Reg IV在炎症性肠病的表达及其对病变活动度的预测作用
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摘要
背景和目的
炎症性肠病(inflammatory bowel disease, IBD)是一组病因未明的非特异性肠道炎症性疾病,包括溃疡性结肠炎(ulcerative colitis, UC)和克罗恩病(Crohn's disease, CD)。近二十余年来,随着人们生活方式的变化和诊断水平的提高,国内炎症性肠病的发病率有逐年上升的趋势。疾病是处于活动期还是静止期直接关系到疾病的正确治疗。IBD临床表现复杂多变,诊断缺乏特异性的指标,而且常因对病情活动的判断不准确而延误治疗。目前临床评价IBD活动与否主要通过临床症状、实验室生化指标、内镜下的表现及病理等综合判断。其中C-反应蛋白(CRP)几乎全部由肝脏产生,短时间内(数小时)即可升高,而机体一旦恢复,CRP又迅速恢复正常(6～7d)。CRP可及时而敏感地反映炎症活动,是目前临床最为推崇的IBD活动性指标之一。另外,各国专家针对IBD制定出疾病活动指数(disease activity index),对疾病的活动性进行量化处理,从而更客观准确地反映疾病的活动性,帮助临床医师作出正确的医疗决策。评价UC活动度常采用Southerland疾病活动指数,也称Mayo指数,较为简单实用,包括腹泻、便血、粘膜表现、医师评估病情几个评价指标。Best的CDAI计算法是国际上广泛应用于临床和科研的评价CD活动度的指标。根据腹痛、腹泻、腹块等8个变量,通过1周观察计分,乘以规定的权重,求得各自分值,再计算积分。但少数患者尤其是某些早期和复发患者,血生化表现不显著,而且在疾病活动的早期内镜也不能发现。有必要寻找新指标以更全面、准确地评价IBD的炎症活动度,为临床应用奠定理论基础。
再生基因(regenerating gene, Reg)家族属钙依赖的植物血凝素超家族,主要在肝、胰和胃肠道发挥作用,是一组分泌性蛋白,与糖尿病、组织损伤、炎症、肿瘤尤其是消化道肿瘤密切相关。根据Reg基因编码蛋白的一级结构, Reg家族可分为四个亚型(Ⅰ、Ⅱ、Ⅲ、Ⅳ)。RegⅣ最早由Hartupee于2001首次从炎症性肠病病人中分离。RegⅣ在正常人的胃肠道即有低表达,包括结肠、小肠、胃和胰腺。另外在结肠、胆囊、前列腺及胃等肿瘤组织中均可发现RegⅣ表达显著升高。目前关于RegⅣ的研究多集中其与胃肠道肿瘤的关系,研究表明RegⅣV起到了促增殖和抗凋亡的作用,但是在IBD中的具体表达及其在炎症性肠病中的机制尚不清楚;炎症性肠病中RegⅣ与炎症指标如C反应蛋白及疾病活动度的关系仍有待研究。因此我们运用免疫组化分析RegⅣ在UC、CD及正常人肠粘膜上皮细胞中的表达情况。并进一步研究了Reg IV表达与CRP、CDAI、Mayo指数的关系；以明确RegⅣ是否可作为评价IBD活动度的新的指标。
方法
1临床取材收集了2008年1月-2010年1月在我院消化内科门诊和住院的IBD患者病变部位肠粘膜活检标本61例,其中CD23例,UC38例。同时还另外收集了25例健康体检者的肠粘膜标本。所有检查对象在征求同意后,行结肠镜或双气囊小肠镜检查并取活检,所有IBD患者的病例资料至少由两位消化专科医师重新复习病史和资料,统一按照中华医学会消化病学分会炎症性肠病学科组制定的诊断指南进行诊断,并经组织病理学活检得到确诊。健康体检者的肠粘膜标本取自排除显著器质性病变且肠镜下诊断为正常肠道的健康体检者。同时采集了所有研究对象的血常规及生化指标。IBD的活动度主要参考CDAI (Crohn's disease activity index)和Mayo指数(也称作Southerland DAI)。
2免疫组织化学：采用免疫组化方法检测RegⅣ在IBD组及健康体检组中的蛋白表达情况。由两名病理科医师在不知道临床资料的情况下独立判定免疫组化染色的结果。染色结果综合染色强度及阳性细胞百分数两个方面进行半定量分析。
3病情分期溃疡性结肠炎及克罗恩病均根据临床资料分为活动期和缓解期。评价UC活动度采用Mayo指数,也称作Southerland疾病活动指数,较为简单实用,包括腹泻、便血、粘膜表现、医师评估病情几个评价指标,求得每例患者的分值。评价CD的活动度采用Best的CDAI计算法。根据腹痛、腹泻、腹块等8个变量,通过1周观察计分,乘以规定的权重,求得各自分值,再计算积分。
4病理学诊断所有活检标本均送南方医院病理科进行处理由两名资深病理医生进行分析。病理诊断标准主要参考中华医学会消化病学分会炎症性肠病协作组于2007年制定的我国炎症性肠病诊断规范的共识意见。
5 RT-PCR检测RegⅣmRNA水平采用逆转录-聚合酶链反应技术(RT-PCR)检测IBD患者及健康体检者中的mRNA表达情况。
6统计学分析结果以平均值±标准差((?)x±s)表示,RegⅣ表达情况在IBD及健康对照者之间的比较、RegⅣ表达情况在UC及CD中的比较、RegⅣ表达情况在活动期及缓解期UC中的比较,RegⅣ表达情况在UC及健康对照者中的比较、RegⅣ表达情况在活动期CD及缓解期CD中的比较均采用Mann-WhitneyU法。多组之间比较采用One-Way ANOVA检验。CRP与CDAI、Mayo指数相关性采用Pearson相关分析。所有的统计结果以P<0.05作为有统计学意义：参数比较前均进行正态性检验和方差齐性检验,在符合正态分布和方差齐性的基础上进行比较。
结果
1.RegⅣ在IBD病人及健康人中的表达情况
我们通过免疫组化检查观察炎症性肠病病人及健康查体者肠粘膜中RegⅣ表达情况。免疫组化结果显示RegⅣ蛋白主要着色于肠粘膜上皮细胞的胞浆。在健康查体者中Reg IV的表达情况是：未表达(0例),弱表达(19例,76%),中度表达(4例,16%),强表达(2例,8%)。在IBD患者中,未表达(0例),弱表达(17例,27.9%),中度表达(17例,27.9%),强表达(27例,44.2%)。
Reg IV在炎症性肠病病人肠粘膜中的表达较健康查体者显著增加(P<0.05)。CD与UC病人Reg IV的表达没有显著差异(P=0.503)。通过比较活动期UC病人较非活动期UC,我们发现前者肠粘膜Reg IV表达显著增加(P<0.05)。而非活动期UC病人与健康查体者之间RegⅣ的表达没有显著差异(P=0.406)。CD无论活动与否,其Reg IV的表达量差异不大(P=0.515)。
RT-PCR结果提示活动期UC中RegⅣ在mRNA水平明显高于缓解期及健康人,而活动期与缓解期CD中RegⅣ在mRNA水平均高于健康人。RT-PCR与免疫组化具有较好的一致性。
2. RegⅣ表达与IBD病人活动程度及C反应蛋白之间的关系
接下来我们研究了RegⅣ表达水平与血清CRP浓度之间的关系。在UC病人中,血清CRP浓度随着RegⅣ表达的增强而升高(P<0.05)：RegⅣ在弱表达的病人CRP为5.59±4.59mg/l,中度表达时CRP为13.73±10.86mg/l,而强表达时,CRP为88.33±47.99mg/l。而在CD病人中,不同RegⅣ表达水平病人所对应的CRP浓度并无显著差异(P=0.455),分别为弱表达：18.84±.60mg/l),中度表达：43.66±34.24mg/l,强表达41.90±41.48mg/l。
同时,我们研究了RegⅣ与炎症性肠病活动度之间的关系。我们发现在RegⅣ不同染色强度中Mayo指数分别为：弱表达4.58±1.88,中度表达6.20±1.32,强表达8.06±2.38。可见,在UC患者中,Mayo指数随着RegⅣ表达的增强而升高。(P<0.05)。在CD病人中, RegⅣ不同染色强度中CDAI指数分别为：弱表达148.40±38.12,中度表达179.14+64.87,强表达241.36±136.95,可见,不同RegⅣ表达水平所对应的CDAI指数并无显著差异(P=0.226)。CRP浓度与UC及CD的炎症活动度指数均呈正相关Mayo指数(r=0.627.,P<0.05),CDAI(r=0.624,P<0.05)。
结论
1、在正常人中,已有低水平的RegⅣ表达,而IBD可伴有RegⅣ表达水平的显著增加。活动期UC较缓解期的UCRegⅣ的表达增强；而CD无论活动与否,其RegⅣ的表达量差异不大。
2、在UC病人中,血清CRP浓度、Mayo指数随着RegⅣ表达的增强而升高；而在CD病人中,不同RegⅣ表达水平病人所对应的血清CRP浓度及CDAI旨数并无显著差异,这表明RegⅣ与CRP、Mayo旨数一样可作为评价UC活动度的指标。
3、肠粘膜活检标本中RegⅣ的免疫组化染色有可能成为一种评价UC活动度的新指标,补充常规内镜病理活检的不足,但尚需大样本研究以进一步证实。
Background
Inflammatory bowel disease (IBD) is a complex inflammatory disease of the gastrointestinal tract with unknown cause that lacks molecular markers for diagnosis. Crohn's disease(CD) and ulcerative colitis (UC) are the two major forms of IBD. The activity and severity of IBD is of great importance in diagnosis and treatment monitoring. CRP is one of the acute-phase proteins mostly studied in IBD, being used as a good inflammation marker for diagnosis, the evaluation of disease activity and risk of complications, the prediction of relapse, and treatment monitoring. Some studies have evaluated the association between CRP and clinical scores in IBD, showing a strong association of CRP with disease activity in Crohn's disease and modest association in ulcerative colitis. Despite having some advantages compared with other indices, CRP is far from an ideal parameter for these conditions. To evaluate the activity and severity of IBD, Many kinds of DAI (disease activity index) were presented.The Mayo Index is the most widely used one in UC,and BEST CDAI in CD.Regenerating gene IV (REG IV), within the calcium depending lectin gene super superfamily, is mainly involved in the liver, pancreatic, gastrointestinal cell proliferation, differentiation or inflammation. Several studies have shown that Reg IV may be involved in inflammatory bowel disease, but its role is not clear.
Material and methods
1.Material
Sixty-one IBD patients (38 ulcerative colitis,23 Crohn's disease] and twenty-five healthy controls were included in the study Biopsy specimens from patients with IBD and healthy controls were used for evaluating the expression of REGⅣ. The clinical activity of disease was determined by the Crohn's disease activity index (CDAI) and the colitis activity index (Mayo Index). The protocol of this study was approved by the Ethics Committee of Nanfang hospital.
2.Immunohistochemistry
Immunohistochemical staining for REGⅣwas performed with an SABC Kit Briefly, the paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded ethanol solutions (100%,95%,80%,70%) and phosphate-buffered saline (PBS). Subsequently they were subjected to heating antigen retrieval using a retrieval solution. Incubation with anti-Reg IV monoclonal antibody (SantaCruz Biotechnology, Santa Cruz, California, USA) was carried out at 1/50-1/100 dilution for 12 hours at 4℃. Detection was achieved using a standard staining system and antigen localization was visualized with DAB. The sections were counterstained with hematoxyline-eosin and mounted. we scored both staining qualities as either negative or weakly, moderately, or strongly positive.
3. The clinical activity of disease
Determination of CRP concentrations was performed at the Department of Clinical Chemistry at Nanfang hospital. Serum CRP concentrations were measured using a latex based immunoassay (N Latex CRP mono, Dade Behring, Behring Laser Nephelometer, Marburg, Germany). At the day of the CRP measurement, the CDAI or Mayo Index was determined in these patients.
4.Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from cells using Trizol (Invitrogen). RNA samples (3μg) were subjected to reverse transcription using a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas). The PCR was initiated by 5 min incubation at 94℃, ended after a 10 min extension at 72℃,36 cycles for denaturation at 94℃for 30s, annealing at 55℃, and extension 72℃for 1 min using PCR kit (SBS Genetech Co. Ltd., Beijing, China).
5. Ethical considerations
All patients in the study consented to participate, and the study was approved by the Regional Ethical Committee (REC) at the Nanfang hospital.
6. Statistical analysis
The Mann-Whitney U-test were used for single comparisons, and One way ANOVA was used for multiple comparisons. The association of CDAI or Mayo Index with CRP was assessed by means of Pearson's correlation coefficient. Data are presented as mean and s.d. if not otherwise stated. P-values less than 0.05 were considered significant.
Results
1. The expression of RegⅣ
Immunohistochemical staining of RegⅣwas performed in normal controls and patients with UC or CD. RegⅣexpression was categorized as 1+(weak); 2+ (moderate); 3+(strong). The immunostaining of RegⅣin normal tissues showed no signal stain in 0 cases, weak stain in 19 cases (76%), moderate stain in 4 cases (16%) and strong stain in 8%(2 cases). In contrast, RegⅣimmunostaining in IBD tissues showed no signal stain in 0 cases (0%), weak stain in 17 cases (27.9%), moderate stain in 17 cases (27.9%) and strong stain in 27 cases (44.2%)
RegⅣexpression was significantly upregulated in IBD, compared with the control group (P<0.05). There were no significantly differences of RegⅣexpression between the UC and the CD (P=0.503). Specimens of active UC had significantly different RegⅣexpression compared with that of inactive UC (P<0.05). RegⅣexpression in inactive UC mucosa had no significantly differences compared with normal controls (P=0.406). Specimens of active CD had no significant difference with that of inactive CD (P=0.515)..
The result of the RT-PCR also showed that the level of the RegⅣmRNA was corresponding with the Immunohistochemical staining of RegⅣ
2. The relationship between RegⅣexpression and activity parameters in IBD
RegⅣexpression and serum CRP concentrations were examined in patients with IBD. Among patients with UC, the serum CRP concentration was 88.33±47.99mg/l for strong RegⅣstaining,13.73±10.86 mg/1 for moderate staining and for weak staining 5.59±4.59mg/l (P<0.05). Among patients with CD,the serum CRP concentration was 41.90±41.48mg/l for strong RegⅣstaining,43.66±34.24 mg/l for moderate staining and for weak staining 18.84±20.60mg/l (P=0.455). For UC patients, CRP concentration was significantly higher in patients with strong RegⅣstaining than in patients with weak staining (P<0.05). With regard to CD patients, there was no significantly difference in patients with strong RegⅣstaining than in patients with moderate or weak staining (P=0.455).
Next, we examined the relationship between clinical disease activities and RegⅣexpression. There was a significant difference in Mayo Index between weak, moderate and strong Reg IV groups(4.58±1.88,6.20±1.32 and 8.06±2.38 respectively, P<0.05). There was no significant difference in CDAI between weak, moderate and strong Reg IV groups(148.40±38.12,179.14±64.87 and 241.16±136.95 respectively, P=0.226). The concentration of CRP positivly correlated with Mayo Index (r= 0.627, P<0.05),and CDAI (r=0.624, P<0.05).
Conclusion
The results of this study showed that RegⅣexpression in mucosa of IBD patients was significantly higher than that of healthy controls.We also found that there was a low expression of RegⅣin normal colonic mucosa, but inflammation caused a significant increase in its expression. The expression of RegⅣincreased with an increasing degree of inflammation in UC.
For UC patients, CRP concentration was significantly higher in patients with strong RegⅣstaining than in patients with weak staining.With regard to CD, There was no significantly difference in patients with strong RegⅣstaining than in patients with moderate or weak staining.
We also examined the relationship between the activity of IBD and RegⅣexpression. There was a significant difference in Mayo Index between weak, moderate and strong RegⅣgroups, There was no significant difference in CDAI between weak, moderate and strong RegⅣgroups.
In our study, we found positive correlation between CRP concentration and the activity of IBD, as represented by Mayo Index and CDAI.
In summary, this study suggested that the expression of RegⅣwas increased in IBD patients compared with that of healthy controls.And RegⅣcould be an independent predictor of disease activity in UC patients.
炎症性肠病(inflammatory bowel disease, IBD)是一组病因未明的非特异性肠道炎症性疾病,包括溃疡性结肠炎(ulcerative colitis, UC)和克罗恩病(Crohn's disease, CD)。近二十余年来,随着人们生活方式的变化和诊断水平的提高,国内炎症性肠病的发病率有逐年上升的趋势。疾病是处于活动期还是静止期直接关系到疾病的正确治疗。IBD临床表现复杂多变,诊断缺乏特异性的指标,而且常因对病情活动的判断不准确而延误治疗。目前临床评价IBD活动与否主要通过临床症状、实验室生化指标、内镜下的表现及病理等综合判断。其中C-反应蛋白(CRP)几乎全部由肝脏产生,短时间内(数小时)即可升高,而机体一旦恢复,CRP又迅速恢复正常(6～7d)。CRP可及时而敏感地反映炎症活动,是目前临床最为推崇的IBD活动性指标之一。另外,各国专家针对IBD制定出疾病活动指数(disease activity index),对疾病的活动性进行量化处理,从而更客观准确地反映疾病的活动性,帮助临床医师作出正确的医疗决策。评价UC活动度常采用Southerland疾病活动指数,也称Mayo指数,较为简单实用,包括腹泻、便血、粘膜表现、医师评估病情几个评价指标。Best的CDAI计算法是国际上广泛应用于临床和科研的评价CD活动度的指标。根据腹痛、腹泻、腹块等8个变量,通过1周观察计分,乘以规定的权重,求得各自分值,再计算积分。但少数患者尤其是某些早期和复发患者,血生化表现不显著,而且在疾病活动的早期内镜也不能发现。有必要寻找新指标以更全面、准确地评价IBD的炎症活动度,为临床应用奠定理论基础。
再生基因(regenerating gene, Reg)家族属钙依赖的植物血凝素超家族,主要在肝、胰和胃肠道发挥作用,是一组分泌性蛋白,与糖尿病、组织损伤、炎症、肿瘤尤其是消化道肿瘤密切相关。根据Reg基因编码蛋白的一级结构, Reg家族可分为四个亚型(Ⅰ、Ⅱ、Ⅲ、Ⅳ)。RegⅣ最早由Hartupee于2001首次从炎症性肠病病人中分离。RegⅣ在正常人的胃肠道即有低表达,包括结肠、小肠、胃和胰腺。另外在结肠、胆囊、前列腺及胃等肿瘤组织中均可发现RegⅣ表达显著升高。目前关于RegⅣ的研究多集中其与胃肠道肿瘤的关系,研究表明RegⅣV起到了促增殖和抗凋亡的作用,但是在IBD中的具体表达及其在炎症性肠病中的机制尚不清楚;炎症性肠病中RegⅣ与炎症指标如C反应蛋白及疾病活动度的关系仍有待研究。因此我们运用免疫组化分析RegⅣ在UC、CD及正常人肠粘膜上皮细胞中的表达情况。并进一步研究了Reg IV表达与CRP、CDAI、Mayo指数的关系；以明确RegⅣ是否可作为评价IBD活动度的新的指标。
方法
1临床取材收集了2008年1月-2010年1月在我院消化内科门诊和住院的IBD患者病变部位肠粘膜活检标本61例,其中CD23例,UC38例。同时还另外收集了25例健康体检者的肠粘膜标本。所有检查对象在征求同意后,行结肠镜或双气囊小肠镜检查并取活检,所有IBD患者的病例资料至少由两位消化专科医师重新复习病史和资料,统一按照中华医学会消化病学分会炎症性肠病学科组制定的诊断指南进行诊断,并经组织病理学活检得到确诊。健康体检者的肠粘膜标本取自排除显著器质性病变且肠镜下诊断为正常肠道的健康体检者。同时采集了所有研究对象的血常规及生化指标。IBD的活动度主要参考CDAI (Crohn's disease activity index)和Mayo指数(也称作Southerland DAI)。
2免疫组织化学：采用免疫组化方法检测RegⅣ在IBD组及健康体检组中的蛋白表达情况。由两名病理科医师在不知道临床资料的情况下独立判定免疫组化染色的结果。染色结果综合染色强度及阳性细胞百分数两个方面进行半定量分析。
3病情分期溃疡性结肠炎及克罗恩病均根据临床资料分为活动期和缓解期。评价UC活动度采用Mayo指数,也称作Southerland疾病活动指数,较为简单实用,包括腹泻、便血、粘膜表现、医师评估病情几个评价指标,求得每例患者的分值。评价CD的活动度采用Best的CDAI计算法。根据腹痛、腹泻、腹块等8个变量,通过1周观察计分,乘以规定的权重,求得各自分值,再计算积分。
4病理学诊断所有活检标本均送南方医院病理科进行处理由两名资深病理医生进行分析。病理诊断标准主要参考中华医学会消化病学分会炎症性肠病协作组于2007年制定的我国炎症性肠病诊断规范的共识意见。
5 RT-PCR检测RegⅣmRNA水平采用逆转录-聚合酶链反应技术(RT-PCR)检测IBD患者及健康体检者中的mRNA表达情况。
6统计学分析结果以平均值±标准差((?)x±s)表示,RegⅣ表达情况在IBD及健康对照者之间的比较、RegⅣ表达情况在UC及CD中的比较、RegⅣ表达情况在活动期及缓解期UC中的比较,RegⅣ表达情况在UC及健康对照者中的比较、RegⅣ表达情况在活动期CD及缓解期CD中的比较均采用Mann-WhitneyU法。多组之间比较采用One-Way ANOVA检验。CRP与CDAI、Mayo指数相关性采用Pearson相关分析。所有的统计结果以P<0.05作为有统计学意义：参数比较前均进行正态性检验和方差齐性检验,在符合正态分布和方差齐性的基础上进行比较。
结果
1.RegⅣ在IBD病人及健康人中的表达情况
我们通过免疫组化检查观察炎症性肠病病人及健康查体者肠粘膜中RegⅣ表达情况。免疫组化结果显示RegⅣ蛋白主要着色于肠粘膜上皮细胞的胞浆。在健康查体者中Reg IV的表达情况是：未表达(0例),弱表达(19例,76%),中度表达(4例,16%),强表达(2例,8%)。在IBD患者中,未表达(0例),弱表达(17例,27.9%),中度表达(17例,27.9%),强表达(27例,44.2%)。
Reg IV在炎症性肠病病人肠粘膜中的表达较健康查体者显著增加(P<0.05)。CD与UC病人Reg IV的表达没有显著差异(P=0.503)。通过比较活动期UC病人较非活动期UC,我们发现前者肠粘膜Reg IV表达显著增加(P<0.05)。而非活动期UC病人与健康查体者之间RegⅣ的表达没有显著差异(P=0.406)。CD无论活动与否,其Reg IV的表达量差异不大(P=0.515)。
RT-PCR结果提示活动期UC中RegⅣ在mRNA水平明显高于缓解期及健康人,而活动期与缓解期CD中RegⅣ在mRNA水平均高于健康人。RT-PCR与免疫组化具有较好的一致性。
2. RegⅣ表达与IBD病人活动程度及C反应蛋白之间的关系
接下来我们研究了RegⅣ表达水平与血清CRP浓度之间的关系。在UC病人中,血清CRP浓度随着RegⅣ表达的增强而升高(P<0.05)：RegⅣ在弱表达的病人CRP为5.59±4.59mg/l,中度表达时CRP为13.73±10.86mg/l,而强表达时,CRP为88.33±47.99mg/l。而在CD病人中,不同RegⅣ表达水平病人所对应的CRP浓度并无显著差异(P=0.455),分别为弱表达：18.84±.60mg/l),中度表达：43.66±34.24mg/l,强表达41.90±41.48mg/l。
同时,我们研究了RegⅣ与炎症性肠病活动度之间的关系。我们发现在RegⅣ不同染色强度中Mayo指数分别为：弱表达4.58±1.88,中度表达6.20±1.32,强表达8.06±2.38。可见,在UC患者中,Mayo指数随着RegⅣ表达的增强而升高。(P<0.05)。在CD病人中, RegⅣ不同染色强度中CDAI指数分别为：弱表达148.40±38.12,中度表达179.14+64.87,强表达241.36±136.95,可见,不同RegⅣ表达水平所对应的CDAI指数并无显著差异(P=0.226)。CRP浓度与UC及CD的炎症活动度指数均呈正相关Mayo指数(r=0.627.,P<0.05),CDAI(r=0.624,P<0.05)。
结论
1、在正常人中,已有低水平的RegⅣ表达,而IBD可伴有RegⅣ表达水平的显著增加。活动期UC较缓解期的UCRegⅣ的表达增强；而CD无论活动与否,其RegⅣ的表达量差异不大。
2、在UC病人中,血清CRP浓度、Mayo指数随着RegⅣ表达的增强而升高；而在CD病人中,不同RegⅣ表达水平病人所对应的血清CRP浓度及CDAI旨数并无显著差异,这表明RegⅣ与CRP、Mayo旨数一样可作为评价UC活动度的指标。
3、肠粘膜活检标本中RegⅣ的免疫组化染色有可能成为一种评价UC活动度的新指标,补充常规内镜病理活检的不足,但尚需大样本研究以进一步证实。
Background
Inflammatory bowel disease (IBD) is a complex inflammatory disease of the gastrointestinal tract with unknown cause that lacks molecular markers for diagnosis. Crohn's disease(CD) and ulcerative colitis (UC) are the two major forms of IBD. The activity and severity of IBD is of great importance in diagnosis and treatment monitoring. CRP is one of the acute-phase proteins mostly studied in IBD, being used as a good inflammation marker for diagnosis, the evaluation of disease activity and risk of complications, the prediction of relapse, and treatment monitoring. Some studies have evaluated the association between CRP and clinical scores in IBD, showing a strong association of CRP with disease activity in Crohn's disease and modest association in ulcerative colitis. Despite having some advantages compared with other indices, CRP is far from an ideal parameter for these conditions. To evaluate the activity and severity of IBD, Many kinds of DAI (disease activity index) were presented.The Mayo Index is the most widely used one in UC,and BEST CDAI in CD.Regenerating gene IV (REG IV), within the calcium depending lectin gene super superfamily, is mainly involved in the liver, pancreatic, gastrointestinal cell proliferation, differentiation or inflammation. Several studies have shown that Reg IV may be involved in inflammatory bowel disease, but its role is not clear.
Material and methods
1.Material
Sixty-one IBD patients (38 ulcerative colitis,23 Crohn's disease] and twenty-five healthy controls were included in the study Biopsy specimens from patients with IBD and healthy controls were used for evaluating the expression of REGⅣ. The clinical activity of disease was determined by the Crohn's disease activity index (CDAI) and the colitis activity index (Mayo Index). The protocol of this study was approved by the Ethics Committee of Nanfang hospital.
2.Immunohistochemistry
Immunohistochemical staining for REGⅣwas performed with an SABC Kit Briefly, the paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded ethanol solutions (100%,95%,80%,70%) and phosphate-buffered saline (PBS). Subsequently they were subjected to heating antigen retrieval using a retrieval solution. Incubation with anti-Reg IV monoclonal antibody (SantaCruz Biotechnology, Santa Cruz, California, USA) was carried out at 1/50-1/100 dilution for 12 hours at 4℃. Detection was achieved using a standard staining system and antigen localization was visualized with DAB. The sections were counterstained with hematoxyline-eosin and mounted. we scored both staining qualities as either negative or weakly, moderately, or strongly positive.
3. The clinical activity of disease
Determination of CRP concentrations was performed at the Department of Clinical Chemistry at Nanfang hospital. Serum CRP concentrations were measured using a latex based immunoassay (N Latex CRP mono, Dade Behring, Behring Laser Nephelometer, Marburg, Germany). At the day of the CRP measurement, the CDAI or Mayo Index was determined in these patients.
4.Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from cells using Trizol (Invitrogen). RNA samples (3μg) were subjected to reverse transcription using a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas). The PCR was initiated by 5 min incubation at 94℃, ended after a 10 min extension at 72℃,36 cycles for denaturation at 94℃for 30s, annealing at 55℃, and extension 72℃for 1 min using PCR kit (SBS Genetech Co. Ltd., Beijing, China).
5. Ethical considerations
All patients in the study consented to participate, and the study was approved by the Regional Ethical Committee (REC) at the Nanfang hospital.
6. Statistical analysis
The Mann-Whitney U-test were used for single comparisons, and One way ANOVA was used for multiple comparisons. The association of CDAI or Mayo Index with CRP was assessed by means of Pearson's correlation coefficient. Data are presented as mean and s.d. if not otherwise stated. P-values less than 0.05 were considered significant.
Results
1. The expression of RegⅣ
Immunohistochemical staining of RegⅣwas performed in normal controls and patients with UC or CD. RegⅣexpression was categorized as 1+(weak); 2+ (moderate); 3+(strong). The immunostaining of RegⅣin normal tissues showed no signal stain in 0 cases, weak stain in 19 cases (76%), moderate stain in 4 cases (16%) and strong stain in 8%(2 cases). In contrast, RegⅣimmunostaining in IBD tissues showed no signal stain in 0 cases (0%), weak stain in 17 cases (27.9%), moderate stain in 17 cases (27.9%) and strong stain in 27 cases (44.2%)
RegⅣexpression was significantly upregulated in IBD, compared with the control group (P<0.05). There were no significantly differences of RegⅣexpression between the UC and the CD (P=0.503). Specimens of active UC had significantly different RegⅣexpression compared with that of inactive UC (P<0.05). RegⅣexpression in inactive UC mucosa had no significantly differences compared with normal controls (P=0.406). Specimens of active CD had no significant difference with that of inactive CD (P=0.515)..
The result of the RT-PCR also showed that the level of the RegⅣmRNA was corresponding with the Immunohistochemical staining of RegⅣ
2. The relationship between RegⅣexpression and activity parameters in IBD
RegⅣexpression and serum CRP concentrations were examined in patients with IBD. Among patients with UC, the serum CRP concentration was 88.33±47.99mg/l for strong RegⅣstaining,13.73±10.86 mg/1 for moderate staining and for weak staining 5.59±4.59mg/l (P<0.05). Among patients with CD,the serum CRP concentration was 41.90±41.48mg/l for strong RegⅣstaining,43.66±34.24 mg/l for moderate staining and for weak staining 18.84±20.60mg/l (P=0.455). For UC patients, CRP concentration was significantly higher in patients with strong RegⅣstaining than in patients with weak staining (P<0.05). With regard to CD patients, there was no significantly difference in patients with strong RegⅣstaining than in patients with moderate or weak staining (P=0.455).
Next, we examined the relationship between clinical disease activities and RegⅣexpression. There was a significant difference in Mayo Index between weak, moderate and strong Reg IV groups(4.58±1.88,6.20±1.32 and 8.06±2.38 respectively, P<0.05). There was no significant difference in CDAI between weak, moderate and strong Reg IV groups(148.40±38.12,179.14±64.87 and 241.16±136.95 respectively, P=0.226). The concentration of CRP positivly correlated with Mayo Index (r= 0.627, P<0.05),and CDAI (r=0.624, P<0.05).
Conclusion
The results of this study showed that RegⅣexpression in mucosa of IBD patients was significantly higher than that of healthy controls.We also found that there was a low expression of RegⅣin normal colonic mucosa, but inflammation caused a significant increase in its expression. The expression of RegⅣincreased with an increasing degree of inflammation in UC.
For UC patients, CRP concentration was significantly higher in patients with strong RegⅣstaining than in patients with weak staining.With regard to CD, There was no significantly difference in patients with strong RegⅣstaining than in patients with moderate or weak staining.
We also examined the relationship between the activity of IBD and RegⅣexpression. There was a significant difference in Mayo Index between weak, moderate and strong RegⅣgroups, There was no significant difference in CDAI between weak, moderate and strong RegⅣgroups.
In our study, we found positive correlation between CRP concentration and the activity of IBD, as represented by Mayo Index and CDAI.
In summary, this study suggested that the expression of RegⅣwas increased in IBD patients compared with that of healthy controls.And RegⅣcould be an independent predictor of disease activity in UC patients.
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