重组腺相关病毒载体介导人血管抑素K1-5基因预防和治疗B16黑素瘤的实验研究  

Prevention and treatment of B16 melanoma by adeno-associated virus-mediated human angiostatin(K1-5) gene transfection

作　　者：曹培国[1] 吴小兵[2] 阳月新[1] 黄程辉[1] 龙林[1] 沈守荣[3] 

机构地区：[1]中南大学湘雅三医院肿瘤科,长沙410013 [2]中国预防医学科学院病毒基因工程国家重点实验室,北京100052 [3]中南大学湘雅三医院消化内科,长沙410013

出　　处：《肿瘤》2006年第5期426-430,共5页Tumor

基　　金：湖南省自然科学基金资助项目(编号:05JJ30167);湖南省卫生厅科研基金资助项目(编号:B2005-070)

摘　　要：目的：研究重组腺相关病毒载体介导人血管抑素K1-5基因（rAAV—AG）对B16F0恶性黑素瘤细胞生长的影响。方法：观察rAAV-AG单次肌肉注射在预防肿瘤生长、血行转移及治疗肿瘤方面的不同作用；Western blot法定性检测血管抑素表达情况；免疫组化法检测肿瘤组织中的微血管密度（MVD）；TUNEL法检测肿瘤凋亡细胞数。结果：rAAV-AG单次肌肉注射后第2周至实验结束时（～70d）小鼠体内均有血管抑素表达；预防肿瘤生长抑瘤率为97％（P〈0．01）；预防肝、肺转移抑瘤率分别为37．5%（P＜0．05）和47．7％（P＜0．01）；低、中、高剂量组治疗肿瘤抑瘤率分别为44％、61％和65％，小鼠中位生存时间分别为58d、66d和68d，与生理盐水组比较，低剂量组无显著性差异（P=0．06），中、高剂量组均有显著性差异（P〈0．01）；基因治疗备组肿瘤MVD减少，凋亡细胞数增加，与生理盐水组比较，统计结果与各组抑瘤率一致。结论：rAAV-AG单次肌肉注射能有效预防和治疗B16恶性黑素瘤的生长及血行转移，延长荷瘤小鼠的生存期；在一定剂量范围内其抑瘤效应呈剂量依赖性；抑瘤作用可能是通过抑制肿瘤血管生成、诱导肿瘤细胞凋亡来实现的。Objective： To study the effect of human angiostatin K1-5（AG） gene transfection mediated by recombinant adeno associated virus（rAAV）on the growth of B16F0 melanoma in Balb/c mice. Methods： The different effects of single intramuscular injection of rAAV AG in prevention of tumor growth and blood metastasis and in tumor therapy were observed. Angiostatin protein expression was examined by Western blot analysis. Microvessel density（MVD）in the tumor tissue was assessed by immunohisto-chemistry with the primary anti vWF antibody. The apoptotic cells were counted by TUNEL staining. Results： The expression of angiostatin was detectable in serum on d 14 and sustained for at least 70 d in skeletal muscle after single intramuscular injection of rAAV-AG. The inhibition rate in preventing tumor growth was 97%（P〈0.01）. The inhibition rate in preventing lung and liver metastasis was 47.7%（P〈0. 01） and 37.5%（P〈0. 05）, respectively. The inhibition rate by rAAV-AG at the low, middie, and high dose in tumor therapy was44%,61%,and 65%, respectively. The median survival time（MST）of each group was 58 d,66 d,and 68 d, respectively. No significant difference was observed between the low dose of rAAV AG group and control group. However compared with normal saline group, the inhibition rate in tumor growth and MST between middle and high dose of rAAV-AG group was significantly different. The amounts of MVD decreased and the number of apoptotic cells increased significantly in all rAAV-AG groups. The results were in accordance with the inhibition rate of tumor growth. Conclusion： Single intramuscular injection of rAAV-AG was effective in prevention and treatment of B16F0 melanoma by inhibiting its growth and blood metastasis and prolonging the survival time of tumor-bearing BALB/c mice. Within a certain dose range, the anti tumor efficacy of rAAV-AG was dose-dependent, rAAV-AG exerts its anti-tumor activity probably through antiangiogenesis and induction of tumor cell apoptosis.

关 键 词：黑素瘤 实验性 血管抑制素类 腺病毒科 基因疗法 

分 类 号：R739.5[医药卫生—肿瘤]