CAJ | 학술논문

目的探讨脑出血后血红蛋白对血脑屏障内皮细胞Rho激酶Ⅱ及紧密连接蛋白claudin-5的影响. 方法将SD大鼠按随机数字表法分为正常对照组(18只)和血红蛋白组(72只),血红蛋白组再按不同取材时间点分为24 h、48 h、3d、7d4个亚组(每个时间点18只).血红蛋白组采用脑内注入血红蛋白法制作脑出血大鼠模型.免疫组化染色观察各组大鼠血脑屏障内皮细胞Rho激酶Ⅱ及磷酸化肌球蛋白轻链的蛋白表达,伊文思蓝浓度测定评估血脑屏障通透性,免疫荧光染色和实时荧光定量PCR检测claudin-5蛋白和mRNA表达. 结果免疫组化染色结果显示:正常对照组血脑屏障内皮细胞Rho激酶Ⅱ及磷酸化肌球蛋白轻链表达较少,血红蛋白组血肿侧血脑屏障内皮细胞呈棕黄色及线条状表达；血红蛋白组48 h、3d时血脑屏障内皮细胞Rho激酶Ⅱ及磷酸化肌球蛋白轻链平均吸光度值明显高于正常对照组,差异有统计学意义(P＜0.05).血红蛋白组各时间点脑内伊文思蓝含量均明显高于正常对照组,差异均有统计学意义(P＜0.05).免疫荧光染色结果显示:与正常对照组相比,血红蛋白组24h、48 h及3d时claudin-5蛋白表达断续和减少.实时荧光定量PCR结果显示:血红蛋白组24 h、48 h、3d及7d时claudin-5 mRNA表达水平明显低于正常对照组,差异有统计学意义(P＜0.05). 结论脑出血后血红蛋白可以导致血脑屏障内皮细胞Rho激酶Ⅱ的表达增高,进而致磷酸化肌球蛋白轻链表达增加、紧密连接蛋白claudin-5表达降低,其可能是导致脑出血后血脑屏障破坏及脑水肿发生的重要机制之一.
목적 탐토뇌출혈후혈홍단백대혈뇌병장내피세포Rho격매Ⅱ급긴밀련접단백claudin-5적영향. 방법 장SD대서안수궤수자표법분위정상대조조(18지)화혈홍단백조(72지),혈홍단백조재안불동취재시간점분위24 h、48 h、3d、7d4개아조(매개시간점18지).혈홍단백조채용뇌내주입혈홍단백법제작뇌출혈대서모형.면역조화염색관찰각조대서혈뇌병장내피세포Rho격매Ⅱ급린산화기구단백경련적단백표체,이문사람농도측정평고혈뇌병장통투성,면역형광염색화실시형광정량PCR검측claudin-5단백화mRNA표체. 결과 면역조화염색결과현시:정상대조조혈뇌병장내피세포Rho격매Ⅱ급린산화기구단백경련표체교소,혈홍단백조혈종측혈뇌병장내피세포정종황색급선조상표체；혈홍단백조48 h、3d시혈뇌병장내피세포Rho격매Ⅱ급린산화기구단백경련평균흡광도치명현고우정상대조조,차이유통계학의의(P＜0.05).혈홍단백조각시간점뇌내이문사람함량균명현고우정상대조조,차이균유통계학의의(P＜0.05).면역형광염색결과현시:여정상대조조상비,혈홍단백조24h、48 h급3d시claudin-5단백표체단속화감소.실시형광정량PCR결과현시:혈홍단백조24 h、48 h、3d급7d시claudin-5 mRNA표체수평명현저우정상대조조,차이유통계학의의(P＜0.05). 결론 뇌출혈후혈홍단백가이도치혈뇌병장내피세포Rho격매Ⅱ적표체증고,진이치린산화기구단백경련표체증가、긴밀련접단백claudin-5표체강저,기가능시도치뇌출혈후혈뇌병장파배급뇌수종발생적중요궤제지일.
Objective To explore the effects of hemoglobin level on Rho kinase Ⅱ of endothelial cells and tight junction protein clandin-5 in blood-brain barrier (BBB) of rats after intracerebral hemorrhage (ICH).Methods Male SD rats were randomized into normal control group (n=18) and hemoglobin inducement group (n=72); hemoglobin inducement group was divided into 4 subgroups (24 and 48 h,and 3 and 7 d after surgery,n=18).Rat ICH models in the hemoglobin inducement group were established by hemoglobin injection into the brain tissues.The expression ofRho kinase Ⅱ of endothelial cells and phosphorylated myosin light chain protein in BBB were observed by immunohistochemical staining,and BBB permeability was evaluated by Evans Blue dye; The protein and mRNA expressions of claudin-5 in the perihematomal brain tissues were detected by immunofluorescence staining and real-time quantitative PCR.Results Immunohistochemical staining indicated that expressions of Rho kinase Ⅱ of endothelial cells and phosphorylated myosin light chain in BBB of normal control group were rare,and those in the hemoglobin inducement group were high; as compared with that in the normal control group,mean optical density of Rho kinase Ⅱ and phosphorylated myosin light chain proteins in the endothelial cells of BBB in hemoglobin inducement group increased significantly at 48 h and 3 d after inducement (P＜0.05).The permeation of Evans Blue in hemoglobin inducement group significantly increased (P＜0.05).Immunofluorescence staining showed that the expression of clandin-5 in hemoglobin inducement group was uncontinuous and low as compared with that in the normal control group at 24 and 48 h and 3 d after inducement.PCR showed that the mRNA expression of claudin-5 in hemoglobin inducement group was significantly lower than that in the normal control group at 24 and 48 h and 3 and 7 d after the inducement (P＜0.05).Conclusion After intracerebral hemorrhage,hemoglobin can lead to increased expression of phosphorylated myosin light chain,and reduce the expression of tight junction protein claudin-5 of BBB by increasing the expression ofRho kinase Ⅱ in endothelial cells of BBB,which may be an important mechanism in the disruption of BBB and the occurrence of brain edema after intracerebral hemorrhage.