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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。
Objective To study the inhibitory effect of recombinant adeno-associated virus-mediated human endostatin on the growth and metastasis of heterotopic xenografts of nasopharyngeal carcinoma in nude mice. Methods Infection efficiency, protein expression and bioactivity of recombinant human adenovirus carrying RECOMBINANT ADENO-ASSOCIATED VIRUSCARRYING HUMAN ENDOSTATIN GENE (RAAV-HENDO) were detected. The animal model of heterotopic xenograft of nasopharyngeal carcinoma in nude mice was established. RAAV-HENDO (HENDO group) carrying human endostatin gene and RAAV-EGFP (RAAV-CARRYING ENHANCED) carrying enhanced green fluorescent protein (EGFP group) and phosphate buffered saline (PBS group). The tumorigenicity, lung metastasis and survival of the liver were observed. Immunohistochemistry was used to detect the tumor microvessel density, TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE-MEDIATED DUTP NICK-END-LABELING, TUNEL method was used to detect tumor cell apoptosis index. Results In vitro experiments showed that the efficiency of in vitro infection of RAAV was 98%. Immunofluorescence staining showed that endostatin protein was mainly expressed in the cytoplasm. The proliferation of ECV304 cells induced by RAAV-HENDO was 67.3%. Animal experiments showed that the tumor inhibition rate of HENDO group was 70.7% compared with that of PBS group; the lung metastasis rates of HENDO group, EGFP group and PBS group were 0 · 0%, 50 · 0% and 66 · 7% X-S, the same below) with densities of (3.67 ± 1.63), (19.67 ± 2.16) and (22.5 ± 3.02), respectively, with significant differences between the groups P <0.01). The mean apoptotic index was (28.83 ± 5.27)%, (6.17 ± 2.79)%, (4.50 ± 2.17)%, respectively. There was significant difference between the two groups (P <0.01). The mean survival time of the three groups were (36.5 ± 8.64) D, (24.00 ± 5.66) D, (21.17 ± 3.92) ) D, the difference between the groups was statistically significant (P <0.01). Conclusions Recombinant adeno-associated virus-mediated endostatin can effectively inhibit the growth and metastasis of heterotopic xenograft in nasopharyngeal carcinoma of nude mice. The possible mechanism is to inhibit tumor angiogenesis and induce apoptosis of tumor cells.