摘要
目的探讨肝素结合性表皮生长因子样生长因子（heparin-binding epidermal growth factor—like growth factor，HB-EGF）在新生儿坏死性小肠结肠炎（neonatal necrotizing enterocolitis，NEC）新生大鼠模型线粒体途径细胞凋亡中的作用。方法新生无特定病原体sprague-Dawley大鼠随机分为3组，每组10只。NEC模型组：采用代乳品人工喂养，并给予100％氮气缺氧90s，4℃冷刺激10min，每天2次，连续3d；HB-EGF干预组：在NEC模型组基础上予HB-EGF灌胃，每次800μg／kg，每天4次，连续3d。正常对照组：鼠乳喂养3d，不予任何干预。大鼠生后72h禁食12h后处死。取回肠末端组织，HE染色观察病理改变并评分；电镜下观察线粒体超微结构变化；免疫组织化学方法检测细胞色素C量；Western印迹技术检测凋亡诱导因子（apoptosis inducing factor，AIF）及凋亡肽酶激活因子-1（apoptotic protease activating factor-1，APAF-1）的表达。组间差异比较采用单因素方差分析，两两比较采用q检验，P〈0．05为差异有统计学意义。结果（1）HB-EGF干预组NEC发生率为2／10，低于模型组（9／10），差异有统计学意义（X^2=7．27，P〈0．01）；对照组未发生NEC。（2）NEC模型组线粒体在肠上皮细胞及肌层细胞中存在明显肿胀，基质内有多数电子透亮区，线粒体超微结构严重损伤。HB-EGF干预组有少量线粒体肿胀，损伤较NEC模型组减轻。（3）NEC模型组回肠组织细胞色素C表达较对照组增强，差异有统计学意义（0．030±0．018与0．002±0．001，q=6．15，P〈0．01），HB-EGF干预组回肠组织细胞色素C表达（0．014±0．018）较NEC模型组减弱，差异有统计学意义（q=3．53，P〈0．05）。NEC模型组的APAF-1表达较对照组增强（1．364±0．299与0．215±0．033，q=15．31，P〈0．05），AIF表达也增强（0．181±0．050与0．127±0．045，q=3．71，P〈0．05）；与NEC模型组比较，HB-EGF干预组APAp1的表达（0．455±0．123）减低（q=4．04，P〈0．05），AIF的表达（0．289±0．045）则明显增强（q=7．32，P〈0．05）。结论H昏EGF能降低新生大鼠NEC发生率，其机制之一可能是通过下调APAF-1表达而减少新生大鼠线粒体途径细胞凋亡。
Objective To investigate the effect ：i heparin-binding epidermal growth factor-like growth factor （HB-EGF） on mitochondrial pathway of apoptosis in rats with neonatal necrotizing enterocolitis （NEC）. Methods Sprague-Dawley neonatal rats were randomly divided into three groups with ten in each. NEC group rats were formula fed, and hypoxia exposed by 100% N2 for 90 s and cold stress at 4 ℃ for 10 rain twice a day for three days. Additionally, rats in HB-EGF group received HB-EGF 800 μg/kg by gavage four times a day for three days. Rats in control group were given breast milk feeding for three days without any interventions. Seventy-two hours after born, all neonatal rats were sacrificed after fasting for 12 h, from which the terminal ileum was removed. HE-staining was done for histologic evaluation. Mitochondrial ultrastructure was observed under electron microscopy. Cytochrome C was detected by immunohistochemical analysis and apoptosis inducing factor （AIF） and apoptotic protease activating factor-1 （APAF-1） were measured by Western blot. Analysis of variance and q test were used to compare the difference among groups. Results （1） The incidence of NEC in HB-EGF group was lower than that in NEC group （2/10 vs 9/10, X^2 = 7.27, P〈0.01）. （2） In NEC group, mitochondria in epithelial cells and muscle cells of intestine were significantly swelling, appearing many electron lucent zones in matrix. Ultrastructure of mitochondria were severely damaged. In HB-EGF group, mitochondria were less swelling and showed milder damage than those in NEC group. （3） The expression of cytochrome C in ileal tissue in NEC group was higher than that in control group （0.0304-0.018 vs 0.002!0.001, q=6.15, P〈0.01）. The expression of cytochrome C in ileal tissue in HB-EGF group was lower than that in NEC group （0.014＋0.018 vs0.0304-0.018, q=3.53,P〈0.05）. The expression of APAF-1 and AIF in NEC group was higher than those in control group （1. 364±0. 299 vs 0. 215-1-0. 033, q=15.31, P〈0.05; 0. 181±0. 050 vs 0.127±0.045, q=3.71, P〈0.05）. Compared to NEC group, the expression of APAF-1 was lower （0. 45510. 123 vs 1. 364±0. 299, q=4.04, P〈0.05） and the expression of AIF was higher （0.289±0.045 vs 0. 181±0.050, q=7.32, P〈0.05） in HBEGF group. Conclusions HB-EGF could reduce the incidence of NEC in neonatal rats by inhibiting the mitochondrial pathway related apoptosis through down regulation of APAF-1.
出处
《中华围产医学杂志》
CAS
北大核心
2013年第4期232-237,共6页
Chinese Journal of Perinatal Medicine
