中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (21): 3381-3386.doi: 10.3969/j.issn.2095-4344.0904

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

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

王 佳1，赵全军1，黑 博2，王兆涛3，孙移坤2，王 涛2，崔绍杰2，王培新2   

  1. 1安徽医科大学解放军第三〇六临床学院，北京市 100101；2解放军第三〇六医院立体定向及脑功能性疾病诊治中心，北京市 100101；3南方医科大学陆军总医院附属八一脑科医院，北京市 100101
  • 修回日期:2018-05-11 出版日期:2018-07-28 发布日期:2018-07-28
  • 通讯作者: 赵全军，博士，主任医师，安徽医科大学解放军第三〇六临床学院，北京市 100101
  • 作者简介:王佳，女，1990年生，安徽省人，汉族，安徽医科大学在读硕士。
  • 基金资助:

    军队后勤科研资助项目(CHJ12J022)；首都临床特色应用研究项目基金(Z141107002514053)

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

Wang Jia1, Zhao Quan-jun1, Hei Bo2, Wang Zhao-tao3, Sun Yi-kun2, Wang Tao2, Cui Shao-jie2, Wang Pei-xin2   

  1. 1The 306th Clinical College of PLA, Anhui Medical University, Beijing 100101, China; 2Stereotactic and Neurofunctional Center of the 306th Hospital of PLA, Beijing 100101, China; 3Affiliated Bayi Brain Hospital of General PLA Hospital, Southern Medical University, Beijing 100101, China
  • Revised:2018-05-11 Online:2018-07-28 Published:2018-07-28
  • Contact: Zhao Quan-jun, M.D., Chief physician, the 306th Clinical College of PLA, Anhui Medical University, Beijing 100101, China
  • About author:Wang Jia, Master candidate, the 306th Clinical College of PLA, Anhui Medical University, Beijing 100101, China
  • Supported by:

    the Military Logistics Research Funding, No. CHJ12J022; Clinical Characteristic Application Research of the Capital, No. Z141107002514053

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摘要：

文章快速阅读：

文题释义：
无血清培养方法：采用不含血清的培养基培养动物细胞的方法。无血清培养基一般是由基础培养基和替代血清的补充因子组成的。
神经干细胞：是一类具有分裂潜能和自我更新能力的母细胞，它可以通过不对等的分裂方式产生神经组织的各类细胞，具有自我更新和多向分化的潜能。

 

摘要
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-tubulin Ⅲ(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。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-6853-9585(赵全军)

关键词: 颅脑手术, 动脉瘤, 脑出血, 成人, 自体, 神经干细胞, 细胞培养, 干细胞库, 分化

Abstract:

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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Tissue Enigneering, Neural Stem Cells, Cells, Cultured, Stem Cells

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