摘要
目的研究Tob基因在胶质瘤细胞系SHG-44中的功能,为下一步进行试验性治疗提供依据。方法按照Tob基因的GenBank登录号D38305,构建带有绿色荧光蛋白(GFP)和新型抗生素Zeocin抗性基因的pTracer-EF/V5-HisA表达载体。利用脂质体将上述表达质粒转入SHG44胶质瘤细胞。在各个不同的培养时段选择2、4、8、16、24、36、48h分别在倒置显微镜下观察,最后在48h时取未转染和转染的细胞大约105个作细胞涂片,进行荧光显微镜摄影和GFAP免疫组化染色分析。同时作体外生长曲线和细胞周期的流式细胞术分析。结果酶切鉴定和序列分析均证实Tob基因被成功克隆,将此基因成功转染到SHG-44细胞后,荧光显微镜观察可见用4μg脂质体和2μgDNA,最后用10μg/mLZeocin筛选可以达到90%的阳性细胞数。在转染Tob基因成功的细胞中GFAP染色的阳性率也同样升高。细胞在转染了Tob基因以后生长速度明显变慢,几乎处于停滞状态。流式细胞术分析显示在体外培养24h后转染阳性细胞G2/M期比例明显减少,而凋亡细胞比例也出现明显的升高。结论Tob基因转染后的胶质瘤细胞生长受到明显抑制,出现向正常胶质细胞的分化和凋亡现象,提示Tob基因确实是一胶质瘤细胞增殖抑制基因,值得进一步做功能研究和实验性基因治疗研究。
Objective To study the function of Tob gene in human glioma cell line SHG-44 by Lipofectamine transfection so as to offer some clues for the further experimental therapy. Methods According to Tob＇s Genbank accession No. D38305, pTracer-EF/V5-His A expression vector with green fluorescent protein （GFP） and Zeocin resistance gene. The above plasmid was introduced into SHG-44 glioma cells by Lipofectamine and the cells were observed under an inverted microscope at 2 h, 4 h, 8 h, 16 h, 24 h, 36 h, 48 h of the different culture time points. The smears of the transfected and non-transfected cells of about 105 each taken at 48 h were prepared and photographed under fluorescent microscope and analyzed using GFAP immunohistochemical staining, external growth curve and flow cytometer. Results Restriction enzyme digestion and DNA sequencing analysis showed that the Tob gene has been cloned and transfected into SHG-44 cells successfully. Mixing 4 μg Lipofectamine and 2 μg DNA and selecting by 10μg/mL Zeocin can get more than 90% transfection-positive cells. After transfection, SHG-44 cells with transfected Tob gene grew slowly and even got into stillness. GFAP positive rate in these cells are higher than ones with non-transfected Tob gene. The growth speed of the cells with transfected Tob gene got slowly, even to a growth arrest. Flow cytometry revealed that after cultured in vitro for 24 h the positive cells in G2/M period decreased remarkably in number and the ratio of apoptotic cells increased notably. Conclusion The growth of SHG-44 glioma cells was suppressed by Tob gene transfection, and the cells tended to differentiate into normal gliocytes and apoptosis. So it was confirmed that Tob gene is really a novel antiproliferative gene of glioma cells, worthy of further functional analysis and research on experimental gene therapy.
出处
《中华神经医学杂志》
CAS
CSCD
2005年第11期1081-1084,共4页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(30070772)
