新生大鼠缺氧缺血性脑损伤后脑组织上清液诱导骨髓基质细胞神经分化

目的:观察缺氧缺血性脑损伤(HIBD)新生大鼠不同时间点脑组织上清液对骨髓基质细胞(BMSCs)向神经细胞分化的影响.方法:7日龄新生SD大鼠结扎左颈总动脉制作HIBD 模型(n=15),另设 15只正常同日龄新生大鼠.两组分别在 HIBD后24h(8日龄)、72h(10日龄)及7d(14日龄)时处死(n=5).分别制备左、右脑组织上清液.将培养3～5代的SD大鼠BMSCs接种于预先置有盖玻片24孔板中,正常培养至细胞达到60%～70%融合后,分别加入上述时间点的脑组织上清液,另正常换液未加上清液组为未干预组.培养3d后行神经元特异性烯醇酶(NSE)、胶质纤维酸性蛋白(GFAP)、少突胶质细胞前体细胞标记O4 的免疫荧光检测,计算阳性率.结果:未干预各组细胞仅有少量NSE,GFAP及O4的表达,正常大鼠各时间点左、右脑上清液共培养各组细胞NSE,GFAP及O4阳性率较未干预组增加(均P＜0.01),但左、右两侧比较差异无统计学意义(P＞0.05).HIBD后24h组、72h组左脑(损伤侧)上清液共培养组细胞NSE,O4的阳性率明显增加,分别与同时间点右侧及正常组同一时间点的左、右侧比较,差异有统计学意义(分别P＜0.05,P＜0.01),HIBD后7d左、右脑上清液共培养组细胞NSE,GFAP及O4的阳性率相近,分别与正常组同一时间点比较差异无统计学意义(P＞0.05).HIBD后24h组左脑上清液共培养组细胞NSE,O4的阳性率较HIBD后72h组及7d组左脑上清液共培养组细胞NSE,O4的阳性率高,差异有统计学意义(均P＜0.01).结论:正常鼠、HIBD鼠脑上清液均能诱导BMSCs发生神经分化,以HIBD后24h损伤侧脑上清液对BMSCs的神经分化率最高.

Differentiation of rat bone marrow stromal cells into neural cells inducedby hypoxic-ischemic brain tissue extracts in neonate rats

Objective To investigate the effect of brain tissue extracts in neonate rats with hypoxic-ischemic brain damage (HIBD) on the differentiation of bone marrow stromal cells (BMSCs) into neural cells. Methods Fifteen 7-day-old neonate rats were induced HIBD by left carotid artery ligation and hypoxia exposure, and another 15-day-old neonate rats were served as normal rats. The left and right brain tissue extracts of the normal and HIBD rats were prepared 24 h after the HIBD (8-day old), 72 h after the HIBD (10-day old), and 7 d after the HIBD (14-day old), respectively (n=5). The rat BMSCs of passage 3~5 were cultured in the medium with or without previous brain tissue extracts. The expressions of neuron specific encolase (NSE), glial fibrillary acidic protein (GFAP) and O4 marked oligodendrocyte were detected after 3 days by immunocytochemistry. Results The expressions of NSE, GFAP and O4 of BMSCs cultured in the medium with left or right brain tissue extracts of different day old normal rats were higher than those of BMSCs cultured without the extracts, respectively (P<0.01), and the expressions of NSE, GFAP and O4 of BMSCs cultured in the medium with left brain tissue extracts of 8-day-old and 10-day-old HIBD rats were higher than those of BMSCs cultured with right brain tissue extracts of the same day HIBD rats and BMSCs cultured with left or right brain tissue extracts of the same day normal rats (P<0.01 or P<0.05). The expressions of NSE, GFAP and O4 of BMSCs cultured in the medium with left brain tissue extracts of 8-day-old HIBD rats were higher than those of BMSCs cultured with left brain tissue extracts of 10-day-old and 14-day-old HIBD rats (P<0.01 or P<0.05). Conclusion The brain tissue extracts of normal and HIBD rats can induce BMSCS into neural cells, and the damaged brain tissue extracts of 8-day-old HIBD rats is the best inductor.