CAJ | 학술논문

目的研究缺氧对星形胶质细胞水通道蛋白4(AQP4)表达水平的影响.方法体外培养星形胶质细胞,用氯化钴建立星形胶质细胞缺氧模型,采用随机数字表法分为对照组和缺氧组,每组再分为15、30 min,1、2、4、6和12 h时间点亚组,共14组,每组6孔.观察星形胶质细胞形态;采用原位杂交、荧光定量、免疫细胞化学、Western blot方法检测AQP4基因及蛋白表达;用Ex-△△Ct表示AQP4基因表达量、D表示AQP4蛋白表达量.结果星形胶质细胞的AQP4基因和蛋白变化具有一致性(r=0.85,P＜0.01).对照组星形胶质细胞呈弱阳性表达.缺氧组星形胶质细胞内AQP4表达从15 min开始随时间的延长而增强,以1～4 h内最明显(mRNA表达缺氧组分别为0.26±0.04、0.31±0.02、0.36±0.04,对照组分别为0.06±0.01、0.09±0.01、0.08±0.01;t=16.51、18.20、15.26,均P＜0.01),6 h后缓慢增强,与对照组对应时间点相比差异均有统计学意义.随缺氧时间延长,细胞肿胀逐渐加重,以1～4 h时间点最显著,电镜下见细胞核增大、内质网和线粒体肿胀,并随时间推移而加重.12 h见少数细胞破裂.结论缺氧所致星形胶质细胞的病理变化是细胞水肿,水肿程度与缺氧时间和AQP4高表达呈正相关,AQP4可能是星形胶质细胞水肿的重要分子基础.
목적 연구결양대성형효질세포수통도단백4(AQP4)표체수평적영향.방법 체외배양성형효질세포,용록화고건립성형효질세포결양모형,채용수궤수자표법분위대조조화결양조,매조재분위15、30 min,1、2、4、6화12 h시간점아조,공14조,매조6공.관찰성형효질세포형태;채용원위잡교、형광정량、면역세포화학、Western blot방법검측AQP4기인급단백표체;용Ex-△△Ct표시AQP4기인표체량、D표시AQP4단백표체량.결과 성형효질세포적AQP4기인화단백변화구유일치성(r=0.85,P＜0.01).대조조성형효질세포정약양성표체.결양조성형효질세포내AQP4표체종15 min개시수시간적연장이증강,이1～4 h내최명현(mRNA표체결양조분별위0.26±0.04、0.31±0.02、0.36±0.04,대조조분별위0.06±0.01、0.09±0.01、0.08±0.01;t=16.51、18.20、15.26,균P＜0.01),6 h후완만증강,여대조조대응시간점상비차이균유통계학의의.수결양시간연장,세포종창축점가중,이1～4 h시간점최현저,전경하견세포핵증대、내질망화선립체종창,병수시간추이이가중.12 h견소수세포파렬.결론 결양소치성형효질세포적병리변화시세포수종,수종정도여결양시간화AQP4고표체정정상관,AQP4가능시성형효질세포수종적중요분자기출.
Objective To investigate the expression of aquaporin-4 (AQP4) in cultured astrocytes after in vitro hypoxia induced by CoCl2. Methods After primary culture and subculture, the astrocytes were placed in a controlled atmosphere culture chamber. Both control group and hypoxia groups were established.These groups were further divided into seven sub-groups according to the different time intervals: 15, 30minutes and 1,2, 4, 6, 12 hours, respectively (6 apertures for each group). The shape of the astrocytes in each group was observed with light microscopy and transmission electron microscopy ( TEM ). All groups were examined using in situ hybridization, real time fluorescence quantitative reverse transcriptase polymerase chain reaction, immunocytochemistry and Western blot. The data was analyzed statistically with SPSS 13.0 software. Results There was significant consistency between the AQP4 mRNA and protein ( r =0. 85, P ＜0. 01 ). There was slight positive expression of AQP4 in a few astrocytes of the control groups. In the hypoxia groups, the expression of AQP4 increased within 15 minutes; the increase was most prominent between 1 and 4 hours( mRNA in hypoxia groups: 0. 26 ± 0. 04, 0. 31 ± 0. 02, 0. 36 ± 0. 04; control groups:0. 06 ±0. 01,0. 09 ±0. 01,0. 08 ±0. 01 )after hypoxia and became less between 6 and 12 hours; There was significant difference in the AQP4 expression between the hypoxia groups and control groups among different time points (t = 16. 51, 18.20, 15.26,all P＜0. 01 ). The corresponding pathological changes were cellular edema, which was most prominent between 1 and 4 hours. Under TEM, increase in size of the nucleolus and swelling of endoplasmic reticulum and mitochondria; these changes became more marked with time.Disruption of a few astrocytes was detected in the hypoxia groups at 12 hours. Conclusions The pathological change of astrocytes is cellular edema following hypoxia. There is a positive relationship between the presence and degree of cellular edema as well as the duration of hypoxia and the up-regulating of AQP4.These results imply that AQP4 expression is an important molecular mechanism of celluar edema of astrocytes.