沙利度胺对儿童克罗恩病的疗效评估及其抑制血管生成的机制研究
发布时间：2018-08-18 07:56
【摘要】：克罗恩病(CD)属于炎症性肠病(IBD),是一种病因不明的慢性肠道非特异性炎性疾病。目前国内报道的IBD发病率越来越高,逐渐受到大家的重视,其中儿童以CD较为多见。尽管治疗IBD的药物种类很多,但难治性1BD的治疗仍是一大难题。IBD的发病机制尚未明确,近期多项研究表明新生血管形成参与整个疾病的发生发展过程,针对促血管生成相关因子在IBD中的表达作用成为研究热点,其中血管内皮生长因子(VEGF)和血管生成素(Ang)被认为在IBD发病机制中发挥着重要作用。我们前期研究发现沙利度胺治疗儿童难治性IBD有良好的临床疗效,另外研究报道沙利度胺有抑制血管生成的能力。本研究拟进行三部分实验：1)临床上观察沙利度胺治疗儿童CD一年内的临床疗效；2)从临床标本水平研究血管生成在儿童CD中的作用及沙利度胺对血管生成的影响；3)在体外细胞水平进一步研究沙利度胺对血管内皮细胞的作用及机制。通过以上实验来揭示沙利度胺治疗儿童CD的临床疗效及安全性,探讨血管生成在儿童CD发病机制中的作用及沙利度胺对血管生成的影响和机制,为沙利度胺的治疗提供理论依据。第一部分沙利度胺治疗儿童克罗恩病的临床疗效和安全性[目的]评估沙利度胺治疗激素不敏感、激素依赖或合并有结核感染的儿童克罗恩病(CD)的临床疗效及其安全性。[方法]收集2006年11月至2012年8月间诊断为克罗恩病并开始使用沙利度胺治疗的儿童或青少年病例资料。沙利度胺的初始剂量均为2mg/kg/day,在治疗初及治疗后1月、3月、6月及12月进行随访观察,采用儿童克罗恩病疾病活动评分指数(PCDAI)评估临床疗效,检测相关实验室检查指标CRP、ESR、PLT和Hb,记录患儿治疗期间激素用药减停情况,采用年龄别体重Z评分评价治疗前后患儿生长发育状况,同时密切观察治疗期间的不良反应。[结果](1)临床特点：共17例CD患儿使用沙利度胺治疗,其中男9例,女8例,治疗初始平均年龄为11.8(1.7～20)岁,诊断CD平均年龄为10.2(1.1～14.1)岁。17例患儿中8例因激素治疗不敏感、5例因激素依赖、4例因合并有结核感染而使用沙利度胺治疗。(2)临床疗效：所有患儿治疗初始平均PCDAI为41.0±11.9,2例患儿随访3月内自行退出试验,余15.例中14例在治疗3月后达到临床缓解(PCDAI≤10),15例患儿治疗12月时均达临床缓解。治疗后1月、3月、6月、12月平均PCDAI为15.2±9.6、5.3±5.0、3.0±3.4、2.3±2.6。治疗前后比较,ESR、CRP、PLT均下降,Hb则逐渐升高。15例患儿治疗后体重均呈明显上升,平均体重增加11.6±6.4kg,治疗前和治疗后12月时年龄别体重Z评分分别为-1.56、0.02,差异具有统计学意义(P0.05)。12例使用激素治疗的患儿在沙利度胺治疗开始后激素逐渐减量,治疗12月时11例患儿停用激素,1例采用小剂量布地奈德维持缓解。(3)不良反应：随访期间2例患儿出现嗜睡、2例有乏力症状,经休息或药物减量后均缓解。1例患儿在用药近一年后出现一过性肝功能轻度异常,给予护肝药物及停用沙利度胺后恢复正常。[结论]沙利度胺治疗激素不敏感、激素依赖或合并结核杆菌感染的CD患儿临床疗效显著,不良反应小,其长期治疗的安全性有待进一步随访观察。第二部分血管生成在儿童克罗恩病中的作用及沙利度胺对其影响[目的]研究血管生成相关因子在克罗恩病患儿肠道黏膜组织中的表达情况以及沙利度胺对血管生成分子的作用。[方法]收集CD患儿采用沙利度胺治疗前、治疗后病例及正常对照组病例肠道黏膜标本各10例,采用免疫组化法检测各组组织标本VEGF、Ang-1、Ang-2、 Tie-2蛋白的表达,同时采用CD31标记血管内皮细胞,计算组织微血管密度(MVD),判断各组间表达水平是否存在差异。采用荧光定量PCR法检测三组间Ang-1、Ang-2 mRNA表达,Western blot法检测沙利度胺治疗前后VEGF、 Ang-1、Ang-2蛋白表达。[结果]沙利度胺治疗前CD患儿肠道黏膜标本中CD31、VEGF、Ang-2表达较正常对照组显著升高(P0.05), Ang-1、Tie-2表达无统计学差异。沙利度胺治疗后达临床缓解CD患儿肠道黏膜组织CD31、VEGF、Ang-2、Tie-2的表达均较治疗前降低(P0.05)。沙利度胺治疗前Ang-2 mRNA表达水平较正常对照组高,治疗后表达水平降低(P0.05),三组间比较Ang-1 mRNA表达水平无显著差异,与免疫组化结果一致。Western blot检测沙利度胺治疗后VEGF、Ang-2蛋白水平下调(P0.05),Ang-1表达无明显差异。[结论]血管生成因子VEGF、Ang-2在儿童克罗恩病的发病机制中起重要作用,沙利度胺可下调VEGF、Ang-2的表达,可能是其治疗有效的机制之一。第三部分沙利度胺对人脐静脉内皮细胞血管生成的作用和机制研究[目.的]研究沙利度胺对HUVEC细胞血管生成功能的影响及作用机制。[方法]体外培养人脐静脉内皮细胞,CCK8法检测空白对照、DMSO对照、和不同浓度沙利度胺干预条件下HUVEC增殖的变化,采用Transwell小室和Matrigel诱导的体外培养法检测在VEGF、Ang-2刺激条件下沙利度胺对内皮细胞迁移和管腔结构形成的影响,荧光定量PCR法检测LPS刺激条件下沙利度胺对VEGF、Ang-1、Ang-2 mRNA表达水平的影响,Western blot法测定沙利度胺对VEGF、Ang-2及血管生成相关信号通路分子表达的影响。[结果]CCK8检测发现沙利度胺浓度在10μ.M以上时能抑制HUVEC的增殖,干预24h后作用明显。沙利度胺对VEGF、Ang-2及两者联合刺激诱导的HUVEC迁移及管腔结构形成具有明显抑制作用(P0.05)。荧光定量PCR结果显示沙利度胺对LPS诱导的VEGF、Ang-2 mRNA水平有抑制作用(P0.05),呈浓度依赖性,对Ang-1水平则无明显抑制作用,Western blot结果显示沙利度胺可抑制VEGF、Ang-2、PI3K/Akt信号分子蛋白水平表达。[结论]体外实验表明,沙利度胺抑制人脐静脉内皮细胞的增殖、迁移及管腔形成,抑制VEGF、Ang-2的表达,抑制PI3K/AKT信号激活,从而抑制血管生成,可能是其治疗儿童克罗恩病有效的作用机制之一。
[Abstract]:Crohn's disease (CD) belongs to inflammatory bowel disease (IBD), is a chronic intestinal non-specific inflammatory disease with unknown etiology. At present, the incidence of IBD reported in China is getting higher and higher, and more attention has been paid to it, especially in children. Although there are many kinds of drugs for IBD, the treatment of refractory 1BD is still a difficult problem. Recent studies have shown that angiogenesis is involved in the pathogenesis of IBD. The role of angiogenesis-related factors in the expression of IBD has become a hot topic. Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) are considered to play an important role in the pathogenesis of IBD. Previous studies have found thalidomide has good clinical efficacy in the treatment of refractory IBD in children. In addition, studies have reported thalidomide has the ability to inhibit angiogenesis. The effect of thalidomide on angiogenesis and the effect of thalidomide on vascular endothelial cells in vitro were further studied. The clinical efficacy and safety of thalidomide in the treatment of children with CD were revealed through the above experiments, and the role of angiogenesis in the pathogenesis of children with CD and the effect of thalidomide on the blood supply of thalidomide were discussed. Objective To evaluate the clinical efficacy and safety of thalidomide in the treatment of childhood Crohn's disease (CD) with steroid insensitivity, steroid dependence or tuberculosis infection. Children and adolescents who were diagnosed with Crohn's disease from November 2006 to August 2012 and started thalidomide therapy were followed up at the initial dose of 2 mg/kg/day for 1 month, 3 months, 6 months and 12 months after treatment. The children's Crohn's disease activity score (PCDAI) was used to assess clinical treatment. Results (1) Clinical features: 17 children with CD were treated with thalidomide, of whom 9 were male. Eight of the 17 children with CD were insensitive to hormone therapy, five were hormone dependent, and four were treated with thalidomide because of concurrent tuberculosis infection. (2) Clinical efficacy: The average initial PCDAI of all the children was 41.0 (+ 11.9), and 2 were followed up. The average PCDAI in 1 month, 3 months, 6 months and 12 months after treatment was 15.2 (+ 9.6), 5.3 (+ 5.0), 3.0 (+ 3.4), 2.3 (+ 2.6). The average weight gain was 11.6 (+ 6.4 kg). The Z score of age-specific body weight before and 12 months after treatment was - 1.56 and 0.02, respectively. The difference was statistically significant (P 0.05). (3) Adverse reactions: During the follow-up period, 2 cases had sleepiness, 2 cases had fatigue symptoms, which were alleviated by rest or reduction of dosage. One case had slight abnormal liver function after nearly a year of medication, and returned to normal after giving hepatoprotective drugs and discontinuing thalidomide. The long-term safety of steroid-dependent or tuberculosis-associated CD patients needs further follow-up observation. Part II The role of angiogenesis in Crohn's disease in children and the effect of thalidomide on it [Objective] To study the role of angiogenesis-related factors in the intestinal mucosa of children with Crohn's disease. [Methods] The expression of VEGF, Ang-1, Ang-2 and Tie-2 in the intestinal mucosa of 10 children with CD were detected by immunohistochemistry, and the blood vessels were labeled by CD31. The expression of Ang-1, Ang-2 mRNA was detected by fluorescence quantitative PCR and the expression of VEGF, Ang-1 and Ang-2 protein was detected by Western blot before and after thalidomide treatment. After thalidomide treatment, the expression of CD31, VEGF, Ang-2 and Tie-2 in intestinal mucosa of children with CD was significantly lower than that before treatment (P There was no significant difference in the expression of Ang-1 mRNA among the three groups, which was consistent with the results of immunohistochemistry. After thalidomide treatment, the levels of VEGF and Ang-2 protein were down-regulated by Western blot (P 0.05), but the expression of Ang-1 was not significantly different. [Conclusion] Angiogenesis factors VEGF and Ang-2 play an important role in the pathogenesis of childhood Crohn's disease, thalidomide can be down-regulated. Part 3 The effect and mechanism of thalidomide on the angiogenesis of human umbilical vein endothelial cells [Objective] To study the effect and mechanism of thalidomide on the angiogenesis of HUVEC cells. Contrast, DMSO control, and thalidomide with different concentrations of thalidomide intervention in the proliferation of HUVEC. Transwell chamber and Matrigel-induced in vitro culture were used to detect thalidomide on the migration of endothelial cells and the formation of lumen structure under the stimulation of VEGF, Ang-2. Fluorescence quantitative PCR was used to detect thalidomide on the stimulation of LPS to VEGF, AN. Western blot was used to determine the effect of thalidomide on the expression of VEGF, Ang-2 and angiogenesis-related signaling pathway molecules. HUVEC migration and lumen formation were significantly inhibited by thalidomide (P 0.05). Fluorescence quantitative PCR showed thalidomide inhibited LPS-induced VEGF and Ang-2 mRNA levels in a concentration-dependent manner, but did not inhibit Ang-1 levels. Western blot showed thalidomide inhibited VEGF, Ang-2, PI3K/Akt signaling molecules. [Conclusion] Thalidomide can inhibit the proliferation, migration and lumen formation of human umbilical vein endothelial cells, inhibit the expression of VEGF and Ang-2, inhibit the activation of PI3K/AKT signal and thus inhibit angiogenesis, which may be one of the effective mechanisms of thalidomide in the treatment of Crohn's disease in children.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.7
本文编号：2188834
[Abstract]:Crohn's disease (CD) belongs to inflammatory bowel disease (IBD), is a chronic intestinal non-specific inflammatory disease with unknown etiology. At present, the incidence of IBD reported in China is getting higher and higher, and more attention has been paid to it, especially in children. Although there are many kinds of drugs for IBD, the treatment of refractory 1BD is still a difficult problem. Recent studies have shown that angiogenesis is involved in the pathogenesis of IBD. The role of angiogenesis-related factors in the expression of IBD has become a hot topic. Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) are considered to play an important role in the pathogenesis of IBD. Previous studies have found thalidomide has good clinical efficacy in the treatment of refractory IBD in children. In addition, studies have reported thalidomide has the ability to inhibit angiogenesis. The effect of thalidomide on angiogenesis and the effect of thalidomide on vascular endothelial cells in vitro were further studied. The clinical efficacy and safety of thalidomide in the treatment of children with CD were revealed through the above experiments, and the role of angiogenesis in the pathogenesis of children with CD and the effect of thalidomide on the blood supply of thalidomide were discussed. Objective To evaluate the clinical efficacy and safety of thalidomide in the treatment of childhood Crohn's disease (CD) with steroid insensitivity, steroid dependence or tuberculosis infection. Children and adolescents who were diagnosed with Crohn's disease from November 2006 to August 2012 and started thalidomide therapy were followed up at the initial dose of 2 mg/kg/day for 1 month, 3 months, 6 months and 12 months after treatment. The children's Crohn's disease activity score (PCDAI) was used to assess clinical treatment. Results (1) Clinical features: 17 children with CD were treated with thalidomide, of whom 9 were male. Eight of the 17 children with CD were insensitive to hormone therapy, five were hormone dependent, and four were treated with thalidomide because of concurrent tuberculosis infection. (2) Clinical efficacy: The average initial PCDAI of all the children was 41.0 (+ 11.9), and 2 were followed up. The average PCDAI in 1 month, 3 months, 6 months and 12 months after treatment was 15.2 (+ 9.6), 5.3 (+ 5.0), 3.0 (+ 3.4), 2.3 (+ 2.6). The average weight gain was 11.6 (+ 6.4 kg). The Z score of age-specific body weight before and 12 months after treatment was - 1.56 and 0.02, respectively. The difference was statistically significant (P 0.05). (3) Adverse reactions: During the follow-up period, 2 cases had sleepiness, 2 cases had fatigue symptoms, which were alleviated by rest or reduction of dosage. One case had slight abnormal liver function after nearly a year of medication, and returned to normal after giving hepatoprotective drugs and discontinuing thalidomide. The long-term safety of steroid-dependent or tuberculosis-associated CD patients needs further follow-up observation. Part II The role of angiogenesis in Crohn's disease in children and the effect of thalidomide on it [Objective] To study the role of angiogenesis-related factors in the intestinal mucosa of children with Crohn's disease. [Methods] The expression of VEGF, Ang-1, Ang-2 and Tie-2 in the intestinal mucosa of 10 children with CD were detected by immunohistochemistry, and the blood vessels were labeled by CD31. The expression of Ang-1, Ang-2 mRNA was detected by fluorescence quantitative PCR and the expression of VEGF, Ang-1 and Ang-2 protein was detected by Western blot before and after thalidomide treatment. After thalidomide treatment, the expression of CD31, VEGF, Ang-2 and Tie-2 in intestinal mucosa of children with CD was significantly lower than that before treatment (P There was no significant difference in the expression of Ang-1 mRNA among the three groups, which was consistent with the results of immunohistochemistry. After thalidomide treatment, the levels of VEGF and Ang-2 protein were down-regulated by Western blot (P 0.05), but the expression of Ang-1 was not significantly different. [Conclusion] Angiogenesis factors VEGF and Ang-2 play an important role in the pathogenesis of childhood Crohn's disease, thalidomide can be down-regulated. Part 3 The effect and mechanism of thalidomide on the angiogenesis of human umbilical vein endothelial cells [Objective] To study the effect and mechanism of thalidomide on the angiogenesis of HUVEC cells. Contrast, DMSO control, and thalidomide with different concentrations of thalidomide intervention in the proliferation of HUVEC. Transwell chamber and Matrigel-induced in vitro culture were used to detect thalidomide on the migration of endothelial cells and the formation of lumen structure under the stimulation of VEGF, Ang-2. Fluorescence quantitative PCR was used to detect thalidomide on the stimulation of LPS to VEGF, AN. Western blot was used to determine the effect of thalidomide on the expression of VEGF, Ang-2 and angiogenesis-related signaling pathway molecules. HUVEC migration and lumen formation were significantly inhibited by thalidomide (P 0.05). Fluorescence quantitative PCR showed thalidomide inhibited LPS-induced VEGF and Ang-2 mRNA levels in a concentration-dependent manner, but did not inhibit Ang-1 levels. Western blot showed thalidomide inhibited VEGF, Ang-2, PI3K/Akt signaling molecules. [Conclusion] Thalidomide can inhibit the proliferation, migration and lumen formation of human umbilical vein endothelial cells, inhibit the expression of VEGF and Ang-2, inhibit the activation of PI3K/AKT signal and thus inhibit angiogenesis, which may be one of the effective mechanisms of thalidomide in the treatment of Crohn's disease in children.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R725.7
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