绿色荧光蛋白标记的人肝癌细胞在Balb/c鼠前房成瘤的观察

Observation of human hepatocellular carcinoma cell expressing green fluorescent protein transplanted in anterior chamber of Balb/c mice

目的观察绿色荧光蛋白(green fluorescent protein,GFP)标记的人肝癌细胞(human hepatocellu-lar carcinoma cell,MHCC97L)在Balb/c鼠眼前房生长成瘤及全身转移情况,探讨前房作为肿瘤细胞培养的活体器官,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GFP的逆转录病毒MP71-GFP-PRE转入人肝癌细胞MHCC97L。于手术显微镜下在20只Balb/c小鼠眼的前房中分别接种2μL细胞密度为1.0×106个/mL的肝癌细胞悬液。术后15,30和45d在荧光显微镜下观察小鼠前房肿瘤生长,冰冻切片在荧光显微镜下观察眼及脑、肝、肺等全身转移情况,并做病理切片HE染色观察肿瘤细胞。结果稳定表达GFP的肝癌细胞基本保持了亲代细胞的特征,在前房的成瘤率为100%,随着时间的推移这些肿瘤团块逐渐增大。术后处死小鼠,在荧光显微镜下未见脑、肝、肺等脏器中有荧光团块或单个荧光点分布。眼球冰冻及病理切片均可见前房、睫状沟、虹膜基质内和玻璃体腔内肿瘤细胞生长。结论前房可以作为有效的培养肿瘤细胞的活体器官。GFP标记瘤细胞后,利用荧光显微镜能在活体观察到肿瘤团块在前房内的生长,以及单个瘤细胞和普通显微镜下无法分辨的微小病灶。

Objective To observe the growth and metastasis of human hepatocellular carcinoma cell MHCC97L expressing green fluorescent protein （GFP） inoculated into the anterior chamber of Balb/c mice, and to explore the possibility of anterior chamber being a living cell culture organ and the superiority of green fluorescent protein as a marker of living cells in tracing the growth and metastasis of the tumor cells. Methods A retrovirus vector MP71-GFP-PRE containing GFP gene was transferred into MHCC97L cell line. Then 2 μL of 1.0 × 10^6 mL^-1 MHCC97L cells was injected into the anterior chamber in one eye of each 20 Balb/c mice. The neoplasms in anterior chamber were observed under the fluorescence microscope on the 15,30 and 45 days post-operatively. Eyes as well as brain, liver and lung were observed under the fluorescence microscope in order to investigate the growth and metastasis of MHCC97L cells. HE stain was also performed. Results Tumor cells expressed GFP steadily and also maintained the feature of parental cells. Neoplasms were formed in all the anterior chambers of mice, which grew gradually. No scattered single or mass tumor cells expressing green fluorescence was observed in brain, lung or liver under the fluorescence microscope post-operatively. Neoplasms in anterior chamber and the place near the ciliary sulcus, and scattered tumor cells in the iris and vitreous were found in iced or HE stained slice. Conclusions Tumor cells could be cultured in anterior chamber, and scattered tumor cells could be observed clearly under the fluorescence microscope when the tumor cell was labeled with GFP.