人羊膜细胞与创伤性脑组织提取液共培养的实验研究  

An experimental study on human amniotic cells co-culture by traumatic brain tissue extracts

作　　者：卢奕[1] 惠国桢[2] 吴智远[2] 李向东[2] 江国华[1] 暨荀鹤[3] 郭礼和[3] 

机构地区：[1]浙江省嘉兴市第一医院神经外科,314000 [2]苏州大学附属第一医院神经外科 [3]中国科学院上海生命科学研究院生物化学与细胞生物学研究所

出　　处：《江苏医药》2008年第9期915-917,F0002,共4页Jiangsu Medical Journal

基　　金：国家自然科学基金资助(30271325);江苏省自然科学基金资助(BK2001170);江苏省卫生厅科研项目资助(H200716)

摘　　要：目的探究将人羊膜细胞(HACs)置于创伤性脑组织提取液的条件培养基中,模拟HACs在创伤性脑损伤微环境下的分化和增殖情况,为HACs脑内移植提供基础研究。方法取无并发症剖宫产羊膜,用0.25%胰酶反复消化3次提取HACs,接种后48h去除未贴壁的HACs。采用改进的Feeney法制作大鼠创伤性脑损伤(TBI)模型,TBI24h后制备创伤性脑组织提取液。将HACs分别接种于RPMI1640培养基(1640)、RPMI1640培养基与正常脑组织提取液(1640+NB)、RPMI1640培养基与创伤性脑组织提取液(1640+TBI)3组。共培养7d后HE染色显微镜下检测各组细胞形态。培养前后分别行Nestin和MAP-2单抗免疫组化检测。将HACs以104/ml密度分别接种上述3种培养基,培养后1、4和7d经酶联免疫检测仪行四氮唑盐(MTT)比色法检测各组HACs活性。结果1640组HACs呈圆形或椭圆形,核大居中;1640+TBI组部分HACs形态出现锥形、三角形等神经元样改变。HACs培养前可见Nestin阳性细胞,培养后1640+TBI组可见MAP-2阳性表达。各组HACs增殖性均不佳。结论HACs与创伤性脑组织提取液共培养后可转化为神经元样细胞。HACs增殖性不佳。Objective To explore the differentiation and proliferation of human amniotic cells （HACs） in the conditioned culture medium with traumatic brain tissue extracts, which mimics the microenvironment of injured brain. Methods A human amniotic sheet was obtained from an uncomplicated elective Cesarean section, and then the separated amnion was treated with 0.25% trypsin for three times to collect HACs within an hour. A rat model of traumatic brain injury （TBI） was established by improved Feeney＇s technique, and the traumatic brain tissue extracts were made 24 hours after TBI. HACs were cultured in RPMI1640 medium （1640）, RPMI1640 and normal brain tissue extracts（ 1640 ＋ NB）, RPMI1640 and traumatic brain tissue extracts （1640＋ TBI）. The cell morphology of each group was observed under phase-contrast microscope within 7 days after co-culture. The immunohistochemical analysis for Nestin and MAP-2 was performed before and after co-culture. HACs were cultured in the three kints of medium by a cell density of 104/ml, then the cellular viability of each group was investigated on the 1st, 4th, and 7th day after co-culture. Results HACs in group 1640 showed round and oval in shape, however in group 1640＋TBI some cells were transformed pyramidal cells like neuron. Cells were immunostained by anti-nestin before culture, and displayed MAP2-positive in group 1640 ＋TBI. The cell proliferation of each group was not significant. Conclusion HACs can transform the neuron-like cell by co-culture with traumatic brain tissue extracts. The proliferation of HACs is not significant.

关 键 词：人羊膜细胞 创伤性脑损伤 神经分化 细胞增殖 

分 类 号：R651[医药卫生—外科学]