沙利度胺對(duì)兒童克羅恩病的療效評(píng)估及其抑制血管生成的機(jī)制研究
發(fā)布時(shí)間：2018-08-18 07:56
【摘要】：克羅恩病(CD)屬于炎癥性腸病(IBD),是一種病因不明的慢性腸道非特異性炎性疾病。目前國(guó)內(nèi)報(bào)道的IBD發(fā)病率越來越高,逐漸受到大家的重視,其中兒童以CD較為多見。盡管治療IBD的藥物種類很多,但難治性1BD的治療仍是一大難題。IBD的發(fā)病機(jī)制尚未明確,近期多項(xiàng)研究表明新生血管形成參與整個(gè)疾病的發(fā)生發(fā)展過程,針對(duì)促血管生成相關(guān)因子在IBD中的表達(dá)作用成為研究熱點(diǎn),其中血管內(nèi)皮生長(zhǎng)因子(VEGF)和血管生成素(Ang)被認(rèn)為在IBD發(fā)病機(jī)制中發(fā)揮著重要作用。我們前期研究發(fā)現(xiàn)沙利度胺治療兒童難治性IBD有良好的臨床療效,另外研究報(bào)道沙利度胺有抑制血管生成的能力。本研究擬進(jìn)行三部分實(shí)驗(yàn)：1)臨床上觀察沙利度胺治療兒童CD一年內(nèi)的臨床療效；2)從臨床標(biāo)本水平研究血管生成在兒童CD中的作用及沙利度胺對(duì)血管生成的影響；3)在體外細(xì)胞水平進(jìn)一步研究沙利度胺對(duì)血管內(nèi)皮細(xì)胞的作用及機(jī)制。通過以上實(shí)驗(yàn)來揭示沙利度胺治療兒童CD的臨床療效及安全性,探討血管生成在兒童CD發(fā)病機(jī)制中的作用及沙利度胺對(duì)血管生成的影響和機(jī)制,為沙利度胺的治療提供理論依據(jù)。第一部分沙利度胺治療兒童克羅恩病的臨床療效和安全性[目的]評(píng)估沙利度胺治療激素不敏感、激素依賴或合并有結(jié)核感染的兒童克羅恩病(CD)的臨床療效及其安全性。[方法]收集2006年11月至2012年8月間診斷為克羅恩病并開始使用沙利度胺治療的兒童或青少年病例資料。沙利度胺的初始劑量均為2mg/kg/day,在治療初及治療后1月、3月、6月及12月進(jìn)行隨訪觀察,采用兒童克羅恩病疾病活動(dòng)評(píng)分指數(shù)(PCDAI)評(píng)估臨床療效,檢測(cè)相關(guān)實(shí)驗(yàn)室檢查指標(biāo)CRP、ESR、PLT和Hb,記錄患兒治療期間激素用藥減停情況,采用年齡別體重Z評(píng)分評(píng)價(jià)治療前后患兒生長(zhǎng)發(fā)育狀況,同時(shí)密切觀察治療期間的不良反應(yīng)。[結(jié)果](1)臨床特點(diǎn)：共17例CD患兒使用沙利度胺治療,其中男9例,女8例,治療初始平均年齡為11.8(1.7～20)歲,診斷CD平均年齡為10.2(1.1～14.1)歲。17例患兒中8例因激素治療不敏感、5例因激素依賴、4例因合并有結(jié)核感染而使用沙利度胺治療。(2)臨床療效：所有患兒治療初始平均PCDAI為41.0±11.9,2例患兒隨訪3月內(nèi)自行退出試驗(yàn),余15.例中14例在治療3月后達(dá)到臨床緩解(PCDAI≤10),15例患兒治療12月時(shí)均達(dá)臨床緩解。治療后1月、3月、6月、12月平均PCDAI為15.2±9.6、5.3±5.0、3.0±3.4、2.3±2.6。治療前后比較,ESR、CRP、PLT均下降,Hb則逐漸升高。15例患兒治療后體重均呈明顯上升,平均體重增加11.6±6.4kg,治療前和治療后12月時(shí)年齡別體重Z評(píng)分分別為-1.56、0.02,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。12例使用激素治療的患兒在沙利度胺治療開始后激素逐漸減量,治療12月時(shí)11例患兒停用激素,1例采用小劑量布地奈德維持緩解。(3)不良反應(yīng)：隨訪期間2例患兒出現(xiàn)嗜睡、2例有乏力癥狀,經(jīng)休息或藥物減量后均緩解。1例患兒在用藥近一年后出現(xiàn)一過性肝功能輕度異常,給予護(hù)肝藥物及停用沙利度胺后恢復(fù)正常。[結(jié)論]沙利度胺治療激素不敏感、激素依賴或合并結(jié)核桿菌感染的CD患兒臨床療效顯著,不良反應(yīng)小,其長(zhǎng)期治療的安全性有待進(jìn)一步隨訪觀察。第二部分血管生成在兒童克羅恩病中的作用及沙利度胺對(duì)其影響[目的]研究血管生成相關(guān)因子在克羅恩病患兒腸道黏膜組織中的表達(dá)情況以及沙利度胺對(duì)血管生成分子的作用。[方法]收集CD患兒采用沙利度胺治療前、治療后病例及正常對(duì)照組病例腸道黏膜標(biāo)本各10例,采用免疫組化法檢測(cè)各組組織標(biāo)本VEGF、Ang-1、Ang-2、 Tie-2蛋白的表達(dá),同時(shí)采用CD31標(biāo)記血管內(nèi)皮細(xì)胞,計(jì)算組織微血管密度(MVD),判斷各組間表達(dá)水平是否存在差異。采用熒光定量PCR法檢測(cè)三組間Ang-1、Ang-2 mRNA表達(dá),Western blot法檢測(cè)沙利度胺治療前后VEGF、 Ang-1、Ang-2蛋白表達(dá)。[結(jié)果]沙利度胺治療前CD患兒腸道黏膜標(biāo)本中CD31、VEGF、Ang-2表達(dá)較正常對(duì)照組顯著升高(P0.05), Ang-1、Tie-2表達(dá)無統(tǒng)計(jì)學(xué)差異。沙利度胺治療后達(dá)臨床緩解CD患兒腸道黏膜組織CD31、VEGF、Ang-2、Tie-2的表達(dá)均較治療前降低(P0.05)。沙利度胺治療前Ang-2 mRNA表達(dá)水平較正常對(duì)照組高,治療后表達(dá)水平降低(P0.05),三組間比較Ang-1 mRNA表達(dá)水平無顯著差異,與免疫組化結(jié)果一致。Western blot檢測(cè)沙利度胺治療后VEGF、Ang-2蛋白水平下調(diào)(P0.05),Ang-1表達(dá)無明顯差異。[結(jié)論]血管生成因子VEGF、Ang-2在兒童克羅恩病的發(fā)病機(jī)制中起重要作用,沙利度胺可下調(diào)VEGF、Ang-2的表達(dá),可能是其治療有效的機(jī)制之一。第三部分沙利度胺對(duì)人臍靜脈內(nèi)皮細(xì)胞血管生成的作用和機(jī)制研究[目.的]研究沙利度胺對(duì)HUVEC細(xì)胞血管生成功能的影響及作用機(jī)制。[方法]體外培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞,CCK8法檢測(cè)空白對(duì)照、DMSO對(duì)照、和不同濃度沙利度胺干預(yù)條件下HUVEC增殖的變化,采用Transwell小室和Matrigel誘導(dǎo)的體外培養(yǎng)法檢測(cè)在VEGF、Ang-2刺激條件下沙利度胺對(duì)內(nèi)皮細(xì)胞遷移和管腔結(jié)構(gòu)形成的影響,熒光定量PCR法檢測(cè)LPS刺激條件下沙利度胺對(duì)VEGF、Ang-1、Ang-2 mRNA表達(dá)水平的影響,Western blot法測(cè)定沙利度胺對(duì)VEGF、Ang-2及血管生成相關(guān)信號(hào)通路分子表達(dá)的影響。[結(jié)果]CCK8檢測(cè)發(fā)現(xiàn)沙利度胺濃度在10μ.M以上時(shí)能抑制HUVEC的增殖,干預(yù)24h后作用明顯。沙利度胺對(duì)VEGF、Ang-2及兩者聯(lián)合刺激誘導(dǎo)的HUVEC遷移及管腔結(jié)構(gòu)形成具有明顯抑制作用(P0.05)。熒光定量PCR結(jié)果顯示沙利度胺對(duì)LPS誘導(dǎo)的VEGF、Ang-2 mRNA水平有抑制作用(P0.05),呈濃度依賴性,對(duì)Ang-1水平則無明顯抑制作用,Western blot結(jié)果顯示沙利度胺可抑制VEGF、Ang-2、PI3K/Akt信號(hào)分子蛋白水平表達(dá)。[結(jié)論]體外實(shí)驗(yàn)表明,沙利度胺抑制人臍靜脈內(nèi)皮細(xì)胞的增殖、遷移及管腔形成,抑制VEGF、Ang-2的表達(dá),抑制PI3K/AKT信號(hào)激活,從而抑制血管生成,可能是其治療兒童克羅恩病有效的作用機(jī)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.7
本文編號(hào)：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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前7條
1 Márcio Rios Leite;Sandra Sousa Santos;André Castro Lyra;Jaciane Mota;Genoile Oliveira Santana;;Thalidomide induces mucosal healing in Crohn's disease: Case report[J];World Journal of Gastroenterology;2011年45期
2 黃瑛;;兒童炎癥性腸病的治療現(xiàn)狀與展望[J];中華兒科雜志;2011年04期
3 Ying-Kit Leung;;Treatment of pediatric refractory Crohn’s disease with thalidomide[J];World Journal of Gastroenterology;2011年10期
4 周云芳;安曉霞;;兒科綜合性醫(yī)院結(jié)核病發(fā)病情況分析[J];臨床兒科雜志;2008年06期
5 中華醫(yī)學(xué)會(huì)消化病學(xué)分會(huì)炎癥性腸病協(xié)作組;歐陽(yáng)欽;胡品津;錢家鳴;鄭家駒;胡仁偉;;對(duì)我國(guó)炎癥性腸病診斷治療規(guī)范的共識(shí)意見(2007年，，濟(jì)南)[J];中華消化雜志;2007年08期
6 Ana Teresa Carvalho;Heitor Souza;Antonio Jose Carneiro;Morgana Castelo-Branco;Kalil Madi;Alberto Schanaider;Flavia Silva;Fernando Antonio Pereira Júnior;Márcia G Pereira;Cláudio Tortori;Ilana Dines;Jane Carvalho;Eduardo Rocha;Celeste Elia;;Therapeutic and prophylactic thalidomide in TNBS-induced colitis: Synergistic effects on TNF-α, IL-12 and VEGF production[J];World Journal of Gastroenterology;2007年15期
7 李輝;季成葉;宗心南;張亞欽;;中國(guó)0～18歲兒童、青少年身高、體重的標(biāo)準(zhǔn)化生長(zhǎng)曲線[J];中華兒科雜志;2009年07期
本文編號(hào)：2188834
本文鏈接：http://sikaile.net/yixuelunwen/eklw/2188834.html
最近更新
教材專著
