腺病毒介导的凝血酶敏感蛋白1抗血管新生衍生物基因转移抑制K562细胞裸鼠体内生长

目的　探讨腺病毒介导的凝血酶敏感蛋白 1(thrombospondin 1,TSP1)抗血管新生衍生物(TSP1f)基因转移对人白血病细胞株K5 6 2裸鼠移植瘤的抑制活性及作用机制。方法　应用RT PCR方法从正常人外周血单个核细胞扩增TSP1fcDNA序列 ;采用AdEasy系统构建TSP1f 重组腺病毒ADV TSP1f;用Western印迹方法检测ADV TSP1f 介导的TSP1f 在K5 6 2细胞中的表达 ;18只皮下移植瘤模型小鼠分为 3组 ,分别于移植瘤内注射 10 9pfuADV TSP1f、10 9pfuADV LacZ及PBS观察对K5 6 2裸鼠体内生长的抑制作用 ;用免疫组化方法比较移植瘤内微血管密度 (MVD)。结果　K5 6 2细胞经ADV TSP1f感染可高效表达 /分泌TSP1f 多肽 ;治疗后 3周ADV TSP1f 治疗组移植瘤体积为 ( 110 8mm3± 179mm3) ,远小于ADV LacZ组 ( 45 18mm3± 45 2mm3)及PBS组 ( 46 6 6mm3± 45 8mm3) ,(P <0 0 1) ;每× 2 0 0倍视野中 ,ADV LacZ、PBS及ADV TSP1f 治疗组组织切片内CD31阳性血管内皮细胞平均数分别为 36± 7,34± 9和 14± 4。结论　ADV TSP1f可显著抑制K5 6 2细胞裸鼠体内生长 ,有望成为治疗恶性肿瘤包括造血系统恶性肿瘤有效的基因治疗药物。

Adenoviral vector expressing an antiangiogenic fragment of thrombospondin 1 inhibits the growth of K562 cell xenografts

OBJECTIVE: To explore the effect of adenovirus-mediated delivery of an antiangiogenic fragment of human thrombospondin 1 (TSP1(f)) on K562 cell growth in nude mice. METHODS: TSP1(f) cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was used as transgene to construct a adenoviral vector (ADV-TSP1(f)). Human leukemia K562 cells were cultured and infected with ADV-TSP1(f) or ADV-LacZ. PBS was used as control. TSP1(f) expression/secretion by these infected K562 cells was demonstrated using Western blot analysis. MTT assay was performed to determine the effect of ADV-TSP1(f) infection on K562 cell growth kinetics. Eighteen female Balb/c nude mice were inoculated subcutaneously with human leukemia K562 cells. When the diameter of the tumor reached 5 approximately 7 mm the rats were randomly divided into 3 groups of 6 rats injected intratumorally with ADV-TSP1(f), ADV-LacZ, and PBS respectively. The volume of K562 xenografts was measured every three days during the 3-week treatment. By the end of the 21st day the mice were killed and the tumors were taken to undergo histological examination. The intratumoral microvessel density (MVD) was determined by immunohistochemical staining. RESULTS: TSP-1f was expressed and secreted efficiently by ADV-TSP1(f)-infected K562 cells. Three weeks after the initial treatment, the volume of K562 xenografts in the mice treated with ADV-TSP1(f), ADV-LacZ, and PBS was (1,108 +/- 179) mm(3), (4,518 +/- 452) mm(3), and (4,666 +/- 458) mm(3) respectively (P < 0.01). The number of CD31+ microvessels counted per x200 field was 34 +/- 9, 36 +/- 7, and 14 +/- 4 in the tumors treated with PBS, ADV.LacZ, and ADV.TSP-1f respectively (P < 0.001). CONCLUSION: Adenovirus-mediated TSP-1f gene transfer greatly inhibits K562-derived tumor growth and angiogenesis in mouse xenograft model, and may serve as a new therapy for hematological malignancies.