CAJ | 학술논문

目的探讨一种简便、稳定、可重复的大鼠成年神经干细胞体外氧糖剥夺/复氧模型的制备方法 .方法以来自于成年Fisher344大鼠的海马神经干细胞系为研究对象,以无血清培养基培养并传代,并用nestin和DAPI免疫荧光双染确认其生物学特性.将三气培养箱氧气浓度调至1％以制备缺氧环境,将培养基换为不含葡萄糖的Earle′s平衡盐溶液,分别缺氧缺糖2h、4h、6h、8h、10h后取出细胞,恢复正常条件继续培养24 h后倒置显微镜下观察细胞形态学变化,CCK-8比色法检测细胞存活率,流式细胞术检测细胞凋亡率.同时设置常氧常糖的正常对照组.结果与正常对照组相比,缺氧缺糖2h组细胞吸光度值明显升高,细胞存活率有一定的增长,但差异无统计学意义(P＞0.05).随着缺氧缺糖时间延长,神经干细胞形态学损伤逐渐加重,细胞吸光度值逐渐下降,缺氧缺糖6h后与正常对照组比较差异有统计学意义(P＜0.05)；缺氧缺糖6h起细胞存活率均较正常对照组明显下降,比较差异有统计学意义(P＜0.05)；神经干细胞凋亡率逐渐增高,且均明显高于正常对照组,差异有统计学意义(P＜0.05),其中缺氧缺糖6h时细胞的凋亡率已超过50％.结论利用三气培养箱物理缺氧方法可成功建立一种简便、有效的神经干细胞体外氧糖剥夺/复氧模型.
목적 탐토일충간편、은정、가중복적대서성년신경간세포체외양당박탈/복양모형적제비방법 .방법 이래자우성년Fisher344대서적해마신경간세포계위연구대상,이무혈청배양기배양병전대,병용nestin화DAPI면역형광쌍염학인기생물학특성.장삼기배양상양기농도조지1％이제비결양배경,장배양기환위불함포도당적Earle′s평형염용액,분별결양결당2h、4h、6h、8h、10h후취출세포,회복정상조건계속배양24 h후도치현미경하관찰세포형태학변화,CCK-8비색법검측세포존활솔,류식세포술검측세포조망솔.동시설치상양상당적정상대조조.결과 여정상대조조상비,결양결당2h조세포흡광도치명현승고,세포존활솔유일정적증장,단차이무통계학의의(P＞0.05).수착결양결당시간연장,신경간세포형태학손상축점가중,세포흡광도치축점하강,결양결당6h후여정상대조조비교차이유통계학의의(P＜0.05)；결양결당6h기세포존활솔균교정상대조조명현하강,비교차이유통계학의의(P＜0.05)；신경간세포조망솔축점증고,차균명현고우정상대조조,차이유통계학의의(P＜0.05),기중결양결당6h시세포적조망솔이초과50％.결론 이용삼기배양상물리결양방법 가성공건립일충간편、유효적신경간세포체외양당박탈/복양모형.
Objective To establish simple,stable and reliable rat models of oxygen glucose deprivation/reoxgenation(OGD/R)in adult neural stem cells(NSCs)in vitro.Methods The NSCs from adult Fisher344 rats were cultured in serum-free medium and identified using nestin and DAPI immunofluorescent double staining.These cells were washed with a Earle′s balanced salt solution without glucose for 2 times,then,incubated for different periods(2,4,6,8 and 10 h)in a trigas incubator with an atmosphere of 1％ O2,5％CO2 and 94％ N2,98％ humidity at 37 ° C.And then,these cells were removed from the anaerobic incubator,washed,and added DEME/F12 containing bFGF supplement.A normoxic-normoglycemic control group was employed.Morphological assessment of NSCs was performed by light microscopy after re-oxgenation for 24 h; CCK-8 colorimetric method was used to determine the survival and proliferation of NSCs,and flow cytometry was employed to detect the apoptosis of NSCs.Results After the setting of oxygen glucose deprivation for 2 h,the OD value and the survival rate in the OGD cells were increased as compared with those in control group without significant difference(P＞0.05).While the morphological damage of NSCs aggravated gradually and the OD value decreased in OGD cells following the prolongation of times; under the setting of oxygen glucose deprivation for 6 h,the OD value in OGD cells obviously decreased as compared with that in the control group(P＜0.05); under the setting of oxygen glucose deprivation for 6 h,the survival rate obviously decreased and the apoptosis rate significantly increased in OGD cells as compared with that in the control group(P＜0.05); under the setting of oxygen glucose deprivation for 6 h,the apoptosis rate of NSCs excessed to 50％.Conclusion By means oftrigas incubator,simple,stable and reliable models of OGD/R in NSCs in vitro can be successfully established.