外源p53对人肺癌细胞生长的抑制

目的　观察外源性野生型p53对有p53基因突变的人肺癌细胞系生长的影响。方法　用PCR SSCP及DNA测序 ,选择p53突变的人肺巨细胞癌系 80 1 D。构建野生型p53表达质粒PZiP p53。用基因枪介导外源基因。经G41 8筛选得到转染细胞系 80 1 D p53。用PCR检测外源基因 ,观察转染细胞恶性生长的变化。结果　转染细胞系 80 1 D p53体外长期传代有外源性p53基因存在 ,转染细胞生长明显受到抑制 ,集落形成抑制率达 96% ,裸鼠异种移植致瘤性降低 ,肿瘤生长明显缓慢。结论　外源性野生型p53经基因枪导入有p53基因突变的人肺癌细胞后可长期存在于转染细胞中 ,且明显抑制所转染的癌细胞的恶性生长。     

Selective biophysical interactions of surface modified nanoparticles with cancer cell lipids improve tumor targeting and gene therapy

Targeting gene- or drug-loaded nanoparticles (NPs) to tumors and ensuring their intratumoral retention after systemic administration remain key challenges to improving the efficacy of NP-based therapeutics. Here, we investigate a novel targeting approach that exploits changes in lipid metabolism and cell membrane biophysics that occur during malignancy. We hypothesized that modifications to the surface of NPs that preferentially increase their biophysical interaction with the membrane lipids of cancer cells will improve intratumoral retention and in vivo efficacy upon delivery of NPs loaded with a therapeutic gene. We have demonstrated that different surfactants, incorporated onto the NPs’ surface, affect the biophysical interactions of NPs with the lipids of cancer cells and normal endothelial cells. NPs surface modified with didodecyldimethylammoniumbromide (DMAB) demonstrated greater interaction with cancer cell lipids, which was 6.7-fold greater than with unmodified NPs and 5.5-fold greater than with endothelial cell lipids. This correlated with increased uptake of DMAB-modified NPs with incubation time by cancer cells compared to other formulations of NPs and to uptake by endothelial cells. Upon systemic injection, DMAB-NPs demonstrated a 4.6-fold increase in tumor accumulation compared to unmodified NPs which also correlated to improved efficacy of p53 gene therapy. Characterization of the biophysical interactions between NPs and lipid membranes of tumors or other diseased tissues/organs may hold promise for engineering targeted delivery of therapeutics.     

肿瘤坏死因子相关凋亡诱导配体基因对人大肠癌细胞株HT29作用的研究

目的 探讨肿瘤坏死因子（TNF）相关的凋亡诱导配体基因（TRAIL）用于人大肠癌细胞株HT29基因治疗的实验研究。方法 将重组腺病毒载体（Ad)介导的TRAIL基因作用于人大肠癌细胞株HT29,通过相差显微镜、MTT比色法和流式细胞仪,研究分析其对HT29细胞作用的效果。结果 Ad/GT-TRAIL能引起HT29细胞出现明显的形态学改变,对HT29细胞的生长抑制率和凋亡诱导率分别为54．3％和11．1％;联合Ad/PGK-GV16后,生长抑制率和凋亡诱导率均显著提高（P＜0．05）,分别为82．7％和24．6％。结论 Ad/GT-TRAIL能有效诱导HT29的凋亡从而抑制HT29的生长,联合Ad/PGK-GV16后将显著提高其疗效。     

逆转录病毒介导p53基因对食管癌细胞株的生长抑制作用

目的观察人类野生型p53(wt p53)基因对人食管癌细胞系的抑制作用.方法用逆转录病毒为载体将外源性wt p53基因导入人食管癌细胞系ECA109,通过体外及小鼠体内实验研究转入基因的表达及对肿瘤的抑制作用.结果 p53在转染细胞ECA109/p53中表达水平提高.外源性wt p53基因的导入和表达能使ECA109细胞的生长速率减低,软琼脂集落形成能力下降,G0 G1期细胞比例增加、S期细胞比例降低,凋亡指数升高,裸鼠体内成瘤能力明显下降.结论逆转录病毒载体介导的外源性野生型p53能够抑制人食管癌细胞生长.     

腺病毒介导p16基因转移及诱导黑色素瘤细胞凋亡

目的 进一步了解Ｐ１６基因对肿瘤细胞的作用。方法 通过腺病毒介导外源性Ｐ１６基因转移到Ｐ１６基因突变的人黑色素瘤细胞秒ＷＭ－９８３Ａ,对Ｐ１６基因的表达,细胞生长的抑制与机制和对肿瘤模型的治疗效果进行分析。结果 外源性Ｐ１６基因在靶细胞高水平的表达显著地抑制了ＷＭ９８３Ａ的生长和集落形成,流式细胞仪检测显示细胞Ｇ１的期阻滞并发生了凋亡,瘤内注Ａｄ－ｐ１６对裸鼠体内移植瘤有一定抑瘤作用。结论 Ｐ１６     

转染NK4基因对人胰腺癌SW1990细胞生物学特性的影响

背景与目的:肝细胞生长因子(hepatocytegrowthfactor,HGF)通过其受体c-Met的激活,在调节肿瘤侵袭和转移和血管形成中具有重要作用。NK4不仅是HGF的拮抗剂,也是血管形成的抑制剂,阻断HGF/c-Met途径和肿瘤血管的形成可成为抗肿瘤治疗的策略之一。为此,我们构建NK4基因真核细胞表达载体并进行转染研究,探讨NK4基因在胰腺癌细胞中的表达及其对胰腺癌细胞生物学特性的影响。方法:对重组pcDNA3/hNK4质粒进行酶切,将NK4基因克隆到真核表达载体pRC/CMV2,应用脂质体将重组pRC/CMV2-hNK4质粒转入胰腺癌SW1990细胞中,采用逆转录聚合酶链反应(RT-PCR)及免疫印迹(Westernblot)分别检测转染的肿瘤细胞中NK4mRNA和蛋白的表达并筛选出高表达的细胞克隆。采用Transwall小室和Matrigel侵袭小室测定转染NK4基因对胰腺癌细胞运动和侵袭的影响。结果:转导NK4基因的SW1990细胞可表达并分泌NK4,RT-PCR扩增出预期的453bp片段,Westernblot显示有50000的NK4蛋白,转染NK4基因对胰腺癌细胞SW1990的生长无抑制作用(P>0.05),但可显著抑制HGF或成纤维细胞所诱导胰腺癌细胞的运动和侵袭(P<0.01),而转染空载体则无此作用(P>0.05)。结论:转染NK4基因可抑制胰腺癌细胞的运动和侵袭,在胰腺癌抗转移治疗中可能具有重要作用。     

白细胞介素-18基因转染的肺癌细胞与树突状细胞融合体的抗肿瘤作用

目的研究白细胞介素-18(IL-18)基因转染的肺癌细胞与树突状细胞(DC)融合体的抗肿瘤作用。方法(1)从人外周血单核细胞诱导培养DC,与IL-18基因转染的NCI-H460肺癌细胞融合,经免疫磁珠筛选。(2)设转染组(GT组)、空载体转染组(PT组)、未转染组(NT组)和对照组(BC组),分别以IL-18转染的融合细胞、pcDNA3.1~ 转染的融合细胞、未转染的融合细胞活化的T细胞为效应细胞,BC组不含效应细胞,用乳酸脱氢酶(LDH)法测定效应细胞对NCI-H460细胞的杀伤作用。(3)荷瘤裸鼠皮下注射上述3种效应细胞,以BC组为空白对照,比较4组裸鼠肿瘤体积和重量。结果3组效应细胞在体外对NCI-H460细胞的杀伤率分别为53.1%,30.1%和31.5%;3组裸鼠肿瘤体积及肿瘤重量明显低于BC组,以GT组最低。结论IL-18基因转染的融合细胞可有效诱导抗肿瘤免疫。     

应用mRNA差异显示方法克隆维甲酸诱导的人肺腺癌GLC-82细胞分化相关基因

目的克隆维甲酸诱导的人肺腺癌ＧＬＣ８２细胞分化相关基因。方法在全反式维甲酸诱导人肺腺癌ＧＬＣ８２细胞系分化的基础上,采用ｍＲＮＡ差异显示技术,对这个细胞系以全反式维甲酸诱导前及诱导后８小时、２４小时和４天的细胞基因表达差异情况进行分析。结果在全反式维甲酸诱导前后的细胞之间存在明显的基因表达差异。有些基因经维甲酸（ＲＡ）诱导后持续表达或瞬时表达,而有些基因经ＲＡ作用后表达水平降低或完全被抑制。经克隆筛选,获得了一些经全反式维甲酸诱导激活或抑制的差异表达基因片段,对其中的３个片段进行了序列分析和同源性比较。结论全反式维甲酸具有调控分化相关基因表达与否的重要作用,由ＲＡ诱导的肿瘤细胞再分化是一个多基因参与的过程     

Inhibition of autophagy by 3-MA enhances IL-24-induced apoptosis in human oral squamous cell carcinoma cells

Background

Interleukin-24(IL-24), also referred to as melanoma differentiation-associated gene-7(mda-7), is a unique member of the IL-10 gene family, which displays nearly ubiquitous cancer-specific toxicity. The most notable feature of IL-24 is selectively induced growth suppression and apoptosis in various cancer cells, with no harmful effects toward normal cells. Autophagy is a self-protective mechanism in many kinds of tumor cells that respond to anticancer treatment. It is reported that autophagy inhibition could enhance the effects of many kinds of anticancer treatments, including gene therapy. However, whether IL-24 is effective to treat oral squamous cell carcinomas (OSCC) and if autophagy inhibition could improve the anticancer effect of IL-24 towards OSCC is has not been detected.

Methods

MTT assays were carried out to determine the cell proliferation; Transfection was used to gene transfer; Western Blot was performed to detect the protein level of LC3II, P62, Beclin 1, Cleaved caspase-3, β-Tubulin and β-actin; Apoptosis rates and cell cycle alteration were analyzed using flow cytometry; Autophagy induction was confirmed by MDC staining, GFP-LC3 staining and transmission electron microscopy. Amount of IL-24 in the culture medium was quantified by ELISA. Apoptosis in vivo was analyzed by TUNEL assay. HE staining was used to observe the morphology of the samples.

Results

In the present study, we proved that IL-24 have a novel anticancer effect towards KB cells and that autophagy inhibition could improve the anticancer effect of IL-24. IL-24 treated cells showed autophagy characteristics and autophagy inhibition by 3-methyladenine (3-MA) significantly enhanced IL-24-induced apoptosis. Similar results were obtained in the KB cells xenograft tumor model.

Conclusions

These results suggest that the combination of autophagy inhibitors and IL-24 based on the AdLTR₂EF1α-mediated gene transfer could be a promising way to cure OSCC.     

EXPERIMENTAL STUDIES ON RADIATION-INDUCIBLE HUMAN TNF GENE THERAPY FOR CANCER

ＥＸＰＥＲＩＭＥＮＴＡＬＳＴＵＤＩＥＳＯＮＲＡＤＩＡＴＩＯＮ－ＩＮＤＵＣＩＢＬＥＨＵＭＡＮＴＮＦＧＥＮＥＴＨＥＲＡＰＹＦＯＲＣＡＮＣＥＲＣａｏＸｕｅｔａｏ曹雪涛ＺｈａｎｇＷｅｉｐｉｎｇ章卫平ＷａｎｇＪｉａｎｌｉ王建莉ＨｕａｎｇＸｉｎ黄欣ＱｉｎＺｈｉｈ...     

Combination therapy of murine liver cancer with il-12 gene and HSV-TK gene

Interleukin-12 (IL-12), produced by antigen-presenting cell, is a heterodimeric cytokine that has multiple immune regulatory functions, various studies have shown that IL-12 has multiple anti-tumor effects and anti-metastatic properties for many tumors.[1] Suicide gene approaches are also widely investigated recently, the gene products are capable of converting the non-toxic pro-drug to the active cytotoxic agent. The most commonly used suicide gene and pro-drug is the herpes simplex virus th…     

EMP-1基因对人食管癌细胞系生长的影响

背景与目的：本室曾用正常食管粘膜上皮细胞和食管癌细胞进行微阵列分析,得到一批在正常食管粘膜上皮细胞和食管癌细胞中差异表达的基因,其中包括上皮膜蛋白－1（EMP－1）基因,EMP－1基因的mRNA水平表达差异有6倍。本文进一步研究EMP－1基因在食管癌发生发展中的作用;方法：构建人EMP－1基因的真核表达载体,并转染人食管癌细胞系,Western blot检测转染效果,RT－PCR检测转染后EMP－1的表达情况,绘制生长曲线,流式细胞检测细胞周期的变化;结果：EMP－1基因成功转入人食管癌细胞系EC9706,EMP－1基因表达量变高,细胞生长变慢,S期减少;结论：EMP－1基因与食管癌发生有一定关系。     

GM—CSF基因修饰的肿瘤细胞疫苗对荷瘤小鼠的抗肿瘤作用

目的研究GM-CSF基因修饰的肿瘤细胞作为瘤苗的可行性.方法通过逆转录病毒载体,将小鼠GM-CSF基因导入EL-4淋巴瘤细胞中,研究EL-4/GM-CSF在同系C57BL/6小鼠中的成瘤性及其诱导抗肿瘤免疫的效果.结果EL-4/GM-CSF细胞在小鼠中的成瘤性下降,50%小鼠无瘤生存.射线灭活的EL-4/GM-CSF瘤苗能有效地保护野生型肿瘤细胞的攻击,在肿瘤生长的早期对实验性荷瘤小鼠进行治疗,能延长荷瘤宿主的存活时间(33.1±1.8)与对照组EL-4/Wt(23.2±1.5天)比差异有显著性(P＜0.01).基因修饰的瘤苗作用明显增强.结论GM-CSF基因修饰的肿瘤疫苗可诱导较强的抗肿瘤免疫反应,导致肿瘤的部分根除,本实验为基因修饰的肿瘤疫苗的临床应用提出了一定的实验依据.     

白细胞介素-24 delE5对人类白血病细胞系K562的抑制作用

 目的　探索白细胞介素（IL）-24 delE5对人类白血病细胞系K562的抑制作用。方法　用TPA诱导白血病细胞系U937和HL-60向单核-巨噬细胞分化后,检测mda-7／IL-24及其选择性剪接体IL-24 delE5的表达。构建IL-24 delE5真核表达载体,稳定转染K562细胞。通过MTT法、集落形成实验、流式细胞术、Annexin-Ⅴ／PI检测及动物实验,观察IL-24 delE5对K562细胞增殖、集落形成、细胞周期、凋亡及体内致瘤性的影响;同时与mda-7／IL-24进行比较。结果　在TPA诱导分化的U937和HL-60细胞中发现IL-24 delE5的表达。稳定转染IL-24 delE5的K562增殖及集落形成明显下降,与空载体相比,G0／G1期细胞比例由（24.46±3.99）%增至（42.69±3.04）%,细胞周期阻滞于G0／G1期,体内实验证实K562细胞移植瘤生长明显被抑制。与mda-7／IL-24相比,上述各实验结果差异均无统计学意义。结论　IL-24 delE5与mda-7／IL-24一样对人类白血病细胞系K562具有明显的体内外抑制作用,该作用可能与IL-24 delE5所引起细胞周期G0／G1期阻滞有关。     

Treatment of rat hepatoma by locally injection of murine IL-12 retrovirus packaging cell

　Objective To investigate the therapeutic effects of the murine IL-12 (mIL-12) retrovirus packaging cell line on hepatoma injected locally. Methods The retrovirus vector encoding mIL-12 gene was constructed and transfected into packaging cell line PA317. The cells were then used to treat the rats with experimental orthotopic hepatoma at different time. The therapeutic effects, immune functions of the hosts, pathological and toxicological responses were documented. Results the results showed that the mIL-12 retrovirus packaging cell line could significantly inhibit the growth of the hepatoma cells injected locally to the hepatoma. The early treatment made the rats survive long, while the medium or late stage treatment could prolong the life time of the rats compared with the bland control group or bland vector control group, though the rats did not survive. The number of NK cells and T cells increased significantly in the treatment group. The effects of the early treatment were superior to those of the medium and late stage treatment. Moreover, the transfection of IL-12 gene locally in the hepatoma tissue could make the hepatoma disappear from other liver lobe. This phenomenon demonstrated that IL-12 could activate the immune cells of the host to kill the untransfected tumor cells. This is very important for IL-12 to be used in gene therapy clinically. Meanwhile, the hepatoma would not recur in the rats that had survived more than 2 months from the early treatment after being re-challenged with tumor cells. Conclusion the results showed that IL-12 gene injected locally in the hepatoma tissue could enhance the anti-tumor immunity of the host.     

SOCS3基因在人肺腺癌细胞株A549中的表达及其对细胞增殖的影响

目的 探讨SOCS3基因在人肺腺癌细胞株A549中的表达及其对细胞增殖的影响。方法 以脂质体为载体将pEFSOCS3和pSV2neo共转染至人肺腺癌细胞株A549，利用G418筛选获得阳性克隆；应用RT-PCR和免疫细胞化学法测定转染前后细胞中SOCS3表达情况；采用四甲基偶氮唑兰(MTT)显色法检测基因转染前后细胞生长情况；应用流式细胞仪进行细胞周期分析。结果 RT-PCR和免疫细胞化学法证实转染前A549细胞中无SOCS3基因表达，转染后A549细胞中SOCS3基因呈稳定表达。MTT检测显示转染pEFSOCS3的A549细胞生长明显受到抑制，抑制率为41．07％。流式细胞仪检测结果显示转染pEFSOCS3的A549细胞Go／G，期细胞比例显著增高，S、G2／M期细胞比例显著降低。结论 SOCS3蛋白可能通过负性调控细胞内信号通路抑制肺癌细胞增殖。     

NGX6对结肠癌细胞系HT-29基因表达谱的影响

背景与目的:结肠癌是最常见的恶性肿瘤之一,其发病机制仍未完全明了。本研究在前期工作的基础上探讨候选抑瘤基因NGX6对结肠癌细胞系HT-29基因表达谱的影响。方法:通过脂质体介导的基因转染构建稳定表达NGX6的结肠癌细胞系pcDNA3.1( )/NGX6/HT-29,以NGX6转染组为实验组,空白质粒转染组为对照组,利用高通量的表达谱芯片BiostarH-80sx1筛选差异表达基因,RT-PCR验证部分基因芯片的实验结果。结果:NGX6基因转染结肠癌细胞系HT-29后引起该细胞系基因表达谱广泛地改变,其中表达差异3倍以上的基因共377个,这些差异表达基因的功能涉及多个方面,包括细胞信号转导﹑细胞周期调控﹑细胞凋亡﹑细胞骨架和运动﹑基因转录和翻译、DNA损伤修复﹑蛋白质合成和代谢等。其中包括一些非常重要的信号转导通路上的分子改变,如Wnt/β-catenin信号通路上的DKK1、ILK、MMP1、COL11A1,ILK信号通路上的ILK,Eph-Ephrins信号通路上的EpHB2,RhoA信号通路上的ROCK2,RanGTPase信号通路上的RANBP1,以及RB/RBBP1信号通路上的RBBP1等。结论:NGX6转染引起了一些非常重要的信号转导通路上的分子改变,可能通过参与调节这些信号转导通路而发挥其抗肿瘤增殖和转移的作用。     

nm23-H1基因转染前后人高转移大细胞肺癌细胞株L9981 PKA活性变化的实验研究

目的探讨nm23-H1基因转染和forskolin对人高转移大细胞肺癌细胞株L9981 PKA活性的影响.方法应用PKA通路特异激动剂forskolin对人高转移大细胞肺癌原代细胞株L9981、转基因细胞株L9981-nm23-H1-pLXSN和空载体转染细胞株L9981-pLXSN共同培养,应用放免法检测forskolin处理后不同时间点三个细胞株PKA的活性变化.结果 (1)forskolin处理前L9981、L9981-pLXSN和L9981-nm23-H1-pLXSN的PKA活性经F检验有显著性差异(P<0.01);两两比较L9981-nm23-H1-pLXSN的PKA活性显著高于L9981和L9981-pLXSN(P<0.01),而L9981与L9981-pLXSN之间比较无显著性差异(P>0.05).(2)在同一作用时间(30 min)下,三个细胞株经不同浓度forskolin处理后PKA活性均显著升高(P<0.01),在forskolin浓度为100 μmol/L时PKA活性最高,呈剂量依赖关系.(3)三个细胞株在同一浓度forskolin(100 μmol/L)处理下,PKA活性随作用时间升高,在forskolin作用时间为30 min时PKA的活性最高,呈时间依赖关系.结论 (1)nm23-H1基因转染能显著上调人高转移大细胞肺癌细胞株L9981的PKA活性,nm23-H1作为一种肿瘤转移抑制基因的作用机理与PKA信号通路可能有一定联系;(2)forskolin能显著上调L9981细胞株中的PKA活性,PKA活性升高呈时间和剂量依赖关系.     

肺癌靶向基因的研究及价值

肺癌的发生发展与众多肺癌相关基因的表达及突变有密切关系.近期国内外肺癌的相关基因研究显示,COPS3基因的表达在刺激肺癌细胞增殖的过程中可能发挥关键作用,棘皮动物微管样蛋白4-间变淋巴瘤激酶融合基因与表皮生长因子受体突变共存,GA733基因与肺癌的生存期密切相关.同时成纤维细胞生长因子受体1、高迁移率蛋白家族、类清道夫受体成员5和表皮生长因子受体为肺癌的基因靶向治疗提供了新思路.肺癌的治疗需要多基因联合治疗和基因治疗联合常规治疗.     

The gene expression profile of highly metastatic human ovarian cancer cell line by gene chip

In this paper, gene chip technique was used to analyze the difference of gene expression patterns in highly metastatic human ovarian tumor cell line HO-8910PM and in normal ovarian epithelial cells to explore the tumor-associated gene-cluster and its function in the process of occurrence an development of ovarian carcinoma. It will be helpful to comprehensively understand the molecular mechanism of cell transformation, to provide the molecular markers and target genes for clinical diagnosis a…