VEGFR2促进脑胶质瘤干细胞增殖和生长的机制研究

Study on the mechanism of VEGFR2 to promote the proliferation and growth of glioma cells

[目的]探讨VEGFR2促进脑胶质瘤干细胞增殖和生长的机制。[方法]通过19例新鲜分离的人脑胶质细胞瘤(Glioblastoma multiforme, GBM)标本进行组织分离、细胞培养,然后对培养的细胞进行慢病毒转染后进行BALB/c(nu/nu)小鼠成瘤实验,并获得神经胶质细胞瘤样本(T556, T1966, T4121, T3691)。分别对分离的人GBM细胞和异种移植神经胶质细胞瘤样本细胞进行流式细胞术CD133干细胞分选及VEGFR2、免疫组化、免疫荧光检测等分析、检测。[结果]CD133^+细胞表面VEGFR2的表达与CD133^-对照组相比富集(CD133^+阳性率19.6%而CD133^-对照的阳性率为4.7%;P<0.001 7);表面VEGFR2阳性的肿瘤细胞比例不同,从1.4%到25.9%(平均13.8±8.3%)不等。对石蜡包埋标本进行免疫组化分析发现GBM细胞表面及细胞浆均有分布VEGFR2,而胞浆VEGFR2则较为显著。对人GBM活检冰冻切片进行的免疫荧光染色证实VEGFR2细胞群聚集靠近血管结构;进一步对标本进行免疫荧光定位、免疫共沉淀和细胞表面蛋白生物素化实验发现VEGFE、VEGFR2和NRP1及磷酸化p-VEGFR2和增值标记蛋白Neuropilin-1在恶性脑胶质瘤病灶和周围正常组织及脑小血管周围的差异表达。[结论]VEGFR2优先表达于CD133人胶质瘤干细胞(GSC)的细胞表面及恶性脑胶质瘤病灶小血管周围,其增殖、生长能力和致瘤性,可能部分依赖于通过VEGFR2-Neuropilin-1 (NRP1)的信号传递,可能在GBM的增殖和生长过程中发挥一定作用。

[Objective]To explore the mechanism of VEGFR2 promoting the proliferation and growth of glioma cells.[Methods]Tissue separation and cell culture were performed on 19 freshly isolated human GBM specimens, the cultured cells were then transfected with lentivirus and subjected to BALB/c(nu/nu) mouse in a tumorigenesis experiment. Glioma samples T556, T1966, T4121, T3691 were obtained. Flow cytometry(FACS), immunohistochemistry and immunohistamycytosis were used to analyze and detect the isolated human GBM and xenograft glioma cells. [Results]The expression of CD133^- cellsurface VEGFR2 was rich compared to CD133^- positive rate compared to CD133^- control group 19.6% and CD133^- control positive rate of 4.7%;P<0.0017);25.9%(average 13.8±8.3%). An immune histification analysis of paraffin encapsulation specimens found that the GBM cell surface and fine plasma were distributed with VEGFR2, while thecytoplasm VEGFR2 was more significant. Immunofluorescent staining of human GBM biopsy frozen slices confirmed that VEGFR2 cell population aggregation was close to vascular structures, and further immunofluorescent prominence. Co-immunoprecipitation(CO-IP) and cell surface protein biotinylation experiments found that VEGFE, VEGFR2 and NRP1 Phosphorylation p-VEGFR2 and the value-added marker protein Neuropilin-1 are expressed in malignant cerebral glioma lesions and around normal tissues and around the cerebral vascular vessels. [Conclusion]The results show that VEGFR2 is preferred on the cell surface of CD133 human glioma stem cells(GSC) and around the small blood vessels of malignant glioma lesions, whose vitality, self-renewal and tumorigenicity may depend in part on signal transmission through the VEGF-VEGFR2-Neuropilin-1(NRP1) axis and may play a role in the proliferation and growth of GBM.