心肺复苏后大鼠皮质区凋亡相关性微小RNA的表达变化

摘要

Objective To observe the expression changes in apoptosis-related microRNA(miRNA) in cerebral cortex after cardiac arrest-cardiopulmonary resuscitation(CA-CPR)in rats and explore the factors that may affect the mechanism of CPR. Methods 24 clean male Sprague-Dawley(SD)rats were randomly divided into three groups,the normal control group,sham operation group and CA-CPR group(each n=8). The animal model of CA induced by asphyxia was established and CPR was performed. In the normal control group,no special management was performed. In the sham operation group,only abdominal cavity anesthesia,tracheotomy,vascular puncture and electrocardiogram(ECG)were performed without clamping the trachea and resuscitating. Normal feeding in normal control group and 24 hours after tracheotomy in sham operation group,at 24 hours after recovery of spontaneous circulation(ROSC)in CA-CPR group,cerebral cortex specimens were obtained for detection of the expression of miRNA by using real time fluorescence quantitative reverse transcription - polymerase chain reaction(RT-PCR). Flow cytometry(FCM)was used to detect the neurocyte apoptotic rate. Results Compared between normal control and sham operation groups,there were no significant differences in the expression of apoptosis-related miRNA and neurocyte apoptosis rate of cerebral cortex(both P>0.05). Compared with sham operation group,in CA-CPR group, 16 miRNA expressions were up-regulated,including Let-7c,miR-15a,miR-21,miR-24,miR-29,miR-29b, miR-34a, miR-103, miR-200a, miR-200b, miR-200c, miR-210, miR-326, miR-338-3p, miR-494 and miR-497,and there were 22 down-regulated,being Let-7a,Let-7b,Let-7d,Let-7e,miR-19a,miR-19b-1, miR-20a,miR-20b,miR-23a,miR-23b,miR-25,miR-98,miR-107,miR-122a,miR-125a,miR-125b, miR-145,miR-181a,miR-181c,miR-335,miR-384-5p and miR-422a. Eight miRNA had significant changes at 24 hours after ROSC,in which miR-15a,miR-21,miR-34a,miR-497 were up-regulated respectively for 6.831±2.625,8.122±3.442,5.349±2.010,6.590±3.689 times,and miR-125b,miR-145,Let-7a,Let-7e were down-regulated respectively for 0.122±0.039,0.199±0.096,0.191±0.069,0.160±0.082 times. The apoptosis rate of cerebral cortex was increased significantly in CA-CPR group〔(32.23±5.31)%〕compared with that in normal control group〔(3.66±1.34)%〕and sham operation group〔(4.98±1.84)%,both P<0.01〕. Conclusions In early period after CA-CPR,obvious neurocyte apoptosis may be found in brain tissue of rats,and in the mean time, changes in apoptosis-related miRNA expression in cerebral cortex occur. The various types of miRNA with significant changes possibly play important roles in cerebral protection after CA-CPR in rats. down-regulated respectively for 0.122±0.039,0.199±0.096,0.191±0.069,0.160±0.082 times. The apoptosis rate of cerebral cortex was increased significantly in CA-CPR group〔(32.23±5.31)%〕compared with that in normal control group〔(3.66±1.34)%〕and sham operation group〔(4.98±1.84)%,both P<0.01〕. Conclusions In early period after CA-CPR,obvious neurocyte apoptosis may be found in brain tissue of rats,and in the mean time, changes in apoptosis-related miRNA expression in cerebral cortex occur. The various types of miRNA with significant changes possibly play important roles in cerebral protection after CA-CPR in rats.%目的：观察心搏骤停-心肺复苏（CA-CPR）后大鼠大脑皮质区凋亡相关性微小RNA（miRNA）的表达变化，探讨影响CPR的机制。方法选择成年雄性SD大鼠24只，按随机数字表法分为正常对照组、假手术组和CA-CPR组，每组8只。采用窒息致CA后CPR法建立CA-CPR大鼠模型；正常对照组不进行任何操作；假手术组仅施行腹腔麻醉、气管切开、血管穿刺及心电监护等操作，不夹闭气管，不复苏。正常对照组于正常饲养时、假手术组于气管切开后24 h时、CA-CPR组于自主循环恢复（ROSC）后各取大脑皮质区提取总RNA，采用实时荧光定量逆转录-聚合酶链反应（RT-PCR）测定大脑皮质区凋亡相关性miRNA的表达；采用流式细胞仪检测神经细胞凋亡率。结果与正常对照组比较，假手术组大鼠miRNA表达及细胞凋亡率差异无统计学意义（均P＞0.05）。与假手术组比较，CA-CPR组大鼠大脑皮质凋亡相关性miRNA有16个上调（分别为Let-7c，miR-15a，miR-21，miR-24，miR-29，miR-29b，miR-34a，miR-103，miR-200a，miR-200b， miR-200c，miR-210，miR-326，miR-338-3p，miR-494，miR-497），22个下调（分别为Let-7a，Let-7b，Let-7d， Let-7e，miR-19a，miR-19b-1，miR-20a，miR-20b，miR-23a，miR-23b，miR-25，miR-98，miR-107， miR-122a，miR-125a，miR-125b，miR-145，miR-181a，miR-181c，miR-335，miR-384-5p，miR-422a），其中有8个miRNA出现变化明显，miR-15a、miR-21、miR-34a、miR-497分别上调了（6.831±2.625）、（8.122±3.442）、（5.349±2.010）、（6.590±3.689）倍；miR-125b、miR-145、Let-7a、Let-7e分别下调了（0.122±0.039）、（0.199±0.096）、（0.191±0.069）、（0.160±0.082）倍。CA-CPR组皮质区细胞凋亡率〔（32.23±5.31）%〕较正常对照组〔（3.66±1.34）%〕和假手术组〔（4.98±1.84）%〕明显升高（均P＜0.01）。结论大鼠CA-CPR后早期脑组织存在明显的细胞凋亡，同期大脑皮质区凋亡相关性miRNA的表达也发生变化，其中发生明显变化的miRNA可能是CA-CPR后脑保护的新靶标。