人结肠癌SW480细胞裸鼠皮下移植瘤DcR3基因的RNA干扰及其作用

目的:观察DcR3基因小干扰RNA(siRNA)对人结肠癌SW480细胞裸鼠皮下移植瘤DcR3基因表达的影响。方法:建立结肠癌SW480细胞裸鼠皮下移植瘤模型,瘤体注射脂质体与DcR3siRNA混合物,转染DcR3siRNA,免疫组织化学及RT-PCR检测观察DcR3基因的表达。结果:建立了结肠癌SW480细胞裸鼠皮下移植瘤模型;治疗后,治疗组移植瘤明显减小,空白对照组、阴性对照组肿瘤体积显著大于治疗组(P<0.01);各组肿瘤组织中DcR3基因均有不同程度的表达,治疗组表达程度明显低于阴性对照组及空白对照组(RT-PCRP<0.05,免疫组化P<0.01)。结论:人结肠癌SW480细胞在裸鼠皮下有良好的成瘤性;脂质体与DcR3siRNA混合物可特异性抑制结肠癌裸鼠皮下移植瘤内DcR3基因的表达。     

抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长的抑制作用

目的:研究抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长的抑制作用,评价其作用为导向治疗药物的临床应用价值.方法:将体外培养的人肝癌细胞系SMMC-7721细胞接种于裸鼠皮下,建立荷人肝癌裸鼠皮下移植瘤动物模型,随机分为hdsFv-hEDN治疗组和对照组,分别给予尾静脉注射hdsFv-hEDN和生理盐水,1次/日,共2周.比较各组裸鼠皮下移植瘤生长速度、肿瘤体积和重量,并计算肿瘤的抑制率.取各组裸鼠肿瘤组织,心,肺,肝,肾组织HE染色,光学显微镜下观察.结果:抗肝癌hdsFv-hEDN治疗组裸鼠皮下移植瘤生长抑制作用显著,肿瘤生长速度减慢,肿瘤体积从42.62±0.57 mm3增加到74.28±2.59 mm3、瘤重为155.82±14.43 mg,而对照组肿瘤体积从41.94±0.91 mm3增加到127.42±4.81 mm3、瘤重为283.28±15.21 mg,两组比较差异非常显著.肿瘤体积抑瘤率和瘤重抑瘤率分别达到41.59±0.02%和45.51±0.09%.组织学观察抗肝癌hdsFv-hEDN组肿瘤组织出现大片坏死,凋亡明显增加,心、肝、肺、肾等重要器官未见明显异常.结论:抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长具有良好的抑制作用.     

siRNA表达载体对SW480细胞原癌基因Pokemon的抑制

观察siRNA表达载体对SW480细胞中Pokemon原癌基因的抑制效应,为进一步研究该基因的功能奠定基础。构建针对Pokemon基因的RNAi质粒表达载体,脂质体法转染人结直肠癌SW480细胞系,观察转染效率及细胞表型变化。稳定转染后,实时荧光定量PCR和Western blot检测SW480细胞中Pokemon mRNA及蛋白质的表达情况;MTT法检测siRNA对SW480细胞恶性增殖的影响;流式细胞仪分析细胞凋亡改变。镜下观察阳性转染率约36%,转染表达载体后细胞形态发生了显著变化;Pokemon mRNA及蛋白质的表达受到明显抑制：与阴性对照组相比,表达质粒产生的siRNA对Pokemon mRNA的抑制率在转染后24 h和48 h分别为34.2%和67.7%;对蛋白的抑制率在48 h和72 h分别为48.3%和73.6%。MTT法检测细胞生长曲线表明Pokemon抑制可使SW480细胞生长速度明显减慢;流式细胞仪分析显示转染Pokemon siRNA表达质粒后SW480细胞凋亡增加。构建的RNAi表达载体可以有效抑制SW480细胞中Pokemon基因的表达,并对SW480细胞的生长具有明显抑制及诱导凋亡作用。     

藻蓝蛋白抑制人结肠癌SW480细胞增殖的初步研究

目的研究藻蓝蛋白对人结肠癌SW480细胞的体外抑瘤作用,为进一步探讨藻蓝蛋白抑制肿瘤的机制提供依据。方法用不同浓度的藻蓝蛋白处理结肠癌SW480细胞后,应用MTT实验来检测藻蓝蛋白对细胞增殖的抑制作用并计算出半数抑制率,利用HE染色法和电镜技术观察凋亡细胞形态结构,采用流式细胞术分析其对SW480细胞周期的影响。结果 MTT试验证明藻蓝蛋白能够抑制SW480细胞的增殖,且呈时间和剂量依赖性;HE染色以及电镜的观察结果显示藻蓝蛋白能够诱导SW480细胞的凋亡,流式细胞术显示SW480细胞被阻滞在G2/M期,G0/G1期细胞比例降低。结论藻蓝蛋白在体外能够抑制结肠癌SW480细胞的增殖,具有可开发人结肠癌治疗光敏剂应用前景。     

Bevacizumab 联合吉西他滨对荷肝癌裸鼠移植瘤生长的抑制作用

目的:探讨抗血管生成药物Bevacizumab联合吉西他滨对人肝癌裸鼠皮下移植瘤生长的抑制作用。方法:构建人肝癌细胞HepG2裸鼠皮下移植瘤模型,随机分为空白对照组、Bevacizumab组、吉西他滨组和联合用药组。观察用药前后肿瘤体积,绘制肿瘤生长曲线;应用免疫组化检测肿瘤微血管密度(MVD);Western Blot检测Bcl-2蛋白的表达。结果:Bevacizumab和吉西他滨单药均能抑制肿瘤生长,两药联合疗效明显增强(P=0.000)。与对照组和吉西他滨组相比,Bevacizumab组和联合用药组能明显抑制肿瘤血管生成,MVD值均明显降低,以联合用药组最为明显(P均0.000)。Bevacizumab和吉西他滨单药均能下调Bcl-2的表达,两药联合下调作用明显增强。结论:Bevacizumab联合吉西他滨能增强对人肝癌裸鼠移植瘤的生长及微血管生成的抑制作用,其机制可能与调控Bcl-2的表达有关。     

靶向FBXL20基因的siRNA片段的筛选及其验证

在人结肠腺癌SW480细胞株及口腔鳞癌HSC3细胞株中,筛选有效干扰fbxl 20的siRNA片段.设计合成3对靶向fbxl 20 mRNA的小干扰RNA片段,通过阳离子脂质体介导转染SW480细胞和HSC3细胞. 结果发现siRNA-1, siRNA-2, siRNA-3均能抑制SW480细胞和HSC3细胞fbxl20 mRNA的表达,siRNA-2抑制作用最明显,抑制率分别为86.8％和100%. MTT试验显示：siRNA-2转染SW480细胞和HSC3细胞后,细胞增殖能力明显降低,细胞增殖抑制率分别为29.5%和43.4%. Transwell小室迁移实验表明,HSC3细胞在转染24 h后,细胞迁移能力明显减弱,迁移抑制率高达72.4%. 流式细胞术检测细胞凋亡结果显示：转染36 h后,SW480细胞实验组细胞凋亡率为21.9%;转染24 h后,HSC3细胞实验组细胞凋亡率为44.7%. 本实验成功的筛选出了特异性的抑制fbxl 20基因的siRNA片段,并初步鉴定了fbxl 20基因的功能.     

RNAi沉默SDF-1基因对人胃癌裸鼠原位移植瘤生物学行为的影响及机制

目的:探讨RNAi技术沉默基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)对人胃癌裸鼠原位移植瘤生物学行为的影响及可能机制。方法:利用RNAi-SDF-1细胞及对照组细胞建立人胃癌裸鼠皮下移植瘤,Western Blot检测裸鼠皮下移植瘤SDF-1蛋白表达情况。利用裸鼠皮下移植瘤建立其原位移植瘤模型,8周后处死裸鼠解剖尸体,检测原位移植瘤生长、凋亡及远处脏器转移情况。Western Blot检测SDF-1沉默对通路蛋白Akt、p-Akt、NF-κB、p-NF-κB以及侵袭转移相关基因E-cadherin、MMP-7表达的影响。结果:成功建立RNAi-SDF-1裸鼠原位移植瘤模型。与空白及阴性对照组比较,RNAi-SDF-1组裸鼠原位移植瘤生长缓慢,体积与质量均明显降低,差异有统计学意义(P0.01)。流式细胞术结果显示,RNAi-SDF-1组肿瘤细胞凋亡率明显高于空白及阴性对照组(P0.01)。尸体解剖结果显示,RNAi-SDF-1组肿瘤腹腔淋巴结及肝脏转移率明显低于空白及阴性对照组(P0.05)。Western Blot结果显示,与空白及阴性对照组比较,RNAi-SDF-1组肿瘤组织中Akt、p-Akt、NF-κB、p-NF-κB、MMP-7表达水平明显降低,E-cadherin表达水平明显升高(P0.01)。结论:RNAi-SDF-1能够有效抑制人胃癌SGC7901细胞裸鼠原位移植瘤的生长,诱导肿瘤细胞凋亡,抑制其腹腔淋巴结、肝脏转移,其机制可能与抑制PI3K-Akt、NF-κB信号通路及MMP-7的表达并上调E-cadherin的表达相关。     

藏红花素对人膀胱移行细胞癌T24细胞裸鼠移植瘤的实验研究

目的检测藏红花素（crocin）对人膀胱移行细胞癌T24细胞株裸鼠移植瘤的作用。方法12只裸鼠右后腿背侧皮下接种T24移植瘤细胞后随机分为PBS对照组（6只）和50mmol/L藏红花素治疗组（6只）。从接种后第28d开始,每3d一次于肿瘤局部注射给予PBS或藏红花素,每3d测量1次肿瘤大小,并绘制移植瘤生长曲线;处理后第21d杀鼠摘取移植瘤,光镜、电镜观察肿瘤组织形态改变;RT-PCR检测Bcl-2、Bax mRNA表达;SP-免疫组织化学法分析sur-vivin、cyclinD1蛋白表达。结果藏红花素对人膀胱癌T24细胞裸鼠移植瘤的生长具有抑制作用,抑制率达28.7%。HE染色结果显示肿瘤标本内出现片状坏死灶;电镜观察可见凋亡的形态学改变;RT-PCR检测结果显示Bcl-2的表达水平下降,而Bax的表达水平上调;免疫组化结果显示经藏红花素处理后survivin、CyclinD1的表达下调。结论藏红花素能够抑制人膀胱癌T24细胞裸鼠移植瘤的生长,作用的分子机制与下调Bcl-2、survivin、cyclinD1和上调Bax基因表达有关。     

shRNA靶向干扰COX-2抑制人肝癌裸鼠皮下移植瘤及其血管生成

目的:探讨重组干扰质粒pshRNA-COX-2对人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成的抑制作用。方法:重组干扰质粒pshRNA-COX-2转染Hep3B细胞并筛选后,RT-PCR和Western blot检测COX-2mRNA和蛋白表达,RT-PCR检测VEGFmRNA表达。将被成功转染的Hep3B细胞种植于裸鼠皮下,测量肿瘤大小,4周后处死裸鼠,免疫组织化学法检测肿瘤组织中COX-2蛋白表达和肿瘤微血管密度(MVD)。结果:与未转染细胞相比,干扰组COX-2mRNA和蛋白表达抑制率分别为65.3%和52.8%(P<0.05),干扰组VEGFmRNA表达抑制率为56.5%(P<0.05)。干扰组瘤体大小明显小于阴性组和空白组(P<0.01)。干扰组COX-2得分和MVD均明显低于阴性组和空白组(P<0.01)。结论:pshRNA-COX-2通过抑制COX-2表达明显抑制人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成。     

封闭Skp2基因表达对大肠癌LS-174T细胞裸鼠皮下移植瘤生长与转移抑制的研究

目的:建立人大肠癌LS-174T细胞的裸鼠皮下移植瘤模型,应用Skp2基因特异性siRNA重组腺病毒治疗,观察其在体内对肿瘤生长和转移的影响,并探讨其相应的作用机制.方法:将大肠癌LS-174T细胞以5×106个/只接种于雌性裸鼠右侧腋窝皮下,待平均瘤体积长至50～150mm3时筛选成模动物分组给药,分别给予重组腺病毒pAD-Skp2/siRNA 5×108和5× 109pfu/只瘤内注射,阳性组给予今又生5×109pfu/只瘤内注射,另设空病毒对照组.每周给药2次,连续4周,以安乐死时肿瘤体积大小和重量来评价治疗效果.然后用Western Blot检测肿瘤组织中Skp2、p27kipl、c-myc、Bcl-2的表达,用免疫组化法检测肿瘤组织中MMP-2的表达.结果:安乐死时阴性对照组平均肿瘤体积与空病毒对照组生长情况基本一致(P≥0.05);今又生组平均肿瘤体积明显小于模型组(P≤0.05);pAD-Skp2/siRNA低、高剂量组平均瘤体积均显著低于模型组(P≤0.01),且有剂量依赖性.今又生组瘤重抑制率为58.16％,pAD-Skp2/siRNA低、高剂量组瘤重抑制率分别为75.59％、86.45％.Skp2蛋白表达在模型组、空病毒对照组和今又生组均有较强表达,而在pAD-Skp2/siRNA低剂量和高剂量组均未见表达;而p27kipl仅在pAD-Skp2/siRNA低剂量和高剂量组有表达,其余三组均未见其表达;c-myc和bcl-2表达趋势一致,均在模型和空病毒对照组有表达,而在今又生组和pAD-Skp2/siRNA两个剂量组均表达明显减弱.模型组和空病毒对照组有棕黄色阳性表达,而今又生组和pAD-Skp2/siRNA两个组均未检测到MMP-2阳性表达.结论:在高转移性大肠癌LS-174T裸鼠移植瘤模型中,封闭Skp2基因可以抑制肿瘤的增殖和转移,其作用机制与细胞周期调控、细胞凋亡中的c-myc和bcl-2以及基质金属蛋白酶MMP-2相关.     

STAT3-siRNA对人结直肠癌细胞的干涉作用

目的:研究STAT3-siRNA对STAT3基因表达阳性的结直肠癌细胞凋亡的影响。方法:应用脂质体转染试剂将STAT3-siRNA表达盒(STAT3-siRNA expression cassettes,STAT3-SECs)体外转染至人结直肠癌SW480细胞及人成纤维细胞中,同时分别设立人成纤维对照组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组。于48h后收集细胞,先经荧光染色方法观察细胞表象变化,再通过流式细胞仪检测人结直肠癌SW480细胞凋亡情况,后分别提取细胞总RNA,用RT-PCR测定STAT3基因在mRNA水平的表达。结果:SW480STAT3-SECs组的细胞可见凋亡小体,出现明显的凋亡现象,而人成纤维对照组、人成纤维STAT3-SECs组、SW480对照组、SW480错配链-SECs组和SW480空转染试剂组未出现明显的凋亡现象。SW480STAT3-SECs组细胞的凋亡比率较SW480对照组、SW480错配链-SECs组和SW480空转染试剂组有明显的增高。RT-PCR所得数据经统计学处理得出:SW480STAT3-SECs组细胞的STAT3基因表达在mRNA水平上显著低于SW480对照组(P0.01);而人成纤维对照组与人成纤维STAT3-SECs组,SW480细胞对照组与SW480错配链-SECs组、SW480空转染试剂组之间无明显差异(P0.05)。结论:应用RNAi技术沉默STAT3基因可以降低人结直肠癌SW480细胞中STAT3的表达,诱导细胞的凋亡。     

A functional variant of IC53 correlates with the late onset of colorectal cancer

The IC53 gene was reported to be upregulated in the colon adenocarcinoma cell line SW480. Here, we show that the expression level of IC53 is positively correlated with the grade and depth of invasion in adenocarcinoma of the colon. Injection of IC53 stably transfected HCT-116 cells into athymic nude mice promoted tumor growth. Furthermore, overexpression of IC53 increased cell invasive growth, which could be dramatically prevented by knocking down IC53 with siRNA. The effects of IC53 on cell-invasive growth were mediated by upregulation of integrins, activation of phosphatidylinositol 3-kinase and phosphorylation of Akt. A single-nucleotide polymorphism rs2737 in the IC53 gene created a potential microRNA379 target site, and microRNA379 expression inhibited IC53 translation. Among 222 patients with colorectal cancer, the C/C rs2737 genotype was associated with late onset of colorectal cancer (median age 63.0 versus 55.3 years, P = 0.003). The frequency of the C/C rs2737 genotype was much lower in patients who developed colorectal cancer below the age of 45 years than in individuals over age 45 years (10.8% versus 26.6%, P = 0.039). These data indicated that IC53 is a positive mediator for colon cancer progression, and IC53-rs2737 may serve as protection from the onset of colorectal cancer.     

Intrahepatically transplanted human cord blood cells reduce SW480 tumor growth in the presence of bispecific EpCAM/CD3 antibody

Humanized mice were generated in order to investigate the anti-tumor efficacy of bispecific antibodies. The engraftment, distribution and differentiation of mononuclear cells (MNC) from cord blood transplanted into the liver of newborn non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were measured. Using a human-specific polymerase chain reaction (PCR), human cells were found to be present in the liver for a time range from 5 min to 5 days. After long-term engraftment of 42 days, the highest level of human cells was measured in mouse thymus, with lower levels in spleen and bone marrow. Engrafted human cells in mouse organs showed T-cell differentiation only, as measured by CD3, CD4 and CD8 expression. The MNC transplanted intrahepatically into newborn mice were tested for T-cell mediated anti-tumor activity in vivo against subcutaneously transplanted human SW480 colon carcinoma in NOD/SCID mice. A delay of SW480 tumor growth in mice in the presence of a bispecific epithelial cell-adhesion molecule (EpCAM)/CD3 antibody was found to be associated with the presence of immunoreactive human CD3 cells within the SW480 tumor. Our data provide evidence that the intrahepatic transplantation of cord blood stem cells into newborn mice represents a valuable model for establishing functionally active human T cells with anti-tumor activity.     

Mediatory role of BLT2 in the proliferation of KRAS mutant colorectal cancer cells

Inflammatory lipid mediators play various roles in colorectal cancer progression through complex pathways. However, the mechanism by which lipoxygenase-derived inflammatory lipid mediators contribute to colorectal cancer progression remains elusive. In this study, we found that BLT2, a cell surface GPCR for leukotriene B₄ and 12‑hydroxyeicosatetraenoic acid, is highly upregulated in KRAS mutant LOVO and SW480 colorectal cancer cells and plays critical roles in mediating proliferation through activation of phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (Akt) and subsequent upregulation of cyclin D1. Exposure to BLT2 siRNA or LY255283, a specific BLT2 inhibitor, clearly suppressed the proliferation of KRAS mutant colorectal cancer cells and markedly increased cell cycle arrest by downregulating the PI3K/Akt-cyclin D1 cascade. Xenograft tumor formation by LOVO and SW480 cells in athymic mice was also substantially reduced by treatment with the BLT2 inhibitor in vivo. Together, our study demonstrates that BLT2 is necessary for the proliferation of LOVO and SW480 cells and thus may be a potential therapeutic target for the treatment of KRAS mutant colorectal cancer.     

AS2O3抑制人结肠癌生长的体内外实验研究

目的：通过研究三氧化二砷（AS2O3）对人结肠癌细胞株LS-174T在体内外实验中增殖情况的影响,探讨As2O3治疗结肠癌的机制。方法：1、试验分组：体外细胞培养及体内动物实验均分三组：对照组,As2O3低剂量组,As2O3高剂量组。2、MTT法测定细胞增殖数的情况。3、丫啶橙/溴乙啶（AO/EB）双荧光染色检测细胞凋亡率。4、免疫组织化学法测定结肠癌细胞和裸鼠实体肿瘤中HIF-1α和VEGF的表达。5、测量各组裸鼠实体肿瘤的重量及体积。结果：体外试验中,由MTT法可以看出,As2O3治疗组细胞生存率明显低于对照组（P〈0.01）,其中AS2O3高剂量组明显低于低剂量组。双荧光染色结果As2O3高剂量组细胞凋亡百分率明显高于低剂量组和对照组（P〈0.01）。免疫组化结果显示结肠癌细胞HIF-1α和VEGF在对照组的阳性表达率明显高于As2O3治疗组（P〈0.01）,低剂量组高于高剂量组（P〈0.05）。体内实验中,治疗组平均瘤重和瘤体积均较对照组明显减低,（P〈0.01）。HIF-1α及VEGF在裸鼠肿瘤组织中均有阳性表达,其中对照组较AS2O3治疗组高（P〈0.01）。结论：As2O3能够抑制体内外人结肠癌细胞的生长,并能明显抑制HIF-1α和VEGF的表达,从而抑制肿瘤的生长。     

胰岛素样生长因子1（IGF-I）通过PI-3K/Akt途径对结肠癌细胞凋亡的影响

目的：探讨胰岛素样生长因子-1（IGF-I）通过磷酯酰肌醇3-激酶/蛋白激酶B（PI-3K/Akt）信号通路对结肠癌细胞株SW480凋亡率的影响及其凋亡抑制蛋白survivin表达水平的变化。方法：培养结肠癌SW480细胞株,实验分成三组：未加IGF-I空白组、IGF-I刺激组、IGF-I＋LY294002阻断组,检测阻断剂LY294002是否阻断PI-3K/Akt通路（Western Blot检测三组P-Akt表达情况）;Western Blot及免疫荧光观察三组survivin蛋白表达变化;MTT法检测细胞增殖活性,流式细胞术检测细胞凋亡情况。结果：Western blot结果显示LY294002可抑制IGF-I诱导的p-Akt的表达（P〈0.05）;阻断IGF-I诱导的PI-3K/Akt通路后MTT显示结肠癌细胞SW480增殖抑制率升高（P〈0.05）,流式细胞术分析显示凋亡率明显上升（P〈0.05）;Western blot及免疫荧光结果显示LY294002可抑制IGF-I诱导的survivin的表达（P〈0.05）。结论：IGF-I可通过PI-3K/Akt通路诱导survivin表达,从而抑制结肠癌细胞SW480的凋亡。     

Antitumor activity of IL-32β through the activation of lymphocytes,and the inactivation of NF-κB and STAT3 signals

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8⁺) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.