携带CD基因骨髓间充质干细胞对C6大鼠胶质瘤体内抑制作用研究

Investigation on The Inhibition Effects of Bone Marrow Mesenchymal Stem Cells With CD Gene on Glioma in C6 Rat

为了研究自杀基因——胞嘧啶脱氨酶基因(cytosine deaminase gene,CD基因)对大鼠脑胶质瘤的体内治疗效果,采用基因转导系统将慢病毒包装的CD基因转染骨髓间充质干细胞(MSC)并使其长时间、高效表达,然后移植到使用颅内立体定向接种法制作的40只SD大鼠胶质瘤模型中.按接种细胞类型将实验SD大鼠分为5组,每组8只:①C6胶质瘤;②C6+MSCs细胞(1∶1);③C6+MSCs细胞(1∶2);④C6+MSC-codA/eGFP细胞(1∶1);⑤C6+MSC-codA/eGFP细胞(1∶2).瘤龄7天后腹腔注射500 mg/(kg.d)5-氟胞嘧啶(5-flucytosine,5-FC),共14天.用磁共振成像(MRI)动态监测肿瘤的体积并进行了大鼠存活期观察、常规病理分析、RT-PCR检测、HE染色.结果显示,第①组瘤龄14天时病灶呈圆形,中心见坏死区,肿瘤平均246 mm3,均存活期15.3天,第②组平均生存期16.0天,第③组平均生存期16.6天,第④⑤组自然存活期均大于30天,14天时病灶平均体积分别为55 mm3、40 mm3,28天时肿瘤抑制率分别为77.24%、83.28%.MRI扫描可清楚显示肿瘤的大小、形态和内部结构,与病理结果高度相关;MRI动态观察可证实携带CD基因的骨髓间充质干细胞及5-FC治疗系统治疗C6颅内胶质瘤有效;RT-PCR检测结果证实肿瘤组织内有胞嘧啶脱氨酶表达.

The in vivo treatment effect of CD（cytosine deaminase） suicidal genetic system on the glioma in rat was explored.The lentivirus carrier which was composed of CD genes was fabricated and the mouse BMMSCs were transfected by such CD genes to obtain constantly expressed cells,then BMMSCs were transplanted into the animal model which was built through an intracranial stereotactic inoculating method using a group of 40 SD rats.The rats were divided into 5 groups uniformly according to the cell type inoculated,i.e.① C6 glioma,②C6＋MSCs（mesenchymal stems cells） cells（1∶1）,③C6＋MSC cells（1∶2）,④C6＋MSC-codA/eGFP cells（1∶1） and ⑤C6＋MSC-codA/eGFP cells（1∶2）.After 7 days of tumor formation,the abdominal cavity of rats were injected with 5-FC at 500 mg/（kg· d） for 14 days.Intensive scanning was carried out weekly to observe tumor volume using MRI.Simultaneously,survival times,routine pathological test,RT-PCR assay and HE staining were also operated.The results of MRI showed that the focus of infection in group ① presented round shape.The necrosis area of tumor found in the center,achieved an average volume of 246 mm3,and the survival time of the center area was 15.3 days.About the group ② and ③,the survival times were 16.0 and 16.6 days respectively.But in group ④ and ⑤,the survival times were both larger than 30 days,the necrosis area of tumor was 55 and 40 mm3 respectively at day 14,and the inhibition efficiency was 77.24% and 83.28% respectively after 28 days of treatment.It was concluded that the MRI scanning could clearly show the volume,shape,and internal structure of the tumor,which was highly related to the results of pathology.The treatment effect of bone marrow MSCs with CD genes and 5-FC therapy system on C6 intracranial glioma could be verified through dynamic MRI observation.Moreover,the RT-PCR test confirmed the expression of cytosine deaminase inside tumor organization.