颅脑手术患者废弃脑组织中神经干细胞的培养与鉴定

目的:研究颅脑手术患者废弃脑组织中神经干细胞的培养与鉴定,为立体定向自体神经干细胞移植治疗脑出血、脑梗塞及颅脑损伤后遗症提供临床前基础。方法:通过改进原代培养方法,从颅脑手术患者废弃脑组织中培养和鉴定神经干细胞(NSCs)。收集颅脑手术患者不同脑区废弃脑组织各约500 mg以上,按改良方法进行原代培养并传代,通过免疫荧光检测NSCs标志物巢蛋白(Nestin)的表达,传代培养5代时开始进行诱导分化。结果:培养约3～10 d后,多数患者废弃脑组织中均可见干细胞球生长,经Nestin免疫荧光检测均呈阳性表达。选取第6代诱导分化后的贴壁细胞分别经β-Tubulin(神经元标志物)、Sox10(少突胶质细胞标志物)与GFAP(星形胶质细胞标志物)免疫荧光检测,可见少量神经元、少突胶质细胞及星形胶质细胞阳性表达。取第3代与第6代细胞行免疫印迹鉴定,第3代细胞仅见Nestin与少量β-Tubulin表达,第6代细胞可见不同程度的Nestin、β-Tubulin、Sox10及GFAP等表达。结论:本研究成功从颅脑手术患者不同脑区废弃脑组织中分离培养获得成人NSCs,并可向神经元及神经胶质细胞分化,使立体定向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。

Culture and identification of autologous neural stem cells from abandoned brain tissue in patients during craniocerebral operation

Objective :To provide the preclinical platform for stereotactic autologous neural stem cells(NSCs)transplantation to treat the sequela of encephalorrhagia,cerebral infarction and craniocerebral injury. Methods :The abandoned brain tissues from different areas were collected during open cranial surgery. Above 500 mg abandoned brain tissues in each case were primarily cultured by the modified methods and handed from generation to generation.The expression of Nestin(the marker of NSCs) was detected by immunofluorescence cytochemistry. The induced differentiation began from the 5^th generation. Results :About 3 to 10 days after inoculation, stem cells sphere growth was observed in most cases. All the stem cell spheres were positive for Nestin. The 6 th generation cells were collected for immunofluorescence cytochemistry with β-Tubulin(the marker of neuron), Sox10(the marker of oligodendrocyte) and GFAP(the marker of astrocyte) respectively, in which a few of the adherent cells were found to be positive as neurons, oligodendrocytes and astrocytes. The Western blotting was performed to 3rd and 6th generation cells. In 3th generation, only Nestin and a few of β-Tubulin were positive. However, in the 6th generation, the Nestin, Sox10,β-Tubulin and GFAP were all found positive on different levels. Conclusion :This study successfully obtains adult NSCs from the abandoned brain tissues in different regions during open cranial surgery and the autologous NSCs could be differentiated into neurons and gliocytes. It is possible for stereotactic autologous NSCs transplantation to repair the neural function from laboratory to clinical applications.