反义hTERT/PTEN联合基因治疗恶性胶质瘤的体内外研究

目的探讨腺病毒介导的反义hTERT和野生型PTEN联合基因转染对恶性胶质瘤细胞生长的影响。方法用细菌内高效同源重组系统构建的含有hTERT反义序列及野生型PTEN的腺病毒在体内外单独或联合转染恶性胶质瘤细胞系U251，检测肿瘤细胞生长情况、端粒酶活性、蛋白表达、细胞周期变化等。结果在体外试验中单独或联合转染都能明显抑制肿瘤细胞的生长，以联合转染效果最明显，转染后第6天联合转染组的细胞生存率为37．6％，端粒酶活性水平和hTERT蛋白表达水平明显下降，分别为28．8TPG、0．2106，PIEN蛋白表达水平上升为0．9630；在体内试验中肿瘤生长也明显减慢。结论腺病毒介导的反义hTERT和野生型PTEN联合转染能明显抑制恶性胶质瘤细胞的生长。     

siRNA特异沉默卵巢癌细胞CD59基因的裸鼠移植瘤研究

目的:通过体内动物实验研究CD59-siRNA对卵巢癌细胞CD59的沉默效应及抑瘤作用,探讨CD59在肿瘤免疫逃逸中的作用.方法:将转染CD59干扰质粒的A2780细胞(T组)、转染空质粒的A2780细胞(V组)及未转染的A2780细胞(C组)分别接种于裸鼠皮下,通过绘制肿瘤生长曲线,裸鼠移植瘤组织切片CD59 mRNA原位杂交及CD59蛋白的免疫组化研究其抑瘤效应及对CD59的沉默效应.结果:肿瘤生长曲线显示,与对照组相比,CD59干扰质粒转染组肿瘤生长明显受抑制(P<0.05).原位杂交及免疫组化结果表明,干扰组的CD59 mRNA及CD59蛋白与对照组相比显著降低(P<0.05).结论:裸鼠体内实验表明,特异性沉默CD59基因的siRNA表达载体可以明显抑制CD59的表达,增加了卵巢癌细胞对补体攻击的敏感性,从而抑制卵巢癌在体内的生长,进一步说明了CD59在肿瘤免疫逃逸中的作用.     

胶质瘤细胞血小板源生长因子B链的纯合二聚体自分泌环活性与肿瘤细胞增殖和凋亡的关系

目的探讨胶质瘤细胞血小板源生长因子Ｂ链的纯合二聚体（ＰＤＧＦＢＢ）自分泌环活性与其细胞增殖和凋亡的关系。方法用原位杂交、原位细胞凋亡检测和免疫组化ＡＢＣ法染色观察了７３例不同级别的胶质瘤组织。结果胶质瘤细胞ＰＤＧＦＢＢ自分泌环活性与其增殖活性呈显著性正相关，两者均随肿瘤恶性程度升高而增强。肿瘤细胞凋亡随肿瘤恶性程度升高而减少，并与肿瘤细胞ＰＤＧＦＢＢ自分泌环活性和增殖活性呈显著性负相关。结论以上指标对评价胶质瘤生物学行为均有参考价值。胶质瘤细胞ＰＤＧＦＢＢ自分泌环活性异常增加可能是刺激肿瘤细胞增殖和抑制其凋亡的重要因素，并在胶质瘤发生及恶性进展过程中起重要作用。     

TNF-α在热疗降低胶质瘤侵袭性过程中的作用

 目的 探讨肿瘤坏死因子-α(TNF-α)在热疗抑制肿瘤侵袭性过程中的作用。方法 热处理大鼠恶性胶质瘤细胞(C6细胞)和胶质瘤大鼠后,放射免疫法监测培养液和脑胶质瘤组织内TNF-α的浓度;免疫组化法检测经热疗/ TNF-α/生理盐水处理过的胶质瘤组织内增殖细胞核抗原(PCNA)蛋白的表达。利用Transwell构建肿瘤侵袭模型,通过结晶紫染色法检测肿瘤侵袭性。电镜观察C6恶性胶质瘤大鼠肿瘤血管内皮细胞的凋亡。结果 热疗可增加C6细胞培养液和胶质瘤大鼠肿瘤组织内的TNF-α含量及降低胶质瘤侵袭性,均于热疗后120min时达高峰(P＜0.01)。热疗与TNF-α单独作用于胶质瘤大鼠后,均可引起胶质瘤大鼠肿瘤血管内皮细胞的凋亡。且TNF-α引起内皮细胞的凋亡水平与热处理后C6细胞培养液中TNF-α含量一致。结论 热疗可能是通过增加TNF-α引起肿瘤血管内皮细胞凋亡而抑制了肿瘤侵袭性。     

仙台病毒 Tianjin 株缺陷干扰颗粒体内外诱导大鼠脑胶质瘤细胞凋亡的研究

目的探讨仙台病毒Tianjin株缺损干扰颗粒（ defective interfering particles ,DI颗粒）体内外诱导大鼠脑胶质瘤细胞C6凋亡的作用。方法将不同滴度仙台病毒Tianjin株DI颗粒分别与大鼠脑胶质瘤细胞C6作用不同时间,以培养基作为阴性对照、完整病毒作为阳性对照,通过DNA片段琼脂糖凝胶电泳、TUNEL染色、AnnexinⅤ-FITC/PI标记流式细胞仪分析等检测细胞凋亡情况。建立大鼠皮下胶质瘤模型,通过测量肿瘤大小观察DI颗粒抑瘤作用,病理切片HE染色观察肿瘤组织病理变化,TUNEL法检测肿瘤组织细胞凋亡情况。结果 C6细胞在体外经DI颗粒诱导后, DNA片段琼脂糖凝胶电泳呈阶梯状;流式细胞仪及TUNEL检测显示,DI颗粒组与完整病毒组细胞凋亡率明显增高,且呈时间-剂量依赖型。动物实验结果显示,DI颗粒和完整病毒均可明显抑制肿瘤生长;肿瘤组织病理切片HE染色显示DI颗粒组和完整病毒组瘤结节内瘤细胞较少;TUNEL原位细胞凋亡检测显示DI颗粒组和完整病毒组凋亡细胞明显增加,以上结果与阴性对照组比较差异均有统计学意义（P＜0．01）。结论仙台病毒Tianjin株DI颗粒在体内外均能引起大鼠脑胶质瘤C6细胞凋亡,且呈时间-剂量依赖型,提示DI颗粒有辅助治疗脑胶质瘤的可能性。     

p16和p27表达对前列腺癌细胞系的作用

目的 探讨p16和p27Kip1 基因蛋白对抑制前列腺癌细胞增殖和调控其凋亡的作用.方法 构建携带人P16和p27基因的腺病毒载体,转染体外培养的前列腺癌细胞系PC-3,采用RT-PCR、Western blot检测目的 基因的表达.通过细胞生长试验、流式细胞仪检测PC-3转染前后细胞增殖和凋亡的变化.结果 病毒滴度Ad-p16为115×10^8 pfuPml 、Ad-p27为112×10^9 pfuPml ,RT-PCR 检测可见p16-mRNA（520bp）和p27Kip12mRNA （320bp）表达,Western blot检测有p16 蛋白（65KD）和P27蛋白（27KD） 特异表达,并可明显抑制PC-3细胞的增殖,诱导其凋亡,联合基因治疗组与单基因组相比差异显著（P＜0.01）.结论 p16和p27可明显抑制前列腺癌细胞株PC-3的增殖,增加细胞的凋亡,转染携带p16和p27的重组腺病毒载体有望成为治疗前列腺癌的有效方法.     

抑癌基因PTEN诱导人脑胶质瘤SHG—44细胞凋亡

目的 研究外源性PTEN基因对人脑胶质瘤细胞系SHG－44凋亡的影响,探讨PTEN基因抑制肿瘤细胞增殖的机制。方法 以携有人PTEN基因的真核表达载体体外转染SHG－44细胞,筛选阳性转染的细胞克隆并扩增培养。以原位杂交和免疫组化染色法检测PTEN基因的表达。采用透射电镜、流式细胞仪和核DNA琼脂糖凝胶电泳,检测转染PTEN基因后SHG－44胶质细胞的凋亡。观察导入入PTEN基因对SHG－44细胞裸鼠致瘤能力的影响。结果 获得PTEN基因稳定转染的胶质瘤细胞SHG－44,并检测到PTEN基因和蛋白的表达。透射电镜下可见转染PTEN基因后细胞核染色质浓缩边集、胞浆浓缩、核碎裂及凋亡小体形成等典型的凋亡表面。流式细胞仪显示,细胞周期从G1期到S期发生抑制,并在G1期峰前出现1个明显的凋亡峰（12．9％）。细胞核DNA琼脂糖凝胶电泳显示凋亡细胞特朋的梯状条带。转染空载体及未转染的SHG－44细胞未见的凋亡表现。转染PTEN基因后,SHG－44细胞对裸鼠瘤能力明显降低。结论 将外源必PTEN基因导入SHG－44肿瘤细胞后,可诱导细胞凋亡,这可能是PTEN基因抑制肿瘤细胞增殖的重要机制之一。     

外源性野生型p53基因在两个肺腺癌细胞系中表达及对p16和p21基因的调控

目的 探讨p53基因在肺癌细胞周期中的作用机制，以及裸鼠体内基因治疗的作用。方法 用以腺病毒为载体的野生型p53基因pAdCMV-p53(Ad-p53)感染高转移肺腺癌95D细胞系和高浸润肺腺癌L-18系，对感染前后各细胞系的细胞生长曲线、p53、p16和p21基因的表达以及调亡进行分析。此外，用Ad-p53对两细胞系进行了裸鼠体内感染实验。结果 体外实验中，导入p53基因细胞系的生长均得到抑制，并且最终都出现凋亡，感染后的细胞中p53和p21基因的mRNA表达量明显增高，p16基因的mRNA表达量则无明显变化。p53基因治疗后的裸鼠，其中95D细胞系的肿瘤全部消失；L-18细胞系的腹腔注射组肿瘤全部消失，而皮下注射组无明显变化。结论 p53基因是一个有效的肿瘤抑制基因，其诱导细胞凋亡的途径与p16基因不同。腺病毒介导的野生型p53基因可以有效地控制肺癌细胞的发展和转移。     

高低转移肺腺癌细胞系中肿瘤抑制基因p16、p21和p53表达研究

目的 探讨肿瘤抑制基因对肺腺癌细胞生长的抑制作用。方法 利用FuGene转染方式分别将p21和p16基因的表达质粒转入－对肺腺癌细胞系Anip973和AGZY83－a中,同时用含野生型p53 基因的腺病毒感染p16基因转染前后的这一对细胞系。对P16和P21蛋白过表达的细胞系进行了细胞生长曲线、克隆形成率、原位末端标记分析和流式细胞仪分析。结果 p16基因的过表达只能使细胞系的G1期细胞比例提高,但细胞生长曲线,克隆形成率均未出现改变,未检测到凋亡信号。P21蛋白过表达的一对细胞系细胞生长曲线斜率降低,克隆形成能力下降,并出现明显的G1期阻滞,但未检测到凋亡信号。p53基因感染AGZY83－a,Anip973及经过p16基因转染的细胞AGZY83－ap16和Anip973p16后呈现时间依赖性表达,细胞生长曲线和四唑盐比色法分析提高,野生型p53基因的大量表达明显抑制以上4种细胞的生长,Anip973和Anip973p16的生长抑制率高于AGZY83－a和AGZY83－ap16;Anip973p16和AGZY83-ap16的生长抑制率高于Anip973和AAGZY83－a。这4种细胞在感染p53后出现典型的凋亡信号。结论p16基因的过表达并不能抑制细胞系的生长,而p21基因的过表达通过G1期阻滞抑制这1对肺腺癌细胞的生长;野生型p53基因在AGZY83－a和Anip973中高效表达可产生明显的细胞生长抑制效应;野生型p53基因对肺腺癌高转移细胞系Anip973抑制作用更为明显。     

siRNA沉默p75神经营养因子受体逆转胶质瘤恶性表型

目的 利用小干扰RNA(siRNA)技术沉默与胶质瘤细胞凋亡和侵袭密切相关的p75神经营养因子受体(p75NTR)基因,观察其逆转胶质瘤恶性表型的治疗效果.方法 设计靶向p75NTR基因的siRNA片段,脂质体转染人U251胶质瘤细胞系,逆转录聚合酶链反应(RT-PCR)和免疫细胞化学方法 检测p75NTR的mRNA和蛋白表达;Transwell细胞侵袭实验检测U251细胞的侵袭力;软琼脂集落形成实验检测细胞集落形成能力;建立裸鼠颅内U251胶质瘤荷瘤模型,原位重复注射siRNA-p75NTR/脂质体复合物3次,MRI检测颅内瘤体积,免疫细胞化学SP法检测p75NTR、神经生长因子(NGF)和细胞周期蛋白(cyclin)D2表达,用TUNEL法做移植瘤细胞原位细胞凋亡检测.结果 转染siRNA基因片段的U251细胞p75NTR mRNA和蛋白质表达水平显著下降(P<0.05),细胞侵袭能力及软琼脂集落形成能力均显著降低(P<0.05);体内实验显示p75NTR的表达程度与NGF的表达呈正相关,与cyclin D2表达及原位细胞凋亡数量呈负相关,MRI示瘤体积增长缓慢,边界轮廓清晰,动物生存期明显延长(P<0.05).结论 靶向p75NTR的siRNA技术可有效降低胶质瘤细胞的侵袭及增殖能力,诱导肿瘤细胞凋亡.     

Antitumor activity of TRAIL recombinant adenovirus in human malignant glioma cells

Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.     

胶质瘤bcd—2基因表达水平与其细胞增殖和凋亡关系的研究

目的 探讨胶质瘤细胞ｂｃｌ－２基因表达水平与肿瘤恶性程度、细胞增殖活性及凋亡程度的关系。方法 以６９例不同级别的人胶质瘤组织为研究对象,用原位杂交及免疫组化染色ＡＢＣ法分别检测ｂｃｌ－２ｍＲＮＡ、ｂｃｌ－２蛋白和增殖细胞核抗原（细胞增殖活性标记物）的表达,并用３’末标记法做原位细胞凋亡检测。结果 ６４例（９２．８％）表达ｂｃｌ－２ｍＲＮＡ,６０例（８７．０％）表达ｂｃｌ－２蛋白,两者的表达水平呈正     

Generation of an effective anti-tumor immunity after immunization with xenogeneic antigens

Central and peripheral tolerance mechanisms are expected to hamper the generation of effective immunity against tumors. To break self tolerance against malignant gliomas, we assessed the therapeutic potential of self/foreign antigen cross-reactivity in an immunocompetent rat glioma model. Immunotherapy of tumors using xenogeneic human glioma membrane proteins (HGP) as a vaccine inhibited tumor growth, whereas no significant effect was obtained with rat glioma membrane proteins (RGP). In contrast to RGP, HGP elicited a specific IgG immune response that cross-reacted with RGP. This immune response was found to be mainly a Th1 type response. On tumor sections stained with hematoxylin and eosin, glioma cells are sparse and apoptotic in HGP-immunized rats, whereas control tumors showed condensed and viable cells. Tumor-specific CTL were induced in HGP-immunized rats. Immunohistochemical analysis revealed that a significant number of CD8(+) and CD4(+) cells infiltrated into tumors from HGP-vaccinated rats, whereas RGP vaccination led to only few tumor-infiltrating T cells. Taken together, the data establish the in vivo applicability of the cross-stimulation between self and foreign antigens as an alternative way to break tolerance against the poorly immunogenic gliomas.     

重组腺病毒介导的p^16与p^53基因联合转移对人肺癌细胞系 …

目的 探讨ｐ＾１６和ｐ＾５３基因对肺癌细胞的协同抑制效应及凋亡诱导作用。方法 首先用同源重组技术构建重组ｐ＾１６和ｐ＾５３腺病毒载体,然后单独或联合感染人肺癌细胞系Ｈ３５８,用免疫组织法及Ｗｅｓｔｅｒｎ ｂｌｏｔ检测腺病毒介导的基因转移效率与表达水平,用克隆形成实验、原位末端标记及流式术观察它们对Ｈ３５８生长特性及凋亡的影响,结果 免疫组织化学染色结果表明重组腺病毒载体可高效地将外源基因ｐ＾５３转     

Malignant transformation of thymoma in recipient rats by heterotopic thymus transplantation from HTLV-I transgenic rats

Transgenic rats expressing the pX gene of human T lymphocyte virus type-I (HTLV-I) under control of the rat lymphocyte-specific protein tyrosine kinase type-I promoter (lck-pX rats) developed benign epithelial thymomas. When the thymuses of newborn lck-pX rats were transplanted into the subcapsular space of the kidney in other thymectomized lck-pX rats, similar tumors developed in the transplanted thymuses. Following the tumor growth, dissemination in the abdominal cavity and distant metastasis occurred. The tumors were histopathologically similar to the original thymomas, but prominent nuclear atypia and high mitotic activity were present. The Ki-67 index was twice as high as that in the originals. The tumors were transplantable into the subcutis of lck-pX rats, although transplantation of the originals never succeeded. All evidence indicated that malignant transformation of thymoma was induced by the heterotopic transplantation. Expression of the pX transgene in the transformed tumors were significantly reduced. Among host genes, the expression of p16ink4a/ARF, which was significantly upregulated in the originals, was never detected in the transformed tumors. Genomic Southern blots and PCR suggest that homozygous deletion of the p16ink4a/ARF gene may play important roles in malignant transformation in this model. Our model described here is a useful unique model for in vivo malignant transformation.     

黏液瘤病毒对大鼠体内胶质瘤作用的研究

目的 探讨黏液瘤病毒对大鼠动物模型体内胶质瘤细胞的作用.方法 采用立体定向法向SD大鼠颅内注射C6细胞建立大鼠额叶胶质瘤模型,明确成瘤后,随机分组,以立体定向方法往瘤腔内注射黏液瘤病毒(MV),5-FU,MV+ 5-FU及灭活黏液瘤病毒(DV),观察不同组别间大鼠体重、肿瘤大小、GFAP表达、Akt表达情况.结果 SD大鼠注射C6细胞后,额叶可见胶质瘤生长.成瘤后瘤腔内注射MV、5-FU及MV+ 5-FU,肿瘤的生长较注射DV减慢,并有缩小趋势,GFAP表达较少.MV组及MV+ 5-FU组PI3k、Akt及mTOR表达较DV组及5-FU组下降.结论 立体定向法注射C6细胞可以建立稳定的胶质瘤模型.MV可以通过调节PI3K-Akt-mTOR通路相关基因的表达,从而增加化疗药物对动物模型体内肿瘤细胞的生物学活性.     

A transporter gene (sodium iodide symporter) for dual purposes in gene therapy: imaging and therapy

Radioiodide uptake (RAIU) in thyroid follicular epithelial cells, mediated by a plasma membrane transporter, sodium iodide symporter (NIS), provides a first step mechanism for thyroid cancer detection by radioiodide injection and effective radioiodide treatment for patients with invasive, recurrent, and/or metastatic thyroid cancers after total thyroidectomy. NIS gene transfer to tumor cells may significantly and specifically enhance internal radioactive accumulation of tumors following radioiodide administration, and result in better tumor control. NIS gene transfers have been successfully performed in a variety of tumor animal models by either plasmid-mediated transfection or virus (adenovirus or retrovirus)-mediated gene delivery. These animal models include nude mice xenografted with human melanoma, glioma, breast cancer or prostate cancer, rats with subcutaneous thyroid tumor implantation, as well as the rat intracranial glioma model. In these animal models, non-invasive imaging of in vivo tumors by gamma camera scintigraphy after radioiodide or technetium injection has been performed successfully, suggesting that the NIS can serve as an imaging reporter gene for gene therapy trials. In addition, the tumor killing effects of 131I after NIS gene transfer have been demonstrated in in vitro clonogenic assays and in vivo radioiodide therapy studies, suggesting that NIS gene can also serve as a therapeutic agent when combined with radioiodide injection. Better NIS-mediated tumor treatment by radioiodide requires a more efficient and specific system of gene delivery with better retention of radioiodide in tumor. Results thus far are, however, promising, and suggest that NIS gene transfer followed by radioiodide treatment will allow non-invasive in vivo imaging to assess the outcome of gene therapy and provide a therapeutic strategy for a variety of human cancers.     

RNA interference targeting enhancer of polycomb1 exerts anti-tumor effects in lung cancer

Background and aim: Lung cancer is one of leading malignant tumor worldwide with a high mortality rate. A new therapy target, enhancer of polycomb1 (EPC1) knocked down by short hairpin RNA (shRNA) interference technology, for lung cancer was established to investigate its effects on lung cancer in present study. Methods: RNA interference technology was applied to down-regulate the expression of EPC1 by specific-shRNA with lentivirus vector in neoplastic human alveolar basal epithelial cells (A549 cells). The survival rate and apoptosis were respectively measured by MTT and Flow Cytometry to evaluate the effects of shRNA EPC1 on cells. Mice xenografts of HCT116 cells with shRNA EPC1 were also established to assess the effect on tumor growth. The levels of AKT and p65 were detected by western blotting. Results: The down-regulation of EPC1 by specific-shRNA with lentivirus vector was significantly decreased the survival rate and apoptosis of A549 cells, and the tumors in EPC1 shRNA transfection group had a significant lower size and weight compared with the ones with control shRNA. The protein expression of p-AKT and p65 was reduced by EPC1 shRNA in both in vitro and in vivo experiments. Conclusion: Silencing EPC1 by shRNA technology had the inhibition effects on cell proliferation and tumor growth in lung cancer, which provided a new potential target for treatment of cancers.