腺病毒介导的内皮抑素基因治疗小鼠肺癌

Inhibitory effect of adenovirus-mediated endostatin gene transfer on transplanted lung carcinoma in mice

目的探讨内皮抑素对小鼠肺癌生长和转移的抑制作用及其对肿瘤内部新生血管的影响。方法在C57BL／6小鼠背部皮下注射2×10^6lewis肺癌（LLC）细胞，建立小鼠肺癌种植瘤模型，2周后，瘤内注射2×10^9pfu内皮抑素腺病毒载体，观察内皮抑素对肿瘤生长、转移及生存率的影响，检测内皮抑素在肿瘤组织的原位表达和血液循环中的表达水平及持续时间。用免疫组化方法，检测肿瘤内部血管密度，观察治疗对肿瘤血管的影响。用透射电镜观察肿瘤细胞的凋亡情况。结果免疫组化检测结果显示，内皮抑素蛋白在内皮抑素组的肿瘤组织中呈强阳性表达，而在空载体对照组和阴性对照组中呈阴性表达或很少量表达。用酶联免疫吸附实验（ELISA）法检测内皮抑素组血清内皮抑素浓度，第2周可达1540±560ng／ml；1个月后，血清内皮抑素浓度降至对照水平。内皮抑素组的肿瘤体积和生存率，与空载体对照和阴性对照组比较，差异有统计学意义（P〈0．05）。抗CD31抗体标记的肿瘤内血管密度（MVD）在内皮抑素组、空载体对照组和阴性对照组中，分别为37．5±4．6、65．2±5．8和68．5±4．5个／200倍视野，抗CD105抗体标记的肿瘤内MVD分别为10．5±3．2、39．7±5．6和42．4±4．8个／200倍视野，内皮抑素组与空载体对照组和阴性对照组比较，差异有统计学意义（P〈0．05）。内皮抑素组的组织在电镜下呈凋亡相的肿瘤细胞多见。结论腺病毒介导的内皮抑素基因可在体内高效、较长时间表达内皮抑素蛋白，对小鼠皮下种植瘤有一定的治疗作用，其作用的靶点是抑制新生血管的生成。

Objective To investigate the effect of adenovirus-mediated endostatin gene transfer on transplanted lung cancer in mice and its mechanism of action. Methods Transplant tumor model was induced by subcutaneous inoculation of 2 × 10^6 Lewis lung cancer （LLC） cells into the back of C57BL/6 mice. The mice were treated by intratumoral injection of 2 × 10^9 pfu Ad-mEndostatin. The expression of endostatin in situ and its maintaining time were detected by immunohistoehemistry and Western Blot, respectively. The endostatin level in serum was determined by ELISA . The inhibition of tumor growth and changes of survival were recorded and the mierovessel density （MVD） was determined by histochemical stainingwith CD31 and CD105 antibodies . The tumor apoptosis was observed by electron microscopy . Results In comparison with controls, intratumoral injection of Ad-mEndostatin significantly inhibited the tumor growth and metastasis, and prolonged the survival rate of mice （ P 〈 0. 05 ）. Strong positive expression of mEndostatin was seen in the tumor tissue after injection of Ad-mEndostatin, immunhistochemieally ostained by mouse endostatin monoelonal antibody, while the control groups showed only very low expression or absence. Serum endostatin concentration was 1540 ± 560 ng/ml at the second week of administration, the expression of endostatin diminished a month later. The mierovessel density （MVD） decreased from 42.4 ± 4.8 to 10.5 ±3.2 per × 200 magnifieetion microscopic field by CD105 staining and from 68.5 ±4.5 to 37.5 ±4.6 by CD3, staining, respectively （P 〈 0.05）. More apoptotie tumor cells were seen under the transmission electron microscope. Conclusion Endostatin gene therapy mediated by adenoviral vector efficiently induces expression of endostatin in vivo, and inhibits the growth and metastasis of tumor. It is concluded that its action is targeted to tumor neovaseulature and the mechanism is inhibition of tumor angiogenesis.