大鼠成年海马神经干细胞体外氧糖剥夺复氧模型的建立.pdf

《大鼠成年海马神经干细胞体外氧糖剥夺复氧模型的建立.pdf》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、大鼠成年海马神经干细胞体外氧糖剥夺/复氧模型的建立谭盛陈健郭阳陈瑞清李粲陈镇洲[摘要]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24h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

2、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、

3、有效的神经干细胞体外氧糖剥夺/复氧模型。氧糖剥夺/复氧;神经干细胞；细胞凋亡;实验模型R-33A1671-8925(2011)12-1238-05Establishmentofratmodelsofoxygenglucosedeprivation/reoxygenationinadultneuralstemcellsinvitroTANShengCHENJianGUOYangCHENRui-qingLICanCHENZhen-zhou*DepartmentofNeurology,ZhujiangHospitalofSouthernMedicalUniversit

4、y,Guangzhou510282,China[Abstract]ObjectiveToestablishsimple,stableandreliableratmodelsofoxygenglucosedeprivation/reoxgenation(OGD/R)inadultneuralstemcells(NSCs)invitro.MethodsTheNSCsfromadultFisher344ratswereculturedinserum-freemediumandidentifiedusingnestinandDAPIimmunofluorescentdo

5、ublestaining.ThesecellswerewashedwithaEarle′sbalancedsaltsolutionwithoutglucosefor2times,then,incubatedfordifferentperiods(2,4,6,8and10h)inatrigasincubatorwithanatmosphereof1％O2,5％CO2and94％N2,98％humidityat37°C.Andthen,thesecellswereremovedfromtheanaerobicincubator,washed,andaddedDEME

6、/F12containingbFGFsupplement.Anormoxic-normoglycemiccontrolgroupwasemployed.MorphologicalassessmentofNSCswasperformedbylightmicroscopyafterre-oxgenationfor24h;CCK-8colorimetricmethodwasusedtodeterminethesurvivalandproliferationofNSCs,andflowcytometrywasemployedtodetecttheapoptosisofN

7、SCs.ResultsAfterthesettingofoxygenglucosedeprivationfor2h,theODvalueandthesurvivalrateintheOGDcellswereincreasedascomparedwiththoseincontrolgroupwithoutsignificantdifference(P＞0.05).WhilethemorphologicaldamageofNSCsaggravatedgraduallyandtheODvaluedecreasedinOGDcellsfollowingtheprolon

8、gationoftime