腺病毒携带的人内皮抑素基因(Ad/hEnd)抑制人舌鳞状细胞癌生长的研究

背景与目的：舌癌是口腔常见的恶性肿瘤,目前常规采用以手术为主结合放疗化疗的综合治疗,总体的5年生存率只有50％左右,抗肿瘤血管生成治疗已成为舌癌治疗的研究方向之～。本实验以5型E1缺陷型腺病毒携带的人内皮抑素基因(Ad／hEnd)感染舌癌细胞(Tca8113)和人脐静脉内皮细胞株(ECV),并对荷瘤裸鼠舌癌的抑瘤效果进行观察,研究其在舌癌细胞中的表达及对舌癌抑制作用。方法：(1)免疫组化法检测内皮抑素蛋白在Tca8113细胞和ECV细胞中的表达及分布。ELISA法检测上清中内皮抑素含量,Western blot检测内皮抑素基因在Tca8113和ECV细胞的表达特征。(2)流式细胞仪检测Ad／hEnd感染ECV后的细胞周期及凋亡,WST-1法检测Ad／hEnd对ECV细胞增殖的抑制。(3)Ad／hEnd对荷瘤裸鼠的舌癌的生长抑制分析。结果：(1)实验结果显示感染Ad／hEnd后。Tca8113细胞和ECV细胞胞浆内可有效合成内皮抑素蛋白,细胞培养液上清中的内皮抑素蛋白表达浓度呈时间剂量依赖关系,最高达到597ng／ml,可持续到第7天,并且表达产物有抑制人体静脉内皮细胞ECV生长特性。呈剂量依赖关系。(2)Ad／hEnd可延长感染后的ECV细胞的S期及G2期。并出现细胞凋亡现象。(3)应用Ad／hEnd后第3天肿瘤体积增长受到抑制,第6天开始肿瘤抑制明显增强,第3周抑瘤率达45．8％。结论：本实验制备的重组腺病毒Ad／hEnd能在ECV和Tca8113细胞中有效表达内皮抑素,表达产物可影响ECV细胞周期、抑制ECV细胞增殖、诱导ECV细胞凋亡及抑制荷瘤裸鼠舌癌的生长。

Inhibition of tongue cancer development in nude mice transfected with adenovirus carrying human endostatin gene

BACKGROUND & OBJECTIVE: The squamous cell carcinoma of tongue is one of the most common malignant tumors of oral cavity. Surgical therapy is now the mainstay of combined treatment for tongue squamous cell Carcinoma with chemotherapy and radiotherapy. The overall 5-year survival rate was about 50%. The antiangiogenesis therapy has become a new approach of the treatment of tongue carcinoma. This paper was designed to study the characteristics of endostatin expression in tongue cancer cell line (Tca8113), human embryonic epithelial cell line (ECV) and the inhibition of carcinogenesis in nude mice, xenografted with Tca8113, after transfected with recombined adenovirus (Ad/hEnd) which was cloned with human endostatin gene in EI mutated region. METHODS: (1) To determine the expression and distribution of endostatin in Tca and ECV cells transfected with Ad/hEnd using immunohistochemistry. To determine the endostatin in supernatants of Tca cells transfected with Ad/hEnd using ELISA method. To examine the characteristics of endostatin gene expression in Tca8113 and ECV cells by Western blot analysis. (2) To determine the inhibition rate of proliferation and apoptosis rate of ECV cells by WST-1 test and flow cytometry (FCM), respectively.(3) To observe the inhibition of tumor growth in xenografted nude mice with Tca8113 cells by Ad/hEnd administration. RESULTS: (1) Immunohistochemistry detection indicated that the endostatin was expressed in cytoplasm of Tca8113 cells and ECV cells transfected with Ad/hEnd. Endostatin expression in the supernatant was dose-dependent with the highest to 597 ng/ml. The expression of endostatin in Tca cells was detectable from 1 day to 7 day. Ad/hEnd inhibited ECV cell growth in dose-dependent manner. (2) FCM showed that Ad/hEnd arrested ECV cells in S and G(2) phase and induced apoptosis.(3) The tumor growth curve showed that Ad/hEnd significantly repressed xenograft tumor growth with Tca cell in nude mice; the inhibition rate on Ad/hEnd administrated groups was 45.8% in the 3rd week. CONCLUSION: Ad/hEnd expressed efficiently in Tca8113 and ECV cells. Ad/hEnd can change the cell cycle distribution of ECV cells and induce apoptosis and inhibit proliferation of ECV cells. Ad/hEnd could inhibit the growth of tongue carcinoma in xenograft nude mice.