东亚钳蝎毒素对大鼠实验性大肠癌的抑制研究

利用盐酸二甲肼(DMH)诱发Wistar鼠大肠癌的动物模型．通过分组、分阶段研究竭毒素对大肠肿瘤发生过程和组织病理改变的作用。结果发现蝎毒素可以减少诱癌的鼠死亡率和诱癌率。20周内单纯诱癌组死亡率达32．69%，蝎毒灌胃组和腹腔注射组分别为23．08%和20．51%，其中竭毒注射组死亡率与单纯诱癌组相比有显著差异(P＜0．05)．20-31周单纯诱癌组诱癌率为81．82％，蝎毒注射组为44%，两组差异显著(P＜0．01)。大肠癌棱仁组成区染色结果发现单纯诱癌AgNOR颗粒明显增加而蝎毒注射组显著减少。每核AgNOR颗粒数两组有显著差异(P＜0．05)，研究提示蝎毒素具有去除DMH毒性，降低实验肿瘤的发生，抑制大脑肿瘤细胞rRNA活性。直接抑杀肿瘤细胞的作用。     

东亚钳蝎毒抑制实验性大肠癌细胞超微结构的观察

东亚钳蝎毒素是东亚钳蝎毒腺细胞分泌的一种具有多种生物活性的小分子多肽类物质。对其药理、毒理研究发现蝎毒素结构复杂,稳定性强。在此基础上,我们应用体外细胞培养的方法,已证实蝎毒素对大肠癌细胞具有显著的抑杀作用。为进一步探讨蝎毒素抑杀肿瘤细胞的机理,我们用东亚钳蝎毒素灌胃治疗大鼠实验性大肠癌,并详细观察了用药前后大肠肿瘤细胞超微结构的变化,结果表明:(1)使用蝎毒后大肠肿瘤体积明显变小,小鼠荷瘤率明显降低。(2)蝎毒对肿瘤细胞膜、细胞核、细胞器均具有直接破坏作用,且能直接影响肿瘤细胞的代谢及核酸活性。     

东亚钳蝎毒抑制大鼠实验性大肠癌细胞核中Ag-NOR的定量研究

东亚钳蝎毒是东亚钳蝎毒腺细胞分泌的一种含多种生物活性成份的小分子肽，对大肠癌细胞有显抑杀作用。为了进一步探讨蝎毒素对大肠癌细胞抑杀的作用机制，我们对蝎毒素对Wistar鼠实验性大肠癌细胞核Ag-NOR的影响进行了研究。     

东亚钳蝎毒抑制实验性大肠癌细胞超微结构的观察

东亚钳竭毒索是东亚钳蝎毒腺细胞分泌的一种具有多种生物活性的小分子多肽类物质。对其药理、毒理研究发现竭毒素结构复杂，稳定性强。在此基础上，我们应用体外细胞培养的方法，已证实竭毒素对大肠癌细胞具有显的抑杀作用。为进一步探讨蝎毒素抑杀肿瘤细胞的机理，我们用东亚钳蝎毒索灌胃治疗大鼠实验性大肠癌，并详细观察了用药前后大脑肿瘤细胞超微结构的变化，结果表明：(1)使用竭毒后大肠肿瘤体积明显变小，小鼠荷瘤率明显降低，(2)蝎毒对肿瘤细胞膜、细胞核、细胞器均具有直接破坏作用，且能直接影响肿瘤细胞的代谢及核酸活性。     

大肠癌旁粘膜核仁组成区嗜银蛋白及癌胚抗原表达的研究

应用胶质银染技术及免疫组化法对31例大肠癌旁粘膜进行核仁组成区嗜银蛋白（AgNOR)形态学定量研究及癌胚抗原（CEA）检测，结果表明：从癌周组织，癌旁组织到癌组织，AgNOR计数逐渐增高（P＜0．01）；CEA阳性表达亦逐渐增强（P＜0．01）。因此，支持大肠癌发生过程中移行粘膜理论。对AgNOR计数高，CEA表达阳性的病例追踪观察可望发现早期大肠癌。     

Ag－NOR计数在判断大肠癌预后中的价值

应用胶质银染技术对５６例根治性手术切除的大肠癌标本进行了定量观察。结果发现，Ａｇ－ＮＯＲ计数与患者的存活时间呈负相关（ｒ＝－０．７７Ｐ＜０．００１）。Ａｇ－ＮＯＲ＜４个／核，５年累积生存率是８０％，Ａｇ－ＮＯＲ４～６个／核为１５％，＞６／核为零。对数秩时序检验Ｐ＜０．００１。恶性程度低的肿瘤其颗粒分布以中央型为主，似恶性程度越高颗粒分布越弥散。大肠癌细胞核Ａｇ－ＮＯＲ计数与ＤＮＡ含量有一定相关性。多因素生存分析显示Ａｇ－ＮＯＲ计数是大肠癌一个重要预后指标。     

东亚钳蝎毒抑制大鼠实验性大肠癌细胞核中Ag-NOR的定量研究

东亚钳蝎毒是东亚钳蝎毒腺细胞分泌的一种含多种生物活性成份的小分子肽,对大肠癌细胞有显著抑杀作用。为了进一步探讨蝎毒素对大肠癌细胞抑杀的作用机制,我们对蝎毒素对 Wistar 鼠实验性大肠癌细胞核 Ag-NOR 的影响进行了研究。     

实验性大肠癌肠粘膜细胞增殖力学的变化规律及其意义

应用流式细胞光度术（ＦＣＭ）和银染核仁形成区（Ａｇ－ＮＯＲ）自动图像分析技术，对二甲基肼（ＤＭＨ）诱发Ｗｉｓｔａｒ大鼠大肠癌发生过程中的细胞增殖力学，进行了定量研究。结果表明，在注射ＤＭＨ第７、１４、２１和２８周时，肠粘膜增殖指数（ＰＩ）分别为１６．８３±１．８６、２４．２２±２６２９、２９．７０±７．１３和３１．８４±３．５５，均显著高于正常大鼠的１２．２０±２．６０（Ｐ＜０．０１）；Ａｇ一ＮＯＲ颗粒数在正常大鼠肠粘膜上皮细胞为３．６±０．７，而在上述诱癌不同时期则分别为５．７±ｌ．０、６．０±０．８、６．８±２．３和５．４±１．０（Ｐ＜０．０１）。这些变化不仅可作为大肠癌发生的早期生物学特征，而且还有助于确定实验动物的患癌易感程度。     

东亚钳蝎毒诱发大肠癌细胞凋亡的电镜观察

东亚钳蝎毒素是东亚钳蝎毒腺细胞分泌的一种具有多种生物活性的小分子多肽类物质。我们应用体外细胞培养的方法,已证实蝎毒素对大肠癌细胞具有显著的抑杀作用。为进一步探讨蝎毒素抑杀肿瘤细胞的机理,我们用东亚钳蝎毒素灌胃治疗大鼠实验性大肠癌,详细研究了用药后大肠肿瘤细胞超微结构的变化,结果表明,蝎毒素能诱发大肠癌细胞程序化死亡,本实验详尽地观察了大肠癌肿瘤细胞程序化死亡的基本过程,这种作用可能是蝎毒抗肿瘤的重要途径之一。     

DNA、核仁组成区相关嗜银蛋白、增殖细胞核抗原定量分析与大肠癌的诊断

近年来大肠癌已经成为我国的常见肿瘤之一，且发病率逐年上升，尤其以结肠癌发病率增长速度迅猛，在大城市增幅更快，上世纪90年代与70年代相比，大肠癌的发病率在城市上升31．95％，在农村上升8．51％。因此，掌握大肠癌的发生发展规律及诊断和预后的方法有重要意义。恶性肿瘤基础研究目前虽已渐进细胞分子遗传学领域，但对大肠癌及癌前病变临床诊断和病理分级，迄今仍主要依靠传统镜下组织结构和细胞形态变化的观察为依据。     

Differences in Carcinogenesis by the Length of Carcinogen Exposure Period in Rat Colon

To clarify the carcinogenic factors—whether it is the kind of carcinogen or their length of exposure—that determine whether colorectal cancer develops from an adenoma or develops de novo in the absence of an adenoma, we histopathologically analyzed a total of 229 rat colon tumors induced by administration of 1,2-dimethyl-hydrazine (DMH) or N-methyl-N"-nitro-N-nitrosoguanidine (MNNG) for three or 15 weeks. In the three-week-exposure groups, 71% of DMH-induced carcinomas and 82% of MNNG-induced carcinomas coexisted with low-grade dysplasia (adenomatous remnant). However, in the 15-week-exposure groups, low-grade dysplasia was observed in only 10% of DMH-induced and 27% of MNNG-induced carcinomas. Even in the tumors smaller than 20 mm³, it was observed in only 10% of DMH-induced and 32% of MNNG-induced carcinomas. Furthermore, carcinomas without low-grade dysplasia predominated from the initial period of tumor occurrence. Next, we investigated association of K-ras and APC gene mutations with these carcinogenesis patterns in 80 tumors. K-ras mutations were not detected in any tumors induced by three weeks of exposure. However, in the 15-week-exposure groups, this mutation was observed in 57% of DMH-induced tumors and 13% of MNNG-induced tumors. APC mutations in the region homologous to the human mutation cluster region were observed in only 6% of tumors. Thus, our results suggest that the carcinogenesis patterns in rat colon are dependent on the length of exposure to carcinogen and that K-ras mutations were partly involved in a subset of them.     

Effects of Cyclooxygenase-2 Inhibition on Serum and Tumor Gastrins and Expression of Apoptosis-Related Proteins in Colorectal Cancer

The objective of the present study was to determine the influence of cyclooxygenase-2 (COX-2) inhibition by Celecoxib (CLX) in humans with distal colorectal adenocarcinoma (CRC) on serum and tumor levels of progastrin and gastrin and serum levels of proinflammatory cytokines (IL-8, TNF-α). In addition, the effects of this CLX treatment on tumor and adjacent mucosa expression of gastrin, its receptors (CCK₂), and COX-1 and COX-2, as well as protein expression of the active form of nuclear factor κ B (NFκ B) and the apoptotic-related proteins Bcl-2 and survivin, have been examined. Ten distal CRC patients were examined twice, once before and then after 14-day treatment with CLX (200 mg bid). Large biopsy samples were taken from the tumor and intact mucosa 10 cm above the tumor. For comparison, 20 age- and sex-matched healthy controls were enrolled and treated with CLX as CRC patients. Serum levels of IL-8 and TNF-α were measured by enzyme-linked immunosorbent assay, and serum levels of amidated gastrins and progastrin, by specific radioimmunoassay. The gene or protein expressions of progastrin, gastrin, CCK₂, COX-1, COX-2, Bcl-2, and survivin as well as NFκ B were determined by RT-PCR or Western blot in biopsy samples of tumor and intact mucosa of CRC patients. Serum IL-8 and TNF-α values were severalfold higher in CRC patients than in controls. The increase in serum proinflammatory cytokines was accompanied by increased expression of the active form of NFκ B. Serum progastrin levels were also found to be significantly higher in CRC than in controls. Treatment of CRC with CLX resulted in a significant decrease in serum levels of progastrin and this was accompanied by an increment in tumor expression of COX-2 with a concomitant reduction in gastrin, Bcl-2, survivin, and NFκ B expression. We conclude that (1) distal CRC patients show significantly higher serum progastrin levels than matched healthy controls, confirming that this hormone may be implicated in rectal carcinogenesis; (2) CRC patients exhibit significantly higher serum levels of IL-8 and TNF-α than healthy controls, probably reflecting more widespread inflammatory reaction in the colonic mucosa in CRC; (3) gastrin, COX-2, Bcl-2, survivin, and NFκ B were overexpressed in CRC tumor compared to intact mucosa, but treatment with CLX significantly reduced serum levels of progastrin and IL-8 and TNF-α, which could mediate the up-regulation of COX-2 in CRC; and (4) CLX also enhanced expression of COX-2, while inhibiting the expression of gastrin, Bcl-2, survivin, and NFκ B, suggesting that COX-2 inhibition might be useful in chemoprevention against CRC, possibly due to suppression of the antiapoptotic proteins and reduction in progastrin-induced and NFκ B-promoted tumor growth.     

罗格列酮对人结肠癌裸鼠移植瘤生长作用研究

目的观察过氧化物酶体增殖物激活受体的配体罗格列酮（ROZ）对人结肠癌细胞系HT-29裸鼠移植瘤的作用,探讨ROZ活化PPARγ,下调NFκB,从而诱导人结肠癌细胞凋亡的作用机制。方法体外培养人结肠癌HT-29细胞,建立人结肠癌细胞HT-29裸鼠移植瘤模型,20只荷瘤裸鼠随机分组进行实验。Western Blot法分析PPARγ、NF-κB、Bcl-2、bax蛋白表达的影响及PPARγ活化依赖性。结果 ROZ能抑制裸鼠移植瘤的生长。结论 ROZ通过上调PPARγ蛋白表达,下调NF-κB蛋白表达,抑制人结肠癌裸鼠移植瘤生长。     

Cyclo-oxygenase-Independent Inhibition of Apoptosis and Stimulation of Proliferation by Leptin in Human Colon Cancer Cells

Obesity increases the risk of colon cancer. Hyperleptinemia is characteristic of obesity and leptin has been reported to be a colonic growth factor. We have examined the involvement of the cyclo-oxygenase (COX) pathways in the proliferation and anti-apoptotic effects of leptin. Leptin stimulated proliferation in HT-29 colon cancer cells: this was unaffected by inhibition of COX-1, COX-2, protein kinase C, or the epidermal growth factor receptor. Leptin did not increase COX-2 mRNA or COX-derived prostaglandin E₂ production. Celecoxib induced apoptosis in a COX-independent manner. Leptin reduced both serum starvation- and celecoxib-induced apoptosis. Inhibition of ERK, p38 MAP kinase, and nuclear factor (NF)-κB abolished the growth-promoting and anti-apoptotic effects of leptin. Treatment of HT-29 cells with leptin stimulated phosphorylation of ERK and p38 MAP kinase and nuclear translocation of active NF-κB. We conclude that leptin stimulates colon cancer proliferation via COX-independent pathways and reduces celecoxib-induced apoptosis via ERK, p38 MAP kinase, and NF-κB pathways.     

Reduction of Squalene Epoxidase by Cholesterol Accumulation Accelerates Colorectal Cancer Progression and Metastasis

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Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.     

SPA免疫便潜血试验和直肠粘液T抗原试验互补普查大肠癌及其随访

本文联合采用人血红蛋白抗血清包被的含A蛋白葡萄球菌(SPA)进行免疫便潜血试验(SPA试验)和直肠粘液T抗原检测(T抗原试验)用于大肠癌普查初筛并对筛检人群进行随访。结果表明,在4843例无症状人群中,SPA和T抗原试验阳性者分别为472例(9.75％)和297例(6.13％).共769例阳性者行纤维结肠镜检查,检出大肠癌4例,腺瘤48例(>1.0cm者17例,占35.4％)。其中,SPA试验中仅3例癌,29例腺瘤阳性,T抗原试验中2例癌,27例腺瘤阳性,提示联合这两种初筛试验可提高大肠癌及其腺瘤的检出率。为验证普查后减少大肠肿瘤发生的效果,2年后对这些人群采用同样的普查方案随访,结果在受检的3641例人群中,共477例阳性者行纤维结肠镜检查,未发现大肠癌病例,腺瘤18例(>1.0cm者仅4例,占22.2％)。将两次检出的腺瘤进行不典型增生程度的比较,第二次检出的腺瘤轻度不典型增生病变占88.89％(16例),中重度不典型增生病变仅占11.11％(2例),而第一次检出腺瘤、中重度不典型增生病变占25％(12例)。上述结果表明利用这两种初筛试验进行互补性普查。可提高大肠癌及癌前病变的检出率,随访结果提示在无症状人群普查,不仅可使大肠癌及腺瘤的再检出率明显减少,且可使中重度不典型增生病变的发病机会明显减少。