CAJ | 학술논문

目的：探讨整合素连接激酶(ILK)/磷酸化蛋白激酶B(Akt)信号通路在新生鼠缺氧缺血性脑损伤(HIBD)修复中的作用。方法采用10日龄SD新生大鼠随机分为假手术组(n=3)和缺氧缺血组(HI组,n=23)，建立HIBD模型，于HI后0、4、6、12、24、48、72 h处死取脑，免疫荧光法检测ILK表达与分布，Western blot检测ILK、Akt、磷酸化蛋白激酶B (p-Akt)、血管内皮生长因子(VEGF)的表达情况。构建靶向ILK RNA干扰的慢病毒载体，抑制新生鼠脑组织中ILK的表达。右侧侧脑室分别注射含有LV-ILK shRNA慢病毒(n=15)和LV-control慢病毒对照(n=3)，建立HIBD模型，于HI后4 h和24 h处死动物取脑，Western blot检测ILK、Akt、p-Akt、VEGF蛋白的表达变化，TUNEL染色检测细胞凋亡。结果ILK主要表达于皮层和海马区，定位于胞浆和胞膜，在假手术组和HI组均有表达。ILK于HI后表达开始逐渐增加，24 h达高峰，之后表达有所降低。p-Akt于HI后4 h明显增加，后逐渐降低，24 h降至最低，后又增加，在48 h达高峰。VEGF于HI后4 h表达开始增加，12 h达高峰，后维持较高水平。构建的靶向ILK RNA干扰的慢病毒载体在体内应用获得成功。注射慢病毒LV-ILK shRNA组在HI 4 h、24 h时所表达的ILK、p-Akt、VEGF均明显低于同一时间点的LV-control组，同时细胞凋亡明显增加。结论新生大鼠HIBD后，ILK、p-Akt、VEGF蛋白表达均增高，通过抑制ILK的表达，能够降低新生大鼠HIBD后p-Akt和VEGF蛋白表达，增加细胞凋亡。提示HIBD后可能通过ILK/Akt信号通路，增加VEGF表达，进而促进神经细胞存活及血管再生，在新生鼠HI脑损伤修复中发挥作用。
목적：탐토정합소련접격매(ILK)/린산화단백격매B(Akt)신호통로재신생서결양결혈성뇌손상(HIBD)수복중적작용。방법채용10일령SD신생대서수궤분위가수술조(n=3)화결양결혈조(HI조,n=23)，건립HIBD모형，우HI후0、4、6、12、24、48、72 h처사취뇌，면역형광법검측ILK표체여분포，Western blot검측ILK、Akt、린산화단백격매B (p-Akt)、혈관내피생장인자(VEGF)적표체정황。구건파향ILK RNA간우적만병독재체，억제신생서뇌조직중ILK적표체。우측측뇌실분별주사함유LV-ILK shRNA만병독(n=15)화LV-control만병독대조(n=3)，건립HIBD모형，우HI후4 h화24 h처사동물취뇌，Western blot검측ILK、Akt、p-Akt、VEGF단백적표체변화，TUNEL염색검측세포조망。결과ILK주요표체우피층화해마구，정위우포장화포막，재가수술조화HI조균유표체。ILK우HI후표체개시축점증가，24 h체고봉，지후표체유소강저。p-Akt우HI후4 h명현증가，후축점강저，24 h강지최저，후우증가，재48 h체고봉。VEGF우HI후4 h표체개시증가，12 h체고봉，후유지교고수평。구건적파향ILK RNA간우적만병독재체재체내응용획득성공。주사만병독LV-ILK shRNA조재HI 4 h、24 h시소표체적ILK、p-Akt、VEGF균명현저우동일시간점적LV-control조，동시세포조망명현증가。결론신생대서HIBD후，ILK、p-Akt、VEGF단백표체균증고，통과억제ILK적표체，능구강저신생대서HIBD후p-Akt화VEGF단백표체，증가세포조망。제시HIBD후가능통과ILK/Akt신호통로，증가VEGF표체，진이촉진신경세포존활급혈관재생，재신생서HI뇌손상수복중발휘작용。
Objective To investigate the possible function of integrin-linked kinase (ILK)/protein kinase B (PKB/Akt) signaling in repair of neonatal rat hypoxia-ischemia brain damage (HIBD). Methods Postnatal day 10 SD rats were randomly divided into hypoxia ischemia (HI) group and sham control group. Rat brains were collected at 0 h, 4 h, 6 h, 12 h, 24 h, 48 h and 72 h after hypoxia ischemia damage. Immunolfuorescence staining was used to observe the distribution and expression of ILK. Western blot was used to detect the expression of ILK, Akt, phosphorylated Akt (p-Akt) and vascular endothelial growth factor (VEGF). Lentiviral vectors expressing ILK shRNA were constructed to inhibit the expression of ILK in neonatal rats. After intracerebroventricular injections of LV-ILK shRNA lentivirus and LV-control respectively, HIBD model was established. Rat brains were collected at 4 h and 24 h after HIBD. Western blot was used to detect the expression of ILK, p-Akt, and VEGF. TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to detect cell apoptosis. Results Immunolfuorescence staining showed that ILK was widely distributed in cortex and hippocampus both in HI group and sham control group. ILK located at cell membrane and cytoplasm. Western blot results demonstrated that ILK protein increased after HI, with a peak at 24 h, and maintained higher level than those in sham control group. The p-Akt protein signiifcantly increased at 4 h after HI, and signiifcantly decreased in the following 24 h, and then increased again, with a peak at 48 h, but the level of p-Akt protein was higher than that of sham control group. The VEGF protein increased at 4 h after HI, with a peak at 12 h, higher than that of sham control group. The expression of Akt protein showed no signiifcant difference between HI group and sham control group. Lentiviral vectors containing RNAi targeting ILK was applied successfully in vivo. At 4 h and 24 h after HIBD model, the expression of ILK, p-Akt, and VEGF proteins in right side brain received LV-ILK shRNA signiifcantly decreased compared with those of right side brain received LV-control at the same time point. And cell apoptosis signiifcantly increased in LV-ILK shRNA group. Conclusions The expression of ILK, p-Akt, VEGF proteins increased after HI. By inhibiting the expression of ILK, the expression of p-Akt and VEGF proteins can be reduced, and cell apoptosis could increase in newborn rats after HIBD. The results suggest that ILK may induce the expression of VEGF through activating the PI3K/Akt signaling pathway, and promote cell survival and angiogenesis after HIBD.