新型脑组织特异性基因LRRC4的功能研究

目的研究LRRC4基因的抑瘤作用,探讨LRRC4基因对U251细胞的生物学功能影响。方法将转染LRRC4基因的U251细胞、转染空白载体PcDNA3.1(+)的U251细胞和阴性对照的U251细胞分别进行HE染色、DNA染色和核仁组成区嗜银蛋白(AgNORs)染色,并采用图像分析系统分别进行平面形态参数、DNA含量、DNA倍体、细胞周期和AgNORs定量分析,流式细胞仪检测各组细胞的细胞周期分布,MTT检测各组细胞的增殖活性。结果转染LRRC4基因U251细胞的面积为(100.6±7.6)μm2,周长为(42.4±2.0)μm,细胞直径为(9.0±1.2)μm,平均DNA含量为(46.8±8.7)pg,AgNORs平均颗粒数为(1.2±0.4)个,AgNORs平均面积为(16.9±2.0)μm2,均显著低于转染空白载体和阴性对照细胞,DNA异倍体也显著低于转染空白载体和阴性对照细胞。转染LRRC4基因的U251细胞的G0/G1期百分比明显增高,而S期和G2/M期细胞减少。转染LRRC4基因的U251细胞增殖活性显著低于转染空白载体和阴性对照细胞(P<0.01)。结论LRRC4基因通过抑制细胞的DNA复制,减少细胞核仁组成区相关蛋白质的合成,使细胞停滞于G0/G1期,抑制细胞的增殖活性,进而发挥抑瘤功能。形态定量检测技术结合流式细胞检测、MTT检测等实验,能更加全面地阐明新基因的生物学功能。

Function of a novel brain-specific gene LRRC4

Objective study the suppressive effect of LRRC4 gene on human glioma U251 cells and further investigate its biological functions. Methods H&E, DNA and AgNORs stainings were performed on LRRC4-transfected U251 cells, mock-transfected U251 cells and non-transfected U251 cells, respectively. Quantitative analysis including cell morphometry, DNA content,DNA ploidy, silver stained argyrophilic nucleolar organizer regions (AgNORs) were investigated by image analysis. Flow cytometry was employed to determine the difference of cell cycle distribution and MTT staining was used to elucidate the activity of the LRRC4-transfected U251 cells. Results The morphological cell parameters such as area, perimeter and diameter, DNA content, chromosomal aneupoloidy, mean area of AgNORs particles and mean nucleus area of the LRRC4-transfected U251 cells were remarkably decreased compared to those of the mock-transfected and non-transfected U251 cells (P< 0.05 , P< 0.01) . Meanwhile, significant accumulation of cells in G_0/G_1 phase but decrease of cells in S and G_2/M phase, was observed in transfected U251 cells compared to those of the mock-transfected and non-transfected U251 cells (P< 0.05 , P<0.01). MTT staining showed that proliferation activity of both the mock-and non-trasfected U251 cells was significantly higher than that of the U251 cells transfected with LRRC4 gene (P< 0.01). Conclusion LRRC4 gene might be involved in tumor suppression by restraining DNA synthesis and the nucleoli organizer regions-associated proteins, keeping the cell cycles in phase G_0/G_1 and reducing proliferation activity of the glioma cells. Morphometry combined with other techniques such as flow cytometry and MTT staining can well elucidate the biological function of novel genes.