大肠上皮异型增生的形态定量研究(细胞异型的定量分析)

用自动化图象分析仪,对慢性结肠炎、大肠上皮良性腺瘤、异型增生和高分化管状腺癌进行形态定量分析,发现与核异型性有关的4项参数(核面积均值、核面积差异度、核平均直径、核浆比率)具有最强的特征性,其中核面积和核平均直径是判断增生级别的主要参素,且呈直线密切相关。因此作者认为这种定量评价提供客观和可重复性的指标有助于大肠上皮异型增生的诊断和分级。

大肠上皮恶性转化不同阶段p53表达与细胞凋亡的关系

目的:观察大肠腺癌,腺瘤恶变区及非恶变区细胞凋亡及其调控基因p53的表达状态,探讨它们在大肠上皮恶性转化进程中的作用及二者的关系。方法:利用DNA缺口末端标记技术,p53蛋白免疫组化染色及双重染色技术,原位观察27例大肠腺癌及21例腺瘤恶变标本中凋亡细胞和p53阳性表达细胞的密度与分布,以8例非肿瘤大肠粘膜作为对照。结果:腺瘤非恶变区凋亡细胞密度分别高于腺癌(P<0.01)、腺瘤恶变区(P<0.01)及非肿瘤粘膜(P<0.01)。腺癌及腺瘤恶变区p53蛋白阳性细胞密度均高于腺瘤非恶变区(P<0.01)及非肿瘤粘膜(P<0.01)。双染原位观察p53蛋白与凋亡细胞的分布发现:p53阳性区凋亡细胞有明显减少趋势,而p53阴性区凋亡细胞分布则有增加趋势。结论:p53蛋白突变在大肠癌发展中具重要的促恶变形成作用,其作用可能是通过抑制细胞凋亡而实现的。

大肠上皮性肿瘤AgNORs及PCNA表达的定量分析

目的 探讨AgNORs计数及PCNA指数在大肠上皮性肿瘤中的诊断意义。 方法 应用AgNORs染色蓝调色技术及SP免疫组化法分别检测 41例大肠癌及 10例大肠腺瘤标本中的AgNORs颗粒及PCNA指数 ,另检测正常大肠组织 6例作为对照 ,并进行统计学处理。 结果 正常大肠组织、大肠管状腺瘤及大肠高、中、低分化腺癌之间AgNORs计数及PCNA指数差异均有显著性 (P <0 .0 1) ,且颗粒分布和形态也不相同。 结论 AgNORs计数及PCNA指数测定为大肠腺瘤及各级大肠腺癌的诊断和鉴别诊断提供了较为客观的依据 ,有重要的参考价值。

大肠上皮增生的动态观察

从大肠上皮增生到大肠癌之间的内在关系,一直是研究的重点课题。我们对56例连续5年以上活检5次以上的大肠增生性病变,进行回顾性临床病理分析,共319例次649枚息肉的观察结果表明,大肠增生性病变的癌变是一个漫长的过程,5年以上的动态变化幅度不大。同时对大肠腺瘤的组织学类型上,提出了从细胞形态角度划分的科学性、实用性问题。

大肠上皮细胞中MDP活化NOD2诱导耐受性CXCL1、CXCL2、CXCL3和CXCL8表达的分子机制

该文探讨肠上皮细胞中胞壁酰二肽(muramyl dipeptide,MDP)活化NOD2诱导趋化因子表达的调控机制。采用RT-PCR和qRT-PCR的方法检测趋化因子、细胞因子及泛素编辑酶A20的mRNA表达水平。Western blot检测A20的蛋白表达水平。ELISA检测趋化因子CXCL8(又称IL-8)的水平。脂质体转染法过表达A20。结果显示,MDP处理HCT116细胞诱导趋化因子CXCL1、CXCL2、CXCL3和CXCL8的表达,但不诱导促炎因子TNFα、IL-1β和IL-6的表达;MDP所诱导的反应具有耐受性,初次处理后的再次处理所诱导的反应程度显著下降;MDP处理上调A20,但过表达A20并不下调MDP诱导的免疫反应;同时,IL-1β上调A20,但IL-1β预处理同样不能下调MDP诱导的反应。总之,大肠上皮细胞中MDP诱导耐受性CXCL1、CXCL2、CXCL3和CXCL8的表达,且A20不参与此耐受机制的形成。

胃和大肠腺上皮分化癌的多态性观察

目的 探讨胃和大肠腺上皮分化癌的组织学类型及组织学分级变异规律。方法 对 2 33例进展期胃和大肠腺上皮分化癌的根治术标本进行回顾性观察分析。结果 2 33例胃和大肠腺上皮分化癌的在向固有肌层浸润时 ,116例发生组织学分型或组织学分级变异而呈两种及两种以上组织学构型 ,117例组织学构型无变异。结论 胃和大肠腺上皮分化癌的组织学构型变异与其生长微环境改变可能有关。

胆囊切除术对二甲基肼诱发小鼠大肠癌的影响

目的 探讨胆囊切除术对大肠癌发生的影响及作用机理。方法 胆囊切除术后以二甲基肼诱发小鼠大肠癌 ,观察大肠肿瘤发生情况 ;流式细胞术测定大肠粘膜上皮细胞S期百分比 ;高效液相色谱仪检测手术前后鼠粪胆酸、鹅脱氧胆酸、脱氧胆酸和石胆酸含量。结果 实验组荷结节鼠大肠平均荷结节数、受检鼠大肠腺瘤发生率及S期百分比均明显高于对照组 (P<0 .0 5 ) ;胆囊切除术后鼠粪石胆酸含量较术前和对照组明显增加 (P=0 .0 0 )。结论 胆囊切除后二甲基肼诱发的小鼠大肠肿瘤发生数量有所增加 ,并且大肠粘膜上皮细胞增殖旺盛 ,提示胆囊切除术对二甲基肼诱发小鼠大肠肿瘤有协同促进作用 ,可能与术后肠道内次级胆汁酸石胆酸含量增加有关。

大肠粘膜上皮良恶性病变AgNOR研究

应用银染技术对56例大肠粘膜上皮良恶性病变活示本石蜡切进行AgNOR计数，其中炎性息肉12例，慢性溃疡性结肠8例，腺瘤性息肉6例，管状腺癌30例（高分化型15例，中等分化型10例，低分化型5例）。结果：炎性息肉、慢性溃疡性结肠炎上皮细胞核内AgNOR颗粒均数与腺癌性息肉、管状腺癌各型均数均存在显著性差异（P＜0．01）。结果显示，本法有助于鉴别大肠粘膜上皮良恶性病变及其预后判定。

Efficacy of the revised Vienna Classification for diagnosing colorectal epithelial neoplasias

AIM： To prospectively investigate the efficacy of the revised Vienna Classification for diagnosing colorectal epithelial neoplastic lesions in cold biopsy specimens.METHODS： Patients were selected for inclusion if they had colorectal epithelial lesions that were not considered suitable for direct endoscopic resection,These included colorectal polyps ≥ 10 mm and lesions suspected of being carcinomas capable of invading the colorectal submucosa or beyond, including strictures, based on the cold biopsies obtained from each lesion prior to resection. We investigated the relationship between diagnoses based on cold biopsy samples using the revised Vienna Classification and resected specimens of the same lesions, and the therapeutic implications of diagnoses made using the revised Vienna Classification. The same cold biopsy specimens were also examined using the Japanese Group Classification guidelines, and compared with the resected specimens of the same lesions for reference.RESULTS： A total of 179 lesions were identified. The sensitivity, specificity, positive and negativepredictive values of the revised Vienna Classification for distinguishing between intramucosal lesions and submucosal invasive carcinomas in cold biopsy specimens was 22.2%, 100%, 100%, and 71.4%,respectively, and for distinguishing between intramucosal lesions and those invading the submucosa or beyond was 59.7%, 100%, 100%, and 37.6%,respectively. The sensitivity, specificity, positive and negative predictive values of the Japanese Group Classification for distinguishing between intramucosal lesions and submucosal invasive carcinomas in cold biopsy specimens was 83.3%, 91.4%, 83.3%. and 91.4%, respectively, and for distinguishing between intramucosal lesions and those invading the submucosa or beyond was 95.1%, 91.4%, 97.9%, and 82.1%, respectively. A total of 137 of 144 carcinomas that had invaded the submucosa or beyond and three high-grade intraepithelial neoplasias were diagnosed as ＂carcinoma＂ using the Japanese Group Classification system.CONCLUSION： The revised Vienna Classification for cold biopsy specimens has high positive predictive value in the diagnosis of colorectal carcinoma invasive to the subrnucosa or beyond.

Suppression of colorectal cancer metastasis by nigericin through inhibition of epithelial-mesenchymal transition

AIM: To evaluate the effect of nigericin on colorectal cancer and to explore its possible mechanism. METHODS: The human colorectal cancer (CRC) cell lines HT29 and SW480 were treated with nigericin or oxaliplatin under the conditions specified. Cell viability assay and invasion and metastasis assay were performed to evaluate the effect of nigericin on CRC cells. Sphereforming assay and soft agar colony-forming assay were implemented to assess the action of nigericin on the cancer stem cell properties of CRC cells undergone epithelial-mesenchymal transition (EMT). RESULTS: Compared with oxaliplatin, nigericin showed more toxicity for the HT29 cell line (IC50, 12.92 ± 0.25 μmol vs 37.68 ± 0.34 μmol). A similar result was also obtained with the SW116 cell line (IC50, 15.86 ± 0.18 μmol vs 41.02 ± 0.23 μmol). A Boyden chamber assay indicated that a significant decrease in the number of HT29 cells migrating through polyvinylidene fluoride membrane was observed in the nigericin-treated group, relative to the vehicle-treated group [11 ± 2 cells per high-power field (HPF) vs 19.33 ± 1.52 cells per HPF, P < 0.05]. Compared to the control group, the numbers of HT29 cells invading through the Matrigel-coated membrane also decreased in the nigericin-treated group (6.66 ± 1.52 cells per HPF vs 14.66 ± 1.52 cells per HPF, P < 0.05). Nigericin also reduced the proportion of CD133+ cells from 83.57% to 63.93%, relative to the control group (P < 0.05). Nigericin decreased the number of spheres relative to the control group (0.14 ± 0.01 vs 0.35 ± 0.01, P < 0.05), while oxaliplatin increased the number of spheres relative to the control group (0.75 ± 0.02 vs 0.35 ± 0.01; P < 0.05). Nigericin also showed a decreased ability to form colonies under anchorage-independent conditions in a standard soft agar assay after 14 d in culture, relative to the control group (1.66 ± 0.57 vs 7 ± 1.15, P < 0.05), whereas the colony numbers were higher in the oxaliplatin group relative to the vehicle-treated controls (14.33 ± 0.57 vs 7 ± 1.15, P < 0.05). We further detected the expression of E-cadherin and vimentin in cells treated with nigericin and oxaliplatin. The results showed that HT29 cells treated with nigericin induced an increase in E-cadherin expression and a decrease in the vimentin expression relative to vehicle controls. In contrast, oxaliplatin downregulated the expression of E-cadherin and upregulated the expression of vimentin in HT29 cells relative to vehicle controls. CONCLUSION: This study demonstrated that nigericin could partly reverse the EMT process during cell invasion and metastasis.

Moxibustion down-regulates colonic epithelial cell apoptosis and repairs tight junctions in rats with Crohn's disease

AIM: To investigate the effects of moxibustion on down-regulation of the colonic epithelial cell apoptosis and repair of the tight junctions in rats with Crohn's disease (CD). METHODS: Sixty male Sprague-Dawley rats were randomly divided into a normal control (NC) group, a model control (MC) group, an herbs-partitioned moxibustion (HPM) group, a mild-warm moxibustion (MWM) group and a salicylazosulphapyridine (SASP) group, with 12 rats in each group. The CD model rats were treated with trinitrobenzene sulphonic acid to induce intestinal inflammation. The rats in the HPM and MWM groups were treated at the Tianshu (ST25) and Qihai (CV6) acupoints once daily for 14 d, and the SASP group was fed SASP twice daily for 14 d. No additional treatment was given to the MC and NC groups. Themicrostructure of the colonic epithelium was observed under a transmission electron microscope, the transepithelial resistance was measured using a shortcircuit current, colonic epithelial cell apoptosis was determined by terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labelling assay, and the expression of occludin, claudin-1 and zonula occludens-l (ZO-1) in the colonic epithelial junction was determined by Western blotting and immunofluorescence staining. RESULTS: Compared with the MC group, the microstructure of the colonic epithelial barrier was signifi-cantly improved in rats treated with HPM, MWM or SASP, meanwhile, the current flow was reduced signifi-cantly, with values of 168.20 ± 6.14 vs 99.70 ± 3.13, 99.10 ± 4.28 and 120.30 ± 3.65 mA, respectively (P = 0.001). However, the HPM and MWM groups had higher current flow rates than the SASP group (99.70 ± 3.13, 99.10 ± 4.28 vs 120.30 ± 3.65 mA, P = 0.001). The number of the apoptotic colonic epithelial cells in HPM, MWM and SASP groups was largely reduced (61.5 ± 16.91 vs 15.5 ± 8.89, 14.8 ± 6.27 and 24.7 ± 9.68, respectively (P = 0.001); and the expression of occlu- din, claudin-1 and ZO-1 in the MWM and HPM groups was signifi cantly enhanced (0.48 ± 0.10, 0.64 ± 0.09 vs 0.18 ± 0.05 for occludin, 0.12 ± 0.02, 0.17 ± 0.03 vs 0.05 ± 0.01 for claudin-1, and 0.08 ± 0.01, 0.11 ± 0.01 vs 0.02 ± 0.01 for ZO-1). And in SASP group, the expression of occludin and ZO-1 was also signifi cantly increased (0.27 ± 0.04 vs 0.18 ± 0.05 for occludin and 0.05 ± 0.01 vs 0.02 ± 0.01 for ZO-1), but there was no significant difference for claudin-1. The HPM and MWM groups had higher expression of occludin, claudin-1 and ZO-1 than the SASP group. CONCLUSION: HPM and MWM treatment can down-regulate apoptosis of colonic epithelial cells, repair tight junctions and enhance colonic epithelial barrier function in rats with CD.

Effect of toll-like receptor 3 agonist poly I:C on intestinal mucosa and epithelial barrier function in mouse models of acute colitis

AIM To investigate potential effects of poly I:C on mucosal injury and epithelial barrier disruption in dextran sulfate sodium (DSS)-induced acute colitis. METHODS Thirty C57BL/6 mice were given either regular drinking water (control group) or 2% (w/v) DSS drinking water (model and poly I:C groups) ad libitum for 7 d. Poly I:C was administrated subcutaneously (20 mu g/mouse) 2 h prior to DSS induction in mice of the poly I:C group. Severity of colitis was evaluated by disease activity index, body weight, colon length, histology and myeloperoxidase (MPO) activity, as well as the production of proinflammatory cytokines, including tumor necrosis factor-a (TNF-alpha), interleukin 17 (IL-17) and interferon-. (IFN-gamma). Intestinal permeability was analyzed by the fluorescein isothiocyanate labeled-dextran (FITC-D) method. Ultrastructural features of the colon tissue were observed under electron microscopy. Expressions of tight junction (TJ) proteins, including zo-1, occludin and claudin-1, were measured by immunohistochemistry/immunofluorescence, Western blot and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS DSS caused significant damage to the colon tissue in the model group. Administration of poly I:C dramatically protected against DSS-induced colitis, as demonstrated by less body weight loss, lower disease activity index score, longer colon length, colonic MPO activity, and improved macroscopic and histological scores. It also ameliorated DSS-induced ultrastructural changes of the colon epithelium, as observed under scanning electron microscopy, as well as FITC-D permeability. The mRNA and protein expressions of TJ protein, zo-1, occludin and claudin-1 were also found to be significantly enhanced in the poly I:C group, as determined by immunohistochemistry/immunofluorescence, Western blot and RT-qPCR. By contrast, poly I:C pretreatment markedly reversed the DSS-induced up-regulated expressions of the inflammatory cytokines TNF-alpha, IL-17 and IFN-gamma. CONCLUSION Our study suggested that poly I:C may protect against DSS-induced colitis through maintaining integrity of the epithelial barrier and regulating innate immune responses, which may shed light on the therapeutic potential of poly I:C in human colitis.

Clinical features of familial adenomas polyps in Chinese and establishment of its immortal lymphocyte cell lines

AIM： To reserve the rare Chinese familial adenomas polyp （FAP） family resource and to investigate the clinical features of FAP in Chinese for its diagnosis. METHODS： Clinical features of patients with FAP were investigated. If there is any question, their medical records were verified. Blood sample was taken and lymphocyte immortal cell lines were established with modified EB-transformation methods. Congenital hypertrophy of retinal pigment epithelium （CHRPE） was checked by an experienced ophthalmologist. RESULTS： Twenty seven families including 21 classical FAP （CFAP） families, 3 attenuated FAP （AFAP） families, and 3 suspected AFAP families were investigated. A total of 116 lymphocyte immortal cell lines were established from 26 families. In all the FAP families, colorectal cancer occurred at the mean age of 42.84 years. Of the 16 families checked, 15 （93.75%） had CHRPE. The mean number of patients suffering from colorectal neoplasm was 3.14 in CFAP families and 2.0 in AFAP families （P 〈 0.01）. The mean oldest age at diagnosis of FAP was 41.75 years in CFAP families, and 58.67 years in AFAP families, respectively （P 〈 0.01）. Mean age of development of colorectal cancer was 42.23 in CFAP and 57.33 years old in AFAP （P 〈 0.01）. Mean of the earliest age at diagnosis of FAP was 29.95 years in the FAP families with a positive family history and 46.80 years in the FAP families with a negative family history （P 〈 0.01）. The ratio of extra-intestinal tumors to colorectal neoplasms was different in the two kinds of families with positive and negative family history （P 〈 0.01）. CONCLUSION： Additional use of ciclosporin will effectively improve to establish lymphocyte immortal cell lines with modified EB- transformation methods. In Chinese FAP, there was a high frequency of CHRPE, and a later age at diagnosis and a later age of development of colorectal cancer in AFAR And earlier age at diagnosis in FAP with positive family history was also found that will help to diagnose various kinds of FAP in Chinese.

The roles of micro RNAs and epithelial-mesenchymal transition in colorectal cancer metastasis

Colorectal cancer(CRC) is the second most common cause of cancer death worldwide. Distant metastasis is the major cause of death in patients with CRC. During progression to metastasis in which malignant cells disseminate from the primary tumor to seeding other organs, a multistep process is involved. Cancer cells proliferate, invade microenvironment, enter into the blood circulation, then survive and colonize into distant organs. Micro RNAs(mi RNAs) and epithelial-mesenchymal transition(EMT) are key regulators and mechanism in tumorigenesis and cancer metastasis. We review the roles of EMT and micro RNAs, especially EMT related micro RNAs in the metastatic pathway of CRC. Micro RNAs provide us a set of potential therapeutic applications and molecular target for CRC.

大肠息肉腺管开口与息肉组织内胰岛素样生长因子-1受体表达的关系

目的探讨大肠息肉腺管开口与息肉组织内胰岛素样生长因子-1受体（IGF-1R）表达的关系及其意义。方法对结肠镜检出的47例大肠息肉，按Kudo标准分类腺管开口；并用免疫组织化学法检测其IGF-1R的表达，进行相关分析。结果47例大肠息肉中，Ⅱ型腺管开口8例，ⅢL型腺管开口6例，Ⅳ型腺管开口30例，V型腺管开口3例；而IGF-1R阴性表达3例，弱阳性表达16例，中等阳性表达17例，强阳性表达11例，与腺管开口分型明显相关（rs=0．432，P〈0．05）。结论大肠息肉组织中IGF-1R过表达可能影响息肉腺窝生长，造成息肉表面腺管开口形态改变，有助于评价息肉的癌变风险。

Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli （UPEC） to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium （K-SFM） with epidermal growth factor （EGF） and bovine pituitary extract （BPE）. Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678- 54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnfl of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

Effect of Yang-activating and stasis-eliminating decoction from Traditional Chinese Medicine on intestinal mucosal permeability in rats with ulcerative colitis induced by dextran sulfate sodium

OBJECTIVE: To evaluate the effect of decoction prepared with Yang-activating and stasis-eliminating medicinals from Traditional Chinese Medicine(TCM) on the intestinal mucosal permeability in rats with ulcerative colitis induced by dextran sulfate sodium(DSS).METHODS: Totally 55 male Wistar rats(body weight of 170-190 g) were randomly divided into the blank group(n = 10) and the model duplication group(n = 45). The blank group was not intervened, while the other was modeled with 5% dextran sulfate sodium by gavaging in a dosage of4 m L per day to induce ulcerative colitis, a total of7 days. Then, the model rats were divided into model blank group, mesalazine group and TCM group,and each group was consisted of 15 rats. They were given retention enema 10 min with normal saline,mesalazine enema(0.036 g/m L), and Yang-activating and stasis-eliminating decoction [0.54 g/m L of a decoction boiled by Puhuang(Pollen Typhae), Xiebai(Bulbus Allii Macrostemonis) and Wulingzhi(Faeces Trogopteri)] for 10 days respectively. Afterwards,all of the rats were evaluated by disease activity index(DAI), histological changes of distal colon, expression of occludin protein and ultrastructure of intestinal epithelial cells. Furthermore, ratio of lactulose to mannitol(L/M) discharged in urine was evaluated.RESULTS: Comparing the results between TCM and model control groups, scores of DAI and histological lesions decreased significantly(P = 0.000 <0.01), ultrastructures of intestinal epithelial cells and tight junctions were more complete. The expression of occludin protein(P = 0.001 < 0.01) increased while the L/M value decreased significantly(P = 0.000 < 0.01) in TCM group. There was no statistical difference between the TCM and mesalazine groups in results of each item(P > 0.05).CONCLUSION: The decoction prepared with Yang-activating and stasis-eliminating TCM medicianls can restore intestinal mucosal epithelial cells and tight junctions the model rats with ulcerative colitis; it can reduce histological lesions and protect the permeability of intestinal mucosa barrier in the rats as well.