体外诱导SH-SY5Y细胞凋亡的氧糖剥离再灌注模型的建立及评价

J4 ›› 2012, Vol. 38 ›› Issue (4): 658-664.

体外诱导SH-SY5Y细胞凋亡的氧糖剥离再灌注模型的建立及评价

刘守跃¹|张影²|胡林森³|罗毅男⁴|关文明¹

（1．吉林大学第二医院神经外科|吉林长春130041；2．吉林大学第一医院儿科|吉林长春 130021；3. 吉林大学第一医院神经内科|吉林长春130021；4. 吉林大学第一医院神经外科|吉林长春130021）

收稿日期:2012-03-28 出版日期:2012-07-28 发布日期:2012-07-28
通讯作者: 关文明 E-mail:（Tel: 0431-88796286,E-mail:lsy2007.2008@yahoo.com.cn）
作者简介:刘守跃（1971-）| 男|吉林省长春市人|主治医师|医学博士|主要从事脑血管病的研究。
基金资助:
吉林省科技厅科研基金资助课题（200905151）

Establishment and evaluation of OGD/R model in vitro inducing apoptosis of SH-SY5Y cells

(1.Department of Neurosurgery,Second Hospital,Jilin University,Changchun 130041,China|2. Department of Pediatrics,First Hospital,Jilin University,Changchun 130021,China|3.Department of Neurology,First Hospital,Jilin University,Changchun 130021,China|4.Department of Neurosurgery,First Hospital|Jilin University,Changchun 130021,China)

Received:2012-03-28 Online:2012-07-28 Published:2012-07-28

摘要/Abstract

摘要：

目的：通过体外模拟缺血-再灌注损伤（I/R）诱导细胞凋亡的氧糖剥离再灌注(OGD/R)模型的建立与评价，探讨成功诱导神经元凋亡发生的可靠时间窗，为进一步阐明神经元凋亡机制奠定基础。方法：选用人神经母细胞瘤系SH-SY5Y细胞分为对照组和实验组，实验组给予不同的OGD/R时间，通过MTT法计算细胞存活率，选定再灌注24 h存活率接近50％的OGD10h/R组和对照组细胞，通过光镜、荧光显微镜及电镜观察细胞形态学改变，采用试剂盒检测培养液中乳酸脱氢酶（LDH）水平、胞浆内丙二醛（MDA）含量及细胞中超氧化物歧化酶（SOD）活力，流式细胞术检测细胞周期和凋亡率。结果：MTT法结果显示，细胞存活率随OGD时间延长显著降低，OGD10h/R24h组为对照组的（44.91±1.21）%，不同OGD/R时间组间细胞存活率比较差异有统计学意义(P<0.05或P<0.01)。倒置显微镜及HE染色显示，OGD10h/R组细胞密度减低，形态多样性，核固缩；AO/EB荧光染色可见凋亡小体形成，电镜显示核内异染色质凝集、趋边等凋亡的改变。实验组培养液内LDH水平及胞浆内MDA含量随再灌注时间延长而增加，LDH水平于再灌注24 h达峰值，MDA含量于再灌注24 h接近平台期，胞浆内SOD活性随再灌注时间延长呈现先降低后回升的趋势，与相同时间对照组比较差异均有统计学意义(P<0.01)。流式细胞术检测细胞周期显示，随再灌注时间延长，OGD10h/R组G0/G1期细胞比例逐渐增加，24 h达峰值（74.09±2.62）%，其后有所降低，G2/M期细胞比例再灌注0 h时最高达（26.85±1.35）%，此后逐渐降低，24 h前均显著高于同时间对照组细胞，S期细胞比例逐渐降低，24 h最低达（19.2±1.58）%，此后逐渐升高，与同时间对照组比较差异均有统计学意义(P<0.05或P<0.01)。结论：体外模拟I/R模型的OGD10h/R组能够成功诱导SH-SY5
Y细胞凋亡的发生，可进一步应用于凋亡机制的研究。

关键词: 氧糖剥离/再灌注；SH-SY5Y细胞；细胞凋亡

Abstract:

Abstract:Objective
To explore time window of inducing neuronal apoptosis successfully via building and evaluating oxygen glucose deprivation/reperfusion (OGD/R) model in vitro to mimic ischemia and reperfusion inducing apoptosis of SH-SY5Y cells,and to build a basis for revealing the potential mechanisms of neuronal apoptosis. Methods SH-SY5Y cells were employed and divided into control group and experimental group treated with OGD/R for different time. The cell viability was calculated by the method of MTT method.The OGD10h/R groups in which the cell viability was decreased approximately 50% 24 h after reperfusion and control groups were selected for further assay. With light microscope,fluorescence microscope and transmission electron microscope the variation of structure and morphous in cells at different time were observed. The changes in lactate dehydrogenase (LDH) levels, malondialdehyde (MDA) contents,superoxide dismutase (SOD) activities were detected at different time of reperfusion. Flow cytometry was used to analyze the cell cycle distribution and the apoptotic rates at different time of reperfusion. Results The MTT results showed that the cell viabilities were decreased significantly with the prolongation of OGD time. At reperfusion 24 h,the cell viability was (44.91±1.21)% in OGD10h/R groups. The cell viabilities in other different OGD groups had significant differences (P<0.05 or P<0.01). In OGD10h/R groups the inverted microscope and HE staining results showed that the cell densities were decreased following varied shape and deep blue pycnotic nuclei.The AO/EB fluorescence staining results showed the apoptotic body emerged.Under electron microscope,the cells showed heterochromatin fringe agglutinated in nucleus which were viewed as the appearance of apoptosis and autophagia. The level of LDH in culture fluid and the contents of intracytoplasm MDA at different reperfusion time were increased with the prolonged time and at reperfusion 24 h respectively reached the peak and platform phase,compared with control groups the differences were significant (P<0.01). The intracytoplasm SOD activities at different reperfusion time were declined at first and recovered later with the prolonged reperfusion time,compared with control groups the differences were significant (P<0.01). By flow cytometry analysis on cell cycle phase at different reperfusion time,in OGD 10h/R groups with the prolonged reperfusion time,the percentage of the cells in G0/G1 phase was increased at first and was decreased later with its peak of (74.09±2.62)% at reperfusion 24 h. The percentage of the cells in G2/M phase reached its peak of (26.85±1.35)% at R 0 h and was declined gradually later. The percentage of the cells in S phase was declined gradually and reached the lowest level of （19.2±1.58）% at reperfusion 24 h. Compared with control groups at the same time,the differences were significant (P<0.05 or P<0.01). Conclusion OGD10h and reperfusion (OGD10h/R) to mimic cerebral ischemia and reperfusion in vitro is a reliable model that induces SH-SY5Y cells apoptosis,which provide a basis for the futher study on neuronal apoptotic mechanisms.

Key words: oxygen glucose deprivation/reperfusion, SH-SY5Y cells, apoptosis

中图分类号:

刘守跃|张影|胡林森|罗毅男|关文明. 体外诱导SH-SY5Y细胞凋亡的氧糖剥离再灌注模型的建立及评价[J]. J4, 2012, 38(4): 658-664.

LIU Shou-yue|ZHANG Ying|HU Lin-seng| LUO Yi-nan| GUAN Wen-ming. Establishment and evaluation of OGD/R model in vitro inducing apoptosis of SH-SY5Y cells [J]. J4, 2012, 38(4): 658-664.