人表皮生长因子的基础和应用研究

本文报道了有关人表皮生长因子（hEGF）及其受体（EGFR）的基础研究结果,以及在此基础上进行新药开发的有关工作;hEGF的基因合成、表达载体的构建和转化宿主细胞、表达产物的纯化和中试规模三批样品笔检验等的有关研究结果、hEGF滴眼液的动物实验和临床实验等新药研制过程。有关基础研究表明,编码51个氨基酸的hEGF可以在α因子前导肽的引导下在酵母体中分泌性表达,表达产物可促进角膜缘上皮细胞的增殖,可促进角膜碱烧伤的愈合,可用于口腔溃疡和皮肢烧伤的治疗,可对大鼠十二指肠溃疡有预防作用等,并用之制备了抗血清以测定血尿中EGF的浓度。研究表明EGF在大于10ng/ml浓度下对神经胶质瘤细胞株BT325和人阴道上皮癌细胞株A431的生长具有抑制作用,并用差异显示方法对此现象进行了分子机制的探讨。研究肾癌EGFR表达和DNA含量检测具有临床意义。对EGF的晶体进行了初步的结构分析。中试产品检测表明hEGF相对分子质量为6000、等电点为4．6、氨基端15个氨基酸顺序正确、具有EGF的免疫原性和生物活性。产品无酵母细胞的残余DNA。动物实验表明EFG在体内无蓄积,有明确的促进角膜上皮细胞增殖的作用,无急毒的长毒副作用,对眼无刺激和局部毒性作用。在四家医院进行的多中心双盲的临床实验以证实该滴眼液安全性、耐受量和有效性。在200例角膜移植和247例翼状胬肉切除术中有明显促进角膜损伤恢复的作用,其效果强于国外产阳性对照素高捷疗（Solcosery Eye Gel)。所有这些研制工作促成了利用酵母体系表达的人表皮生长因子制成的滴眼液获得了国家I类新药证书,这是我国首次批准的酵母体系表达的基因工程药物。

The basic and applied study on the epidermal growth factor

This article reviews the results of the basic research about epidermal growth factor and its receptor, and the development of the novel drug, EGF eyedrop, that containing chemically synthesized EGF gene, the construction of EGF expression vector, the transformation of the host cells, the purification of the recombinant protein EGF, the preparation of three batches of the EGF product and identification, the preclinical and clinical trials. Relevant studies show that recombinant EGF consisting of 51 amino acids can be secreted into the medium under the control of the alpha factor leading sequence in the yeast cells. The EGF can accelerate the growth of corneal-limbal epithelial cells and the healing of an alkali burned corneal. The EGF can be used in curing oral cavity ulcer and skin burned wound. And it has the preventive effects on experimental duodenal ulcer of rat. The antiserum was made for test of the concentration of blood EGF and urine EGF by RIA. Data from studies demonstrate the inhibition effect of EGF on the growth of tumor cells, such as A431 and BT325 cells in the presence of high EGF concentration (> 10 ng/ml). The expression of EGFR and DNA ploidy in renal carcinoma has clinical significance. Crystallization and preliminary x-ray diffraction studies of the EGF has been made. The MW of the EGF product is 6000, and the pI is about 4.6 and it has correct N-terminal amino acids sequences, immunogenicity and biological activity. There is no vestige of the DNA of the yeast cells. Animal experiments reveal that there is no cumulation of the EGF in the body, and EGF can promote corneal epithelial healing. There is no toxicological effect during cornea wound healing of rabbit. A randomized, double-blind, placebo-controlled, multi-center clinical trial was conducted in four hospitals to assess safety, ocular tolerance and efficacy of an ophthalmic solution of EGF for 200 cases of cornea transplantation and 247 cases of nebulae. Unequivocal results were obtained as the eyedrop really accelerate the wounded cornea healing. So, the EGF eyedrop as a novel drug of class I is approved by the National Drug Administration, and this is the first gene engineering drug that come from yeast expression system in China.