目的 探索内源性神经干细胞在大鼠海马可溶性因子中的体外发育归宿及分化鉴定。方法 显微镜下分离Wistar大鼠海马组织放置于低温DMEM/12培养基,低温振荡2小时后高速离心(15000 g),获取实验所用海马组织可溶性因子。取材出生1天的Wistar乳鼠海马中的内源性神经干细胞(endogenous neural stem cells, ENSCs),将ENSCs分别于含海马可溶性因子终浓度为0(对照组)、50、100、200、400 μl/mL的无血清DMEM/F12培养基中培养6天并每日观察,使用免疫细胞化学、Western Blot印记技术比较各组ENSCs中Nestin、CD133的表达量;同时计量并比较各组ENSCs成球个数,以探索在模拟颅内微环境情况下,ENSCs发育、归宿及分化。进一步于最适宜的海马可溶性因子终浓度中分化神经球,对分化的细胞行神经元特异性蛋白入(如:β-tubullin III、MAP2)及胶质细胞特异性蛋白(如:GFAP、S100及p75 NGFR)免疫细胞化学检测。结果 大鼠ENSCs在培养基中呈单细胞漂浮生长,球形; ENSCs于海马可溶性因子各实验分组中培养第2天呈细胞球状态,对照组中无细胞球形成(与100 μl/mL组比较,P₁=0.00),100 μl/mL组与对照组比较有统计学意义(P₁=0.00<0.05);至第6天,在100 μl/mL组中的细胞球数量明显多于其余各组(P₁'=P₂'=P₃'=P₄'=0.00)。在免疫细胞化学检测中,100 μl/mL组中细胞球表达干细胞高亲和蛋白Nestin、CD133阳性,Western Blot免疫印迹检测其中Nestin、CD133蛋白高于对照组。进一步分化试验中,细胞球呈贴壁生长的单细胞状态、有突起伸出、长梭形,免疫细胞化学检测分化的细胞表达胶质细胞特异性蛋白GFAP、S100、p75NGFR阳性,但不表达神经元特异性蛋白β-tubullin III与MAP2。结论 大鼠ENSCs在终浓度为100 μl/mL的HSF作用下,可促进 ENSCs的增殖分裂;ENSCs在同样浓度下的HSF中可进一步分化为表达GFAP、S100、p75NGFR阳性的胶质样细胞;100 μl/mL的HSFS是ENSCs的一种生理性诱导剂或参与促进ENSCs增殖、分化及通过细胞替代或因子分泌等机制修复神经损伤。

Objective The aim of this study was to explore induction and differentiation of endogenous neural stem cells(ENSCs) in the hippocampus soluble factors(HSF) from the hippocampus of adult Wistar rats by mimicking an intracranial microenvironment. Methods After Wistar rats sacrificed, the hippocampus tissue was obtained in cold DMEM/F12. After centrigued and filtered, the HSF was stored at -20℃. The ENSCs was obtained from the hippocampus tissue of a neonate Wistar rat. Collected the tissue, digested and obtained the ENSCs. After we observed the morphology, the ENSCs were cultured in different concentration (0、50、100、200、400 μl/mL) of HSF for 6 days, and compared the expression of Nestin and CD133 by immunocytochemistry. Meanwhile,we compared the Nestin and CD133 protein by western blot. And then we explored the optimal concentration of HSF by the numbers of all groups on the second and sixth day. Furthermore, we did the differentiated experiment using the same concentration of HSF. Results The number of neurospheres in the 100 μg/mL group was significantly higher than those in the other groups on the 6th day. Immunofluorescence revealed that the neurospheres from ENSCs in the 100 μg/mL group more highly expressed nestin and CD133 than control. This result was confirmed by western blot analysis. The neurospheres can differentiate into glia-like cells in 100 μg/mL HSF and 1% FBS expressing GFAP, S100 and P75 NGFR by immunofluorescence. Conclusion These data indicated that HSF alone, mimicking a destination of ENSCs in vitro, could induce and differentiate neurospheres from ENSCs, as a new method to get NSCs and glia-like cells differentiated from ENCs to repair the diseases of center nervous system.

目的 探讨颈动脉残端压(SP)联合电生理监测在颈动脉内膜切除术(CEA)中的应用价值。方法 回顾性分析19例CEA患者临床资料,通过监测SP、体感诱发电位(SEP)和运动诱发电位(MEP),以确定术中是否放置转流管;比较术前和术后6月美国国立卫生院卒中量表(NIHSS)评分和改良Rankin量表(mRS)评分变化情况。结果 10例患者SP≥50mmHg,SEP和MEP监测无异常,术中未放置转流管;5例患者SP<50 mmHg,SEP波幅下降>50％,MEP监测正常,予以放置转流管;3例患者SP≥50 mmHg,SEP波幅下降>50％,MEP监测正常,予以放置转流管;1例患者SP>50 mmHg,SEP监测正常,MEP波幅下降>50％,未放置转流管。所有患者手术均获得成功,无手术死亡率。患者术后6月NIHSS评分和术前无统计学差异(P>0.05),但术后6月mRS评分较术前下降(P<0.05)。结论 通过术中SP、SEP和MEP联合监测,有助于避免CEA术后缺血性脑卒中的发生,提高CEA手术的安全性。

Objective To investigate the value of stamp pressure(SP), somatosensory and motor evoked potentials(SEP,MEP) monitoring in carotid endarterectomy. Methods 19 patients with carotid endarterectomy were retrospectively analyzed.SP, SEP and MEP were monitored during the operation.National Institutes of Health Stroke Scale (NIHSS) and Modified Rankin Scale (mRS) were used to evaluate the neurological function before and 6 months after surgery. Results Intraluminal shunting was not performed in 10 patients with normal SP, SEP and MEP.However, intraluminal shunting technique was used in 5 patients (SP<50 mmHg and SEP fluctuation decreased by 50％) and 3 patients (SP≥50 mmHg and SEP fluctuation decreased by 50％).1 patient showed the fluctuation of MEP decreased by 50％, while SP and SEP was normal, no shunting was performed.All patients were successfully operated, and no mortality occurred.The mRS score, not the NIHSS score, revealed statistically difference between preoperation and 6 months after surgery (P<0.05). Conclusion The combination of SP, SEP and MEP monitoring maybe useful for preventing ischemic stroke after carotid endarterectomy, increasing the safety of surgery.