腺病毒介导PTEN基因抗脑胶质瘤作用的实验研究

目的：探讨10号染色体上缺失的磷酸酶和张力蛋白同源物(phosphatase and tensin homology deleted on chromosome ten，PTEN)对人脑胶质瘤细胞的抗增殖作用，包括细胞周期改变、抗血管生成的作用。方法：用含PTEN基因的重组腺病毒载体，体外转染人脑胶质瘤细胞U87，用RTPCR、Western blot检测基因及蛋白的表达。通过细胞生长试验、流式细胞仪分析技术检测转染细胞的增殖、细胞周期的改变，用MTT法检测PTEN对人脐静脉内皮细胞生长的影响，体内实验检测其致瘤能力。结果：RT-PCR、Western blot检测转染AdPTEN病毒后的U87细胞PTEN表达由阴性转阳性，转染后对U87细胞的体外生长有明显抑制作用，使细胞出现G0～G1期阻滞，并抑制其体内肿瘤生长。含PTEN的上清对内皮细胞生长有明显抑制作用。体内实验表明，AdPTEN治疗组可明显抑制肿瘤的生长。结论：重组腺病毒载体介导的PTEN基因在体内外对人脑胶质瘤细胞L187的生长有抑制作用，并抑制肿瘤血管生成，具有显著的抗胶质瘤作用。     

尼莫地平增强HyTK/ACV自杀基因对脑胶质瘤细胞的杀伤作用

目的 :探讨尼莫地平增强阿昔洛韦 (ACV)介导的转HyTK基因治疗诱导脑胶质瘤细胞的死亡方式及旁观者效应机制。方法 :构建含HyTK基因的逆转录病毒载体LNSHyTK ,并转染人脑胶质瘤BT32 5和SCW细胞。单用ACV和联合尼莫地平应用体外试验观察对BT32 5 tk和SCW tk的杀伤效果。同时应用转基因前和转基因后瘤细胞共培养观察其旁观者效应。结果 :低浓度尼莫地平联合ACV作用下转tk基因细胞的凋亡率明显高于单独使用ACV ,并明显提高旁观者效应。结论 :尼莫地平能增强HyTK ACV自杀基因对脑胶质瘤细胞的杀伤作用 ,为钙拮抗剂应用于转tk基因治疗提供了实验依据。     

尼莫地平增强HyTK／ACV自杀基因对脑胶质瘤细胞的杀伤作用

目的：探讨尼莫地平增强阿昔洛韦(ACV)介导的转HyTK基因治疗诱导脑胶质瘤细胞的死亡方式及旁观者效应机制。方法：构建含HyTK基因的逆转录病毒载体LNSHyTK，并转染人脑胶质瘤BT325和SCW细胞。单用ACV和联合尼莫地平应用体外试验观察对BT325／tk和SCW／tk的杀伤效果。同时应用转基因前和转基因后瘤细胞共培养观察其旁观者效应。结果：低浓度尼莫地平联合ACV作用下转tk基因细胞的凋亡率明显高于单独使用ACV，并明显提高旁观者效应。结论：尼莫地平能增强HyTK／ACV自杀基因对脑胶质瘤细胞的杀伤作用，为钙拮抗剂应用于转tk基因治疗提供了实验依据。     

腺病毒介导的大鼠增殖抑制基因抑制大鼠胶质瘤的生长

目的:探讨腺病毒介导的大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)对大鼠胶质瘤生长的抑制作用.方法:建立大鼠C6胶质瘤动物模型,分别在接种C6胶质瘤细胞后第4和11天,于肿瘤原位注射0.9％氯化钠溶液(对照组)、重组腺病毒Adv-rHSG-GFP和腺病毒Adv-GFP,并于接种C6胶质瘤细胞后第9、15和21天取脑胶质瘤观察肿瘤的生长情况,免疫组织化学法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和rHSG蛋白的表达以及微血管密度(microvessel density,MVD),RT-PCR和蛋白质印迹法检测rHSG mRNA及蛋白的表达.结果:Adv-rHSG-GFP组肿瘤体积明显小于对照组和Adv-GFP组(P＜0.01).Adv-rHSG-GFP组PCNA标记指数和MVD均低于对照组和Adv-GFP组.Adv-rHSG-GFP组rHSG mRNA和蛋白的相对表达量均明显高于对照组和Adv-GFP组(P＜0.01).结论:Adv-rHSG-GFP能够抑制胶质瘤血管生成和肿瘤细胞增殖,对恶性胶质瘤的生长具有明显的抑制作用.     

干细胞介导溶瘤腺病毒治疗脑胶质瘤研究进展

溶瘤腺病毒治疗具有肿瘤特异性、安全性和反复表达治疗基因等特点,通过溶瘤腺病毒治疗胶质细胞瘤已获得令人鼓舞的成果,但局部应用溶瘤腺病毒无法作用于散在分布的肿瘤细胞,影响了治疗效果.干细胞对脑胶质瘤具有内在趋化作用,介导溶瘤腺病毒治疗胶质瘤可进一步提高疗效.     

冷冻联合rhTNF-α治疗C6大鼠脑胶质瘤的实验研究

目的探讨冷冻联合rhTNF-α治疗C6大鼠脑胶质瘤的可行性。方法借助于立体定位技术,将C6胶质瘤细胞接种于40只雄性Wister大鼠脑S1区,待肿瘤长至第15天,直径约6mm时,随机将荷瘤鼠分为四组:G1、G2、G3、G4分别为盐水对照组、冷冻治疗组、rhTNF-α治疗组及联合治疗组,每组10只,同时分别行相应的治疗。治疗后第15天,行核磁共振(MRI)检查,测量肿瘤体积和抑瘤率。动态观察各组荷瘤鼠生存期和rhTNF-α对荷瘤鼠的毒副作用。结果联合治疗组肿瘤体积和生存期与其它各组相比差异有显著性,且该组对肿瘤生长的抑制率(89.5%)明显大于冷冻组(66.3%)和rhTNF-α治疗组(49.0%),且大于理论抑瘤率(82.8%)。同时观察到rhT-NF-α对实验动物有一定毒副作用。结论冷冻和rhTNF-α联合应用,具有协同抗肿瘤作用,为进一步临床研究提供了实验依据。     

人脑胶质瘤中PTEN基因突变和蛋白表达的研究

目的 :研究人脑胶质瘤中PTEN蛋白表达和PTEN基因突变情况。方法 :应用免疫组化技术和聚合酶链反应 -单链构象多态性分析 (PCR SSCP)技术 ,检测 10 2例人脑胶质瘤中PTEN蛋白表达及PTEN基因突变。结果 :PTEN阳性染色主要定位于细胞质中。 10 2例中PTEN蛋白表达 5 5 / 10 2(5 3 9% ) ,高分化组 (Ⅰ级和Ⅱ级 ) 32 / 39(82 1% )与低分化组 (Ⅲ级和Ⅳ级 ) 2 3/ 6 3(36 5 % )之间差异有极显著意义 ,P <0 0 1;4 2例胶质母细胞瘤中共有 11例发生PTEN基因突变 ,突变率为 2 6 % (11/ 4 2 ) ;6 0例其他胶质瘤中仅 1例发生突变 ,胶质母细胞瘤突变率显著高于其他胶质瘤 ,χ2 =11 6 2 ,P <0 0 1。结论 :PTEN基因突变或缺失在人脑胶质瘤的发生发展中起重要作用 ,与肿瘤恶性分化程度密切相关     

腺病毒介导PTEN基因对胶质瘤增殖与侵袭力的影响

脑胶质瘤为神经外科的常见病、多发病 ,约占颅内肿瘤的 45 % ,其主要治疗手段包括手术切除、化学治疗与放射治疗 ,其生存时间短、预后差 ,平均生存时间不足 1年 ,这与其具有高增殖性及高侵袭性有关。本实验应用腺病毒作为载体介导抑癌基因PTEN体外转染人脑胶质瘤细胞U87,检测其细胞增殖力与体外侵袭力的改变 ,进而探讨PTEN作为胶质瘤基因治疗靶点的可能性。分别用的Ad PTEN(实验病毒 )、Ad LacZ(对照病毒 )感染人胶质瘤细胞U87(病毒滴度为 5× 1 0 8pfu/ml,MOI为 5 0 ) ,同时用无病毒感染的U87作空白对照 ,首先用RT PCR检测PTE…     

脑胶质瘤基因治疗载体

基因载体是将治疗基因导入肿瘤细胞的载体,在基因治疗中起关键作用。目前常用的基因治疗载体分为生物载体和非生物载体两大类。生物载体包括病毒载体、细菌载体和干细胞载体。研究显示生物载体虽然靶向性好,转导效率高但存在遗传安全隐患;非生物载体包括脂质体和纳米材料类载体,这类载体制备简单,安全可控但转导效率低。建立安全有效的脑胶质...     

Chemosensitivity of human malignant glioma: modulation by p53 gene transfer

Loss of wild-type p53 activity is one of the most common molecular abnormalities in human cancers including malignant gliomas. The p53 status is also thought to modulate sensitivity to irradiation and chemotherapy. Here, we studied the effect of a p53 gene transfer on the chemosensitivity of three human glioma cell lines with different endogenous p53 status (LN-229, wild-type; LN-18, mutant; LN-308, deleted), using the murine temperature-sensitive p53 val¹³⁵ mutant. Expression of mutant p53 enhanced proliferation of LN-308 cells but reduced proliferation in the other cell lines. Expression of wild-type p53 caused reversible growth arrest of all cell lines but failed to induce apoptosis. Growth arrest induced by wild-type p53 was associated with strong induction of p21 expression. Strong induction of BAX expression and loss of BCL-2 expression, which are associated with p53-dependent apoptosis rather than growth arrest, were not observed. Wild-type p53 failed to sensitize glioma cells to cytotoxic drugs including BCNU, cytarabine, doxorubicin, teniposide and vincristine. The combined effects of wild-type p53 gene transfer and drug treatment were less than additive rather than synergistic, suggesting that the intracellular cascades activated by p53 and chemotherapy are rebundant. Unexpectedly, forced expression of mutant p53 modulated drug sensitivity in that it enhanced the toxicity of some drugs but attenuated the effects of others. These effects may represent a dominant negative effect of mutant p53 in LN-229 cells which have wild-type p53 activity but must be considered a gain of function-type effect in the other two cell lines which have no wild-type p53 activity. Importantly, no clear-cut pattern emerged among the three cell lines studied. We conclude that somatic gene therapy based on the reintroduction of p53 will limit the proliferation of human malignant glioma cells but is unlikely to induce clinically relevant sensitization to chemotherapy in these tumors.     

子宫内膜癌及其癌前病变组织中PTEN基因突变的研究

目的:研究抑癌基因PTEN在子宫内膜癌及其癌前病变组织中的突变,并探讨其与子宫内膜癌发生发展的关系。方法:应用聚合酶链反应-单链构象多态性分析(PCR-SSCP)方法检测60例子宫内膜癌、32例子宫内膜癌前病变(复杂型增生12例和不典型增生20例)和20例正常子宫内膜癌组织中PTEN基因的突变情况。结果:子宫内膜癌、子宫内膜癌前病变和正常子宫内膜组织中PTEN基因的突变率分别为36·67%(22/60)、9·38%(3/32)和0(0/20),三者之间差异有统计学意义,P<0·05。统计分析表明PTEN基因突变与子宫内膜癌的组织学分级、组织病理类型和肌层浸润深度有关,P<0·05。子宫内膜癌组织中,PTEN基因突变率在G1~G2级、浅肌层浸润分别高于G3级、深肌层浸润的子宫内膜癌。结论:PTEN基因突变主要发生在组织学分化好、肌层浸润浅的子宫内膜癌中。PTEN基因突变可能是子宫内膜癌发生过程中的早期分子事件。     

Gene therapy of malignant glioma: Recent advances in experimental and clinical studies

Recent advances in molecular tumor biology and gene technology have provided the possibility to treat patients with malignant brain tumors by altering gene expression in tumor cells. Tumor development and progression involves alterations in a wide spectrum of genes, therefore a variety of gene therapy approaches for malignant gliomas have been proposed. In this review article, we discuss some principles of current gene therapeutic strategies that are under investigation in laboratories and in clinics. In addition, some general issues that remain to be resolved for clinical application of gene therapy in patients with malignant gliomas will be addressed.     

PTEN抑癌基因异常与肿瘤靶向治疗

PTEN在肿瘤中存在基因组、基因转录、翻译和翻译后等多个水平的异常,从而导致PI3K/Akt通路的过度激活.这不仅是肿瘤发生的重要机制,也与某些靶向治疗后的耐药相关.克服PTEN异常所致耐药的方法包括直接应用作用于P13K/Akt通路的新型靶向药物以及重新恢复PTEN基因的表达和功能.     

Strategies for cancer gene therapy

Gene therapy offers new opportunities for cancer treatment and prevention through the use of targeted, relatively nontoxic treatments that can identify, disable, and destroy malignant cells. This article reviews the principles behind oncogene inactivation, tumor suppressor gene replacement, inhibition of angiogenesis, immunopotentiation, molecular chemotherapy, and addition of drug resistance genes. The adcantages and limitations of viral and nonviral vectors for delivery of the therapeutic genes are presented.     

Radiation-inducible caspase-8 gene therapy for malignant brain tumors

PURPOSE: Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue. METHODS AND MATERIALS: We constructed the plasmid pEGR-green fluorescence protein that included the radiation-inducible early growth response gene-1 (Egr-1) promoter and evaluated its characteristics. The pEGR-CSP8 was constructed and included the Egr-1 promoter and CSP8 complementary DNA. Assays that evaluated the apoptosis inducibility and cytotoxicity caused by CSP8 gene therapy combined with RT were performed using U251 and U87 glioma cells. The pEGR-CSP8 was transfected into the subcutaneous U251 glioma cells of nude mice by means of in vivo electroporation. The in vivo effects of CSP8 gene therapy combined with RT were evaluated. RESULTS: The Egr-1 promoter yielded a better response with fractionated RT than with single-dose RT. In the assay of apoptosis inducibility and cytotoxicity, pEGR-CSP8 showed response for RT. The pEGR-CSP8 combined with RT is capable of inducing cell death effectively. In mice treated with pEGR-CSP8 and RT, apoptotic cells were detected in pathologic sections, and a significant difference was observed in tumor volumes. CONCLUSIONS: Our results indicate that radiation-inducible gene therapy may have great potential because this can be spatially or temporally controlled by exogenous RT and is safe and specific.     

霍奇金淋巴瘤R-S细胞PTEN基因的表达与意义

目的：研究PTEN基因在霍奇金淋巴瘤（Hodgkin＇s lymphoma,HL）R-S细胞中的表达和意义。方法：PCR检测HL R-S细胞PTEN基因的外显子以证明PTEN基因是否存在,用原位杂交和免疫组化染色方法检测HL R-S细胞PTEN的表达。结果：20例HL中,PCR均检测到PTEN基因的3、4、5外显子,75.0%（15/20）HL R-S细胞中表达PTEN mRNA较强,同样地,70.0%（14/20）HL R-S细胞中PTEN蛋白表达强,而周围小的淋巴细胞不表达或弱表达PTEN mRNA和PTEN蛋白。结论：PTEN的强表达与R-S细胞形成有密切的关系。     

Vaccination with tumor cell lysate-pulsed dendritic cells augments the effect of IFN-beta gene therapy for malignant glioma in an experimental mouse intracranial glioma

Interferon-beta (IFN-beta) has been used as an antitumor drug against human glioma, melanoma and medulloblastoma since the 1980s. Recently, we developed a new gene therapy using the IFN-beta gene against malignant gliomas and then began clinical trials in 2000. Since stimulation of immune system was one mechanism of antitumor effect induced by IFN-beta gene therapy, we hypothesized that combination of IFN-beta gene therapy with immunotherapy might increase its effectiveness. In the present study, we tested whether combination therapy with IFN-beta gene therapy and immunotherapy using tumor cell lysate-pulsed dendritic cells (DCs) would increase the efficacy of IFN-beta gene therapy. In an experimental mouse intracranial glioma (GL261), which cannot be cured by either IFN-beta gene therapy or DC immunotherapy alone, IFN-beta gene therapy following DC immunotherapy resulted in a significant prolongation in survival of the mice. Moreover, when this combination was performed twice, 50% of treated mice survived longer than 100 days. Considering these results, this combination therapy may be one promising candidate for glioma therapy in the near future.     

外源型P14ARF基因对人脑胶质瘤细胞生长的影响

目的：研究Ｐ１４ＡＲＦ基因对人脑胶质瘤细胞株（Ｕ－２５１）生长的影响。方法：用脂质体介导携带Ｐ１４ＡＰＦ的真核表达载体转染Ｕ－２５１细胞,观察其对该细胞株的生长、增殖周期等生物学行为的影响。结果：导入Ｐ１４ＡＰＦ胶质瘤细胞Ｕ－２５１的生长有明显的抑制作用。     

PTEN基因蛋白在恶性黑素瘤中的表达及意义

目的探讨PTEN基因蛋白在恶性黑素瘤中的表达及其意义.方法采用免疫组化EnVision检测PTEN基因蛋白在51例恶性黑素瘤以及30例皮内痣组织中的表达情况.结果恶性黑素瘤组织中PTEN表达强度低,总体阳性率52.9%(27/51),其中Ⅰ、Ⅱ、Ⅲ各期阳性率分别为59.4%(17/32)、38.5%(5/13)、33.3%(2/6);而皮内痣中PTEN均呈强阳性,表达阳性率为100%(30/30),二者差异具有显著性(P＜0.01).结论恶性黑素瘤的发生可能与PTEN基因蛋白的低表达有关,PTEN基因蛋白表达缺失或低下者更易发生远处转移,标记PTEN可作为判断黑素瘤预后的一项指标.