VEGF受体KDR胞外Ⅴ～Ⅶ区的克隆、单抗制备及KDR在不同来源肿瘤组织中的表达

Cloning and monoclonal antibody preparation of VEGF receptor KDR extracellular Ⅴ～Ⅶ domain and KDR expression in carcinomas of different origins

目的利用制备的血管内皮细胞增殖因子（ＶＥＧＦ）受体ＫＤＲ抗体，通过免疫组化，检测ＫＤＲ在不同来源肿瘤组织中的表达，为探讨ＶＥＧＦ及其受体对肿瘤生长及转移的作用提供可能。方法采用ＲＴＰＣＲ方法，从人胎儿脐静脉内皮细胞克隆ＫＤＲ胞外Ⅴ～Ⅶ区基因片段，在大肠杆菌中表达，并用传统的杂交瘤融合技术制备小鼠ＫＤＲ单克隆抗体。通过组化及Ｗｅｓｔｅｒｎｂｌｏｔ鉴定ＫＤＲ单抗的特异性，最后采用ＳＰ免疫组化法，分别对不同来源的１１５例肿瘤组织及相应正常组织进行了ＫＤＲ表达分析。结果不同来源的肿瘤组织中，ＫＤＲ受体的表达率及强度存在差异，移行上皮来源的膀胱癌１００％表达ＫＤＲ，表达强度最高；乳腺癌及肠道腺癌细胞表达ＫＤＲ次之；肺鳞癌表达最弱。此外，肿瘤组织中不仅血管内皮细胞表达ＫＤＲ受体，且肿瘤细胞、血管平滑肌细胞及某些间质细胞也有ＫＤＲ表达，而相应正常组织中ＫＤＲ表达相对较弱。结论ＶＥＧＦ受体ＫＤＲ不仅表达于肿瘤血管内皮细胞，而且表达于肿瘤细胞。

Objective To examine the expression of VEGF receptor KDR in carcinomas of different origins and provide possibility of exploring its relation to tumor growth and metastasis. Methods VEGF receptor KDR cDNA (Ⅴ～Ⅶ) fragment was cloned from human umbilical vein with RT PCR and expressed in E.coli Jm109. The purified GST KDR fusion protein was used to immunize Balb/c mice and monoclonal antibody against KDR was prepared. With this monoclonal antibody, tumor tissue and related normal tissue of different origins were examined immunohistochemically for KDR expression. Results The frequency and intensity of KDR expression in different tumor tissues were obviously different. Transitional cell cancer of urinary bladder was 100% KDR positive and with highest intensity, while KDR expression in breast and intestinal adenocarcinoma was weaker and that in squamous cell carcinoma of the lung was weakest. Moreover, KDR was also weakly expressed in other cells, such as smooth muscle cells and interstitial cells. Conclusion VEGF receptor is expressed not only in the endothelial cells of tumor vasculature, but also in tumor cells. Tumor cell expression of KDR varies in intensity with tumors of different origins. The possible significance of VEGF receptor expression with regard to tumor cell proliferation and metastasis is discussed.