论文部分内容阅读
背景与目的:LRRC4是作者最近克隆的一个新基因,该基因在原发性脑肿瘤活检标本中明显表达下调。本研究旨在研究LRRC4基因是否具有抑制脑肿瘤生长的能力。方法:LRRC4基因的全长编码区被亚克隆至表达载体pcDNA3.1中,应用脂质体转染的方法将重组的质粒载体导入胶质母细胞瘤细胞系U251,经G418筛选,建立稳定表达LRRC4基因的U251的细胞系。采用细胞增殖实验、软琼脂实验、肿瘤形成实验来考察LRRC4基因表达对于细胞生长和肿瘤形成的影响。结果:经过脂质体转染和筛选,建立了稳定表达LRRC4全长编码区的U251细胞系,用于进一步实验。比较未转染组和转染空白载体组,Northernblot实验证实转染了LRRC4基因的细胞LRRC4mRNA的表达增强。细胞增殖一定时间后,转染LRRC4基因的细胞较未转染细胞的生长速度明显减慢,克隆形成率明显降低。将这些细胞注射入无胸腺裸鼠体内,40天后处死裸鼠,测量肿瘤大小,结果显示转染LRRC4基因的细胞形成的肿瘤明显小于对照组。结论LRRC4基因可转染于人脑胶质母细胞瘤细胞系U251。LRRC4在U251细胞的表达有抑制瘤细胞增殖和抑制裸鼠移植瘤的形成和生长的作用。
BACKGROUND & AIM: LRRC4 is a new gene cloned recently by the authors. The gene was significantly down-regulated in primary brain tumor biopsy specimens. This study aimed to investigate whether LRRC4 gene has the ability to inhibit the growth of brain tumors. METHODS: The full-length coding region of LRRC4 gene was subcloned into the expression vector pcDNA3.1. The recombinant plasmid vector was transfected into glioma cell line U251 by lipofectamine. The recombinant plasmid was selected by G418 and stably expressed LR25 gene of U251 cell line. Cell proliferation assay, soft agar assay and tumor formation assay were used to investigate the effect of LRRC4 gene expression on cell growth and tumor formation. Results: U251 cell line stably expressing LRRC4 full-length coding region was established by lipofection and screening for further experiments. Compared with untransfected cells and blank transfected cells, Northern blot analysis confirmed that the expression of LRRC4 mRNA in cells transfected with LRRC4 gene was enhanced. After a certain period of cell proliferation, the growth rate of LRRC4 transfected cells was significantly slower than that of untransfected cells, and the clonogenic rate was significantly decreased. The cells were injected into athymic nude mice and the nude mice were sacrificed 40 days later to measure the size of the tumors. The results showed that the cells transfected with LRRC4 gene were significantly smaller than the control group. Conclusion LRRC4 gene can be transfected into human glioblastoma cell line U251. The expression of LRRC4 in U251 cells has the effect of inhibiting tumor cell proliferation and inhibiting the formation and growth of xenografts in nude mice.