD2的影响,在病毒感染Caco-2细胞48 h后,采用real-time Q-PCR及Western blot检测Caco-2细胞HBD1及HBD2在转录、蛋白表达水平的变化。结果显示Ad-CYP27B1组Caco2细胞HBD-1蛋白及其mRNA显著高于Con组(P<0.05);HBD-2蛋白及其mRNA显著高于Con组(P<0.01, P<0.05).结论: CYP27B1基因促进了HBD-1及HBD-2在Caco-2细胞的mRNA和蛋白表达,从而可能增强结肠上皮的抗菌功能,对维持结肠黏膜屏障功能完整起到保护作用。第三部分维生素D对溃疡性结肠炎维持缓解的作用目的:溃疡性结肠炎(ulcerative colitis, UC)是临床常见复发性疾病,目前尚无满意的缓解期治疗措施,标准治疗方案仅能将1年内累积复发率降为50%,研究显示UC发病和维生素D缺乏相关联,本研究的目的是对5-氨基水杨酸方案联合维生素D对维持缓解的效果进行前瞻、单盲的随机对照观察,探索其做为缓解期维持治疗方案的可行性,为UC缓解期治疗提供新的思路。方法:将52例诱导缓解的溃疡性结肠病人,随机分为维生素D+美沙拉秦组(26人)和美沙拉秦组(26人),分别给予罗钙全0.25μg 1/日+美沙拉秦500mg 3/日或单独给予美沙拉秦500mg 3/日。终点事件为疾病复发。采用Kaplan - Meier法和Log–rank检验等统计方法对两组数据进行比较。结果:试验截尾,两组之间的基本临床资料、复发率及累积缓解率无显著差异;对两组高复发率患者进行分层统计后,两组间缓解时间(V+M组,224±39天;MSL组134±27天,Student t test P=0.026)和累积缓解率(Log-rank test P =0.039)表现出统计学差异。结论:维生素D联合美沙拉秦不能显著降低一般患者的累积复发率,但对高复发率患者,可能延长其缓解时间及累积缓解率。更多还原

【Abstract】 Ulcerative colitis (UC) is a group of chronic intestinal inflammatory disease, the etiology of which is still unknown. The natural history of UC is characterized by chronic relapsing and remitting episodes. Currently, the goal of the available treatments is only to induce and to maintain remission of symptoms and mucosal inflammation. Therefore, it is an urgent task to find an effective approach to maintain remission of ulcerative colitis.Recent understanding of UC is a result of a genetic predisposition to environmental triggers. The onset and reactivation of UC are triggered by environmental factors that transiently break the impaired mucosal barrier, which make pathogenic enteric bacteria can not be eliminated timely, leading to pathogenic chronic intestinal inflammation. The intestines are situated in a complex environment of bacteria, and prior to the onset, where exist an increased mucosal permeability in UC patients. The weakening of intestinal barrier is probably the initiating factor in the relapse of UC. Therefore, to remove the surface bacteria adhesion to the intestinal wall might be the key to the prevention of UC recurrence.Antibiotic peptides, which have been demonstrated with activity against a wide range of microorganisms including bacteria, protozoa, yeast, fungi, viruses and even tumor cells,contribute to the maintenance of intestinal mucosal barrier of bacterial invasion. Human defensins are the most important antibiotic peptide in gut, playing a crucial role in controlling pathogen invasion in IBD. It mainly expresses Humanβ-defensin 1 (HBD-1) and humanβ-defensin 2 (HBD-2) in colonic tissue.It has been proved that active vitamin D, 1,25(OH) 2D3, regulated the expression and activation of antibiotic peptides. Vitamin D deficiency is highly prevalent among patients with IBD, irrespective of established or new-onset IBD, or even IBD is well controlled. Compelling data in mice have shown that vitamin D and signaling through the vitamin D receptor (VDR) dictated the outcome of experimental UC, and that vitamin D prevented and ameliorated symptoms of experimental IBD murine. But the required 1,25-(OH) 2D3 concentration for biological effect is far higher than the plasma concentrations of 0.03～0.06 ng/ml,which suggests there may have a form of local activation of vitamin D. 25-Hydroxyvitamin D3 1-alpha -hydroxylase(CYP27B1), the key rate-limiting enzyme of vitamin activation, has been detected in colonby Zehnder D in 2001. We speculated that CYP27B1 may play a key role in the regulation of the expression of human defensins in colonic tissue. Therefore, we conducted the following study:(1) Investigate the expression of CYP27B1, HBD-1 and HBD-2 on the protein and transcription level and to explore the relationship between CYP27B1 and human defensins in UC patients and normal intestinal biopsy specimens; (2) By over-expression strategy, construct recombinant adenovirus vector containing CYP27B1 gene and analysis of expression of HBD1 and HBD2 in vitro; (3) Investigate the efficacy of vitamin D combined with mesalazine in maintaining remission of UC, evaluation of the feasibility of active vitamin D as a maintenance therapy for UC patient in quiescent period. The project contain three parts as below:Part 1: The expression of CYP27B1 in colonic mucosa of UC and the correlation with HBD1 and HBD2Objective: To investigate the expression of CYP27B1, HBD1 and HBD2 on the protein and transcription level in colonic mucosa of UC, and to explore the correlation between CYP27B1 and human defensins.Methods: The UC patients were final diagnosed according to clinical data, biochemical indicator and endoscopic characteristics. The expressions of CYP27B1, HBD1 and HBD2 in colon mucosa of UC patients and healthy people were determined by immunohistochemical staining. Then, the mRNA and protein expression level of CYP27B1, HBD1 and HBD2 were assessed by real-time fluorescent qunatitative reverse transcription-polymerase chain reaction (real-time Q-PCR) and Western blot, respectively. The groups were as follows: contorl group: normal colon mucosa; uninflammatory group: noninflammatory colon mucosa of UC; infl group: inflammatory colon mucosa of UC.Results: (1) A total of 35 patients (19 men and 16 women)were collected and divided into mild(8), moderate(18) and heavy(9) group; (2) The results of immunohitochemical assay of colonic mucosa showed that CYP27B1 protein located in the membrane of epithelium, near the of cavity surface, and in the nuclears of Inflammatory cells, respectively, HBD-1 was expressed in surface and crypt epithelium in uninflamed and inflamed colon and in crypt. HBD-2 protein expressed abundantly in the cytoplasm of epithelium with inflammation, and nucleus of Inflammatory cells, respectively. CYP27B1 expressed in the three groups, and the expression in inflammatory group was significantly higher than the control group and uninflammatory group (P<0.05). The expression of HBD1 in control group was significantly higher than that of uninflammation group and inflammatory group (P<0.05, P<0.01, respectively). The expression of HBD2 in inflammatory group was conspicuous and higer than that in contorl group and uninflammatory group in which there were almost no expression. Pearson’s correlation analysis showed that the expression of CYP27B1 in inflammatory mucosa was positively correlated with HBD2; (3) The data measured by real time PCR showed the mRNA expressions of HBD1 in uninflammatory group and inflammatory group were significantly lower than that in Con group (P<0.01). Meanwhile, mRNA expressions of HBD2 and CYP27B1 in inflammatory group were significantly higher than that in control group (P<0.01); (4) The data measured by western blot showed the protein expressions of HBD1 in uninflammatory group and inflammatory group were significantly lower than that in control group (P<0.05, P<0.01, respectively). Meanwhile, protein expressions of HBD2 and CYP27B1 in inflammatory group were significantly higher than that in control group (P<0.01, P<0.05, respectively).Conclusions: (1) There was significant difference in the expression of CYP27B1, HBD-1 and HBD-2 between normal colon mucosa and colon mucosa of UC. The level of HBD-1 was lower in noninflammatory and inflammatory colon mucosa of UC than in normal colon mucosa. On the contrary, the level of CYP27B1 and HBD-2 were higher in noninflammatory and inflammatory colon mucosa of UC. The results suggested that CYP27B1, HBD1 and HBD2 may be involved in the pathogenetic procedure of UC; (2) The expression of CYP27B1 in inflammatory colon mucosa was positively correlated with HBD2. The correlation between HBD-1and CYP27B1 need to be further proved.Part 2: Construction of CYP27B1 recombinant adenovirus vector and its effexts on expression of defensins in Caco-2 cellsObjective: To investigate regulatory effects of over-expression of CYP27B1 on expression of HBD-1and HBD-2 in Caco-2 cells.Methods: (1) To construct the CYP27B1 adenovirus vector, genomic fragment containing CYP27B1 was generated by RT-PCR using total RNA from Caco-2 cells. The cDNA was first cloned into pTA2 vector and then subcloned into pAdTrack-CMV, resulting in pAdTrack-CYP27B1. Subsequently, CYP27B1 was constructed into backbone plasmid pAdEasy-1 by homologous recombination in competence BJ5183. The positive recombinant pAdEasy-1 containing CYP27B1 gene was linearized by PacⅠand transfected into 293 cells to package recombinant adenovirus. All PCR-amplified fragments and cloning junctions were verified by DNA sequencing and enzymatic digestion. An adenovirus encoding green fluorescent protein(Ad-GFP)was used as control. (2) To confirm the recombinant adenovirus particles containing CYP27B1 gene, The expression of CYP27B1 in infected Caco-2 cells was measured by western blot and real-time PCR.③To gain the over-expression of CYP27B1 in Caco-2 cells, the cells were infected by the recombinant adenovirus particles containing CYP27B1. HBD1 and HBD2 expression in Caco-2 was measured by western blot and real-time PCR. Cells were grouped as follows: (1)control group, cells were cultured in DMEM medium containing 10% FBS; (2)Ad-GFP group, Caco-2 cells were infected with adenovirus expressing green fluorescent protein alone; and (3)Ad- CYP27B1 group, Caco-2 cells were infected with adenovirus harboring genes CYP27B1 and GFP.Results: (1) Construction of recombinant adenovirus vector containing CYP27B1 gene: The size of the amplified PCR product was 1527bp. The results of sequencing were consistent with those from GenBank. CYP27B1 gene was subcloned into adenoviral shuttle vector pAdTrack-CMV. Then, CYP27B1 was successfully constructed into backbone plasmid pAdEasy-1 by homologous recombination in competence BJ5183. Recombinant adenovirus containing CYP27B1 were obtained by transfecting into 293 cells. The adenovirus virus titer was about 1011U/mL; (2) The expression of CYP27B1 in Caco-2 cells was detected after recombinant adenovirus particles infected cells for 48 hours. The mRNA expression of CYP27B1 in Caco-2 cells was measured by real time Q-PCR. The relative values of CYP27B1 mRNA were calculated and compared with untreated control group(The relative value in con group was assigned to 1 ). Obviously, the mRNA expression of CYP27B1 in the Ad-CYP27B1 group was significantly higher than that in the control group (P < 0.01). The result of western blot analysis showed that protein expression levels of CYP27B1 in Ad-CYP27B1 group were significantly higher than that in control group (P<0.01). No significant differences were observed in the mRNA and protein expressions of CYP27B1 between control group and Ad-GFP group (P > 0.05). Overall, the cells were successfully infected by the recombinant adenovirus particles containing CYP27B1; (3) Infected Caco-2 cells were observed by fluorescent microscope after the cells were infected by recombinant adenovirus. The transfection efficiency was about 48% at 24h post-transfection and 80% at 48h post-transfection; (4) To evaluate the effect of CYP27B1 on induction expression of HBD-1 and HBD-2 in Caco-2 cells, the expressions of HBD-1 and HBD-2 in Caco-2 were detected by real-time PCR and western blot after Ad-CYP27B1 infected the cells post 48 h. The results showed that the mRNA and protein expressions of HBD-1 in Ad-CYP27B1 group were significantly higher than that in control group (P < 0.05). The mRNA and protein expressions of HBD-1 in Ad-CYP27B1 group were also significantly higher than that in control group (P<0.01, P<0.05). Additionally, there were no significant differences in the mRNA and protein expression of HBD1 and HBD2 between Ad-GFP group and control group (P>0.05).Conclusions: CYP27B1 can improve the expression of HBD-1 and HBD-2 in the Caco-2 cells, and promote the antibacterial activity of colonic mucosa, and further protect the colon mucosa barrier. Part 3: Efficacy of Vitamin D in Maintaining Remission of Ulcerative ColitisObjective: At present there is no satisfactory remission maintenance therapy for Ulcerative colitis (UC). Evidence indicated that there existed a correlation between incidence and vitamin D deficiency. The goal of this work was to evaluate the efficacy of vitamin D combined with mesalazine in maintaining remission of UC.Methods: A total of 52 eligible patients were enrolled in a single-blind prospective randomized controlled observation. Patients were randomized to receive either mesalazine 1500 mg/d alone (MSL group, n=26) or combination with Rocaltrol 0.25μg/d (V+M group, n=26). The endpoint of the study was defined as the incidence of relapse.Results: For frequent relapse patients, although relapse rates were similar between the V+M group (69.2%) and the MSL group (83.3%; P=0.409), patients in the V+M group were found to relapse later than patients in the MSL group (Log-rank P=0.039) by comparing the duration of remission. The differences in maintaining remission time for these two groups are statistically significant (224±39 vs 134±27, respectively; P=0.026)Conclusions: The efficacy of combined treatment with vitamin D and mesalazine in maintaining remission of UC is superior to treatment with mesalazine alone, and also can prolong the remission time and improve the cumulative remission rates for frequent relapse patients.更多还原