腺相关病毒介导的内皮抑素对鼻咽癌移植瘤生长和转移的抑制作用

目的研究重组腺相关病毒介导的人内皮抑素对裸鼠鼻咽癌肝脏异位移植瘤生长和转移的抑制作用。方法检测携带人内皮抑素的重组腺相关病毒(RECOMBINANT ADENO-ASSOCIATED VIRUSCARRYING HUMAN ENDOSTATIN GENE,RAAV-HENDO)体外感染效率、蛋白表达及生物活性。建立裸鼠鼻咽癌肝脏异位移植瘤动物模型,经尾静脉分别注射携带人内皮抑素基因的RAAV-HENDO(HENDO组)、携带增强型绿色荧光蛋白基因的RAAV-EGFP(RAAV-CARRYING ENHANCED GREEN FLUORESCENT PROTEIN GENE,RAAV-EGFP,EGFP组)和磷酸缓冲液(PBS组),观察动物肝脏成瘤、肺转移及生存期等情况,免疫组化检测肿瘤微血管密度,原位末端标记(TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE-MEDIATED DUTP NICK-END-LABELING,TUNEL)法检测肿瘤细胞凋亡指数。结果体外实验表明:RAAV体外感染效率达98%;免疫荧光染色显示内皮抑素蛋白主要表达于细胞质;RAAV-HENDO感染细胞培养上清对ECV304细胞72H增殖抑制率为67·3%。动物实验表明:相对PBS组,HENDO组抑瘤率为70·7%;HENDO组、EGFP组、PBS组肺转移率分别为0·0%、50·0%、66·7%;微血管平均(X-±S,以下同)密度分别为(3·67±1·63)、(19·67±2·16)、(22·50±3·02),组间比较差异有统计学意义(P<0·01);凋亡平均指数分别为(28·83±5·27)%、(6·17±2·79)%、(4·50±2·17)%,组间比较差异有统计学意义(P<0·01);3组动物平均生存期分别为(36·50±8·46)D、(24·00±5·66)D、(21·17±3·92)D,组间比较差异有统计学意义(P<0·01)。结论重组腺相关病毒介导的内皮抑素能有效抑制裸鼠鼻咽癌肝脏异位移植瘤的生长和转移,其可能机制为抑制肿瘤新生血管形成,诱导肿瘤细胞凋亡。

Adeno-associated virus-mediated gene transfer of endostain for inhibiting growth and metastasis of human nasopharyngeal carcinoma in nudemice

OBJECTIVE: To investigate the inhibitory effect on growth and metastasis of human nasopharyngeal carcinoma in nude mice by adeno-associated virus-mediated gene transfer of human endostatin. METHODS: The infectious efficiency of recombinant adeno-associated virus (rAAV) in vitro was detected. The endostatin expressed in human umbilical vein endothelial cell ECV304 was detected by immunofluorescence staining. MTT was used to assay inhibitory effect of the infecting supernatant of recombinant adeno-associated viral vectors carrying human endostatin gene (rAAV-hEndo)on ECV304 cell. Heterotopic implantation model of human nasopharyngeal carcinoma cell C666-1 in nude mice was established. rAAV-hEndo or rAAV-EGFP or PBS were injected into the tail vein of tumor bearing mice. Three weeks after implantation, the volumes of tumor, inhibition rate, the percentage of lung metastases, microvessel density (MVD) and apoptotic index (AI) were evaluated respectively. RESULTS: The infectious efficiency of rAAV was 98% in vitro. Immunofluorescence staining showed the humam endostatin protein was expressed mainly in cytoplasm. ECV304 cell proliferation was obviously inhibited by the infecting supernatant of rAAV-hEndo. The inhibitory rate was 67.3% when the supernatant was used 72 h later. Compared with PBS group, the restrained percentage of tumor in hEndo group was 70.7%. The percentage of lung metastases in hEndo group, EGFP group and PBS group was 0.0%, 50.0% and 66.7% respectively. The average MVD of hEndo group (3.67 +/- 1.63) was significantly lower than that of EGFP group (19.67 +/- 2.16) and PBS group (22.50 +/- 3.02) (P < 0.01). The apoptotic index increased significantly in hEndo group(28.83 +/- 5.27)% versus EGFP group (6.17 +/- 2.79)% and PBS group (4.50 +/- 2.17)% (P < 0.01). The survival time of tumor bearing mice in hEndo group (36.50 +/- 8.46) d was significantly longer than EGFP group (24.00 +/- 5.66) d and PBS group (21.17 +/- 3.92) d (P < 0.01). CONCLUSIONS: The gene transfer of human endostatin mediated by adeno-associate virus can inhibit the growth and metastasis of human nasopharyngeal carcinoma in nude mice effectively. The mechanism may be due to the effect of antiangiogensis and inducement of tumor cell apoptosis.