U251胶质瘤荷瘤鼠皮下模型的建立及其表皮生长因子受体通路成员异常表达

The establishment of U251 glioma subcutaneous model in nude mice and its abnormal expression of members of EGFR pathway

目的建立稳定可靠的荷瘤鼠皮下U251胶质瘤实验动物模型,使其良好地用于胶质瘤分子机制的研究。方法取生长状态良好的U251胶质瘤细胞悬液按1×106个细胞／50μl接种于10只荷瘤鼠腹腔皮下,待其成瘤后将肿瘤组织块接种于20只实验鼠,观察并记录肿瘤皮下生长情况,于成瘤后第5周处死荷瘤鼠检测是否存在肿瘤远处转移;同时对肿瘤组织行HE和免疫组织化学染色检测胶质瘤的病理学特征,观察表皮生长因子受体、磷酰肌醇3-激酶和丝氨酸／苏氨酸蛋白激酶-2基因阳性表达率,结果与体外培养的U251胶质瘤细胞系的基因异常表达进行比较。结果细胞悬液接种可成功获得皮下U251胶质瘤模型,但潜伏期延长(2-3周),成功率低(6／10);若将其生成的肿瘤组织块再次接种于荷瘤鼠皮下则可获得高效、稳定的成瘤率(20／20)且潜伏期明显缩短(3d-5d)。荷瘤鼠生长状态良好,无恶病质变化,未发生其他脏器转移。大体标本观察显示,肿瘤体积>750mm3时其内部多见坏死伴囊性变,部分肿瘤尚有脂肪浸润或纤维化;肿瘤有假包膜形成,瘤体部分呈鱼肉状,是胶质瘤生发和再移植取材的部位。免疫组织化学检测显示,荷瘤鼠皮下胶质瘤组织和体外培养的U251胶质瘤细胞对表皮生长因子受体、磷酰肌醇3-激酶以及丝氨酸／苏氨酸蛋白激酶-2基因通路主要成员的基因表达情况相似。结论将U251胶质瘤细胞悬液接种于荷瘤鼠皮下形成的胶质瘤再移植到荷瘤鼠皮下建立U251移植瘤实验模型的方法简便、潜伏期短、成功率高、稳定性好,可以作为胶质瘤体内实验研究的动物模型。

Objective To establish a stable, dependable U251 glioma subcutaneous model in nude mice in order to study the molecular pathogenesis of glioma. Methods Well cultured U251 glioma cell suspension was inoculated into the inguinal groove subcutaneous tissue of 10 nude mice by 106 cells/50μl, and when tumors appeared, another 20 nude mice were inoculated with the formed tumor tissue pieces, and then observed and recorded the growth of transplanted tumors. Executed the nude mice at the fifth week after tumors established, and resocted their five main organs and the brain tissues. Performed HE stain and immunohistochemistry examination to detect pathological characteristics of glioma, and the expression level of the epidermal growth factor receptor （EGFR）, phosphatidylinositol 3-kinase （PI3K）, and serine/threonine protein kinase-2 （AKT-2） gene. The results were compared with the same gene expression of U251 glioma cell line cultured in vitro. Results U251 glioma subcutaneous model was successfully established by cell suspension inoculation, but with long latent period （2-3 weeks） and low success rate （6/10）. Subcutaneous inoculation of tumor tissue pieces can obtain an effecient and stable tumor formation rate （20/20）, and the latency can be obviously shortened （3 d-5 d）. The status of nude mice was good, without cachexia changes and other organs metastasis. In gross specimen, when tumor 〉 750 mm^3, necrosis with cystic degeneration was often seen. Adipose infiltration or fibrous degeneration could be found in some tumors. Tumors possessed pseudocapsule, and its parenchyma presented as fish flesh which could be the substantial part for glioma regeneration and transplantation. The immunohistochemistry findings indicated that there were no significant differences in the expression of the main members in EGFR- PI3K-AKT pathway between glioma tissue from nude mice and the cultured U251 glioma cells in vitro. Conclusion Glioma tumor was generated by means of inoculating U251 glioma cell suspension in nude mice, then their tumor tissue pieces were transplanted to other nude mice to reform new tumor. This method was convenient, with low latency, high success rate and good stability, so it can be used to establish reliable animal models for glioma experimental study in vivo.