恶性胶质瘤靶向治疗研究进展

目的 恶性胶质瘤是一种最常见的原发恶性脑肿瘤,是癌症治疗中最具挑战性疾病之一.因为手术切除后肿瘤易复发和治疗抵抗性,患者预后普遍较差.胶质瘤的分子靶向治疗正逐渐引起广泛关注.本研究总结恶性胶质瘤发病相关的分子病理改变和靶向药物的临床应用与研究进展.方法 采用PubMed文献检索系统,以“恶性胶质瘤”和“分子靶向治疗”为关键词,检索2007-01-01-2015-12-31的相关文献.纳入标准:(1)与恶性胶质瘤分子靶向通路相关的文献;(2)与恶性胶质瘤抗血管生成治疗相关的文献;(3)与恶性胶质瘤分子靶向药物的Ⅰ期及Ⅱ期临床研究相关的文献;(4)与恶性胶质瘤分子靶向耐药相关的文献.根据纳入标准分析文献43篇.结果 胶质瘤靶向治疗方向主要集中在RTK/RAS/PI3K通路、促血管生成通路和一些其他重要的细胞内信号转导通路.然而,一些因素如信号通路之间的串扰、瘤内异质性和胶质瘤干细胞的治疗抵抗性限制了单一药物的活性.各种分子靶向药物单药治疗未能表现出更好的生存获益,还需与其他治疗方法联合应用.目前对于恶性胶质瘤患者多靶点激酶抑制剂治疗的研究还处于起始阶段.结论 分子靶向药物在恶性胶质瘤的治疗中具有重要临床意义和应用潜力,但由于胶质瘤的复杂的分子生物学特性,分子靶向治疗面临诸多挑战,还需进一步探索与研究.     

胶质瘤干细胞的分子靶向治疗

胶质瘤干细胞（GSC）被认为是恶性胶质瘤迁移、复发和放化疗抵抗的根源。结合分子靶向药物在其他肿瘤领域走向临床的背景,针对 GSC 的分子靶向药物有望取得成功。目前,相关研究主要集中在 Hedgehog、PI3K 等通路,代表药物主要有环巴胺及其衍生物、雷帕霉素及其衍生物,经典药物三氧化二砷、二甲双胍也有抗 GSC 作用。     

Silencing stathmin gene expression by survivin promoter-driven siRNA vector to reverse malignant phenotype of tumor cells

Stathmin gene overexpression has been shown to play an important role in maintenance of malignant phenotype in tumor cells, and the blocking efficacy and tumor specificity of this target has been concerned in clinical trails. In this report, we designed survivin promoter-driven siRNA eukaryotic expression vector that expressed the small interfering RNA targeting stathmin gene to selectively knock down the stathmin gene expression in two different kinds of tumor cell lines while sparing normal cell lines. The therapeutic potential of this recombinant vector was tested in human cervical cancer Hela cells and osteosarcoma SSOP-9607 cells, and in human umbilical vein endothelial cell line ECV304 cells as control. The siRNA vector- transfected Hela cells and SSOP-9607 cells revealed marked inhibition of stathmin expression and a dramatic growth inhibition comparing with ECV304 cells, parental-vector transfected cells and untransfected cells. Cell cycle analysis of siRNA vector transfected tumor cells by Flow Cytometry showed G(2)/M phase block, while morphologic analysis by TURNEL staining method showed marked increase of apoptosis. Our study indicates that survivin gene promoter-driven stathmin siRNA expression vector may have potential use in tumor gene therapy with targeted tumor gene silencing effect.     

骨髓增生异常综合征骨髓细胞端粒酶活性和端粒酶逆转录酶的表达

目的　探讨骨髓增生异常综合征 ( MDS)骨髓细胞端粒酶活性与端粒酶逆转录酶表达的关系及临床意义。方法　采用端粒重复序列扩增 -酶联免疫吸附试验 ( TRAP- EL ISA)检测端粒酶活性 ,RT- PCR法检测端粒酶逆转录酶 ( h TERT) m RNA水平的表达。结果　高危组 MDS患者骨髓细胞端粒酶活性较低危组 MDS患者骨髓细胞端粒酶活性明显增高 ( 0 .2 0 0± 0 .2 2 5 vs 0 .0 2 7± 0 .0 13,P=0 .0 37) ;正常人、缺铁性贫血 ( IDA)患者和低危组 MDS患者骨髓细胞 h TERT m RNA表达检测结果均阴性 ,而高危组 MDS患者和急性白血病 ( AL )患者骨髓细胞均表达h TERT基因 ;h TERT m RNA表达和端粒酶活性呈正相关 ( r=0 .774 ,P=0 .0 2 4 )。结论　端粒酶活性与 h TERT基因 m RNA水平的异常表达能反映出 MDS病情的进展。定期检测 MDS患者骨髓细胞端粒酶活性和 h TERTm RNA水平的表达可作为观察 MDS患者病情变化的指标之一。     

Stem cell-based therapy for malignant glioma

Stem cells have been extensively investigated as tumour-tropic vectors for gene delivery to solid tumours. In this review, we discuss the potential for using stem cells as cellular vector systems in gene therapy for malignant gliomas, with a focus on neural stem cells, and multipotent mesenchymal stromal cells. Tumour cell-derived substances and factors associated with tumour-induced inflammation and tumour neovascularisation can specifically attract stem cells to invasive gliomas. Injected stem cells engineered to produce anti-tumour substances have shown strong therapeutic effects in experimental glioma models. However, the potential caveats include the immunosuppressive functions of multipotent mesenchymal stromal cells, the contribution of stem cells to the pro-tumourigenic stroma, and the malignant transformation of implanted stem cells. In addition, it is not yet known which stem cell types and therapeutic genes will be most effective for the treatment of glioma patients. Here, we highlight the possibilities and problems for translating promising experimental findings in glioma models into the clinic.     

人端粒酶逆转录酶启动子介导的靶向性肿瘤基因治疗

由于人端粒酶逆转录酶(hTERT)启动子区域已被克隆,其特点也已被鉴定,hTERT启动子介导的靶向性肿瘤基因治疗成为研究的热点.现综述hTERT启动子介导的靶向性肿瘤基因治疗最新研究进展.     

Retrovirus-mediated gene transfer targeted to malignant glioma cells in murine brain.

A murine model for meningeal metastasis of malignant glioma was developed to study selective gene transfer into tumor cells and to establish a reliable means of determining the rate of tumor cell infection. A murine ecotropic retroviral vector was created in which the Escherichia coli beta-galactosidase gene served as a marker for gene expression from the integrated retrovirus. This retrovirus exhibited a high rate of infectivity in RSV-M mouse glioma cells in vitro. The recombinant retrovirus was injected directly into the cisterna magna of the mice. Staining of beta-galactosidase showed that the rate of gene integration was high in the disseminated glioma cells. These results suggest the possibility of retrovirus-mediated gene therapy for meningeal dissemination of malignant glioma.     

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.     

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...     

Radioimmunoconjugates for targeted alpha therapy of malignant melanoma

Effective targeted cancer therapy requires high selectivity and cytotoxicity. To this end we have prepared and tested a new alpha-emitting radioimmunoconjugate (RIC) against malignant melanoma. The melanoma antibody 9.2.27 is specific for most melanoma cell lines. This antibody was labelled with an a emitter, bismuth-213 (213Bi), and a positron emitter, terbium-152 (152Tb), which is an analogue of the alpha-emitting radioisotope terbium-149. The chelators cDTPAa (a cyclic anhydride of diethylenetriamine pentacetic acid) and CHX-A" (a 2-(p-SCN-Bz)-cyclohexyl-DTPA ligand) were used in order to obtain high labelling yields for both isotopes with either chelator. The labelling efficiency with 213Bi was found to be 96% and 92% with cDTPAa and CHX-A", respectively. With 152Tb it was 93% and 89%, respectively. Serum stability studies showed 20% leaching with 213Bi over a period of 2.5 half-lives. For 152Tb the leaching was 13%. There was no difference in the melanoma cell binding of the labelled and unlabelled antibodies. DNA synthesis data were compared for both isotopes with either chelator. Based on these results, the therapeutic activity ratio for 213Bi a particles and 152Tb positrons for the same endpoint was calculated to be 120. The stability of the bismuth and terbium RICs, together with the outstanding cytotoxicity of the alpha emitter, provides the basis for a new approach to the potential control of micrometastatic melanoma.     

重组腺病毒携带钠碘转运体基因介导放射性碘治疗小鼠胶质瘤

目的 验证转染人钠碘转运体基因(hNIS)介导放射性碘治疗肿瘤的有效性.方法 利用重组腺病毒将hNIS基因及人甲状腺过氧化物酶(hTPO)基因转染入人胶质瘤细胞系U251中,使肿瘤细胞获得hNIS和 hTPO基因,然后进行体外摄125I实验、过氯酸盐抑制实验、体外125I反流及内流实验.应用转染hNIS和hTPO基因及未转染hNIS和hTPO基因的细胞株,分别建立荷瘤裸鼠模型,并检测131I对肿瘤的抑制作用.结果 利用腺病毒可以将hNIS和hTPO基因成功转染到U251细胞系中,转染上述基因的肿瘤细胞系可以摄取碘,而且这种摄碘的功能是由hNIS基因所介导的,转染后的hNIS-U251细胞系摄碘能力是U251细胞的121.2倍,hNIS-hTPO-U251细胞系摄碘能力是U251细胞的172.0倍.131I对裸鼠移植瘤的治疗结果表明,在131I作用下,对照组肿瘤均继续迅速生长,而hNIS转染组及hNIS和hTPO共转染组移植瘤体积均有所减小.结论 在肿瘤细胞中转染hNIS和hTPO基因后,可以提高其摄取12I的活性.131I 可以有效杀伤荷瘤裸鼠体内的中瘤细胞.

Abstract：

Objective To explore the possibility of tranfecting hNIS and hTPO genes into gliomas cells by recombinant adenovirus for radioactive iodide treatment. Methods To tranfect hNIS gene into human glioma cell line U251 by recombinant adenovirus.The biological functions of the cells stably expressing hNIS and hTPO genes were assessed by 1251 uptake assay,125I influx-course and 12I-effluxcourse.A glioma model was established with inoculation of the U251 cells in nude mice,and the inhibiting effect of 131 I on the tumor growth was tested in the mouse models. Results The hNIS and hTPO genes were successfully transfected into human gliomas cell line U251 cells by recombinant adenovirus.The radioactive iodide could be intaken by the tumor cells mediated by hNIS gene.The uptake of 125I was higher in cell lines hNIS-U251 and hNIS-hTPO-U251 cells than in cell line U251 cells.The tumor volume of the mice after 131I treatment was significantly decreased in comparison with that before treatment.Conclusion Radioactive 131I treatment after HNIS-based gene transfer can be enhanced and effectively inhibite the tumor growth in nude mice.