来源于胚胎干细胞的神经前体细胞移植修复脊髓损伤

背景:在一定条件下,胚胎干细胞可诱导分化成为神经前体细胞,当移植到健全或损伤的中枢神经系统后,可以与宿主细胞有效整合,修复重建损伤的神经组织。目的:观察胚胎干细胞诱导的神经前体细胞移植对小鼠脊髓损伤神经功能恢复的影响。设计:随机对照动物实验。单位:上海第二医科大学发育生物学研究中心。材料:选用28只6～8周龄C57/BL6J小鼠,雌雄不限。小鼠胚胎干细胞株S8及携带LacZ标记基因由上海市发育生物学研究中心提供。高糖DMEM、2-巯基乙醇、小鼠白血病抑制因子、丝裂霉素C均来自GIBCO贴壁诱导培养液由上海市发育生物学研究中心提供。方法:实验于2003-04/2004-04在上海第二医科大学发育生物学研究中心实验室完成。采用贴壁诱导法将ES细胞培养诱导分化为神经前体细胞,将小鼠麻醉,在T9～10椎骨平面制成脊髓半横断模型,随机摸球法将大鼠分为3组,假手术组(n=9):仅剪除T9-10棘突和椎板后,逐层缝合。干细胞移植组(n=10):脊髓半横断后,行距损伤区域以远约1cm的椎管内注射细胞。模型组(n=9):脊髓半横断后在损伤邻近区注射DMEM。各组分别于实验后第1,2,4,6,8周采用BBB评分观察各组小鼠神经功能恢复情况,实验后第8周(BBB评分后)利用x-gal染色观察各组脊髓损伤区胚胎干细胞存活和分化情况。X-gal染色干细胞移植组阳性的损伤脊髓部位切片进行荧光免疫组化检测。主要观察指标:①各组小鼠神经功能恢复情况。②各组脊髓损伤区胚胎干细胞存活和分化情况。③荧光免疫组化检测结果。结果:①干细胞移植组第1,2,4周BBB得分,高于模型组(P<0.01)。②脊髓损伤区胚胎干细胞存活和分化情况:干细胞移植组中小鼠的脊髓损伤处组织切片可以发现x-gal染色阳性的细胞,胞浆呈现蓝色,即胚胎干细胞来源的衍生细胞,而假手术组和模型组的脊髓均未见该现象。干细胞移植组损伤脊髓部位植入的胚胎干细胞来源的衍生细胞分布损伤区周围并与周围组织整合,与周边细胞在形态上类似。③干细胞移植组损伤脊髓部位,X-gal染色阳性细胞可表达神经元特异性标志NF,但没有表达GFAP标志。结论:将ES细胞的培养诱导分化为神经前体细胞移植后,能够存活、迁移、并分化为神经元,但改善神经功能情况不明显。

Transplantation of neural precursors derived from embryonic stem cells for repairing spinal cord injury

BACKGROUND: Embryonic stem cells can be induced to differentiate into neural precursors under certain conditions, and they can effectively integrate with host cells after transplanted into healthy or injured central nervous system, and then repair and rebuild the injured nerve tissue.OBJECTIVE: To observe the effects of transplantation of neural precursors induced by embryonic stem cells on the recovery of neurological function in mice with spinal cord injury.DESIGN: A randomized controlled animal trial.SETTING: Research Center of Developmental Biology, Shanghai Second Medical University.MATERIALS: Twenty-eight C57/BL6J mice of 6-8 weeks old, both sexes, were used. Mice embryonic stem cell strain S8 and carrier LacZ labeling genes were provided by Shanghai Research Center of Developmental Biology. High-glucose Dulbecco's modified Eagle media (DMEM), β-mercaptoethanol (BME), mice leukemia inhibitory factor (LIF) and mitocin-C were all from GIBCO attachment induction medium, which were provided by Shanghai Research Center of Developmental BiologyMETHODS: The experiment was carried out in the central laboratory of developmental biology of Shanghai Second Medical University from April 2003 to April 2004. The embryonic stem cells were cultured and induced to differentiate into neural precursors by means of attachment induction. The mice were anesthetized and made into models of spinal cord hemisection on the T9-T10 plan. The mice were randomly divided into three groups: sham operated group (n =9): Only T9-T10 spinous process and corresponding lamina of vertebra were removed, then the skin was sutured layer by layer;ransplantation group (n =10): After spinal cord hemisection, embryonic stem cells were injected into the vertebral canal about 1 cm away from the injured site; model group (n =9): DMEM was injected into the region around the injured site.The mice were evaluated with Basso, Beattie, Bresnahan (BBB) locomotor rating scale to observe the recovery of neurological function at 1, 2, 4, 6 and 8 weeks (the score ranged from 1 to 21 points, the higher the scores, the better the recovery of neurological function). At 8 weeks, the survival and differentiation of embryonic stem cells at the injured site of spinal cord were observed using X-Gal staining in each group. The positively stained sections with X-Gal at the injured site of spinal cord were detected with fluorescent immunohistochemical staining.MAIN OUTCOME MEASURES: ① Recovery of neurological function; ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord; ③ Results of fluorescent immunohistochemical staining.RESULTS: ① The BBB scores in the transplantation group at 1, 2 and 4 weeks were higher than those in the model group (P ＜ 0.01). ② Survival and differentiation of embryonic stem cells at the injured site of spinal cord: In the transplantation group, there were X-Gal positively stained cells in the tissue sections of the injured spinal cord of mice,the cytoplasm was blue with nucleoli in it, i.e. the cells derived from embryonic stem cells, which were not observed in the sham-operated group and model group. In the transplantation group, the cells derived from embryonic stem cells, which were implanted to the injured spinal cord, distributed around the injured sites, and integrated with the surrounding tissue and had similar form with the surrounding cells. ③ At the injured site, X-Gal positively stained cells in the transplantation group aiso expressed neurofilaments (the specific marker of neurons), but did not express GFAP.CONCLUSION:The embryonic stem cells were cultured and induced to differentiate into neural progenitors, and they could survive, migrate and differentiate into neurons after transplantation, but there was no obvious improvement of neurological function.