低氧／复氧后星形胶质细胞对永生化神经前体细胞增殖与分化的影响

Effects of astrocytes on proliferation and differentiation of immortalized neural progenitor cells after hypoxia/reoxygenation

目的探讨低氧／复氧后星形胶质细胞对永生化神经前体细胞增殖与分化的影响。方法24h内新生SD大鼠，断头处死，分离、培养和传代星形胶质细胞，取本室构建的永生化神经前体细胞，根据培养条件的不同分为3组，每组24孔，对照组永生化神经前体细胞在正常培养条件下培养（O217％．CO25％-N278％）；共培养组取第三代星形胶质细胞孵育24h后接种永生化神经前体细胞，培养条件同对照组；低氧／复氧后共培养组取第三代星形胶质细胞，先置入低氧培养箱（O22％．CO25％．N293％）中孵育12h后，恢复正常培养条件12h，再接种永生化神经前体细胞。3组以相同密度接种永生化神经前体细胞（1×10^4／cm^2），隔天半量置换培养基，培养时间为6d。共培养6d后每组取6孔，采用免疫荧光细胞化学法，观察共培养后永生化神经前体细胞的增殖与分化情况，计算其增殖倍数、神经元阳性率和星形胶质细胞阳性率。结果与对照组和共培养组相比，低氧／复氧后共培养组永生化神经前体细胞增殖倍数升高（P〈0．05），神经元阳性率和星形胶质细胞阳性率差异无统计学意义（P〉0．05）。结论低氧／复氧后星形胶质细胞可促进永生化神经前体细胞增殖，但对其分化无影响。

Objective To investigate the effects of astrocytes on proliferation and differentiation of immortalized neural progenitor cells after hypoxia/reoxygenation （H/R）. Methods Primary astrocytes were isolated from hippocampus of neonatal SD rats. Immortalized neural progenitor cells constructed by our lab were divided into 3 groups： group Ⅰ control; group Ⅱ co-culture and group Ⅲ H/R ＋ co-culture. In control group the immortalized neural progenitor cells were cultured in normal culture atmosphere （17% O2-5 % CO2-78 % N2 ）. In group Ⅱ after being cultured for 24 h the third generation astrocytes were co-cultured with immortalized neural progenitor cells in the same culture condition as in control group. In group Ⅲ after being exposed in hypoxic air （2%O2-5% CO2- 93% N2 ） for 12 h the astrocytes returned to normal culture atmosphere （17% O2-5% CO2-78% N2 ） and were then co-cultured with immortalized neural progenitor cells for 6 days. Immortalized neural progenitor cells were labeled with nestin, MAP-2, GFAP, SV40 and Hoechest 33342 by immuno-cytochemistry. The growth curves were then drawn by counting the positive cells to detect the proliferation of immortalized neural progenitor cells and the ratio of neurons derived from progenitor cells was calculated. Results The number of progeny generated from immortalized neural progenitor cells was significantly increased in group Ⅲ （H/R ＋ co-culture） as compared with group Ⅰ and Ⅱ （ P 〈 0.05）, but the ratio of neurons generated from progenitors was not significantly changed （ P 〉 0.05）, Conclusion The proliferation of immortalized neural progenitor cells is promoted by co-culture with astrocytes having been exposed to hypoxia but the differentiation of neural progenitor cells to neurons is not significantly influenced in vitro.