连接蛋白CX43与水通道蛋白4联合诱导胶质瘤性脑水肿形成的偶联机制

The coupling mechanism of connexin 43 and aquaporin 4 together inducing brain edema formation in glioma

目的:探讨细胞间连接蛋白43(connexin 43,CX43)与水通道蛋白4(aquaporin 4,AQP4)在联合诱导成胶质细胞瘤性脑水肿形成过程中的功能偶联机制。方法:在大鼠颅内建立C6胶质瘤脑移植模型,磁共振成像检测成瘤及水肿情况,免疫荧光法和蛋白质印迹法检测肿瘤内部及瘤周CX43和AQP4的表达特点。体外采用低渗环境培养C6胶质瘤细胞和正常胶质细胞后,反转录PCR法比较2种细胞中CX43和AQP4的mRNA表达变化趋势;采用小干扰RNA(small interference RNA,siRNA)技术分别沉默C6胶质瘤细胞和正常胶质细胞中AQP4和CX43的表达,检测另一基因的表达变化。结果:C6胶质瘤脑移植模型中,胶质瘤内部CX43表达缺失和AQP4表达水平轻度增高,瘤周则表现为CX43表达水平轻度增高和AQP4明显高表达。体外实验发现,低渗环境下正常胶质细胞和C6胶质瘤细胞中AQP4和CX43的mRNA水平随时间的延长均有不同程度的升高,但正常胶质细胞的变化程度明显低于肿瘤细胞,且CX43的变化时间窗落后于AQP4。siRNA沉默C6细胞的AQP4表达后,其CX43表达水平的升高程度明显下降(P<0.05);沉默正常细胞的CX43表达后,其AQP4水平则无明显变化(P>0.05)。结论:AQP4和CX43在胶质瘤内部及其边缘存在2种不同的致脑水肿机制,CX43可能是AQP4的潜在下游调控因子。

Objective： To search for the coupling mechanism of connexin 43 （CX43） and aquaporin 4 （AQP4） together inducing brain edema formation in glioma. Methods： Intracranial C6 glioma xenograft models were established in the rats, then magnetic resonance imaging detection was used to find the tumor and brain edema, and immunofluorescence and Western blotting method were used to detect CX43 and AQP4 expressions in both intratumoral and peritumoral tissues. C6 glioma cells and normal glial cells were cultured in hypotonic medium, then RT-PCR （reverse transcription-PCR） was used to detect andcompare CX43 and AQP4 mRNA expression trends. AQP4 or CX43 expression was silenced in C6 glioma cells and normal glial cells by siRNA （small interference RNA） technology, then the other genetic change was detected by RT-PCR. Results： In C6 glioma xenograft models, CX43 expression was missed and AQP4 expression was slightly elevated in the intratumoral tissues. But in the peritumoral tissues, CX43 expression was slightly elevated and AQP4 expression was significantly increased. In vitro, the hypotonic environment induced elevation of AQP4 and CX43 mRNA levels with time-varying degrees in both normal glial cells and C6 glioma cells, but the change degree of normal glial cells was significantly lower than that of tumor cells, and the time window of CX43 changes was backward of AQP4. The elevated degree of CX43 was significantly decreased when AQP4 expression was silenced by siRNA in C6 cells （P 〈 0.05）, while the AQP4 expression was not affected by CX43 expression silencing in normal cells （P 〉 0.05）. Conclusion： There are two different mechanisms of AQP4 and CX43 inducing brain edema formation intratumorally and in the edqe of qlioma. CX43 may be one of the potential downstream regulatory factors of AQP4.