CAJ | 학술논문

目的从原代培养的人脐静脉血管内皮细胞(HUVEC)提取细胞总RNA,采用逆转录PCR(RT-PCR)方法得到VEGF受体KDR全长胞外区cDNA 片段,检验其体内抗肿瘤血管生成的作用.方法将获得的受体基因克隆到AAV基因治疗载体pSNAV中,得到重组质粒pSNAV/KDR.重组质粒转染BHK细胞,加入辅助病毒后,得到表达目的蛋白的重组AAV.重组AAV表达的KDR具有与VEGF结合的活性,重组AAV感染人膀胱癌EJ细胞,皮下注射Balb-c裸鼠.Ⅷ因子免疫组化染色进行微血管密度测定.结果重组AAV感染人膀胱癌EJ细胞形成的肿瘤血管化程度明显低于对照组.结论重组AAV介导的KDR胞外区基因可有效的抑制裸鼠人膀胱癌组织的新生血管生成.
목적종원대배양적인제정맥혈관내피세포(HUVEC)제취세포총RNA,채용역전록PCR(RT-PCR)방법득도VEGF수체KDR전장포외구cDNA 편단,검험기체내항종류혈관생성적작용.방법장획득적수체기인극륭도AAV기인치료재체pSNAV중,득도중조질립pSNAV/KDR.중조질립전염BHK세포,가입보조병독후,득도표체목적단백적중조AAV.중조AAV표체적KDR구유여VEGF결합적활성,중조AAV감염인방광암EJ세포,피하주사Balb-c라서.Ⅷ인자면역조화염색진행미혈관밀도측정.결과중조AAV감염인방광암EJ세포형성적종류혈관화정도명현저우대조조.결론중조AAV개도적KDR포외구기인가유효적억제라서인방광암조직적신생혈관생성.
Objective To verify whether the extracellular domain of kinase domain region (KDR) has anti-angiogenesis activity in vivo.Methods cDNA was cloned into adeno-associated virus (AAV) vector pSNAV and transfected to baby hamster kidney (BHK) cells. Recombinant AAV was obtained from the cell culture supernatant after adding helper virus. Recombinant AAV-infected human bladder cancer EJ cell line (EJ cells) were injected subcutaneously into Balb-c nude mice. Tumor specimens were removed from the mice, paraffin-embedded and sliced, then stained by immunohistochemistry. Microvessel density (MVD) was determined under a microscope.Results The tumor volume developed by EJ cells transfected with the extracellular domain of KDR was significantly smaller (1.70±0.18 cm3) compared with that in the control (5.62±0.67 cm3) (P<0.05), although tumor developed to be detectable on almost the same time (14.7±2.4 days vs 14.1±3.2 days). Further, MVD in the experimental group was lower than that in the control (41.3±4.8 vs 6.2±2.1, P<0.05).Conclusion The extracellular domain of KDR could be expressed in nude mouse bladder cancer tissue and inhibit tumor angiogenesis.