[A new orthotopic retinoblastoma model expressing green fluorescent protein]

In order to observe the growth and metastasis of the tumor directly, a new orthotopic retinoblastoma model was established with human RB cells expressing green fluorescent protein (GFP).

pEGFP-N(1), the eukaryotic expressive plasmid of GFP, was transferred into human RB cell line HXO-RB(44) by liposome Dosper. Then, the cell clones expressing GFP steadily were selected by means of neomycin, fluorescence microscope, and flow cytometer. Two microliters of RB cells (density at 4.5 x 10(8) - 5.5 x 10(8) cells per ml) were injected into the subretinal space of 30 nude mice (60 eyes) under binocular operating microscope. The growth of transplanted RB was observed in vivo using fluorescence stereomicroscope. The nude mice were killed at different times post-operatively to investigate the metastasis process of the tumor to the optic nerve, brain, and other organs including lung, liver and kidney.

The spread process of the tumor in the subretinal space was successfully observed under stereomicroscope. Transplanted RB developed into extra-ocular stage phase at 34 - 37 days after the operation. And metastasis to the cranium along the optic nerve was observed, with green RB cells distributing along the optic nerve sheath and long posterior ciliary artery. The histopathological characteristics of the transplanted tumor were similar to the human RB. Immunohistochemical staining showed positive expression of GFP in the tumor cells.

The established orthotopic RB model expressing GFP via injection of human RB cells into the subretinal space of nude mice provides a new approach to exploring the growth and metastasis processes of RB in natural situations.