Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma~*

Objective： To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods： The expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and human pancreatic carcinoma cell lines （MIAPaCa-2） was detected by using RT-PCR. Results： The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion： The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines （Hep-2） is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

The impact on secreted VEGF of Hep-2 human laryngeal cancer cell induced by Rg3

Objective：The aim of this study was to investigate the af ecting of Rg3 to secreted VEGF of human laryngeal carcinoma Hep-2 cells and its mechanism of inhibition to tumor angiogenesis. Methods：Cultured human laryngeal cancer cellline Hep-2 and human vascular endothelial cells in vitro, cells got into the period of exponential phase of growth, was diviced into 3 groups：group I （control group）, group II （DDP group）, group III （Rg3 group）. Added to the Hep-2 cells Rg3 and DDP, made Rg3 final concentration was 300μg/mL, and DDP was 3μg/mL. 48 h later, specimens from sample to be done immunocytochemistry, and the protein of VEGF in Hep-2 cells to be detected. Col ecting Hep-2 cells supernatant, some was used to measure the protein level of VEGF in Hep-2 cells supernatant by ELISA. Some was used to culture HVEC. 24 h later, cellgrowth inhibition rate of human vascular endothelial was determined by MTT. Results：The protein level of VEGF was evi-dently higher in group I compared to group II and group III, it was not only in Hep-2 cells, but also in supernatant of Hep-2 cells. There was no significantly dif erent between group II and group III. MTT results showed that, the human vascular endothelial cellgrowth inhibition rate of group I was significantly lower than that of group II and group III （P〈0.05）. At the same time the HVEC growth inhibition rate of group II was significantly lower than that of group III （P〈0.05）. Conclusion：The inhibition to tumor angiogenesis of Rg3 is stronger than traditional chemotherapy drug cisplatin. It worke by reducing the biological ef ects of secreted VEGF, But the ef ecting worke by reducing the activity of secreted VEGF itself or af ecting endothelial function of VEGF receptor or some other ways to be further studied.

Effect of DRB on the Biological Characteristics of Human Laryngeal Carcinoma Hep-2 Cell Line

In order to study the effect of 5, 6-Dichloro-l-13-D-ribofuranosyl-benzimidazole （DRB） on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry （FCM） and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose- and time-dependent manner. After being treated with 0, 10, 20, 40, 80 μmmol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was （0.68±0.19）%, （1.95±0.12）%, （8.51±0.26）%, （11.26±0.17）% and （14.99±0.32）%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 μmmol/L, and with the increase of DRB concentration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.

The impact of ^(60)Co γ-ray on apoptosis to Hep-2 human laryngeal cancer cell in the condition of reoxygenation after hypoxia

Objective： The aim of the study was to establish models of Hep-2 laryngeal cancer cell line of different oxygen supplying, trying to investigate the impact of normoxia, hypoxia, reoxygenation after hypoxia on apoptosis and expression of proteins HIF-1α and p53 to Hep-2 human laryngeal cancer cell line induced by ^60Co γ-ray. Methods： Human laryngeal cancer Hep-2 cells were divided into 3 groups： group A （normoxia）, group B （hypoxia）, and group C （reoxygenation after hypoxia）. All of the cells were exposed to 5 Gy dosage of γ-ray. Flow cytometry （FCM） was used tomeasure the protein levels of HIF-1α and p53 and to detect cell apoptosis. The protein levels of HIF-la and p53 were also determined by immunohistochemistry and Western blotting. The expression of HIF-la mRNA was determined by RT-PCR. Results： The protein levels of HIF-1α and p53 were evidently increased in group B compared to group A. The protein levels of HIF-1α and p53 in group C were lower compared to group B; the rate of apoptosis in group C was higher than that in group B. Conclusion： Hypoxia decreased the effect of apoptosis induced by ^60Co γ-ray in Hep-2 human laryngeal cancer cell line. The apoptosis pathway maybe related to some other genes or proteins but not p53 in the conditions of hypoxia and reoxygenation after hypoxia.

The impact of ^(60)Co γ-ray on cycles to Hep-2 human laryngeal cancer cell in the condition of hypoxia

Objective： The aim of the study was to investigate the impact of 60Co y-ray on apoptosis, cell cycles and the expression of protein hypoxia-inducible factor-1α （HIF-1α） to Hep-2 cell line in the conditions of normoxia and hypoxia. Methods： Hep-2 cell were divided into 2 groups： group A （normoxia） and group B （hypoxia）. All of the ceils were exposed to y-ray with dosage being 0, 1, 3, 5, 10, 20, and 40 Gy. Flow cytometry was used to measure the protein level of HIF-1α and to detect apoptosis and cell cycles. The protein level of HIF-1α was also determined by immunohistochemistry and Western blotting. Results： The protein level of HIF-1α in group B was significantly higher than that in group A. In group A, low doses （1-5 Gy） of y-ray had caused G0/G1 cell cycle arrest and high doses （10-40 Gy） had caused G2/M cell cycle arrest. In group B, without exposure of y-ray （0 Gy） had caused G0/G1 cell cycle arrest, all of the different dosage of y-ray could cause G2/M cell cycle arrest. The curve of apoptosis rate in group A was a parabola, the apoptotic rate was related to the dosage of y-ray in a dosage dependent manner. The peak was at the point of 5 Gy. The apoptosis rate in group A was significantly higher than that in group B. Conclusion： Different doses of y-ray could cause different cell cycles arrest then make different impact on apoptosis to Hep-2 ceil. The lower apoptosis rate in condition of hypoxia maybe has a relationship with G2/M cell cycle arrest. Up-regulated HIF-1α protein may be one of the reasons for G2/M cell cycle arrest.

MTA2对喉癌细胞系Hep-2的增殖、迁移和侵袭的影响

目的研究转移相关基因2(Metastasis-associated gene 2,MTA2)对喉癌细胞系Hep-2的增殖、迁移和侵袭的影响。方法采用化学合成的针对MTA2基因的小干扰RNA(siRNA-MTA2)下调该基因的表达,采用Real timePCR(RT-PCR)和Western blot检测转染效果,通过MTT、细胞划痕实验和Transwell法分别检测MTA2对Hep-2细胞增殖、迁移和侵袭的影响。结果将siRNA-MTA2转染入Hep-2后,Hep-2细胞中MTA2mRNA和蛋白表达水平都明显降低,细胞增殖能力显著降低,细胞的迁移和侵袭能力显著降低。结论在喉癌细胞系Hep-2中MTA2与恶性增殖和侵袭性相关,有望成为喉癌早期诊断、分子治疗的靶点。

mTOR信号通路调控1,25二羟维生素D_3抑制喉癌细胞Hep-2细胞增殖的研究

目的研究1,25二羟维生素D3抑制喉癌细胞Hep-2细胞增殖作用以及对雷帕霉素靶蛋白(mTOR)信号通路的影响。方法用不同剂量1,25二羟维生素D3(10-8 mol/L、10-7 mol/L、10-6 mol/L)分别处理Hep-2细胞24、48、72h,四甲基偶氮唑蓝法(MTT)检测Hep-2细胞的增殖情况,并计算抑制率;采用流式细胞仪分析1,25二羟维生素D3对Hep-2细胞周期分布的影响,Western blot检测1,25二羟维生素D3对mTOR信号通路的影响。结果不同浓度1,25二羟维生素D3均可抑制Hep-2细胞增殖,改变细胞周期分布,使G0/G1期Hep-2细胞比例增高。1,25二羟维生素D3干预后Hep-2细胞TSC1、TSC2蛋白表达较对照组增高(P<0.01),Rheb蛋白表达明显降低;mTOR蛋白及其磷酸化水平表达与对照组比较均降低(P<0.01),mTOR蛋白磷酸化表达降低尤为明显(P<0.01);4EBP-1蛋白表达较对照组增高(P<0.01)。结论 1,25二羟维生素D3改变喉癌细胞Hep-2细胞周期分布,影响mTOR信号通路蛋白表达,从而抑制细胞增殖。

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells

Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.

基于压缩感知理论的HEp-2细胞识别

针对HEp-2细胞识别中低特征描述质量及低识别率等问题,提出一种基于压缩感知理论的识别6类HEp-2(human epithelial type 2)细胞的方法。对HEp-2细胞图像进行二维离散小波变换(two-dimensional discrete wavelet transform,2D-DWT);在小波变换域中,采用奇异值分解(singular value decomposition,SVD)方法对HEp-2细胞图像进行提取特征值;采用压缩感知分类器对HEp-2细胞做出类别判断。仿真结果表明,基于压缩感知理论的HEp-2细胞识别方法识别率高,具有高质量特征描述、低计算复杂度及易于实现等特性。

白藜芦醇对喉癌Hep-2细胞顺铂敏感性的增强作用及其机制

目的:探讨白藜芦醇提高喉癌Hep-2细胞株对顺铂(DDP)敏感性的作用,并阐明其作用机制。方法:Hep-2喉癌细胞随机分为空白对照组和DDP组(终浓度分别为3、6、12和24μmol·L-1)培养6、12、24及48h。CCK-8法检测各组细胞生存率;选出DDP的最佳作用浓度6μmol·L-1和作用时间24h。Hep-2喉癌细胞株经培养后随机分为对照组、DDP组、白藜芦醇组、sirtinol组、白藜芦醇+sirtinol组、白藜芦醇+DDP组、sirtinol+DDP组和白藜芦醇+sirtinol+DDP组。CCK-8法检测各组细胞生存率;采用免疫荧光法观察各组细胞中Sirt1蛋白的表达强度;AnnexinⅤ-FITC Kit法检测各组细胞凋亡率。结果:CCK-8检测,与对照组比较,DDP组和白藜芦醇组细胞生存率明显下降(P<0.01);与DDP组比较,白藜芦醇+DDP组细胞生存率明显下降(P<0.01);与白藜芦醇+sirtinol+DDP组比较,白藜芦醇+DDP组细胞生存率明显下降(P<0.05)。细胞免疫荧光法检测,与DDP组比较,白藜芦醇+DDP组细胞中Sirt1蛋白的平均免疫荧光强度明显升高(P<0.01);与白藜芦醇+sirtinol+DDP组比较,白藜芦醇+DDP组细胞中Sirt1蛋白的平均免疫荧光强度明显升高(P<0.01)。AnnexinⅤ-FITC Kit检测,与DDP组比较,白藜芦醇+DDP组细胞凋亡率均明显升高(P<0.01);与白藜芦醇+sirtinol+DDP组比较,白藜芦醇+DDP组细胞凋亡率明显升高(P<0.01)。结论:白藜芦醇提高Hep-2喉癌细胞株对DDP的敏感性,其机制可能与提高喉癌细胞中Sirt1蛋白表达有关联。

Inhibitory Effects of Celecoxib and Sc-58125 on Proliferation of Human Carcinoma of Larynx Hep-2 In Vitro

Summary: The inhibitory effects of two kinds of selective cyclooxygenase-2 inhibitors on the proliferation of human carcinoma of larynx Hep-2 in vitro and their corresponding mechanisms were investigated. Hep-2 cells were cultured with two kinds of selective cyclooxygenase-2 inhibitors (Sc-58125 and Celecoxib) at various concentrations for 24 h. Morphological changes were observed under the phase microscopy and the growth suppression was detected by using MTT colorimetric assay. Apoptotic DNA fragments were observed by agarose gel electrophoresis, and the cell cycle and apoptotic rate were detected by flow cytometry (FCM) respectively. Hep-2 cells became rounded and detached from the culture dish after being treated with Celecoxib for 24 h, however, they remained morphologically unchanged with Sc-58125. Sc-58125 could increase G 2 phase cells, whereas, Celecoxib rose G 1 phase cells. Both of the two effects were dose-dependent. Moreover, the Hep-2 cells cultured with 50 μmol/L and 100 μmol/L Celecoxib showed obvious apoptosis, with the nuclear DNA of cells exhibiting characteristic DNA ladder. So Sc-58125 could inhibit the proliferation of Hep-2 cells by altering the G 2 phase cells. However, Celecoxib had the same effect by changing the G 1 phase cells and inducing apoptosis at higher concentration.

In Vitro Study on the Effect of Bee Venom on Some Cell Lines and Lumpy Skin Disease Virus

Bee venom （BV） was used from long time ago in the medical field as treatment of chronic joint affections. In the recent decades, the screening process of new sources of antimicrobials discovers its high advantageous characteristics for combating various types of microbes, as well as trials to discover its anti-cancer medicinal fields. Lumpy skin disease virus （LSDV） causes disease in cattle of economic importance, and this work aimed to find treatment as well as alternative inactivant for LSDV. The use of bee venom as antiviral was experimented in this work and exhibited satisfied inhibitory effects on LSDV, meanwhile, the antigenic properties was still intact. The viability of virus was tested in tissue culture cells lines and in embryonated chicken eggs. According to doses and time of exposure, the cell lines of Hep-2 （human larynx carcinoma） and MCF7 （breast carcinoma cell line） were treated with different concentrations of BV and examined after 24 h post-inoculation. The Hep-2 and MCF7 cell lines were treated with various concentrations of BV in descending doses as follow： 25, 20, 15, 10, 5 and 0.5 ug/mL of BV. Then bee venom pathological effects on Hep-2 cells and MCF7 cells were observed, such as apoptosis, retarded growths and cytolysis. The results indicate the possibilities of using bee venom as anti-neoplastic and antiviral.

Transfection,overexpression and clinical application of human 60 kDa Ro/SSA autoantigens in HEp-2 cells

Objective To develop an improved substrate for indirect immunofluorescence test (IIF) for detecting anti-Ro60/Sjogren's syndrome A (Ro/SSA) autoantibodies.Methods 60-kDa Ro/SSA autoantigens (Ro60) cDNAs were obtained from human placental cDNA library using polymerase chain reaction (PCR) and were cloned into the mammalian expression vector-pEGFP-C1. Then, the recombinant plasmids were transfected into HEp-2 cells. We confirmed the overexpression, localization and antigenicity of fusion proteins in transfected cells by means of immunoblotting, confocal fluorescence microscopy and IIF. HEp-2 and HEp-Ro60 were analyzed by IIF using a panel of 10 precipitin-positive anti-Ro human sera simultaneously.Results Stable expression of Ro60-green fluorescent protein (Ro60-GFP) fusion proteins were maintained ten more generations. Ro60-GFP kept the antigenicity of Ro while demonstrating its own characteristic immunofluorescent pattern in HEp-Ro60 cells. The transfectants dramatically increased the sensitivity of IIF testing (a mean increase of 6.7-fold in endpoint titer). Eight overten (8/10) positive anti-Ro sera showed characteristic immunofluorescent patterns for HEp-Ro60, including two sera that were anti-nuclear antibodies (ANA) negative for untransfected HEp-2. IIF-ANA in all healthy sera was negative for HEp-Ro60. Conclusions As a new substrate for IIF, the Ro60 transfectants can be used to detect anti-Ro antibodies. In addition, transfected HEp-2 cells keep the immunofluorescent properties of HEp-2 cells in IIF-ANA tests and can be employed as a substrate for routine IIF-ANA detection.

鸦胆子油乳对Hep-2细胞增殖与凋亡诱导作用的研究

目的:探讨鸦胆子油乳注射液对喉癌Hep-2细胞的增殖抑制及调亡诱导作用。方法:利用MTT比色法检测5个不同药物浓度(0.1、0.2、0.4、0.6和0.8g/L)对细胞的抑制率;采用倒置显微镜观察细胞的形态变化;流式细胞仪检测细胞凋亡率;实时荧光定量PCR检测凋亡基因Bcl-2和Bax的表达。结果:MTT显示,鸦胆子油乳对Hep-2细胞的生长抑制作用呈剂量和时间依赖性。倒置显微镜下可见细胞凋亡的形态学改变,显示加药组细胞染色体固缩,聚集呈块状、新月状,出现凋亡小体。流式细胞仪检测显示,0.2、0.4和0.6g/L药物作用48h后凋亡率分别为(42.58±4.36)%、(57.71±3.07)%和(75.50±3.93)%,显著高于对照组(0.96±0.17)%,差异有统计学意义,F=8.82,P=0.003;实时荧光定量PCR结果显示,随着鸦胆子油乳注射液的浓度增加,Bcl-2mRNA的表达减少,Bax mRNA的表达量逐渐上升,与对照组比较差异有统计学意义,P<0.01。结论:鸦胆子油乳能够抑制Hep-2细胞的增殖并诱导其凋亡,作用呈剂量和时间依赖性。

姜黄素促进紫杉醇抑制肿瘤细胞的增殖

培养的人喉癌细胞Hep-2及肝癌细胞HepG-2,分别用不同浓度姜黄素（CUR,0~50μmol/L）和紫杉醇（PTX,0~5nmol/L）单独处理或联合处理（5μmol/L CUR＋0.5nmol/L PTX、10μmol/L CUR＋0.5nmol/L PTX）.处理48h后,用WST-1细胞增殖试剂盒检测细胞增殖情况,再用结晶紫染色检测活细胞的密度,最后用Hoechst 33258凋亡染色试剂盒检测细胞凋亡情况.结果显示：姜黄素或紫杉醇单独处理对Hep-2细胞和HepG-2细胞的增殖均有抑制作用,并呈现出一定剂量效应关系;姜黄素与紫杉醇联合处理后细胞的增殖速率要比紫杉醇单独处理的明显降低;同时,姜黄素与紫杉醇联合处理后细胞的凋亡要比紫杉醇单独处理的更为明显.上述研究结果说明,姜黄素对紫杉醇抑制肿瘤细胞Hep-2和HepG-2的增殖有促进作用,并能促进紫杉醇诱导细胞的凋亡.

MCPH1基因对喉癌Hep-2细胞迁徙及侵袭影响

目的探讨MCPH1(microcephalin1)基因过表达对人喉癌Hep-2细胞迁移、侵袭的影响。方法将质粒pc DNA3.1(-)/MCPH1(实验组)和pc DNA3.1(-)(阴性对照组)分别转染人喉癌Hep-2细胞,采用Realtime PCR法检测转染48h后的细胞内MCPH1的表达;采用细胞划痕试验及细胞Transwell试验分别检测MCPH1过表达对喉癌细胞迁移、侵袭能力的影响。结果实验组Hep-2细胞中MCPH1的RNA水平明显比阴性对照组高,两组比较有统计学意义(P<0.05);实验组细胞迁移及侵袭能力较阴性对照组明显降低,差异有统计学意义(P<0.05)。结论 MCPH1基因过表达可抑制喉癌Hep-2细胞的迁移侵袭能力。

Chemoresistance of CD133＋ cancer stem cells in laryngeal carcinoma

Background Mounting evidence suggests that tumors are histologically heterogeneous and are maintained by a small population of tumor cells termed cancer stem cells. CD133 has been identified as a candidate marker of cancer stem cells in laryngeal carcinoma. This study aimed to analyze the chemoresistance of CD133＋ cancer stem cells.

MICA在喉癌组织和细胞中的表达及意义

目的:探讨MICA在喉癌细胞Hep-2和喉鳞状细胞癌组织中的表达及意义。方法:RT-PCR和Western-blot方法检测MICA mRNA和蛋白在喉癌细胞和喉癌组织中的表达情况,并以喉外伤组织为对照。结果:RT-PCR检测结果显示MICA在Hep-2细胞中强势表达。与对照相比,MICA在喉癌组织中的表达均有增强(P<0.01),MICA表达强度与临床分期无关。Western-blot检测结果显示MICA蛋白在Hep-2细胞呈强势表达,但在喉癌组织中MICA蛋白随临床分期增高而表达降低。结论:MICA mRNA在Hep-2细胞和喉癌组织中均强势表达,其表达水平与临床分期无关。MICA蛋白在Hep-2细胞呈强势表达,但在喉癌组织中MICA蛋白随临床分期增高而表达降低。由此可见,MICA可能通过多种机制参与喉癌的发生、发展。