腺病毒载体介导的血小板第4因子p17-70 cDNA对荷瘤裸鼠抗血管新生的作用

目的　以腺病毒做载体研究血小板第 4因子 (PF4)氨基末端改构体cDNA(p1 7 70cDNA)的荷瘤裸鼠体内、外抗血管新生作用。方法　将p1 7 70cDNA克隆至AdEasyTM系统 ,转染 2 93细胞包装成含p1 7 70cDNA的腺病毒载体。用RT PCR方法证明有p1 7 70cDNA外源基因插入 ,Western印迹分析KB细胞 (人上皮细胞癌 )表达的目的蛋白P1 7 70肽。用此病毒直接转导人脐静脉内皮细胞(HUVEC)并将此病毒注射到负有KB细胞的荷瘤裸鼠体内。以内皮细胞的增殖情况及瘤块体积、质量、瘤体内微血管数分析P1 7 70肽的抑制血管新生的作用。结果　转导p1 7 70cDNA病毒后的HUVEC增殖较含空载体病毒对照减低 58% ,注射病毒 2周后的实验组、空载体组及PBS对照组瘤块质量分别为 (0 0 86± 0 .0 54)g ,(0 .1 71± 0 .0 76)g和 (0 .1 95± 0 .0 67)g ,体积分别为 (1 6 .7± 5 .2 )mm3 、(36 .5± 2 3 .7)mm3 和 (41 .5± 1 2 .2 )mm3 ,免疫组化示微血管计数分别为 9.5± 1 .2 ,30 .6± 2 .6 ,31 .3± 2 .5 ,实验组、空载体病毒组及PBS对照组之间差异有显著性 (P <0 0 5) ,而空载体病毒与PBS两个对照组之间差异无显著性 (P >0 .0 5)。结论　腺病毒载体介导的P1 7 70肽体外具有抑制内皮细胞增殖活性 ,裸鼠体内具有抑制血管新生从而抑制肿瘤生长

Inhibition of tumor angiogenesis in nude mice by adenovirus-mediated PF4 p17-70 cDNA transfection

OBJECTIVE: To investigate the in vivo effect of modified platelet factor 4 (PF4)-p17-70 cDNA on tumor angiogenesis in nude mice. METHODS: The p17-70 cDNA was cloned into the AdEasy system to transfect packing cell line 293 and produce viral particles encoding p17-70cDNA (Ad p17-70). The integration of p17-70 cDNA was confirmed by RT-PCR and the P17-40 peptide Western blot. The biological activity of purified recombinant adenovirus was determined by umbilical veinal endothelial cell proliferation assay in vitro and in vivo tumor angiogenesis suppression of nude mice bearing human head and neck carcinoma. RESULTS: p17-70 significantly inhibited in vitro proliferation of endothelial cells being 58% lower than that of empty vector and reduced tumor volume in vivo. The tumor mass was (0.086 +/- 0.054) g, (0.171 +/- 0.076) g and (0.195 +/- 0.067) g, the tumor volume was (16.7 +/- 5.2) mm(3), (36.5 +/- 23.7) mm(3) and (41.5 +/- 12.2) mm(3) in p17-70 cDNA transfected group, empty vector group and PBS group, respectively. Immunohistochemical staining demonstrated a decreased number of blood vessels in the tumors. CONCLUSION: P17-70 peptide mediated by adenoviral vector could inhibit the endothelial proliferation in vitro and the tumor growth in vivo.