人胚神经干细胞的长期培养与基因修饰  

The Long-term Culture and Gene Modification of Human Neural Stem Cell Line

作　　者：林月秋[1] 阳运康[2] 汤逊[1] 徐永清[1] 

机构地区：[1]成都军区昆明总医院骨科,云南昆明650032 [2]泸州医学院附属医院骨科,四川泸州646000

出　　处：《昆明医学院学报》2008年第3期41-46,共6页Journal of Kunming Medical College

基　　金：云南省自然科学基金资助项目(2005C0070M)

摘　　要：目的通过逆转录病毒将增强绿色荧光蛋白(EGFP)基因转染人胚神经干细胞,观察其生物学特性与体内表达情况.方法分离孕10～20周人胚脑皮层来源的神经干细胞并连续传代培养,通过克隆试验、免疫组织化学等方法检测其生长特性及增殖能力;使用构建了EGFP基因的逆转录病毒转染神经干细胞并检测其生物学特性;制备兔T9全横断脊髓损伤模型,观察携带EGFP基因的人胚神经干细胞在体内的表达情况.结果从胎龄10～20周的人胚脑皮层中成功分离出人胚神经干细胞,该细胞具有连续克隆能力及多分化潜能,表达干细胞及分化细胞的特异性抗原.转染EGFP基因后,仍保持未分化状态,能够自我更新形成新的神经球;移植入兔脊髓损伤模型后,仍高效表达转染基因蛋白,并在体内发生增殖、迁移.结论人胚脑皮层中存在能长期传代培养的神经干细胞;转染EGFP基因后仍保留神经干细胞的基本特性;移植入体内能持续、高效表达EGFP,有利于观察其存活、迁移情况,并为功能基因修饰与移植研究提供了方法.Objective To isolate human neural stem cells （hNSCs） and establish hNSCs line through long-term culture in vitro. To observe the boilogical features and protein expressions in vitro/vivo after enhanced green fluorescent protein （EGFP） gene was transfected into the hNSCs by using retroviral. Methods HNSCs were isolated from human fetuses aged from 10 to 20 weeks following abortion. Following culture and clone, the morphological changes, the growth condition, the differentiation status, the immunohistochemistry features were observed and analyzed. Then, the retroviral-mediated EGFP gene was transfected to the hNSCs. After observed the biological features and protein expression in vitro, the hNSCs was transplanted to the adult rabbit models of spinal cord injury. Results Human neural stem cell line from fresh embryonic cortex at E10 to E20 weeks was established successfully, the cells could be cloned and passed continuously, expressing Nestin antigen. After induced to differentiate, it expressed phenotypic markers for neurons and astrocytes. It had been passagedcontinuously 17 passages （ 10 months） keeping strengthened capacity of proliferation our labs. These hNSCs can be genetically engineered to express foreign transgenes in vitro. It still remained undifferentiated and had self-renewing capacity and potential muhipotency of differentiation after transfected. After transplanted to the adult rabbits models of spinal cord injury, it were viable. It could passage, migrate, and efficiently express foreign transgenes. Conclusions The hNSCs isolated from the human embryonic tissues retain their biological features after long-term culture in vitro. It keeps the essential features of hNSCs after being transfected with retroviral-mediated EGFP gene. It offers one effective methord for further gene modification and transplantation therapy research.

关 键 词：神经干细胞 人胚脑皮层 培养 逆转录病毒 转染 

分 类 号：R511.5[医药卫生—内科学]