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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 glioma cell line SHG-44 and provide the basis for further experimental treatment. Methods According to the GenBank accession number D38305 of Tob gene, pTracer-EF / V5-HisA expression vector with green fluorescent protein (GFP) and novel antibiotic Zeocin resistance gene was constructed. The above expression plasmids were transfected into SHG44 glioma cells using liposomes. In each of the different training period selected 2,4,8,16,24,36,48h were observed under an inverted microscope, and finally at 48h to take untransfected and transfected cells about 105 for cell smear fluorescence Microscopic photography and GFAP immunohistochemical staining. At the same time for in vitro growth curve and cell cycle flow cytometry analysis. Results The restriction endonuclease analysis and sequence analysis confirmed that the Tob gene was successfully cloned. After transfection of this gene into SHG-44 cells successfully, 4μg liposome and 2μg DNA were observed by fluorescence microscopy. Finally, 10μg / mL Zeocin screening could achieve 90% The number of positive cells. The positive rate of GFAP staining in cells transfected with the Tob gene was also increased. After the Tob gene was transfected, the growth rate of cells was obviously slowed down, almost at a standstill. Flow cytometry analysis showed that the proportion of transfected cells in G2 / M phase was significantly decreased and the percentage of apoptotic cells was significantly increased after cultured in vitro for 24h. Conclusion The growth of glioma cells transfected with Tob gene was significantly inhibited and the differentiation and apoptosis of glioma cells were observed. It is suggested that Tob gene is indeed a suppressor of glioma cell proliferation, which is worth further study on function and experiment Sexual gene therapy research.