颅内动脉瘤手术废弃脑组织中成人自体神经干细胞的培养与鉴定

Culture and identification of adult autologous neural stem cells from abandoned brain tissues during surgery in patients with intracranial aneurysm

背景:对胎儿、胎鼠及成鼠神经干细胞培养已有大量研究,但成体人自体神经干细胞培养相关研究较少。目的:通过改进原代培养方法,从颅内动脉瘤破裂脑出血患者血肿腔周边废弃脑组织中培养和鉴定神经干细胞,并进行冻存与复苏研究,建立成人自体神经干细胞库。方法:收集3例动脉瘤破裂脑出血(2例大脑中动脉瘤及1例前交通动脉瘤)手术中血肿腔周边的废弃脑组织各约500 mg以上,采用细小组织块接种法进行原代无血清悬浮培养成人自体神经干细胞,倒置显微镜下观察细胞生长情况,免疫荧光细胞化学技术检测神经干细胞标志物巢蛋白(Nestin)的表达。培养获得的神经干细胞球以体积分数4%胎牛血清诱导其贴壁生长,待细胞增殖达到一定数量后传代培养。传代后部分细胞予以冻存建库,并复苏继续传代及诱导分化培养。体积分数10%胎牛血清诱导神经干细胞分化,通过免疫荧光细胞化学技术检测胶质纤维酸性蛋白(标记星形胶质样细胞),β-tubulinⅢ(标记神经元样细胞),SOX10(标记少突胶质样细胞)蛋白的表达来测定神经干细胞的分化能力。结果与结论:改进培养方法后,3例患者废弃脑组织中,在无血清培养液中均可形成Nestin阳性表达的神经球,并可在体外大量扩增和连续传代。在给予体积分数10%FBS条件下神经球可分化为神经元样细胞、少突胶质细胞及星形胶质细胞,即β-tubulin Ⅲ,SOX10及胶质纤维酸性蛋白阳性表达。经传代培养至5代后,仍呈Nestin阳性即可维持其神经干细胞特征。冻存细胞复苏后生长状态良好且呈Nestin阳性,可继续传代扩增。结果表明,从动脉瘤破裂脑出血患者不同脑区(颞极及额底)血肿腔周边废弃脑组织中可成功培养获得自体神经干细胞,并可向神经元样细胞、少突胶质样细胞及星形胶质样细胞分化,使自体神经干细胞移植促进神经功能修复从实验室到临床应用成为可能。

BACKGROUND： A large number of studies on neural stem cell culture have been carried out in aborted fetus, fetal and adult mice, but there are few studies on adult human neural stem cell culture. OBJECTIVE： With the improvement of primary cell culture, to culture and identify adult autologous neural stem cells from the abandoned brain tissues around hematoma cavity during surgery in patients with aneurysm rupture, and then to study the cell cryopreservation and recovery so as to establish the adult autologous neural stem cell bank.METHODS： Over 500 mg abandoned brain tissues were collected during surgery from the surrounding tissues of hematoma cavity in three cases of aneurysm rupture. Adult autologous neural stem cells were cultured in serum-free medium with small tissue piece culture method. Cell growth was investigated by inverted phase contrast microscope. The expression of nestin, the specific antigen of neural stem cells, was confirmed by immunofluorescence method. Culture medium containing 4% fetal bovine serum was used to induce adherent growth of neural stem cells. Subculture was carried out when the cells proliferated to a certain number. Neural stem cells partially cryopreserved were used to establish the adult autologous neural stem cell bank. Recovered cells continued to subculture and induce differentiation. The cell differentiation was induced in the culture medium containing 10% fetal bovine serum. Expression of glial fibrillary acid protein （GFAP; marker for astrocytes）, β-tublin （marker for neurons） and SOX10 （marker for oligodendrocytes） was detected by immunofluorescence method.RESULTS AND CONCLUSION： Nestin-positive neurospheres were successfully obtained from the abandoned brain tissues in the three cases through the improved serum-free medium culture. The cells could be amplified in vitro and handed from generation to generation. When cultured in the medium containing 10% fetal bovine serum, neurospheres were differentiated into β-tublin-positive neurons, SOX10-positive oligodendrocytes and GFAP-positive astrocytes. After subculture, passage 5 cells were still positive for nestin and maintained their characteristics of stemness. After recovery, the cryopreserved cells could grow well, still be positive for Nestin and be passed and amplified continuously. It is proved that neural stem cells can be obtained from abandoned brain tissues in different regions （lower frontal lobe and temporal lobe） around the hematoma cavity in patients with aneurysm rupture. These cells can differentiate into neurons, oligodendrocytes and astrocytes. Autologous neural stem cells transplantation is likely to promote the repair of neural function from laboratory to clinical application.