绿色荧光蛋白表达对检测胶质细胞瘤体内侵袭的意义

利用体外转染绿色荧光蛋白基因的胶质瘤细胞大鼠移植瘤模型,研究胶质瘤体内侵袭行为。方法：携有增强型绿色荧光蛋白（ＥｎｈａｎｃｅｄＧｒｅｅｎＦｌｕｏｒｅｓｃｅｎｔＰｒｏｔｅｉｎ,ＥＧＦＰ）基因的ｐＥＧＦＰ－Ｎ３质粒体转染Ｃ６胶瘤细胞,筛选稳定表达绿色荧光蛋白的细胞克隆,并作流式细胞仪和电镜检测,以立体定向法植入ＳＤ大鼠脑实质内建立大鼠移植模型。４周后处死大鼠并作鼠脑连续石蜡切片,相邻切片分别作苏木素－     

二甲基甲酰胺对胶质瘤细胞基因蛋白水平表达的影响

目的：观察诱导分化剂二甲基甲酰胺（ＤＭＦ）地胶质瘤细胞Ｃ６的增殖抑制作用,探讨作用机理中ＤＭＦ对ｃ－ｍｙｃ、ｐ１６基因蛋白水平表达的影响。方法：「＾３Ｈ」－Ｔ掺入法测增殖抑制率;免疫组化ＳＰ法测Ｃ－ｍｙｃ、ｐ１６基因蛋白水平的表达。结果：应用二甲基甲酰胺后,胶质瘤细胞ＣＰＭ值降低,ＤＮＡ合成总量减少。Ｃ－ｍｙｃ基因胞浆阳性表达下降,ｐ１６基因胞浆阳性表达升高。结论：二甲基酰胺对胶质瘤细胞增殖存在剂量依赖性抑制,作用机理与降低了癌基因Ｃ－ｍｙｃ对Ｃ６细胞恶性增殖的转录调节作用、增强了抑制基因ｐ１６活性有关。     

苯乙酸对胶质瘤细胞C6增殖的抑制及对P16基因的激活作用

目的：观察诱导分化剂苯乙酸对胶质瘤细胞Ｃ６增殖的抑制作用，及对抑癌基因ｐ１６的蛋白水平表达变化的影响。方法：〔＾３Ｈ〕－ＴｄＲ掺入法测细胞增殖率；免疫组化ＳＰ法检测ｐ１６基因蛋白水平的表达。结果：应用苯乙酸后，细胞ＣＰＭ值降低；ｐ１６基因胞浆阳性表达显著升高。结论：苯乙酸对胶质瘤细胞存在剂量依赖性抑制，增强了抑癌基因ｐ１６表达活笥，促使Ｃ６细胞向凋亡方向转化。     

中国人血管抑制素Endostatin基因的分子克隆与序列鉴定

目的：内抑素（Ｅｎｄｏｓｔａｔｉｎ）是一种有效的血管生成抑制因子,分离、克隆和测定中国人的Ｅｎｄｏｓｔａｔｉｎ基因,为进一步研究Ｅｎｄｏｃｔａｔｉｎ的功能奠定了基础。方法：从中国人胎肝组织中提取ｍＲＮＡ,用ＲＴ－ＰＣＲ方法将Ｅｎｄｏｓｔａｔｉｎ的ｃＤＮＡ扩增出,克隆到ｐＧＥＭ－Ｔ载体中,测定其ＤＮＡ序列。结果：中国人的Ｅｎｄｏｓｔａｔｉｎ ｃＤＮＡ序列被成功克隆,序列分析证实为 Ｅｎｄｏｓｔａｔｉｎ基因。结论：中国人的Ｅｎｄｏｓｔａｔｉｎ ｃＤＮＡ基因编码序列与国外文献报道的相应序列完全一致。     

肾癌组织中ras基因产物p21表达和K－ras基因点突变分析

目的研究ｒａｓ基因激活在肾癌发生中的意义。方法应用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）和免疫组织化学检测肾癌组织Ｋ－ｒａｓ基因第１２位密码子突变及其基因产物ｐ２１蛋白的表达水平。结果４２例肾癌中６例Ｋ－ｒａｓ基因第１２位密码子点突变，１９例ｐ２１蛋白阳性表达，正常肾组织和癌旁组织均阴性。ｐ２１表达与肿瘤分期和分级有关。ｐ２１阳性患者的手术后生存率明显低于ｐ２１阴性患者。结论肾癌Ｋ－ｒａｓ基因第１２位密码子点突变并不常见，ｐ２１表达可能成为肾癌恶性程度和预后评价的指标。     

脑胶质瘤p15基因缺失及5‘CPG岛甲基化研究

目的 探讨ｐ１５基因变异及其与脑胶质瘤的发生、恶性进展的关系。方法 利用ＰＣＲ和ＰＣＲ－ｂｓｅｄ甲基化检测技术检测了５６例脑胶质瘤中ｐ１５基因外显子１缺失及５’ＣＰＧ岛甲基化情况。结果 ４３例高级别的脑胶质瘤中，１４例发生了ｐ１５基因缺失（３２．６％），而１３例低级别的脑胶质瘤中无一例发生ｐ１５基因缺失，差异具有显著性（Ｐ〈０．０５）。１例低级别的脑胶质瘤、３例高级别的脑胶质瘤发生了ｐ１５基因５’ＣＰＧ岛甲基化。结论 ｐ１５基因异常可能参与脑胶质瘤的发生、恶性进展。基因纯合缺失是脑胶质瘤中ｐ１５基因失活的主要机制。     

p16基因对脑胶质瘤细胞作用的实验研究

目的 探索ｐ１６基因在脑胶质瘤发生发展过程中的作用及ｐ１６基因在脑胶质瘤临床诊断、治疗中的应用前景。方法 采用脂质体转染的方法,将外源野生型ｐ１６基因导入胶质瘤细胞株Ｕ２５１、Ｃ６,筛选阳怀克隆。同时以空载体质粒ｐＣＤＮＡ３为对照,免疫组化检测ｐ１６基因表达,用ＭＴＴ 测定瘤细胞生长曲线及对转染的瘤细胞在裸鼠体内形成瘤块的变化进行分析。结果 转染ｐ１６基因的Ｕ２５１、Ｃ６细胞有外源ｐ１６基因的整合     

睾丸癌组织C—myc蛋白及ras基因产物p21蛋白表达评价

本文探讨了人睾丸癌组织中Ｃ－ｍｙｃ蛋白及ｒａｓ基因产物Ｐ２１蛋白表达的临床意义。方法：应用Ｃ－ｍｙｃ和Ｐ２１单克隆抗体，通过免疫组织化学方法检测５例正常睾丸组织和２７例睾丸癌组织中ｒａｓ癌基因产物ｐ２１物Ｃ－ｍｙｃ蛋白的表达状况。结果：５例正常睾丸组织中未发现ｐ２１和Ｃ－ｍｙｃ蛋白阳性表达。阳性表达的ｐ２１和Ｃ－ｍｙｃ蛋白分别定位于肿瘤的细胞膜上和细胞核内。     

IL-4基因治疗脑胶质瘤的实验研究

目的 探索一条细胞因子ＩＬ－４基因治疗脑胶质瘤的新途径。方法 用脂质体转染的方法,以重组逆转录病毒做载本,将目的基因鼠ＩＬ－４基因转入包装细胞和大鼠Ｃ６胶质瘤细胞,通过Ｇ４１８抗性筛选,得到分泌ＩＬ－４的细胞株Ψ２ ＩＬ－４和Ｃ６ＩＬ－４。Ｅｌｉｓａ法检测病毒上清中ＩＬ－的含量,通过Ｓｏｕｔｈｅｒｎ ｂｏｌｔ 和Ｄｏｔ ｂｌｏｔ证明ＩＬ－４基因的整合和表达。体外观察并比较了野生型和转基因瘤细胞的细     

D-苧烯对人胰腺癌细胞法尼基蛋白转移酶、p21~(ras)膜结合的抑制作用的体外试验

目的探讨ｄ－艹宁烯在化学诱发实验性肿瘤及人实体瘤的防治作用及其机制。方法采用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ方法观察了ｄ－艹宁烯对胰腺癌细胞中与膜结合的ｐ２１ｒａｓ蛋白表达和法尼基蛋白转移酶活性的抑制作用。同时,采用免疫组化法观察了ｄ－艹宁烯对ｐ２１ｒａｓ在细胞膜上定位的影响。并以Ｎｏｒｔｈｅｒｎｂｌｏｔｉｎｇ方法检测ｄ－艹宁烯对Ｈ－ｒａｓ癌基因在人胰腺癌细胞系中的表达。结果ｄ－艹宁烯对人胰腺癌细胞呈现的抑制作用,其机制可能与其抑制法尼基蛋白转移酶活性,从而降低ｐ２１Ｈ－ｒａｓ蛋白的异戊二烯化修饰有关。ｄ－艹宁烯可降低ｐ２１ｒａｓ蛋白的膜结合,增加胞浆ｐ２１ｒａｓ蛋白的积蓄。在经ｄ－艹宁烯处理过的胰腺癌细胞加入抗ｐ２１ｒａｓ抗体,采用免疫组化法也可观察到上述现象。结论法尼基蛋白转移酶的抑制和ｐ２１ｒａｓ蛋白膜结合与ｐ２１ｒａｓ定位的改变密切相关。ｐ２１ｒａｓ法尼基化的抑制作用改变了它在细胞内的定位,从而影响其生物学活性,但尚未发现与Ｈ－ｒａｓ癌基因表达的密切相关。     

罗勒多糖对肿瘤转移行为的影响

目的:研究腺病毒介导的鼠内皮抑素基因对胃癌的治疗作用.方法:利用病毒重组技术将内皮抑素克隆入增殖缺陷型腺病毒基因组中,观察体外转染表达后的生物学活性.通过建立荷人胃癌的裸鼠动物模型,分析基因转导后动物肿瘤细胞中内皮抑素的表达情况及对肿瘤的抑制作用.结果:构建了表达内皮抑素的重组腺病毒载体pCA13-mEndo;检测到内皮抑素在体外的mRNA水平和蛋白质水平均有表达;鸡胚绒毛尿囊膜实验表明其对血管生长起抑制作用;荷瘤裸鼠体内肿瘤生长抑制明显.结论:所构建的pCA13-mEndo重组腺病毒载体可有效表达具有生物学活性的内皮抑素,使得肿瘤内微血管生成减少,肿瘤细胞增殖减慢,为抗血管生成方法治疗实体瘤的临床应用奠定基础.     

外源性RGS16基因稳定转染对胶质瘤C6细胞生长的影响

背景与目的:有研究表明野生型p53可以诱导RGS16表达,而RGS16可能与胶质瘤的发生有关。本研究旨在探讨RGS16基因转染对大鼠胶质瘤C6细胞生长的影响。方法:构建真核表达载体pIRES2-EGFP-RGS16,脂质体法转染C6细胞,经G418筛选后荧光显微镜观察细胞中增强型绿色荧光蛋白(enhancedgreenfluorscentprotein,EGFP)的表达,RT-PCR证实RGS16的mRNA表达,免疫细胞化学方法检测细胞中RGS16蛋白表达。最后利用流式细胞仪、生长曲线、平板克隆形成等方法研究RGS16基因对胶质瘤细胞周期、生长及增殖的影响。结果:成功构建了稳定表达RGS16和表达空载体的细胞系C6-RGS16、C6-GFP。C6-RGS16、C6-GFP和C6经流式细胞仪检测S期的细胞比例分别为28.5%、18.9%和14.3%(P<0.05);生长曲线表明C6-RGS16生长的速度明显快于C6-GFP及C6,但其平板克隆形成率分别为12%、25%和25%(P<0.05)。结论:RGS16促进C6细胞周期从G1期向S期过渡,加快细胞生长速度,但是并不促进细胞克隆形成。     

IL-24基因对大鼠胶质瘤细胞生长状况的影响

目的　探讨IL 2 4基因对C6大鼠胶质瘤细胞生长状况的影响。方法　应用逆转录病毒载体 ,将IL 2 4基因导入C6细胞 ,经G4 18筛选后获得表达IL 2 4分子的阳性细胞克隆C6 /IL 2 4 ;用RT PCR方法检测目的基因表达 ;四甲基偶氮唑蓝 (MTT)法检测细胞体外增殖状况 ,流式细胞技术检测细胞的增殖活性 ,并制作荷瘤动物模型 ,观察C6 /IL 2 4和C6细胞的体内致瘤性。结果　RT PCR检测表明 ,外源IL 2 4基因于mRNA水平在C6 /IL 2 4细胞已获得稳定表达。C6 /IL 2 4细胞系的体外增殖性较亲代C6细胞明显下降 ,流式细胞术检测其细胞增殖指数 (PI)为 (2 9.71± 0 .89) %。 9只接种C6 /IL 2 4细胞的实验组大鼠中 ,6只颅内成瘤 ,肿瘤体积为 (14 .0 8± 9.81)mm3 ,明显小于接种C6细胞大鼠的肿瘤体积 (P <0 .0 5 )。结论　外源性IL 2 4基因可部分抑制胶质瘤细胞异常增殖的肿瘤特性。     

CT7 (MAGE-C1) antigen expression in normal and neoplastic tissues

Angiogenesis is a vital component of the development and progression of many human solid tumors. Glioblastoma multiforme is one of the most highly vascularised class of solid tumors. Thus, we have investigated the potential antitumourigenic activity of endostatin, an angiogenic inhibitor, in the rat C6 glioma model. We have engineered C6 cells that endogenously express mouse endostatin in order to assess the growth of C6 tumors in vivo when endostatin is constitutively expressed. Endostatin secreted by stably transfected C6 cells is biologically active as shown by its inhibition (26%) of bFGF-stimulated proliferation of BAECs in culture. The subcutaneous implantation of endostatin-C6 cells in athymic (nu/nu) mice resulted in a reduced tumor growth rate (90% inhibition) compared to control cell lines throughout the duration of our experiments. Tumor inhibition was associated with a 50% reduction in the number of vessels, which were also smaller in morphology. However, endostatin-C6 tumors were no more necrotic than control tumors. The implantation of endostatin-C6 cells into immunocompetent Wistar rat brains also resulted in reduced tumor volumes (71% inhibition) compared to controls. Tumor cells were sparsely localised along the injection tract but had not formed discrete tumors. Despite the inhibitory response mediated by endostatin on C6 growth, complete tumor inhibition or dormancy was not observed in either the athymic or immunocompetent tumor models. These findings demonstrate that the endogenous expression of endostatin by C6 glioma cells results in a reduced tumor growth rate in vivo that is associated with an inhibition of tumor angiogenesis. Our data suggest that endostatin should be developed as an adjuvant gene therapy for the effective treatment of gliomas.     

RI基因真核表达载体的构建及其对C6胶质瘤细胞生长的影响

目的通过构建核糖核酸酶抑制因子(ribonuclease inhibitor, RI)基因的真核表达载体——pLNCX-ri, 并转染C6神经胶质瘤细胞,探讨RI抑制肿瘤生长的作用机制.方法用Nde I / Xho从已构建的pET-ri上切下1.4 kb的RI基因片段,再构建到pLNCX上,获得真核表达载体(pLNCX-ri),采用Lipofect AMINE辅助转染大鼠C6神经胶质瘤细胞,经G418筛选获得稳定转染的细胞克隆,用Western blotting 检测RI基因的表达水平.将转染阳性的C6神经胶质瘤细胞接种于大鼠皮下,观察肿瘤的生长情况.结果在转染的C6神经胶质瘤细胞中,RI基因的表达量明显高于未转染的C6神经胶质瘤细胞,转染阳性的C6神经胶质瘤细胞在大鼠体内的成瘤潜伏期23±5.7天(对照组14±3.5天),瘤组织重量转染组1.35±0.43g比对照组2.40±0.61g(P＜0.01)明显下降,瘤组织的血管密度转染组27.2±4.31比对照组47±6.54(P＜0.01)明显减少.结论RI基因的转染对肿瘤的生长有明显抑制作用,其作用机制可能是抑制肿瘤组织血管的形成.     

脂质体介导的鼠内皮细胞抑制素基因治疗人肝癌的研究

目的：利用内皮细胞抑制素进行肿瘤抗血管基因治疗的尝试。方法：用ＲＴ－ＰＣＲ从鼠地脏扩增出内皮细胞抑制素ｃＤＮＡ,经测序证实后,装入分泌表达载体ｐＳＥＣ－ｈｙｇｒｏｍｙｃｉｎ,用ＤＥＡＥ－葡聚糖将其转染ＣＯＳ－７细胞,从ｍＲＮＡ水平及蛋白水平检测内皮细胞抑制素的表达,并观察分泌上肖是否具有特异性抑制ｂＦＧＦ刺激的内皮细胞增殖作用,并通过ＴＤＴ介导的ｄＵＴＰ缺口末端标记法和琼脂糖电泳来探讨其作用机制。     

Construction of double suicide genes system controlled by MDR1 promoter with targeted expression in drug-resistant glioma cells

The multiple drug resistance protein (MDR1) is frequently overexpressed in human glioma. The aim of this study is to clone the MDR1 promoter from C6/ADR, construct the double suicide genes expressive vector controlled by MDR1 promoter, and explore its targeted expression in C6/ADR cells. MDR1 promoter from C6/ADR genomic DNA, which was linked with T vector, was amplified by using Polymerase chain reaction (PCR). After cut by NdeI and HindIII, MDR1 promoter was cloned into pcDNA3-TK (thymidine kinase) plasmid. The cytosine deaminase (CD) gene from pcDNA3-CD-TK plasmid was directly cloned into the above vector to construct pcDNA3-MDR1-promoter-CD-TK vector. Then this vector was transfected into C6 and C6/ADR cells respectively by liposome. After selection by G418, the tumor cell lines were stably established. Then these cell lines were examined through PCR and RT-PCR to respectively detect the integration and expression of TK and CD genes. The results showed the length and sequence of MDR1 promoter amplified by PCR were confirmed by DNA sequencing. The pcDNA3-MDR1-promoter-CD-TK expression vectors were constructed successfully. PCR indicated the double suicide genes were integrated into C6 and C6/ADR cells. RT-PCR reveled that CD and TK genes expressed in C6/ADR/CD-TK cells, whereas not in C6/CD-TK cells. In conclusions, construction of expressive vector containing double suicide genes controlled by MDR1 promoter with targeted expression in C6/ADR will provide a sound basis for targeted gene therapy for multidrug resistance (MDR) glioma.     

V806M突变型Axin基因真核表达载体的构建及其在神经胶质细胞瘤内的表达

目的构建V806M突变型Axin基因真核表达载体pIRES2-EGFP-MT-Axin(V806M),并稳定表达于大鼠神经胶质瘤细胞系C6。方法用分子克隆技术,构建真核表达载体pIRES2-EGFP-MT-Axin(V806M),经NheⅠ和SmaⅠ双酶切鉴定后,用脂质体法稳定转染神经胶质瘤细胞C6。结果构建了真核表达载体pIRES2-EGFP-MT-Axin(V806M),稳定转染后,经荧光显微镜和免疫细胞化学染色法检测,可见细胞内有EGFP及Axin的表达。结论成功构建真核表达载体pIRES2-EGFP-MT-Axin(V806M),并在神经胶质瘤细胞C6中表达,为研究此种突变体Axin是否影响细胞的生物学行为以及是否参与胶质瘤的发生发展奠定了实验基础。     

苯乙酸对胶质瘤同源盒基因表达的上调作用

目的 观察苯乙酸（PA）诱导分化胶质瘤细胞C6过程中,同源盒（Hox)基因表达的变化。方法 根据引物的不同,将已知的22种Hox基因分为P1、P2、P3三组。用逆转录PCR（RT－PCR）及图像分析法,分组检测应用PA前后Hox基因组在C6细胞中的表达。对应用PA前后表达显著不同的Hox基因组,检测其特异Hox基因的表达。结果 C6细胞中,P2组基因表达明显弱于P1、P3组。应用PA后,P2组基因及P2组中Hox B2基因表达显著增强（P＜0．001）。PA在分化诱导胶质瘤过程中,对Hox基因mRNA水平的表达有明显上调作用。结论 PA抑制胶质瘤细胞增殖的作用机理与调节胶质瘤细胞转录过程有关。     

Antitumor effect of endostatin overexpressed in C6 glioma cells is associated with the down-regulation of VEGF

Endostatin is an anti-angiogenic agent that blocks endothelial cell proliferation, tumor growth, and metastasis. Several lines of direct evidence show that gliomas express high levels of endostatin. However, the anti-angiogenic activity and cellular mechanisms of endostatin from tumor cells have not been completely elucidated. In this study, we established C6 glioblastoma (GBM) xenografts in nude mice by subcutaneously injecting glioma cell lines, C6-null cells, and stable transfected-C6 cells overexpressing mock vector (C6-mock) and endostatin (C6-endo). We found that overexpression of endostatin in C6-endo cells significantly suppressed the expression of VEGF in tumor cells in vivo as well as in vitro. Furthermore, the tumor growth derived from C6-endo cells was inhibited. Our data demonstrate that endostatin could play a direct role in inhibiting tumor cells, and suggest that enhancing endostatin expression in gliomas could be an effective treatment strategy.