眼科研究 2000年第2期第18卷 实验研究
作者:吴静 徐锦堂
单位:510632 广州,暨南大学医学院眼科
关键词:层粘连蛋白;凋亡;角膜上皮细胞;培养
摘要 目的 观察层粘连蛋白(LN)对离体角膜上皮细胞存亡的影响,以寻找预防角膜上皮细胞凋亡的途径。方法 应用流式细胞仪(FACStarplus)检测凋亡细胞的亚2倍体DNA的方法,对基础培养的、与LN共育的以及阻断LN受体后的兔角膜上皮细胞的凋亡情况进行了对比观察及分析。结果 基础培养的第6代角膜上皮细胞出现明显的细胞凋亡;与LN共育的角膜上皮细胞凋亡不明显;阻断LN受体后,角膜上皮细胞出现显著的凋亡现象。结论 LN对离体培养的角膜上皮细胞的凋亡有保护作用。
分类号 R 772
Laminin prevents apoptosis of corneal epithelial cells in vitro
Wu Jing,Xu Jintang.
Department of Ophthalmology,Medical College of Jinan University,Guangzhou 510632
Abstract ObjectiveTo prevent apoptosis of corneal epithelial cells cultured in vitro,the effects of laminin upon cell survival was studied.MethodsCorneal epithelial cells from New Zealand white rabbits were cultured respectively in basic culture medium (DMEM-Ham’s F-12 medium 1∶1),in basic culture medium in which laminin was added and in basic culture medium in which antibody against laminin was added.The cells were passaged every 48 h.The cells form passages 1 to 6 were fixed with 70% ethanol after cultured for 48 h.After staining with propidium iodide(PI),apoptosis was measured by the presence of degraded DNA(< 2 n DNA) in a flow cytometric assay.ResultsFlow-cytometric analysis of propidium iodide-stained cells indicated that 7.00%±2.23% of the primary cells contained < 2 n DNA while cultured in basic culture medium.Sixth passage cells grown in basic culture medium showed significantly increased amounts of degraded DNA (18.74%±3.93%,P<0.01).A significantly higher percentage of cells survived in the 6th passage when cells were cultured in the presence of laminin.In contrast,an increased amount of apoptosis(30.80%±7.90%)was found when cells were cultured in the presence of with anti-laminin antibody.ConclusionLaminin can prevent apoptosis of corneal epithelial cells cultured in vitro.
Key word slaminin apoptosis corneal epithelial cell culture
利用培养的角膜上皮行角膜移植以改善眼表,是治疗角膜表面疾患的新尝试。体外能获得长期存活并增殖的角膜上皮细胞是此方法实施的前提,可是在我们对角膜缘上皮细胞进行离体培养时,历经几次细胞传代培养,上皮细胞的增殖力逐渐减弱,最后停止增殖而死亡。为探索细胞死亡的原因,我们应用基础培养、与层粘连蛋白(laminin,LN)共育以及阻断LN受体法,对离体角膜缘上皮细胞的存活情况进行了观察及对比分析,以寻找预防细胞凋亡的途径。
1 材料与方法
1.1 角膜上皮细胞培养法
1.1.1 基础培养法 取新鲜兔眼球,无菌处理后,取浅层带有角膜上皮的角膜缘组织,以质量浓度0.25%胰蛋白酶-0.01 mol/L EDTA孵育消化法获取角膜上皮细胞(1×106/ml),在含有10%小牛血清的F12-DMEM(Gibco,USA)培养液中,5% CO2,37℃,饱和湿度培养箱中培养。48 h 传代1次。
1.1.2 与LN共育法 将96孔培养板底涂一层LN液(25 μg/cm2),-70℃冻干后,将角膜上皮细胞(1×106/ml)种于上。5% CO2,37℃,饱和湿度培养箱中培养48 h。
1.1.3 阻断LN受体法 将角膜上皮细胞种植在含有10 μg/ml抗-LN单克隆抗(Sigma,USA)培养液中,5% CO2,37℃,培养48 h。
1.2 细胞凋亡的流式细胞仪检测法
用质量浓度0.25%胰蛋白酶、0.01 mol/L EDTA消化培养48 h后的各组角膜上皮细胞,收集细胞,调细胞浓度在1×106个/ml;取1 ml细胞悬液,离心800~1 000 r/ min,10 min,弃上清;重悬细胞于100 μl的PBS中;慢慢滴注细胞悬液 到(事先预冷-20℃)5 ml的70%的乙醇中固定;放置4℃中待用。用前离心去除固定液;加PNAase 200 μl 37 ℃水浴30 min;再加入800 μl碘化丙啶(prooidium iodide,PI)染色液,混匀,4 ℃,避光30 min;在FACStarplus流式细胞仪上分析细胞凋亡(亚2倍体DNA)的情况。实验获取的数据经SPSS 4.0统计学软件处理分析。
2 结果与分析
2.1 离体角膜上皮细胞在基础条件培养下的细胞凋亡检测结果 角膜原代上皮细胞被检测出7.00%±2.23%的凋亡细胞。第1代到第5代各组细胞与原代相比,细胞凋亡率并无明显变化(P>0.01);从第6代开始,角膜上皮细胞凋亡率为18.74%±3.93%,与原代相比,细胞凋亡率有显著升高(P<0.01)。
2.2 与LN共育的第6代角膜上皮细胞凋亡率明显低于基础条件培养下的同代细胞(P<0.01),见表1。
表1 LN对离体培养的角膜上皮细胞
凋亡率的影响( ±s,n=5)
Tab.1 The effect of laminin on the apoptosis rate of corneal
epithelial cells cultured in vitro( ±s,n=5)
|
Apoptosis rate(%) |
t |
P |
Passage 6 (control) |
18.82±3.91 |
Passage 6 in LN |
7.4±1.49 |
6.106 9 |
0.002 92 |
t test P<0.012.3 阻断LN受体后,角膜上皮细胞贴壁障碍,原代细胞即被检测出30.80%±7.90%的凋亡DNA。见表2。
表2 阻断LN受体对离体培养的角膜上皮细胞
凋亡率的影响( ±s,n=5)
Tab.1 The effect of anti-laminin receptors on the apoptosis rate
of corneal epithelial cells cultured in vitro( ±s,n=5)
|
Apoptosis rate(%) |
t |
P |
Passage 0 (control) |
6.3±1.7 |
|
|
Passage 0 in ant i-LN |
30.8±7.9 |
6.779 6 |
0.001 9 |
t test P<0.01
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