【作者】 向丽；

【导师】 金先庆；

【作者基本信息】 重庆医科大学 ， 儿外科学， 2012， 博士

【摘要】 目的：采用免疫组织化学方法在结直肠息肉，腺瘤，腺癌渐变过程中检测出有重要意义的分子标记，并通过跨胎盘RNAi技术干扰其在子一代小鼠的表达，构建BMP息肉小鼠模型,初步证实BMP、TLR3、TLR4在结直肠息肉发生、发展、恶变的机制中有重要作用。方法：1）应用免疫组织化学方法（sP法）检测MBP2,TLR4, TLR3,COX2在儿童结直肠幼年性息肉，儿童结直肠错构瘤样息肉，成人结直肠炎性息肉，结直肠腺瘤，结直肠腺癌组织中的表达情况；2）将孕鼠随机分Ringer’s液空白对照组（空白组）、pSES阴性对照组（阴性组）、pSES-SiBMP4实验组（实验组），采用跨胎盘RNAi技术，在孕鼠的尾静脉注射沉默BMP4基因的质粒，通过跨胎盘RNAi干扰子一代小鼠的BMP4的表达，分别于出生1周，8周，10周处死子代小鼠，解剖观察子一代小鼠的结直肠肠道息肉位置及数量情况。结果：一、免疫组织化学结果：1．BMP2在腺体组织中呈现弥漫均一细胞浆的棕黄色颗粒的表达，在儿童幼年性息肉组（OD68.65±18.15）,儿童错构瘤样息肉组（OD34.8±18），成人炎性息肉组（OD42.9±10.24），结直肠腺瘤组(OD50.3±10.2)中的表达明显高于结肠腺癌组(OD19.1±5.3)，在结肠腺癌组中明显降低的表达确有显著差异（p<0.05）。2. TLR4的表达情况：1）儿童幼年性息肉组（OD4.05±1.36）与儿童错构瘤样息肉组之间的表达有显著差异性（p<0.05）；儿童幼年性息肉组与结直肠腺癌组中的表达有显著差异性（p<0.05）。2）儿童错构瘤样息肉组(OD29.5±11)和结直肠腺瘤组的表达之间有显著差异（p<0.05）。3）结直肠腺瘤组（OD16.2±6.5）的表达与结直肠腺癌组(OD47.6±11.5)之间的表达有显著差异（p<0.05）。3. TLR3在儿童幼年性息肉组（OD270±90.42），儿童错构瘤样息肉组(OD191.3±47)，结直肠腺瘤组(OD147.1±58)，结直肠腺癌组(OD94.9±39.8)中的表达呈逐步下降趋势，在结直肠腺癌组中明显降低的趋势在相互比较中有显著差异（p<0.05）。4.COX2的表达主要是弥散的表达在腺上皮的细胞胞浆和细胞核膜上，在儿童幼年性息肉组(OD60.7±20.7)，儿童错结肠腺瘤组(OD69.1±31.5)，结直肠腺瘤组(OD53.8±16.4)，结肠腺癌组(OD60.5±20.4)中的表达均高于成人结直肠息肉组(OD12.65+4.21)，但相互比较无显著差异（p>0.05）。二、BMP息肉小鼠模型特点：1．运用RT-PCR,蛋白印迹法检测子一代小鼠结肠粘膜的BMP4的表达均较同周龄正常对照组显著降低；2.在出生后1周未见确切息肉样组织形成，第8周时可见包块样突起，经HE染色可见腺体上皮增生，病理证实为息肉形成，第10周包块突起明显，数量较8周增多，HE染色同样证实为结直肠息肉形成，未见腺瘤及腺癌样改变。结论：1）通过免疫组织化学的方法，证明在结直肠息肉，腺瘤，腺癌的渐变过程中有意义的三种分子标记：BMP缺失影响肠道的腺上皮的分化和增殖过程，导致结直肠息肉，腺瘤，腺癌的渐变过程；TLR3的低表达可能预示息肉恶变的高倾向；TLR4是结直肠息肉，腺瘤，腺癌的渐变过程的重要分子标记。2）通过跨胎盘RNAi技术首次成功构建了BMP息肉小鼠模型，为研究结直肠息肉发生、发展及恶变的机制奠定了实验室基础。通过本实验初步证实BMP,TLR3，TLR4参与了结直肠息肉的发生、发展和恶变机制。目的：通过免疫组织化学的方法检测结直肠息肉，结直肠腺瘤，结直肠腺癌中不同分子标记的表达情况，为结直肠息肉，结直肠腺瘤，结直肠腺癌的渐变过程找到有意义的分子标记物。方法：应用免疫组织化学方法（sP）法检测MBP2、TLR3、TLR4、COX2在儿童结直肠幼年性息肉，儿童结直肠错构瘤样息肉，成人结直肠炎性息肉，结直肠腺瘤，结直肠腺癌组织中的表达情况。结果：1．BMP2在腺体组织中呈现弥漫均一细胞浆的棕黄色颗粒的表达，在儿童幼年性息肉组（OD68.65±18.15）,儿童错构瘤样息肉组（OD34.8±18），成人炎性息肉组（OD42.9±10.24），结直肠腺瘤组(OD50.3±10.2)中的表达明显高于结肠腺癌组(OD19.1±5.3)，在结肠腺癌组中明显降低的表达确有显著差异（p<0.05）。2. TLR4的表达情况：1）儿童幼年性息肉组（OD4.05±1.36）与儿童错构瘤样息肉组之间的表达有显著差异性（p<0.05）；儿童幼年性息肉组和结肠腺瘤组之间的表达无显著差异（p>0.05）；儿童幼年性息肉组与结直肠腺癌组中的表达有显著差异性（p<0.05）。2）儿童错构瘤样息肉组(OD29.5±11)和结直肠腺瘤组的表达之间有显著差异（p<0.05）；儿童错构瘤样息肉组与结直肠腺癌组的表达之间无显著差异（p>0.05）。3）结直肠腺瘤组（OD16.2±6.5）的表达与结直肠腺癌组(OD47.6±11.5)之间的表达有显著差异（p<0.05）。3. TLR3在儿童幼年性息肉组（OD270±90.42），儿童错构瘤样息肉组(OD191.3±47)，结直肠腺瘤组(OD147.1±58)呈逐步下降趋势，在分别与结直肠腺癌组(OD94.9±39.8)的比较中有显著差异（p<0.05）。4.COX2的表达主要是弥散的表达在腺上皮的细胞胞浆和细胞核膜上，在儿童幼年性息肉组(OD60.7±20.7)，儿童错结肠腺瘤组(OD69.1±31.5)，结直肠腺瘤组(OD53.8±16.4)，结肠腺癌组(OD60.5±20.4)中的表达均高于成人结直肠息肉组(OD12.65+4.21)，但相互比较无显著差异（p>0.05）。结论：在结直肠息肉，结直肠腺瘤，结直肠腺癌的渐变过程中，BMP的缺失影响肠道的腺上皮的分化和增殖过程，导致结直肠息肉形成，甚至恶变；TLR3的低表达可能预示息肉恶变高倾向；TLR4是结直肠息肉，腺瘤，腺癌的渐变过程的重要分子标记。目的：采用跨胎盘RNAi的方法沉默孕鼠的BMP4，导致子一代小鼠结直肠多发性息肉形成从而建立BMP结肠息肉小鼠模型，证明BMP参与小鼠结直肠息肉发生机制。方法：将孕鼠随机分Ringer’s液空白对照组（空白组）、pSES阴性对照组（阴性组）、pSES-SiBMP4实验组（实验组），采用跨胎盘RNAi技术，在孕鼠的尾静脉注射沉默BMP4基因的质粒，通过跨胎盘作用干扰子一代小鼠的BMP4的表达，分别于出生1周，8周，10周处死子代小鼠，解剖观察子一代小鼠的结直肠的息肉位置及数量。结果：1．运用RT-PCR，蛋白印迹法检测子一代小鼠的结直肠BMP4的表达均较正常同龄对照组显著降低；2.在出生后第1周未见确切息肉样组织形成，第8周时可见包块样突起，经HE染色可见腺体上皮增生病理证实为息肉形成，第10周包块突起明显，数量较第8周增多，HE染色同样证实为结直肠息肉形成，未见腺瘤及腺癌样改变。结论：1.首次成功建立了BMP结直肠息肉小鼠模型，证明BMP参与了结直肠息肉形成机制。2.跨胎盘RNAi技术在干扰基因表达方面有广阔的应用前景，值得进一步推广和完善。更多还原

【Abstract】 Objective: To clarify the mechanism of the formation and malignanttransformation of colorectal polyps by testing the expression of somemolecular markers in the different pathological types of colorectal polypsand establishing a BMP colorectal polyps mice model with the new RNAinterference (RNAi) technology, which was targeted to postimplantationstaged mouse embryos by using tail vein injection. Methods:1） detecte theexpression of MBP2,TLR4, TLR3, COX2in children’s colorectal juvenilepolyps, children’s colorectal hamartoma polyps, adult colorectalinflammatory polyps, colorectal adenomas, colorectal adenocarcinoma byusing immunohistochemical methods.2） deliver siRNA targeted to BMP4to postimplantation staged mouse embryos using tail vein injection.Pregnant mice were completely random delivered three groups：Ringer’sliquid blank control group (blank group), pSES negative control group(negative group), pSES-SiBMP4group (experimental group). Results:一、outcomes of histochemistry and immunohistochemistry tests:1. BMP2was present in uniform diffuse cell plasma particles, the expression of BMP2 in children’s juvenile polyps (OD68.65±18.15), children’s hamartomapolyps (OD34.8±18), adult inflammatory polyps (OD42.9±10.24),colorectal adenomas (OD50.3±10.2) was higher than that in colorectalcancer (OD19.1±5.3), the expression of BMP2in colorectal cancer wassignificantly decline (p <0.05).2. TLR4expression:1) The express betweenchildren’s juvenile polyps (OD4.05±1.36) and children’s hamartoma polypswas significant difference (p <0.05); The express between children’sjuvenile polyps and colorectal adenocarcinoma polyp was significantdifference (p <0.05).2) There was a significant difference betweenchildren’s hamartoma polyps (OD29.5±11) and colorectal adenomas (p <0.05);3) There was a significant difference between colorectal adenomas(OD16.2±6.5) and colorectal adenocarcinoma (OD47.6±11.5)(p <0.05).3. The expressions of TLR3in children’s juvenile polyps (OD270+90.42),children’s hamartoma polyps (OD191.3±47), colorectal adenomas (OD147.1±58), colorectal adenomas (OD94.9±39.8) have been graduallydecline, the decline trend in colorectal adenocarcinoma significantlydifference (p <0.05).4. The expression of COX2is mainly diffuse inglandular epithelium cells of the cytoplasm and cell membrane, theexpression in children’s juvenile polyps (OD60.7±20.7), children’shamartoma polyps (OD69.1±31.5), colorectal adenomas (OD53.8±16.4),colorectal cancer (OD60.5±20.4) was higher than that in colorectal polyps(OD12.65±4.21), but compared each other no significant difference (p> 0.05).二、the characteristics of BMP polyps mice model:1. Using RT-PCR,western blot method to detect the generation mice, the expressions of BMP4are significantly lower than normal control group;6. Generation mice of oneweek have not been seen the exact polypoid organization in the colon, thereare visible plypoid organization in the colon of eight weeks and ten weeks ofgeneration mice. the polypoid organization in the colon was polyp testedby hematoxylin-eosin staining.Conclusion:1)In the evolution process ofcolorectal polyps from colorectal polyps, colorectal adenomas, to colorectaladenocarcinoma, there are significant difference molecular markers: thelack of BMP may lead to the formation and malignant transformation ofcolorectal polyps by affecting the proliferation and differentiong ofglandular epithelium; the lower expression of TLR3may indicate thetendency of malignant transformationof colorectal polyps; TLR4may bethe important molecular marker in the gradual transformation ofcolorectal polyps.2) we successfully established the colon polyps micemodel by delivering siRNA targeted to BMP4to postimplantation stagedmouseembryos using tail vein injection. Objective: To find the meaningful molecular markers for theformation and malignant transformation of colorectal polyps by testingthe expression of some molecular markers in the colorectal polyps,colorectal adenomas and colorectal adenocarcinoma. Methods: detectethe expression of MBP2,TLR4, TLR3, COX2in children’s colorectaljuvenile polyps, children’s colorectal hamartoma polyps, adultcolorectal inflammatory polyps, colorectal adenomas, colorectaladenocarcinoma by using immunohistochemical methods. Results:1.BMP2was present in uniform diffuse cell plasma particles, theexpression of BMP2in children’s juvenile polyps (OD68.65±18.15),children’s hamartoma polyps (OD34.8±18), adult inflammatorypolyps (OD42.9±10.24), and colorectal adenomas (OD50.3±10.2)was higher than that in colorectal cancer (OD19.1±5.3), the expressionof BMP2in colorectal cancer was significantly decline (p <0.05).2.TLR4expression:1) The express between children’s juvenile polyps(OD4.05±1.36) and children’s hamartoma polyps was significant difference (p <0.05); The express between children’s juvenile polypsand colonic adenoma was no significant difference (p>0.05); Theexpress between children’s juvenile polyps and colorectaladenocarcinoma polyp was significant difference (p <0.05).2) Therewas a significant difference between children’s hamartoma polyps (OD29.5±11) and colorectal adenomas (p <0.05); there was no significantdifference between children’s hamartoma polyps and colorectaladenocarcinoma (p>0.05).3) There was a significant differencebetween colorectal adenomas (OD16.2±6.5) and colorectaladenocarcinoma (OD47.6±11.5)(p <0.05).3. The expressions ofTLR3in children’s juvenile polyps (OD270+90.42), children’shamartoma polyps (OD191.3±47), colorectal adenomas (OD147.1±58), colorectal adenomas (OD94.9±39.8) have been gradually decline,the decline trend in colorectal adenocarcinoma significantly difference(p <0.05).4. The expression of COX2is mainly diffuse in glandularepithelium cells of the cytoplasm and cell membrane, the expression inchildren’s juvenile polyps (OD60.7±20.7), children’s hamartomapolyps (OD69.1±31.5), colorectal adenomas (OD53.8±16.4),colorectal cancer (OD60.5±20.4) was higher than that in colorectalpolyps (OD12.65±4.21), but compared each other no significantdifference (p>0.05). Conclusion: In the evolution process of colorectalpolyps from colorectal polyps, colorectal adenomas, to colorectal adenocarcinoma, there are significant difference molecular markers. Inthe evolution process of colorectal polyps from colorectal polyps,colorectal adenomas, to colorectal adenocarcinoma, there aresignificant difference molecular markers: the lack of BMP may lead tothe formation and malignant transformation of colorectal polyps byaffecting the proliferation and differentiong of glandular epithelium;the lower expression of TLR3may indicate the tendency of malignanttransformationof colorectal polyps; TLR4may be the importantmolecular marker in the gradual transformation of colorectalpolyps.We wish to understand the malignant transformation ofcolorectal polyps by testing the expression of molecular markers. Objective: To establish a BMP colorectal polyp’s mice model with thenew RNA interference (RNAi) technology, which was targeted to postimplantation staged mouse embryos by using tail vein injection. and toprove that the BMP is related to the formation of the colorectal polyps. Methods: deliver siRNA targeted to BMP4to post implantation stagedmouse embryos using tail vein injection. Pregnant mice were completelyrandom delivered three groups, Ringer’s liquid blank control group (blankgroup), pSES negative control group (negative group), pSES-SiBMP4group(experimental group). Results:1. Using RT-PCR, western blot method todetect the generation mice, the expressions of BMP4are significantly lowerthan normal control group;2. generation mice of one week have not beenseen the exact polypoid organization in the colon, there are visible polypoidorganization in the colon of eight weeks and ten weeks of generation mice.The polypoid organization was polyps by hematoxylin-eosinstaining.Conclusion:1. we successfully established the colon polyps’ micemodel by delivering siRNA targeted to BMP4to post implantation stagedmouse embryos using tail vein injection. We also prove that the BMP isrelated to the formation of the colorectal polyps.2. The new RNAinterference (RNAi) technology which was targeted to post implantationstaged mouse embryos using tail vein injection is worth further promote andperfect.更多还原