人脑胶质瘤新生血管的细胞来源与血液流通功能的初步评价

Preliminary studies on cell derivation of in human glioma and its functional evaluation

目的探讨胶质瘤中部分新生血管是否源于胶质瘤干细胞转分化，并初步评价其血液运输功能。方法将SHG44人脑胶质瘤细胞系接种于30只NC裸小鼠皮下和20只NC裸小鼠脑内尾状核，将SU-2细胞接种于12只表达绿色荧光蛋白的NC／C57BL／6J-绿色荧光蛋白裸小鼠的尾状核内。取移植瘤组织，石蜡切片后，分别进行CD34-PAS双染及CD31、CD34、CD133、胶质纤维酸性蛋白（GFAP）、人类白细胞抗原（Ki67）、HLA免疫组化染色，并结合HLA免疫荧光共聚焦显微成像对移植瘤血管进行分类，对肿瘤血管起源细胞进行鉴定。每组取3只荷瘤鼠，以活性炭颗粒混悬液行心脏灌注，对移植瘤切片进行相应的免疫组化染色，并在光镜下观察炭颗粒在肿瘤血管中的分布。结果裸小鼠移植瘤中，同时存在CD34一PAS双阳性的内皮细胞依赖型血管、CD34-PAS^＋的血管生成拟态和由肿瘤细胞与CD34^＋细胞嵌合而成的马赛克血管，炭颗粒随机分布在这些血管腔内。在人脑胶质瘤干细胞移植瘤中，CD31^＋细胞依附于血管壁腔侧面；CD34^＋细胞亦沿血管分布，但在形态上介于肿瘤细胞与内皮细胞之间；在血管壁上可见HLA^＋和人Ki67^＋细胞，提示构成血管壁的这些细胞来自人源细胞。HLA免疫荧光共聚焦显微成像可见，移植瘤中的血管以表达HLA（红色）的人源肿瘤细胞为主，兼有表达GFP（绿色）的宿主细胞，或人鼠融合细胞。结论在移植性人脑胶质瘤组织中存在具有血液运输功能的内皮细胞依赖型血管、血管生成拟态和马赛克血管3类血管，人脑胶质瘤干祖细胞有通过分化和转分化机制自主形成后两类肿瘤血管的潜能。

Objective The finding of vasculogenic mimicry （VM） in many solid tumors indicates that tumor cells themselves could participate in the construction of tumor vessels. However the origin of these cells is still not fully elucidated, and whether these vessels have the ability of blood-supply is still unclear. Preliminary studies were performed to investigate whether part of tumor neovascularity is derived from tumor stem cells （TSCs） and whether TSCs-derived vessels are functional. Methods Transplanted glioma tissues obtained from subcutaneous and orthotopic transplantation nude mouse models were processed into paraffin sections. In order to identify the cell origin and types of tumor vessels, sections were stained with CD31, CD34, CD133, GFAP, Ki67 and HLA, respectively. CD34-PAS staining was performed as well. A part of tumor-bearing mice were perfused with activated carbon through the systemic circulation and the distribution of activated carbon was observed. Results CD34-PAS staining showed that endothelium-dependent vessels （ CD34 ^＋ , PAS ^＋ ）, VM vessels （ CD34-, PAS ^＋ ）, and the MVs （ CD34 ^＋ , PAS- ） could be seen in the transplantated tumors. Activated carbon particles were observed in all three types of vessels. CD31 ^＋ cells adherent to the luminal surface of vessel wall. CD34 ^＋ cells distributed along the vessels as well, but morphologically were more like a transition type between tumor cells and endothelial cells. Human specific Ki67 and HLA positive cells could be seen in the tumor vessels indicating that these vessels were derived from human tumor cells. Moreover, cells of tumor vessels were proved to be constructed by human tumor cells mainly and fusion cells of host cells and tumor cells under confocal microscope. Conclusions Three types of blood supply sources including endothelium-dependent vessels, vasculogenic mimicry （VMs） and mosaic vessels （MVs） exist in transplantation tumors of human glioma. Glioma stem and progenitor cells （GSCPs） have the potential to differentiate and transdifferentiate into VMs and MVs.