CAJ | 학술논문

目的探讨内皮抑素和血管生成抑素( Endo - Angio)融合基因修饰的单纯疱疹病毒(VAE)对胶质瘤干细胞(GSCs)的体外溶瘤作用。方法新鲜高级别（WHOⅢ级、Ⅵ级）人脑胶质瘤标本经原代培养获得GSCs，采用流式细胞术检测其中CD133阳性细胞数，单克隆形成实验检测GSCs的自我更新能力，免疫荧光技术检测未分化GSCs的Nestin及分化后GFAP、NF和MAG的表达；MTS法检测VAE对GSCs增殖活性的影响，RT - PCR和Western blot检测Endo - Angio融合蛋白的表达并检测其对人脑微血管内皮细胞(HBMEC)增殖的影响；最后观察病毒处理后仍存活细胞的自我更新和诱导贴壁情况。结果 (1)从20例高级别胶质瘤标本中培养出4例GSCs，能够表达CD133及Nestin，具有自我更新能力和多向分化能力。(2)VAE能够感染GSCs，4例GSCs增殖活性明显下降(P＜0.05)。(3)VAE感染GSCs 48 h后，基因水平及蛋白水平都有Endo- Angio融合蛋白表达，该融合蛋白使HBMEC增殖活性明显下降(P＜0.05)。(4)VAE感染后仍存活的细胞不再具有形成GSCs球的能力，加入血清诱导后也不再贴壁生长。结论在体外，VAE能够显著抑制GSCs活性，能够表达具有生物活性的Endo-Angio融合蛋白，为脑胶质瘤溶瘤病毒治疗开辟了新的途径。
목적 탐토내피억소화혈관생성억소( Endo - Angio)융합기인수식적단순포진병독(VAE)대효질류간세포(GSCs)적체외용류작용。방법 신선고급별（WHOⅢ급、Ⅵ급）인뇌효질류표본경원대배양획득GSCs，채용류식세포술검측기중CD133양성세포수，단극륭형성실험검측GSCs적자아경신능력，면역형광기술검측미분화GSCs적Nestin급분화후GFAP、NF화MAG적표체；MTS법검측VAE대GSCs증식활성적영향，RT - PCR화Western blot검측Endo - Angio융합단백적표체병검측기대인뇌미혈관내피세포(HBMEC)증식적영향；최후관찰병독처리후잉존활세포적자아경신화유도첩벽정황。결과 (1)종20례고급별효질류표본중배양출4례GSCs，능구표체CD133급Nestin，구유자아경신능력화다향분화능력。(2)VAE능구감염GSCs，4례GSCs증식활성명현하강(P＜0.05)。(3)VAE감염GSCs 48 h후，기인수평급단백수평도유Endo- Angio융합단백표체，해융합단백사HBMEC증식활성명현하강(P＜0.05)。(4)VAE감염후잉존활적세포불재구유형성GSCs구적능력，가입혈청유도후야불재첩벽생장。결론 재체외，VAE능구현저억제GSCs활성，능구표체구유생물활성적Endo-Angio융합단백，위뇌효질류용류병독치료개벽료신적도경。
Objective Exploring the effect of endostatin and angiostatin(Endo-Angio) fusion genemodified herpes simplex virus ( VAE ) on glioma stem cells ( GSCs ) in vitro. Method Surgical specimens of human high grade glioma ( WHO Ⅲ, Ⅵ) were collected, and GSCs were isolated under the culture conditionsoriginally designed for selective expansion of neural stem cells. In order to identify GSCs,the number of CD133 positive cells in GSCs were detected by flow cytometry; self - renewal capacity and the expression of Nestin, GFAP, NF, and MAG of GSCs were detected by monoclonal forming and immunofluorescence assay respectively. After that, GSCs viability was detected by MTS assay. Expression of Endo - Angio fusion gene at mRNA and protein level was detected by RT - PCR and Western blot. At last,the efficacy of fusion protein to human brain microvascular endothelial cells (HBMEC) and the biological properties of GSCs were detect after infected by VAE. Results ( 1 )4 GSCs isolated from 20 surgical specimens could suspend growth and had the ability of self-renewal and multipotential differentiation,and could express neural stem cells marker,CD133 and Nestin. (2)VAE could infect GSCs and significantly inhibit the viability of GSCs( P ＜0.05). (3) Significant expression of Endo-Angio fusion gene was observed and it could inhibit HBMEC proliferation( P ＜0.05). (4) Residual viable cells lost the ability of self - renewal and adherent differentiation. Conclusions In vitro, VAE can inhibit the activity of GSCs and the expression of exogenous Endo - Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novd virus -gene therapy strategy for glioma.