辐射对L929细胞生长及CCN1基因表达的影响

Effects of radiation on growth and CCN1 expression of mice fibroblast cell line L929

目的通过观察辐射对小鼠皮肤成纤维细胞系L929生长及CCN1基因表达的影响,探讨CCN1基因与辐射损伤的关系。方法体外培养L929细胞系,以4Gy60Coγ射线照射为辐射组,并设对照组,MTT法测细胞增殖变化,平板克隆形成实验检测克隆形成能力,流式细胞术测细胞周期,RT-PCR及免疫细胞化学法检测L929细胞中CCN1基因的表达情况。结果辐射组细胞辐照2d增殖抑制明显(P<0.01),克隆形成也明显受到抑制(P<0.05);辐射后24h,细胞出现G2期停滞;辐射后6hL929细胞CCN1的mRNA表达水平明显降低(P<0.01),至24h开始回调(P<0.05);辐照后24～48h,细胞CCN1蛋白的表达水平明显下降(P<0.01)。结论因辐照生长受到抑制的小鼠成纤维细胞L929,其CCN1基因的表达水平下调,提示CCN1基因的表达水平低下是辐射导致细胞生长抑制的因素之一。

Objective To observe the effects of radiation on the growth and expression of cysteine,rich 61 （Cyr61/CCN1） of L929 cells and investigate the relationship between CCN1 expression and radiation injury. Methods L929 cells were cultured and divided into 2 groups, cells irradiated with 4 Gy γ-irradiation as radiogroup and untreated cells as control group. The cell proliferation was measured by MTT assay and plate colony formation testing. Flow cytometry was utilized to quantify the cell cycle distribution. CCN1 expression at protein and mRNA levels were determined by immunocytochemistry （ICC） and RT-PCR respectively. Results Significant inhibition of proliferation （ P 〈 0.01 ） and colony formation （ P 〈 0.05 ） were found in cells of radiogroup. Meanwhile, cells were arrested at G2 phase at 24 h after radiation. The expression of CCN1 mRNA was down-regulated at 6 h （P 〈0.01 ）, but moderately increased at 24 h （P 〈0.05）. The expression of CCN1 protein was obviously decreased at 24 to 48 h after radiation （ P 〈 0.01 ）. Conclusion Radiation inhibits the growth and CCN1 expression of cultured fibroblasts, indicating CCN1 may be involved in the suppression of fibroblasts