不同转移潜能食管癌细胞株裸鼠模型的建立

背景：食管癌的转移率较高,是导致患者死亡的主要原因,但其机制仍不完全清楚。目的：建立具有不同转移潜能的高侵袭能力食管癌细胞株亚系裸鼠模型。方法：应用Transwell侵袭小室从食管癌细胞株Eca-109中筛选出高侵袭能力的食管癌细胞株亚系。观察母系和亚系细胞形态,MTT法检测细胞增殖能力的变化,划痕法检测细胞迁移能力。将食管癌Eca-109细胞及其亚系Eca-109 T4细胞分别皮下注射于裸鼠体内诱导移植瘤模型,观察成瘤率、成瘤时间、肿瘤生长情况。结果：成功从食管癌Eca-109细胞株中筛选出高侵袭能力的食管癌细胞株亚系Eca-109 T4,两者细胞形态无明显差异。与Eca-109细胞相比,Eca-109 T4细胞增殖能力和划痕愈合能力均明显增强。Eca-109组和Eca-109 T4组裸鼠成瘤率均为100%,与Eca-109组相比,Eca-109 T4组裸鼠成瘤时间更早且瘤体生长更快。结论：成功建立了不同转移潜能的高侵袭能力食管癌细胞株亚系裸鼠成瘤模型,为食管癌转移的进一步研究提供了理想模型。     

玉竹提取物B对人食管癌细胞Eca-109增殖与凋亡的影响

目的观察玉竹提取物B（EB-PAOA）对人食管癌细胞Eca-109增殖与凋亡的影响。方法将体外培养的Eca-109细胞与不同浓度的EB-PAOA共育,采用MTT法检测Eca-109细胞增殖抑制率,采用流式细胞仪检测Eca-109细胞凋亡率。结果随着EB-PAOA浓度增大、作用时间延长,Eca-109细胞的增殖抑制率逐渐升高（P均〈0.05）,呈时间、剂量依赖性;随着EB-PAOA浓度增加,Eca-109细胞凋亡率逐渐增加,呈一定浓度依赖性（P均〈0.05）。结论EB-PAOA能够抑制人食管癌细胞Eca-109的增殖,并诱导其凋亡。     

Effect of 2-（3-carboxy-1-oxopropyl） amino-2-deoxy-D-glucose on human esophageal cancer cell line

AIM:To determine whether 2-(3-carboxy-l-oxopropyl) amino-2-deoxy-D-glucose(COPADG),a derivative of D- amino-glucose,inhibited the growth of human esophageal cancer cell line Eca-109. METHODS:Effects of COPADG on Eca-109 cells cultured in RPMI 1640 medium were examined by a tetrazolium- based colorimetric assay(MTT assay). RESULTS:COPADG inhibited the growth of Eca-109 cells in a dose-and time-dependent manner;the maximum inhibition rate was 83.75%. CONCLUSION:COPADG can directly inhibit the proliferation of Eca-109 cells,which may serve as the experimental evidence for development of new drugs for esophageal cancer therapy.     

Effect of 2-(3-carboxy-1-oxopropyl) amino-2-deoxy-D-glucose on human esophageal cancer cell line

AIM: To determine whether 2-(3-carboxy-1-oxopropy1)amino-2-deoxy-D-glucose (COPADG), a derivative of Damino-glucose, inhibited the growth of human esophageal cancer cell line Eca-109.METHODS: Effects of COPADG on Eca-109 cells cultured in RPMI 1640 medium were examined by a tetrazoliumbased colorimetric assay (MTT assay).RESULTS: COPADG inhibited the growth of Eca-109 cells in a dose- and time-dependent manner; the maximum inhibition rate was 83.75%.CONCLUSION: COPADG can directly inhibit the proliferation of Eca-109 cells, which may serve as the experimental evidence for development of new drugs for esophageal cancer therapy.     

构建shRNA真核表达质粒抑制CXCR4对人食管癌细胞Eca-109迁移的影响

目的 构建人CXCR4 mRNA的shRNA真核表达载体质粒,特异性抑制人食管癌细胞CXCR4的表达,并筛选抑制效果理想的质粒,研究对食管癌细胞迁移的影响.方法 以人CXCR4 mRNA编码区中3条不同序列作为RNA干扰靶点,分别构建3个shRNA真核表达载体质粒GYJ-1、GYJ-2、GYJ-3,并进行测序.用脂质体转染人食管癌细胞株Eca109,实时定量逆转录-聚合酶链反应（RT-PCR）检测mRNA水平.应用细胞划痕试验研究干扰质粒对食管癌细胞迁移的影响.结果 GYJ-1呈高效特异地抑制人CXCR4的表达;而GYJ-2及GYJ-3抑制效果较GYJ-1差.GYJ-1显著抑制食管癌细胞的迁移.结论 通过构建CXCR4的shRNA真核表达载体导入食管癌细胞,可有效抑制人食管癌细胞中CXCR4的表达,并影响食管癌细胞的迁移.     

ShRNA-mediated gene silencing of β-catenin inhibits growth of human colon cancer cells

AIM: To observe the gene silencing mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205.METHODS: Two shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with LipofectamineTM2000. The down-regulations of β-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two β-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry.RESULTS: These two shRNA vectors targeted against β-catenin efficiently suppressed the expression of β-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level.The shRNA-mediated gene silencing of β-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing.CONCLUSION: These specific shRNAs targeted against β-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against β-catenin is of potential value in gene therapy of colon cancer.     

JNK MAPK信号通路在食管癌细胞系Eca-109细胞中的作用机制

目的探讨c-jun氨基末端激酶1/2（c-jun N-teuninal kinase,JNK 1/2）信号通路在食管癌细胞系Eca-109细胞中的作用。方法体外培养Eca-109细胞,以特异性JNK信号转导通路抑制剂SP600125处理Eca-109细胞;RT-PCR的方法检测JNK1和JNK2基因的表达,Western blot法检测JNK和p-JNK蛋白的表达,MTT（3-（4,5-Dimethylthiazol-2-yl）-2,5-di-phenyltetrazolium bromide）法检测细胞增殖,流式细胞术检测细胞凋亡。结果 Eca-109细胞经SP600125分别处理24h和48 h后,分别与对照组比较,JNK1 mRNA的表达无统计学差异（均P〉0.05）,JNK2 mRNA的表达也无统计学差异（均P〉0.05）,但活化的JNK即P-JNK1/2蛋白的表达显著减少,细胞的增殖显著被抑制,细胞的凋亡率有统计学差异（均P〈0.05）。结论 JNK信号通路可能在Eca-109细胞的发生发展中发挥重要作用。     

125I粒子组织间植入对裸鼠食管癌移植瘤的影响

目的 研究125I放射性粒子组织间植入对于食管癌移植瘤的影响.方法 将人食管癌细胞Eca-109注入裸鼠腋侧皮下建立食管癌裸鼠移植瘤模型,随机分成对照组(牛理盐水+空心粒子)、125I粒子组(剂量22.2 MBq×1)和顺铂组(1 ms/kg).观察人食管癌移植瘤的生长情况和病理学改变.结果 术后16 d,对照组、125I粒了组、顺铂组的瘤块平均重量分别为(0.20±0.06)g,(0.12±0.03)g,(0.12±0.05)g,3组间差异有统计学意义(P<0.05).病理结果,125I粒子组、顺铂组镜下可见变性、坏死的食管癌肿瘤细胞.125I粒子组、顺铂组肿瘤组织坏死范围大于对照组(P<0.05).结论 125I粒子植入可以引起食管癌移植瘤肿瘤组织变性、坏死,抑制肿瘤组织的生长.     

下调CD44基因表达对人胃癌SGC7901细胞裸鼠移植瘤的抑制作用及其机制

目的探讨靶向CD44的短发夹RNA(shRNA)对人胃癌裸鼠移植瘤生长抑制作用及其机制。方法利用CD44 shRNA细胞及对照细胞株建立裸鼠原位移植瘤及皮下种植瘤模型,监测肿瘤生长变化,采用RT-PCR及免疫组织化学方法检测瘤体内CD44表达的变化,并进一步检测上皮-间质转化(EMT)相关因子表达的变化。结果成功构建了CD44 shRNA转染荷瘤皮下及原位裸鼠模型。与对照shRNA组、空白对照组分别比较,CD44 shRNA组荷瘤裸鼠肿瘤生长速度减慢,皮下种植瘤及原位移植瘤质量减轻,差异均有统计学意义(P均0.01)。RT-PCR结果发现,CD44 shRNA组CD44mRNA表达在皮下种植瘤及原位移植瘤均显著下调,差异均有统计学意义(P均0.01)。CD44 shRNA组细胞EMT相关基因E-cadherin表达增加,N-cadherin、Vimentin表达降低。结论 CD44 shRNA可以有效抑制人胃癌SGC7901细胞裸鼠移植瘤生长,并下调CD44的表达水平,这可能与CD44基因参与EMT相关。     

腺病毒介导的凋亡素、内皮抑素双基因对食管癌的实验研究

目的探讨携带有凋亡素(vp3、Apoptin)、内皮抑素(Endostatin)双基因的重组腺病毒载体在食管癌等多种肿瘤细胞内的表达情况及致凋亡作用,为进一步的食管癌治疗应用研究打下基础。方法将已纯化的携带凋亡素和内皮抑素基因的腺病毒Ad-vp3-IRES-sEndo-His感染食管癌细胞Eca-109、小鼠肝癌细胞Hepa1-6、结肠癌细胞LoVo提取各种细胞RNA,针对凋亡素、内皮抑素基因的表达进行RT-PCR检测,同时对感染腺病毒Ad-vp3-IRES-sEndo-His、Ad-vp3的食管癌细胞通过流式细胞仪、Hoechst33258染色等方式进行细胞凋亡率的检测。结果 RT-PCR检测显示被感染的多种肿瘤细胞均可表达凋亡素、内皮抑素基因的mRNA,Ho-echst33258染色显示被Ad-vp3-IRES-sEndo-His感染的食管癌细胞出现凋亡形态学改变,用流式细胞仪测定时段最高凋亡率达47.7%,与对照组相比具有统计学意义(P<0.05)。结论携带凋亡素及内皮抑素双基因的腺病毒载体能在多种肿瘤细胞中表达,感染食管癌细胞后,能有效诱导其凋亡且其致凋亡率高于仅携带凋亡素的单基因腺病毒载体。     

5-LOX基因沉默对人胰腺癌细胞SW1990裸鼠移植瘤生长的影响

目的 观察裸鼠的人胰腺癌细胞SW1990移植瘤内注射靶向5-脂氧合酶(5-LOX)的短发夹RNA(shRNA)对移植瘤以及瘤组织5-LOX和血管内皮生长因子(VEGF)表达的影响,探讨其临床应用价值.方法 将胰腺癌细胞SW1990接种至裸鼠背部皮下,待移植瘤生长至100 mm3左右后随机分为shRNA1组、shRNA2组、阴性对照shNC组和脂质体组.每组6只,雌雄各半.实验组瘤内注射相应shRNA 50μg/次,1次/3 d,共7次.对照组瘤内注射脂质体液100 μl/只.每3 d测体重及移植瘤长、短径一次.第29天处死动物,取肿瘤组织,称瘤重.应用免疫组化和RT-PCR法检测移植瘤组织5-LOX、VEGF的mRNA和蛋白的表达.结果 shRNA1组、shRNA2组、shNC组和脂质体组的瘤重分别为(32.5±19.0)mg、(30.1±14.1)mg、(50.5±15.6)mg和(71.7±25.4)mg,shRNA1组、shRNA2组、shNC组抑瘤率分别为54.7%、58.0%和29.5%,shRNA1组、shRNA2组较shNC组和脂质体组相差显著(P值均＜0.05).shRNA1组和shRNA2组移植瘤的5-LOX、VEGF mRNA和蛋白的表达较shNC组和脂质体组均显著减少,但两治疗组之间未见明显差异,shNC组和脂质体组之间也无明显差异.结论 靶向5-LOX的RNA干扰治疗通过直接抑制5-LOX的表达、间接抑制VEGF的表达而抑制SW1990裸鼠移植瘤的生长.     

Ribonucleic acid interference targeting S100A4 (Mts1) suppresses tumor growth and metastasis of anaplastic thyroid carcinoma in a mouse model

CONTEXT: The characteristic feature of malignant neoplasm is invasion and metastasis. Despite advances in the management of thyroid carcinoma and other solid tumors, metastasis continues to be the most significant cause in cancer mortality. OBJECTIVE: Our objective was to examine the effects of S100A4 expression knockdown by RNA interference on the growth and metastasis of human anaplastic thyroid carcinoma cells (ARO) and the sensibility of ARO to paclitaxel after S100A4 knockdown. DESIGN: A plasmid construct was made that expressed small hairpin RNA (shRNA) specific for S100A4. The construct was stably transfected into ARO cells (ARO/S100A4-shRNA). The tumorigenicity, metastatic potential, and sensibility of ARO/S100A4-shRNA to paclitaxel were investigated. RESULTS: S100A4 expression was reduced by 71.3 +/- 4.7% in ARO/S100A4-shRNA by real-time RT-PCR analysis. The growth rate of ARO/S100A4-shRNA was reduced by 46 +/- 7.6% in a cell proliferation assay. Cell cycle analysis showed increased G(2)/M accumulation in ARO/S100A4-shRNA. Tumor formation and growth induced by sc injection of 5 x 10(6) ARO/S100A4-shRNA into the nude mice were significantly reduced, and no tumor metastasis was found in any of the mice. We also demonstrated significant induction of apoptosis in ARO/S100A4-shRNA after incubation with 15 nm paclitaxel, indicating that tumor cells were sensitized to chemotherapy as a result of S100A4 knockdown. CONCLUSIONS: These data suggest that reduction of S100A4 by RNA interference is a viable approach to inhibit tumor growth and metastasis. Given that S100A4 is overexpressed in many kinds of tumors, the current study provides the proof of concept in its therapeutic potential.     

VEGF165 antisense RNA suppresses oncogenic properties of human esophageal squamous cell carcinoma

AIM: To investigate the effect of antisense RNA to vascularendothelial growth factor165 (VEGF165) on human esophagealsquamous cell carcinoma call line EC109 and the feasibilityof gene therapy for esophageal carcinoma.METHODS: Using subclone technique, the full length ofVEGF165 amino acid cDNA, which was cut from pGEM-3Zf( + ), was cloned inversely into the eukaryotic expressionvector pCEP4 . The recombinant plasmid pCEP-AVEGF165was transfected into EC109 cell with Iipofectamine. After astable transfection, dot Blot, enzyme-linked immunosorbentassay (ELISA), laser confocal imaging system analysis,transmission electron microscopy and flow cytometry wereperformed to determine the biological characteristics ofEC109 cell line before and after transfection in vitro andwhether there was a reversion in the tumorigenic propertiesof the EC109 cell in vivo.RESULTS: The eukaryotic expression vector pCEP-AVEGF165was successfully constructed and transfected into EC109cells. The expression of VEGF165 was significantly decreasedin the transfected cells while the biological characteristics ofthe cells were not influenced by the expression of antisensegene. The tumorigenic and angiogenic capabilities weregreatly reduced in nude mice, as demonstrated by reducedtumorend volume (820 ± 112.5)mm3 vs (7930 ± 1035) mn3and (7850 ± 950) mm3, P ＜ 0.01) and microvessel density(8.5 ± 1.2)mm-2 vs (44.3 ± 9.4)mm-2 and (46.4 ± 12.6)mm-2, P ＜ 0.01) in comparison between experimentalgroup, empty vector transfected group and control group.CONCLUSION: The angiogenesis and tumorigenicity ofhuman esophageal squamous cell carcinoma were effectivelyinhibited by VEGF165 antisense RNA. Antisense RNA toVEGF1, can potentially be used as an adjuvant therapy forsolid tumors.     

Silencing of EphA2 inhibits invasion of human gastric cancer SGC-7901 cells in vitro and in vivo

Receptor tyrosine kinases (RTKs), the common products of transforming oncogenes, have been widely used as indicators in the genesis and progression of human tumors. Until now, the erythropoietin-producing human hepatocellular (Eph) receptors have been recognized as the largest family of RTKs. EphA2, one member of Eph receptors, locates on human chromosome 1p36.1 which is a hot region for cancer research. It has been reported that high EphA2 expression levels were correlated with the tumor metastasis and poor prognosis. Increased expression of EphA2 can promote tumor growth and enhance the metastatic potential. To further define the function of EphA2 in malignant invasion, we employed the small interference RNA (siRNA) technique to knockdown gene expression of EphA2 in the gastric cancer SGC-7901 cell. Our results showed that the expression of double stranded RNA led to the efficient and specific inhibition of endogenous EphA2 expression in SGC-7901 cells. Silencing of EphA2 expression inhibited cell proliferation, caused cell cycle arrest, and decreased cell invasion in vitro. In addition, intratumoral injection EphA2 siRNA plasmid suppressed the growth of SGC-7901 cells xenografts in nude mice. Furthermore, knockdown of EphA2 expression reduced the expression of matrix metalloproteinase-9 (MMP-9) in vitro and in vivo. In conclusion, our findings demonstrate that silencing of EphA2 inhibits gastric cancer SGC-7901 cell proliferation, invasion and MMP-9 expression, which indicate that the specific inhibition of EphA2 may be a potential approach for gastric cancer therapy.     

人参皂甙Rh2对食管癌Eca-109细胞caspase3、caspase8基因的影响

目的:探讨人参皂甙Rh2诱导人食管癌Eca-109细胞凋亡过程中caspase3、caspase8凋亡调节基因的相互关系及可能的作用机制.方法:应用MTT法测定其对细胞的生长抑制作用,流式细胞术分析人参皂甙Rh2作用后细胞凋亡及增殖的变化,应用免疫细胞化学及Western blot技术检测用药前后凋亡相关基因caspase3、caspase8蛋白表达的变化.结果:人参皂甙Rh2对人食管癌Eca-109细胞有生长抑制作用,并呈时效和量效依赖关系.流式细胞仪分析结果发现,食管癌Eca-109细胞在DNA组方图上出现典型的亚二倍体峰即凋亡峰,在细胞周期中的分布也发生了明显的变化,其48h凋亡率明显高于对照组(19.10%±2.12% vs 2.10%±0.87%,P<0.01).免疫细胞化学及Western blot技术结果显示,20mg/L人参皂甙Rh2作用72h后食管癌Eca-109细胞caspase3、caspase8蛋白表达明显升高(0.35±0.04 vs 0.10±0.02,0.84±0.06 vs 0.31±0.11,均P<0.05).结论:人参皂甙Rh2具有诱导食管癌Eca-109细胞凋亡和抑制细胞...     

Effects of cyclooxygenase-2 on human esophageal squamous cell carcinoma

AIM:To study the relationship between the cyclooxy-genase(COX)-2 gene and the proliferation and apopto-sis of esophageal squamous carcinoma EC109 cells.METHODS:The techniques of RNA interference(RNAi)and cell transfection,as well as the levels of oncogenic-ity in nude mice,were used to study the role of COX-2 in the esophageal squamous carcinoma cell(ESCC)line EC109.Following RNAi and transfection,Western blot-ting analysis was used to determine the expression of the COX-2 protein.The 3-(4,5-dimethylthiazol...     

Mitochondrial pathway of the lysine demethylase 5C inhibitor CPI-455 in the Eca-109 esophageal squamous cell carcinoma cell line

BACKGROUNDEsophageal cancer is a malignant tumor of the digestive tract that is difficult to diagnose early. CPI-455 has been reported to inhibit various cancers, but its role in esophageal squamous cell carcinoma (ESCC) is unknown.AIMTo investigate the effects and mechanism of the lysine demethylase 5C inhibitor, CPI-455, on ESCC cells.METHODSA methyl tetrazolium assay was used to detect the inhibitory effect of CPI-455 on the proliferation of Eca-109 cells. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential were assessed by flow cytometry. Laser confocal scanning and transmission electron microscopy were used to observe changes in Eca-109 cell morphology. The protein expression of P53, Bax, lysine-specific demethylase 5C (KDM5C), cleaved Caspase-9, and cleaved Caspase-3 were assayed by western blotting.RESULTSCompared with the control group, CPI-455 significantly inhibited Eca-109 cell proliferation. Gemcitabine inhibited Eca-109 cell proliferation in a concentration- and time-dependent manner. CPI-455 caused extensive alteration of the mitochondria, which appeared to have become atrophied. The cell membrane was weakly stained and the cytoplasmic structures were indistinct and disorganized, with serious cavitation when viewed by transmission electron microscopy. The flow cytometry and western blot results showed that, compared with the control group, the mitochondrial membrane potential was decreased and depolarized in Eca-109 cells treated with CPI-455. CPI-455 significantly upregulated the ROS content, P53, Bax, Caspase-9, and Caspase-3 protein expression in Eca-109 cells, whereas KDM5C expression was downregulated.CONCLUSIONCPI-455 inhibited Eca-109 cell proliferation via mitochondrial apoptosis by regulating the expression of related genes.