【摘要】 目的目的研究Cx43在新生大鼠HIBD的表达,探讨其在HIBD的作用及意义。方法7d龄健康SD大鼠128只,随机分为对照组和缺氧缺血组,每组64只。两组大鼠根据处死时相点每组再随机分为6h、24h、48h、72h四个小组,每组16只。每组大鼠取8只,采用HE染色、DNA原位末端标记法、免疫组化染色方法观察各组新生大鼠脑组织病理变化、细胞凋亡及Cx43表达、分布情况;每组另外8只应用Western-blot定量分析Cx43蛋白在皮层及海马的表达。结果对照组各时相点皮层、海马未见DNA原位末端标记法阳性细胞;缺氧缺血组24h组开始细胞凋亡细胞逐渐增多,缺氧缺血组24h、48h、72h组的凋亡阳性细胞分别为15.12±2.68、30.24±3.65、68.72±5.60,各组差异有显著性意义,P<0.01;对照组均有Cx43表达,6h、24h、48h、72h组Cx43染色阳性面积分别为(8.38±0.16)%、(8.38±0.19)%、(8.39±0.03)%、(8.35±0.15)%,各时相点组Cx43阳性表达差异无显著意义,P>0.05;缺氧缺血后6h、24h、48h、72h组Cx43染色阳性面积分别为(18.64±0.30)%、(29.65±0.26)%、(35.34±0.21)%、(42.05±0.31)%,随着缺氧缺血后时间的推移,Cx43阳性表达逐渐增加,各组对比,差异有显著意义,P<0.01;缺氧缺血组与对照组对比,缺氧缺血组各时相点Cx43阳性表达均高于对照组,P<0.01;Western-blot结果:各组电泳均可显示在43～46KD范围的条带,Cx43/GAPDH电泳条带密度比值对照组6h、24h、48h、72h分别为1.00±0.01、0.99±0.02、1.01±0.01、1.01±0.01;缺氧缺血组6h、24h、48h、72hCx43/GAPDH电泳条带比值分别为1.11±0.04、1.25±0.02、1.37±0.01、1.50±0.01;缺氧缺血组与对照组对比,同一时相点Cx43蛋白的表达均较对照组高,P<0.01;缺氧缺血6h组Cx43表达已开始增加,随着缺氧缺血后时间的推移逐渐增加,至72h组最明显,缺氧缺血各组间比较,差异有显著意义,P<0.01;对照组各时相点比较,Cx43蛋白的表达差异无显著意义,P>0.05。结论缺氧缺血后Cx43的表达逐渐增加,其变化规律与神经细胞凋亡严重程度及光镜观察到的脑损伤进展的时间框架相吻合,且发生时间早于细胞凋亡,提示Cx43表达的增加参与了新生大鼠HIBD的病理形成过程,推测其通过缝隙连接增强传播和放大细胞损伤,在HIBD早期发挥“姐妹杀伤效应”,导致和加重脑损害。更多还原

【Abstract】 Objective To investigate the expression of connexin-43(Cx43) in brain tissue of hypoxic-ischemic neonatal rats and explore its role in hypoxic-ischemic brain damage(HIBD) .Methods Totally 128 seven-day-old neonatal rats were randomly divided into two groups:control group and hypoxic-ischemia(HI) group with 64 rats in each.According to the time of sacrefice,64 rats of every group were further randomly divided into four groups including six hour(6h) ,twenty-four hour(24h) ,forty-eight hour(48h) and seventy-two hour(72h) ,with 16 rats in each group.In each group,8 rats were used for observing pathologic changes,neural cell apoptosis,and the expression of Cx43 through the techniques of HE staining,TUNEL staining and immunohistochemical staining,and the brains another 8 rats was used for measuring the expression levels of Cx43 protein of rat cortex and hippocampus by Western-blot analysis.Results In control group,apoptotic cells were not observed by TUNEL staining.In HI 4h group,apoptotic cells also were not observed.After HI,the level of apoptotic cells increased progressively at 24h to 72h(24h:15.12±2.68,48h:30.24±3.65,72h:68.72±5.60) .There was a highly significant difference among them(P<0.01) .The expression of Cx43 by immunohistochemical staining:The positive cells of Cx43 existed in all the control and HI groups.Cx43 distributed in cellular membrane and ecptoma of astrocytes.In control group,the expression of Cx43 has no difference at 6h to 72h[6h:(8.38±0.16) %,24h:(8.38±0.19) %,48h:(8.39±0.03) %,72h:(8.35±0.15) %].There was no significant difference among them(P>0.05) .After HI,the expression of Cx43 increased progressively at 6h to 72h[6h:(18.64±0.30) %,24h:(29.65±0.26) %,48h:(35.34±0.21) %,72h:(42.05±0.31) %].There was a highly significant difference among them(P<0.01) .At 6h,24h,48h and 72h,there was a highly significant difference between HI and control group(P<0.01) .The expression of Cx43 by Western-blot analysis:Proteins of Cx43 are extracted at 43～46KD position in all the control and HI groups.In control group,the expression of Cx43 has no difference at 6h to 72h(6h:1.00±0.01,24h:0.99±0.02,48h:1.01±0.01,72h:1.01±0.01) .There was no significant difference among them(P>0.05) .After HI,the expression of Cx43 increased progressively at 6h to 72h(6h:1.11±0.04,24h:1.25±0.02,48h:1.37±0.01,72h:1.50±0.01) .There was a highly significant difference among them(P<0.01) .At 6h,24h,48h and 72h,there was a highly significant difference between HIBD and control group(P<0.01) .Conclusion The expression of Cx43 protein increased progressively after HI,regularity of which was consistent with the time frame for development of brain damage and apoptosis.It implied Cx43 may play important roles in the pathogenesis of HIBD in neonatal rats through enhancing gap junction intercellular communication which killed the adjacent cells and increased brain damage after hypoxic-ischemia.更多还原