摘要
目的研究嗅鞘细胞培养液(OECCM)对海仁藻酸(KA)损伤后原代培养脊髓神经元的作用,进一步探讨其保护的作用机理。方法采用原代细胞培养法,分别培养了胚胎小鼠脊髓神经元和成年大鼠的嗅神经鞘细胞(OECs),收集OECCM。通过给予KA建立体外的损伤模型,KA分别作用6,8,12h后,加入OECCM继续培养24￣36h。然后进行抗神经元型一氧化氮合酶(ncNOS)免疫细胞化学染色,观察细胞ncNOS表达情况,并对各组ncNOS阳性神经元数目进行统计,同时运用四唑盐(MTT)比色法检测神经细胞活性;最后对不同组的一氧化氮合酶(NOS)阳性细胞染色情况、阳性细胞数目和细胞活性进行比较。结果KA作用后,一氧化氮(NO)都出现异常表达,但与实验组(OECCM干预组)相比,OECCM处理组NO表达水平明显低于单纯KA损伤组。OECCM处理组NOS阳性细胞数目(6h组,34.3±5.25;8h组,40.6±3.68),明显低于对照组(6h组,42.3±3.48;8h组,54.5±6.30)(P<0.01),在KA损伤12h时,两组NOS阳性细胞数(52.3±5.23vs61.32±6.45)(P<0.05);而细胞活性(6h,0.372±0.034;8h,0.312±0.026)明显高于对照组(6h,0.283±0.041;8h,0.219±0.033)(P<0.01),12h时两组细胞活性没有明显差异(0.199±0.012vs0.202±0.032)(P>0.05)。结论OECCM中可能含有一些保护脊髓神经元免遭KA损伤的因子,这些因子可能在参与抑制NOS异常表达起了重要作用。
Objective To investigate the effects of olfactory ensheathing cells culture medium （OECCM） on the primarily cultured neuronal cells of spinal cord and explore its protective mechanisms after kainic acid excitotoxic injury. Methods Primarily cultured neurons and olfactory ensheathing cells were prepared from embryonic mouse spinal cord and adult rat olfactory bulb, respectively. At same time OECCM was harvested for next step experiment. Injury model was established by addition of KA into the neuronal cultures in vitro. After the neuronal cultures were treated with KA for 6, 8 and 12 h in vitro, respectively, OECCM was added into cultures and cells were cultured for 24-36 h. The neuronal constructive nitric oxide synthase （ncNOS） protein levels were assayed with immunocytochemical methods. The number of NOS-positive cells was counted. In addition, the viability of neurons was assessed by MTT assay. Eventually, the number of positive cells and their viability were compared between different groups. Results Abnormal expression ofncNOS was induced in cultured neurons in K.A-induced injury groups, as compared with that in control group. The levels ofncNOS protein in OECCM group were significantly lower as compared with that in injured group. The number of NOS-positive cells （6 h group, 34.3±5.25; 8 h group 40.6±3.68） was smaller than that of control groups （6 h group, 42.3±3.48; 8 h group, 54.5±6.30）（P〈0.01）. After KA injury for 12 h, the number of NOS positive cells was 52.3±5.23 , 61.32±6.45, respectively （P〈 0.05）, and the viability of spinal cord neurons were significantly higher in OECCM treatment groups （6 h, 0.372±0.034; 8 h, 0.312±0.026）than that of control groups （6 h, 0.283±0.041; 8 h, 0.219±0.033） （P〈0.01）.As for 12 h, the viability of two groups were 0.199±0.012, 0.202±0.033 with no marked difference between them （P〉0.05）. Conclusion OECCM may contain some factors which can protect spinal cord neurons from KA-induced injury. One of mechanisms is th
出处
《中华神经医学杂志》
CAS
CSCD
2005年第12期1189-1193,共5页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(30371466)
关键词
嗅鞘细胞
海仁藻酸
细胞培养
脊髓
一氧化氮
Olfactory ensheathing cells
Kainic acid
Cell culture
Spinal cord
Nitric oxide
