CAJ | 학술논문

目的探讨钾通道在体外培养的新生大鼠星形胶质细胞缺氧缺血性水肿中的作用机制.方法体外培养出生3 d新生大鼠的星形胶质细胞;采用RNA干扰技术制作水通道蛋白4(AQP4)敲低型(AQP4-/-)细胞模型;用放射性[3H]标记的甲基D-葡萄糖摄取,测定缺氧缺血性AQP4-/-和野生型(AQP4+/+)星形胶质细胞体积;利用全细胞膜片钳技术记录培养的星形胶质细胞电压依赖性钾通道(Kv)的电流特性,并记录缺氧缺血性星形胶质细胞Kv通道的电流变化.结果 AQP4+/+和AQP4-/-星形胶质细胞在缺氧缺血时均较其对照组细胞体积明显增加(AQP4+/+和AQP4+/+组细胞在缺氧缺血0.5、1、2、4 h组占所对应的对照组D-葡萄糖摄取值的百分数分别为104±7、109±6、126±12、152±9和97±7、105±9、109±7、132±6,均P<0.05),但相同缺氧缺血时间点AQP4-/-介导细胞水肿程度明显减轻(均P<0.05),而且细胞电流密度随着缺氧缺血时间延长,进行性下降(对照组和缺氧缺血0.5、1、2、4 h组细胞电流密度值分别为116±8,107±9,91±10,76±6,37±11,均P<0.05).结论在细胞缺氧缺血时,细胞外向性钾通道下调,可能阻止细胞内堆积的钾离子流出细胞外,引起渗透性改变而导致水通过AQP4流入细胞内,从而出现细胞水肿.
목적 탐토갑통도재체외배양적신생대서성형효질세포결양결혈성수종중적작용궤제.방법 체외배양출생3 d신생대서적성형효질세포;채용RNA간우기술제작수통도단백4(AQP4)고저형(AQP4-/-)세포모형;용방사성[3H]표기적갑기D-포도당섭취,측정결양결혈성AQP4-/-화야생형(AQP4+/+)성형효질세포체적;이용전세포막편겸기술기록배양적성형효질세포전압의뢰성갑통도(Kv)적전류특성,병기록결양결혈성성형효질세포Kv통도적전류변화.결과 AQP4+/+화AQP4-/-성형효질세포재결양결혈시균교기대조조세포체적명현증가(AQP4+/+화AQP4+/+조세포재결양결혈0.5、1、2、4 h조점소대응적대조조D-포도당섭취치적백분수분별위104±7、109±6、126±12、152±9화97±7、105±9、109±7、132±6,균P<0.05),단상동결양결혈시간점AQP4-/-개도세포수종정도명현감경(균P<0.05),이차세포전류밀도수착결양결혈시간연장,진행성하강(대조조화결양결혈0.5、1、2、4 h조세포전류밀도치분별위116±8,107±9,91±10,76±6,37±11,균P<0.05).결론 재세포결양결혈시,세포외향성갑통도하조,가능조지세포내퇴적적갑리자류출세포외,인기삼투성개변이도치수통과AQP4류입세포내,종이출현세포수종.
Objective To investigate the mechanism of potassium channel in brain edema caused by hypoxia-ischemia (HI). Methods Astrecytes were obtained from 3-day-old SD rats, cultured, and randomly divided into 2 groups: normoxia group, cultured under normoxic condition, and hypoxic-ischemic group, cultured under hypoxic-ischemic condition. The cell volume was measured by radiologic method. Patch-clamp technique was used to observe the electric physiological properties of the voltage-gated potassium channels (Kv) in a whole cell configuration, and the change of voltage-gated potassium channel current (IKv) was recorded in cultured neonatal rat astrocyte during HI. Aquaporin 4 (AQP4) expression vector was constructed from pSUPER vector and transfected into the astrocytes (AQP4 RNAi) to construct AQP4 knockdown (AQP4-/-) cells, cellular volume was determined using [3H]-3-O-methyld-D-glucose uptake in both AQP4-/- and AQP4+/+ cells under the condition of HI. Real time PCR and Western blotting were used to detect the mRNA and protein expression of AQP4. Results The percentages of the AQP4+/+ and AQP4-/- astrocyte volumes in the condition of HI for 0. 5, 1,2, and 4 h were 104±7, 109±6, 126±12, and 152±9 times, and 97±7, 105±9, 109±7, and 132±6 times as those of their corresponding control groups (all P < 0. 05), thus showing that the cellular volume of both AQP4+/+ and AQP4-/- astrocytes significantly increased during HI and the degrees of edema mediated by AQP4 knockdown at different time points were all significantly milder (all P < 0.05). The current density values at the time points 0. 5, 1,2, and 4 h of the HI group were 107±9, 91±10, 76±6, 37±11, respectively, compared to that of the control group of 116±8, showing a tendency of time-dependent decreasing manner (all P < 0. 05). Conclusion During HI, the dowuregnlation of outward potassium (K+) conductance may prevent the emission of interacellularly accumulated K+ ions, thus resulting in osmotically drived water influx into astrocytes via aquaporin-4 and then cell swelling.