裸鼠视网膜下注射HXO-Rb_（44）细胞建立视网膜母细胞瘤动物模型

目的建立一种生物学行为更接近临床的视网膜母细胞瘤（Rb）动物模型。方法 Rb细胞取HXO-Rb44细胞系,培养于含质量分数10%胎牛血清的改良型RPMI-1640培养基内,细胞悬液密度调至（5.0～6.0）×106/mL备用。30只裸鼠,分为视网膜下组和玻璃体腔组,每组各15只鼠30只眼。2组注射HXO-Rb44细胞悬液后每天裂隙灯下观察裸鼠眼内Rb肿物生长情况,并于注射后21、28、35、42d,根据组织病理学检查统计成瘤情况,比较2组成瘤率。行Rb特异性标志神经元特异性烯醇酶（NSE）及酸性钙结合蛋白（S100）和胶质纤维酸性蛋白（GFAP）免疫组织化学检测。结果视网膜下组和玻璃体腔组在注射后均观察到肿瘤在裸鼠眼内生长并逐渐增大。注射后21、28、35、42d,视网膜下组成瘤率高于玻璃体腔组,差异均有统计学意义（P〈0.05）。免疫组织化学染色提示,Rb细胞质NSE表达较强,S100表达较弱,GFAP表达最弱。结论在同等条件下建立Rb动物模型,视网膜下注射的成瘤率高于玻璃体腔内注射者,且视网膜下注射建立的是原位肿瘤模型,生物学行为更接近临床。

Establishment of animal model for retinoblastoma in nude mice by subretinal injection of HXO-Rb44 cells

Background Retinoblastoma（Rb） is common intraocular malignant tumor in infants.It is important for researcher to imitate a process of Rb in animal in order to better understand the development of tumor.Objective This study was to establish an animal model of retinoblastoma which biological behavior was closer to the clinical patient.Methods Rb cell line,HXO-Rb44,was cultured in modified RPMI-164030 containing 10% fetal bovine serum and the cell suspension was prepared with the cell density（5.0-6.0）×106/mL.Cell suspension was subretinally or intravitreously injected in 15 BALB/C（nu/nu） nude mice respectively to establish the Rb models.The growth of tumor in the eyes of nude mice were observed once a day after the injection of HXO-Rb44cells under the slit lamp.Tumor formation rate in the mice of different injection fashion groups were compared at 21,28,35 and 42 days after injection upon the histopathological diagnosis of the tumor.The identification of Rb was performed by the label of neurone specific enolase（NSE）,S protein（S100） and glial fibrillary acidic protein（GFAP）.Results The Rb grew in eyes after subretinal injection and intravitreous injection of suspension and gradually enlarged with the prolong of time.The tumor formation rate was significantly increased in subretinal injection group compared with intravitreous injection group at 21,28,35 and 42 days after injection,showing the statistically significant differences（P0.05-0.01）.The Rb cells presented the positive response for NSE,S100 and GFAP,showing the brown-yellow staining in the cytoplasm with the strongest reaction in NSE and weakest one in GFAP.Conclusion The subretinal injection of Rb cell suspension has a higher tumor formation rate than intravitreal injection.The Rb animal model by subretinal injection of Rb cell suspension is reliable,and the biological behavior of Rb mouse model is resemble to one of clinical patient.