小鼠endostatin基因的克隆与C6鼠脑胶质瘤的治疗

Cloning of mouse endostatin gene for treatment of C6 brain glioma in rats

目的 克隆小鼠 endostatin基因 ,检测其表达蛋白的生物学活性 ,应用该蛋白治疗大鼠 C6脑胶质瘤 .方法 从小鼠胶原 c DNA用 PCR法克隆 endostatin基因 ,重组入p U C19,测序后构建非融合表达载体 p DH- endostatin,使其在DH5α内经温度诱导表达 ,纯化该表达蛋白并用鸡胚绒毛膜尿囊膜实验和内皮细胞抑制实验检测其活性 .经荷瘤大鼠皮下注射该蛋白治疗其脑胶质瘤 .结果 成功获得 5 5 1bp的endostatin基因 ,经测序正确 .该诱导表达蛋白 Mr2 0 0 0 0 ,具有抗血管生成活性 .应用该蛋白 10或 2 0 mg· kg- 1· d- 1可抑制荷瘤大鼠脑胶质瘤血管生成和肿瘤生长 .结论 Endostatin基因的表达和初步应用 。

AIM To clone mouse endostatin gene, detect its biological activities and use it to cure C6 gliomas of the rat brain. METHODS Endostatin gene was cloned from the mouse collagen ⅩⅧ cDNA by PCR and recombined into the vector of pUC19. The gene was sequenced and a non fusionexpression vector pDH endostatin was constructed. The recombinant mouse endostatin gene was expressed in DH5α at under the condition of temperature induction. The protein activities were tested by chicken chorio allantoic membrane (CAM) assay and endothelial cell proliferation inhibitory assay. Endostatin was applied to the rat with tumor through hypodermic injection in order to cure the C6 glioma of the rat brain. RESULTS The acquired 551 bp endostatin gene sequence was correct and its protein size was M r 20 000 . The protein possessed the anti angiogenesis activity and could inhibit the growth of C6 glioma in rat brain. 10 or 20 mg·kg -1 ·d -1 endostatin could inhibit the angiogenesis of glioma and tumor growth. CONCLUSION The expression and clinical application of mouse endostatin gene lay the foundation of the antiangiogenesis therapy of brain gliomas.