Pu P, Liu X, Liu A, Cui J, Zhang Y
Department of Neurosurgery, Tianjin Medical University General Hospital, People's Republic of China.
J Neurosurg. 2000 Jan;92(1):132-9. doi: 10.3171/jns.2000.92.1.0132.
The goal of this study was to evaluate the effect of antisense epidermal growth factor receptor (EGFR) RNA on the growth of rat glioma cells in vitro and in vivo and to determine the feasibility of targeting the EGFR gene for gene therapy in gliomas.
Antisense EGFR complementary (c)DNA was transfected into C6 glioma cells by using lipofectamine. In vitro studies, Southern and Northern blot analyses, in situ hybridization, and immunohistochemical staining were designed to examine the integration and expression of antisense EGFR constructs. The 3'(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the average number of argyrophilic nuclear organizer regions (Ag-NORs) were used to evaluate cell proliferation, whereas the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method and microscopy were used to observe cell apoptosis. As part of the in vivo studies, parental C6 cells and C6 cells transfected with EGFR antisense cDNA were implanted stereotactically into the right caudate nucleus of Wistar rats (C6-injected animals and transfected C6-injected animals). Rats with well-established cerebral C6 glioma foci were treated intratumorally with either antisense EGFR cDNA or empty-vector DNA by using lipofectamine (treated-C6 and control treated group). The general behavior and survival of the rats, findings on magnetic resonance images of their brains, histopathological changes, proliferation activity, and apoptosis of the cerebral gliomas in each group of rats were examined. Exogenous antisense EGFR cDNA was integrated into the genome of C6 cells and expressed. In clones with a high expression of the antisense construct, there was a dramatic decrease in endogenous EGFR messenger RNA and protein levels, reduced proliferation activity, and induction of apoptosis in vitro. The mean survival time of rats injected with C6 cells was 17.3 days. The mean survival time of rats injected with C6 cells followed by treatment with empty vector in lipofectamine was 15.4 days. Survival time was significantly prolonged in 100% of the rats injected with antisense-transfected C6 cells and in two thirds of the rats injected with C6 cells followed by antisense EGFR cDNA. Magnetic resonance imaging revealed distinct cerebral tumor foci in C6-injected rats and in control rats of the treated group, but none were found in the rats injected with transfected C6 cells. Furthermore, tumor foci disappeared completely in C6-injected rats treated with antisense EGFR cDNA. The cerebral gliomas of the rats treated by injection of antisense EGFR RNA were characterized by reduced proliferation activity and the induction of apoptosis.
The results of this study indicate that EGFR plays an important role in the genesis of malignant gliomas. It may, therefore, be an effective target of antisense gene therapy in patients with gliomas.
本研究旨在评估反义表皮生长因子受体（EGFR）RNA对大鼠胶质瘤细胞体外和体内生长的影响，并确定针对EGFR基因进行胶质瘤基因治疗的可行性。
利用脂质体将反义EGFR互补（c）DNA转染至C6胶质瘤细胞。体外研究采用Southern和Northern印迹分析、原位杂交及免疫组织化学染色，以检测反义EGFR构建体的整合与表达。采用3'-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法及嗜银核仁组成区（Ag-NORs）平均数评估细胞增殖，而采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记（TUNEL）法及显微镜观察细胞凋亡。作为体内研究的一部分，将亲代C6细胞及转染了EGFR反义cDNA的C6细胞立体定向植入Wistar大鼠的右侧尾状核（注射C6细胞的动物及转染C6细胞后注射的动物）。对已形成脑内C6胶质瘤灶的大鼠，通过脂质体瘤内注射反义EGFR cDNA或空载体DNA进行治疗（治疗C6组和对照治疗组）。检查每组大鼠的一般行为和生存情况、脑部磁共振成像结果、组织病理学变化、脑胶质瘤的增殖活性及凋亡情况。外源性反义EGFR cDNA整合入C6细胞基因组并表达。在反义构建体高表达的克隆中，内源性EGFR信使RNA和蛋白水平显著降低，体外增殖活性降低且诱导了细胞凋亡。注射C6细胞的大鼠平均生存时间为17.3天。注射C6细胞后用脂质体中的空载体治疗的大鼠平均生存时间为15.4天。注射反义转染C6细胞的大鼠100%及注射C6细胞后再注射反义EGFR cDNA的大鼠三分之二生存时间显著延长。磁共振成像显示注射C6细胞的大鼠及治疗组对照大鼠脑内有明显肿瘤灶，但注射转染C6细胞的大鼠未发现肿瘤灶。此外，用反义EGFR cDNA治疗的注射C6细胞的大鼠肿瘤灶完全消失。注射反义EGFR RNA治疗的大鼠脑胶质瘤的特征为增殖活性降低及诱导细胞凋亡。
本研究结果表明EGFR在恶性胶质瘤的发生中起重要作用。因此，它可能是胶质瘤患者反义基因治疗的有效靶点。
