胞嘧啶脱氨酶自杀基因转染U251恶性脑胶质瘤细胞及其表达研究

目的探讨胞嘧啶脱氨酶（CD）自杀基因转染U251恶性脑胶质瘤细胞的方法及其表达效果。方法采用脂质体法将CD基因转染U251恶性脑胶质瘤细胞，G418筛选阳性克隆（U251-CD），采用RT—PCR、免疫细胞化学染色、流式细胞仪（FCM）凋亡分析和高效液相色谱（HPLC）等方法检测CD基因的表达及其功能。结果CD基因成功转染U251细胞。G418筛选获得阳性克隆．RT—PCR证实阳性细胞有CD基因表达，抗-CD-抗体免疫细胞化学法显示细胞浆染色阳性．U251-CD细胞加入5-氟胞嘧啶（5-FC）后，FCM显示细胞凋亡峰，HPLC可检测到5-FC培养液内有5-氟尿嘧啶（5-FU）产生。结论CD基因能够在U251细胞表达．并具备将5—Fc转变为5-Fu的功能，造成肿瘤细胞凋亡．CD-5-FC系统可作为恶性脑胶质瘤辅助治疗手段之一。

Cytosine deaminase suicide gene transfection and its expression on U251 malignant brain glioma cells

Objective To explore the methods of cytosine deaminase (CD) suicide gene transfection and its expression in U251 malignant brain glioma cells. Methods CD gene was transfected into U251 malignant brain glioma cells using liposome-mediated method, positive clone (U251-CD) was screened with G418 presence. CD gene expression and its function were identified by RT-PCR, immunocytochemical staining, FCM analysis of apoptotic cells and HPLC. Results CD gene was successfully transfected into U251 cells, and positive clone was screened out after G418 presence. RT-PCR indicated that positive cells could express CD gene, and immunocytochemical staining with CD antibody showed positive reaction in the cell plasma. FCM analysis showed apoptotic cells after gene transfection with 5-FC presence. HPLC detection showed that 5-FU was detected in U251-CD cell medium with 5-FC incubation. Conclusion CD gene can transfect into U251 cells. U251-CD cells can change 5-FC into 5-FU, and induce tumor cell apoptosis. The CD-5-FC suicide gene therapy system can act as the main assistant strategy in treatment of malignant gliomas.