绿色荧光蛋白标记的CY15瘤细胞眼前房移植瘤早期转移的观察  

Early Metastasis of CY15 Cell Expressing Green Fluorescent Protein in Anterior Chamber

作　　者：聂莉[1] 李永平[1] 李亮平 张文忻[1] 张波[1] 

机构地区：[1]中山大学中山眼科中心//眼科学教育部重点实验室,广东广州510060 [2]Max-Delbruck分子医学中心,德国柏林13092

出　　处：《中山大学学报（医学科学版）》2006年第4期414-418,434,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省科技计划资助项目(2003A3020302)

摘　　要：【目的】观察绿色荧光蛋白(GFP)标记的恶性组织细胞增生症CY15细胞在Balb/c鼠眼前房生长及全身转移的规律,探讨GFP作为活细胞标记追踪肿瘤细胞生长、转移的优势并初步探讨肿瘤转移的机制。【方法】将携带绿色荧光蛋白的逆转录病毒MP71-GFP-PRE转入CY15细胞,G418筛选阳性克隆。20只Balb/c小鼠(20只右眼),手术显微镜下每只小鼠眼前房接种2μL细胞密度为2.0×106个/mL的CY15GFP细胞悬液。术后15d、20d和30d分别处死5只小鼠在荧光显微镜下观察全身转移情况。采用流式细胞仪分析术后30d处死的另外4只小鼠和4只对照小鼠外周血单个核细胞中瘤细胞的比率。【结果】稳定表达绿色荧光蛋白的CY15GFP细胞基本保持了亲代细胞的特征,在前房的成瘤率为100%。术后15d处死小鼠在荧光显微镜下观察可见肝、肺、胃肠壁等多个脏器中有散在的单个荧光点分布,为转移的单个肿瘤细胞,这些荧光点在肝脏中分布的数量最多。术后20d、30d分别处死5只小鼠,发现随着时间的推移这些微小转移灶逐渐增大。流式细胞仪分析术后30d处死的小鼠外周血单个核细胞中瘤细胞的平均比率为0.048%±0.021%。【结论】绿色荧光蛋白标记瘤细胞后,利用荧光显微镜能有效地观察到肿瘤发生远距离转移在继发脏器中分布的单个瘤细胞和普通显微镜下无法分辨的微小转移灶,这些微小转移灶逐渐增大,为肿瘤转移的克隆起源学说提供了有力的证据。流式细胞仪能准确定量瘤细胞在外周血中的分布情况。[Objective ] To observe the growth and metastatic rule of malignant histocytosis CY15 cell expressing green fluorescent protein inoculated in the anterior chamber of Balb/c mice,and to further explore the superiority of green fluorescent protein as the marker of living cells to trace the growth and metastasis of the tumor cells. On the basis of this, we discussed the mechanism of metastasis preliminarily. [Methods] MP71-GFP-PRE, a retrovirus vector that contained green fluorescent protein gene, was transferred into CY15 cell line and the cell clones expressing green fluorescent protein steadily selected by means of G418. 2 μL of CY15GFP cells （2.0×10^6 cells per milliliter） was injected into the anterior chamber in the right eye of 20 Balb/c mice under the operating microscope. Five mice were sacrificed respectively on the 15^th, 20^th, and 30^th day pest-operatively to investigate the metastasis under the fluorescence microscope. The ratio of tumor cells in the peripheral blood mononuclear cells （PBMC） was analyzed by flow cytometer for four mice killed on the 30^th day and four mice as control. [Results] Malignant histoeytosis CY15GFP cell expressed green fluorescence protein steadily and kept the same characters as its parent cell essentially. Neoplasms were displayed in all the mice in the anterior chamber. On the 15^th day pest-operatively, scattered single tumor cells expressed green fluorescence were observed in many organs under the fluorescence microscope ,such as the liver, lung and the digestive tract, etc. And there were the most fluorescent dots in the liver. Five mice were sacrificed respectively on the 20^th and 30^th day post-operatively which showed the micrometastasis augmented gradually with the time developed. The average ratio of tumor cells in the peripheral blood mononuclear cells （PBMC） was 0.048%×0.021% by flow cytometer analysis in the 30^th day group. [Conclusion] The single tumor cells and micrometastasis undetectable by ordinary microscope in the metastatic organs cou

关 键 词：恶性组织细胞增生症 绿色荧光蛋白 肿瘤转移 眼 

分 类 号：R77[医药卫生—眼科]