摘要
目的:观察人胚神经干细胞(hNSCs)、人脐血干细胞(hUCBCs)治疗脑缺血大鼠的效果及其在缺血大鼠脑内的增殖、分化情况。方法:从自然流产的孕10～13周的人胚脑组织中分离、培养hNSCs;采集足月新生儿脐带血60～100ml,分离出其中的单个核细胞;治疗前hNSCs、hUCBCs均经5-溴脱氧嘧啶尿苷(BrdU)标记48h。采用线栓法制作大鼠脑缺血模型,1天后经尾静脉注射未分化的hNSCs、hUCBCs至脑缺血大鼠体内,对治疗后大鼠进行神经损害严重程度评分(NSS),用免疫组化方法观察hNSCs、hUCBCs的存活、迁移、分化状况。结果:从人胚脑中成功培养出hNSCs,培养条件下呈悬浮状态生长,形成神经球;hUCBCs在体外也具有增殖能力。两治疗组大鼠均自治疗后21天起其NSS显著低于对照组(P<0.05),两治疗组间比较各时间点NSS无显著性差异(P>0.05)。治疗后14、21、28、35天脑组织切片中均可见BrdU染色阳性细胞,缺血侧明显多于对侧(P<0.05),治疗后21、28、35天明显多于治疗后14天(P<0.05);hNSCs组BrdU染色阳性细胞数多于hUCBCs组(P<0.05);治疗组各时间点脑组织切片中均可见nestin染色阳性细胞;在BrdU阳性细胞群中,hNSCs组73.8%为胶质纤维酸性蛋白(GFAP)染色阳性细胞,16.7%为2,3-环核苷酸磷酸二脂酶(CNPase)染色阳性细胞,9.5%为神经元特异性烯醇化酶(NSE)染色阳性细胞;hUCBCs组74.5%为GFAP染色阳性细胞,15.4%为CNPase染色阳性细胞,10.1%为NSE染色阳性细胞;两组间差异无显著性(P>0.05)。结论:hNSCs、hUCBCs均具有多分化潜能,受缺血部位微环境信号的影响分化成3种主要类型的神经细胞;静脉注射hNSCs、hUCBCs能有效改善脑梗死大鼠的神经功能评分;除了hNSCs外,hUCBCs也是治疗缺血性脑血管病的一种可能手段。
Objective：To explore the effects of the human neural stem cells （hNSCs） and human umbilical cord blood cells （hUCBCs）on the treatment of cerebral ischemic rats and the proliferation and differentiation status of these cells in the ischemie brain tis sue of rats. Methods：HNSCs were separated from 10-13 weeks brains of spontaneous abortion human embryo and cuhured;hUCBCs were gotten from the placentas of full-term babies;both hNSCs and hUCBCs were marked with BrdU（5 μmol/L） for two days. The middle cerebral artery occlusion （MCAO） rat models were made and the stem cells were injected into the tail vein one day later. The Neurological Severity Scores （NSS） tests were undertaken in all rats of the three groups at 0-35 days after injection. Immunohistoehemical method was used to check the differentiation and migration of stem cells in vivo. Results：Neural stem cells from human embryonic brains had been successfully cultured, and found to form typical neurospheres in suspension. The hUCBCs had the capacity of proliferation in vitro. Twenty-one days later,the neurologic function of rats with injection recovered much better than those without injection, as evidenced by the NSS （P 〈 0.05）. There was no statistical difference of NSS between the two injected groups at every time point before and after injection （P 〉 0.05）. Within the brain tissue,BrdU positive cells were distributed throughout the damaged brain of recipient rats,the majority of these cells localized in the ischemic hemisphere, few of these cells were observed in the contralateral hemisphere （P 〈 0.05）. Significantly more BrdU positive cells were found in the brain tissue at the 21,28 and 35 days points respectively than that at the 14th days after injection （P 〈 0.05）. More Brdg positive cells were found in the rats received hNSCs the rats injected with hUCBCs. Nestin positive cells were found within the damaged brains at every time point. Among all the BrdU positive cells of hNSCs group,73.8% of these cells were positive for the astrocyte marker glial fibrillary acidic protein（GFAP）, 16.7% were positive for the CNPase and 9.5% expressed the neuronal markers neuron specific enolase （NSE）. Among all the BrdU positive cells of hUCBCs group,74.5% of these cells were positive for GFAP, 15.4% were positive for CNPase and 10.1% expressed NSE. There was no statistical difference between the two groups （P 〉 0.05）. Conclusions：HNSCs and HUCBCs had muhipotential differentiation properties in vivo and differentiated into three main types of neural cells by the influcence of the ischemic microenvironmental signals. Intravenous injection of hNSCs and hUCBCs could effectively improve the neurologic function of ischemic rats. Besides of hNSCs, hUCBCs injection may be an efficient method to treat ischemic cerebrovascular disease.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第11期1508-1513,共6页
Journal of Nanjing Medical University(Natural Sciences)
关键词
人胚神经干细胞
人脐血干细胞
脑缺血
human neural stem cells
human umbilical cord blood cells
cerebral ischemia
