早产儿支气管肺发育不良不同时期基质金属蛋白酶表达及其调控的研究

目的　探讨新生儿呼吸窘迫综合征 (NRDS)向支气管肺发育不良 (BPD)发展过程中c Jun蛋白、c Fos蛋白、基质金属蛋白酶 2 (MMP 2 )及其特异性组织抑制物 (TIMP 2 )的动态表达情况及其相关关系。方法　应用免疫组化方法检测 2 5例经临床和病理确诊为NRDS向BPD转变的尸检肺组织标本中c Jun蛋白、c Fos蛋白、TIMP 2以及MMP 2的表达强度 ,并结合计算机图像处理系统进行分析。结果　急性期c Jun、c Fos表达即达峰值 ,以后两者表达开始下降 ,但c Jun仍保持较高水平 ,至慢性期时 ,其表达水平与对照组相比差异有显著性 (P <0 0 1) ;而c Fos的表达至再生期后与对照组相比差异无显著性 (P >0 0 5 )。MMP 2表达与c Jun表达基本一致 ,且与c Jun、c Fos的表达呈显著性正相关 (P <0 0 1)。MMP 2 /TIMP 2的比值则呈下降趋势 ,但仍显著高于对照组水平 (P <0 0 1或 0 0 5 )。结论　MMP 2 /TIMP 2表达失衡参与了NRDS向BPD进展的过程 ;MMP 2表达与c Jun蛋白、c Fos蛋白表达密切相关。     

胰岛素样生长因子结合蛋白2在新生鼠肺发育中的作用

目的探索胰岛素样生长因子结合蛋白(IGFBP)2在新生鼠肺发育中的作用。方法SD孕鼠80只分为4组,即A:对照组;B:地塞米松(Dex)1组;C:Dex2组;D:维A酸(RA)组。A、B、D组分别于孕18～20d皮下注射生理盐水、Dex,腹腔注射RA;C组于生后1～3d皮下注射Dex。各组分别于孕18、20、21d(C组孕期不取)、生后1、3、5、7、10、14、21d取肺标本作形态学检查、免疫组织化学、Westernblot及RT PCR检测。结果1.肺组织形态学:B、C组早期肺泡发育提前,数目多,壁薄,晚期则明显落后于A、D组。2.IGFBP2肺表达:A组中IGFBP2主要在胎肺组织中表达,孕18d左右表达最强,随后表达渐减弱。B、C组表达趋势同A组,但生后各时间点均较A组增强;D组生后各时间点均较A组减弱。3.Westernblot:IGFBP2多肽表达强度各组孕18d表达最强,随后渐降低;B、C组生后各时间点表达强度明显高于A组(P均<0.01);D组各时间点浓度均明显低于A组(P均<0.01)。4.RT PCR:IGFBP2mRNA表达的强度变化规律与其肽浓度变化相似。结论IGFBP2在肺发育过程起重要作用,其浓度过高则影响肺发育。     

维甲酸对大鼠高氧肺损伤的保护机制及其与调控丝裂原活化蛋白激酶的关系

目的 探讨维甲酸(RA)对高氧肺损伤保护作用机制及其与调控丝裂原活化蛋白激酶(MAPKs)关系.方法 建立高氧(85%)暴露早产SD大鼠肺损伤模型,运用RT-PCR方法检测基质金属蛋白酶2,9(MMP-2、-9)mRNA表达,采用明胶酶谱法检测MMP-2和MMP-9酶原及活酶表达,采用Western blot技术检测磷酸化和非磷酸化总的ERK1/2、JNK1/2和038蛋白质表达.结果 与空气组比较,高氧暴露4、7、14 d,MMP-2和MMP-9 mRNA的表达均显著提高(P均<0.01),MMP-2活酶(除4 d外)、MMP-9酶原及活酶的表达明显上调(P<0.05或P<0.01),磷酸化ERK1/2、JNK1/2和p38表达显著增加(P均<0.01);与高氧组比较,4、7、14 d时,RA明显下调高氧暴露后MMP-2(除14d外)、MMP.9 mRNA的表达(P<0.01或P<0.05),不同程度降低MMP-2活酶、MMP.9酶原及活酶的表达和显著下调磷酸化JNK1/2、038水平,进一步上调磷酸化ERK1/2表达.结论 高氧暴露显著提高MMP-2和MMP-9表达,是造成肺损伤的重要因素;JNK1/2和p38信号途径可能参与了高氧暴露下早产大鼠肺组织MMP-2和MMP-9的表达调控;RA可通过降低JNK1/2和p38磷酸化水平,抑制MMP-2和MMP-9的高表达和活化,从而发挥高氧肺损伤保护作用.     

Effect of Amygdalin on the Proliferation of Hyperoxia-exposed Type Ⅱ Alveolar Epithelial Cells Isolated from Premature Rat

Summary: The pathogenesis of hyperoxia lung injury and the mechanism of amygdalin on type 2 alveolar epithelial cells (AEC2) isolated from premature rat lungs in vitro were investigated. AEC2 were obtained by primary culture from 20-days fetal rat lung and hyperoxia-exposed cell model was established. Cell proliferating viability was examined by MTT assay after treatment of amygdalin at various concentrations. DNA content and the proliferating cell nuclear antigen (PCNA) protein expression of AEC2 were measured by using flow cytometry and immunocytochemistry respectively after 24 h of hyperoxia exposure or amygdalin treatment. The results showed that hyperoxia inhibited the proliferation and decreased PCNA protein expression in AEC2 of premature rat in vitro. Amygdalin at the concentration range of 50-200 μmol/L stimulated the proliferation of AEC2 in a dose-dependent manner, however, 400 μmol/L amygdalin inhibited the proliferation of AEC2. Amygdalin at the concentration of 200 μmol/L played its best role in facilitating proliferation of AEC2s in vitro and could partially ameliorated the changes of proliferation in hyperoxia exposed AEC2 of premature rat. It has been suggested that hyperoxia inhibited the proliferation of AEC2s of premature rat, which may contribute to hyperoxia lung injury. Amygdalin may play partial protective role in hyperoxia-induced lung injury.     

血小板衍生生长因子对肺发育的影响

目的探讨血小板衍生生长因子(PDGF)在胚胎和生后新生大鼠肺发育中的作用机制,研究其对肺分支形态形成,肺实质和间质发育调控原理。方法应用免疫组织化学法和RT-PCR法半定量分析和检测各时间点(胚胎18、20、21d和生后1、4、7、14、21d)PDGFmRNA及其表达水平。结果在胚胎晚期,PDGF表达于肺间质细胞和肺实质细胞胞浆。出生后随日龄增加,PDG-FmRNA及其表达呈动态变化,组间比较差异显著(P<0.05,P<0.01)。结论PDGF在肺分支形态发育和肺组织细胞增殖中起着关键作用,它不仅参与肺泡数量调节,而且是肺间质细胞和肺实质细胞分化成熟不可缺少的因子。     

Association of Insulin-like Growth Factors with Lung Development in Neonatal Rats

To explore the relationship between Insulin-like growth factor (IGF)-Ⅰ,-Ⅱ and lung development in neonatal rats. 80 timed pregnant Sprague-Dawley (SD) rats were randomly divided into 4 groups (n=20): group A (Control group), group B (Dexamethasone (DEX) 1 group),group C (DEX 2 group), group D (retinoic acid (RA) group). 20 pregnant rats in group A, B and D were injected subcutaneously or intraperitoneally with vehicle (NS), DEX, or RA respectively during gestational day 16 to 18. All newborn rats in group C were subcutaneously injected with DEX at day 1 to 3 after birth. The lung tissue was obtained at the following times: fetuses at gesta-tional ages of 18, 20 and 21 days, and 1, 3, 5, 7, 10, 14 and 21 days after birth. Lung tissues were used for histopathological study, the polypeptides analysis of IGF-Ⅰ, -Ⅱ(immunohistochem-istry and Western blot) and mRNA analysis ( RT- PCR). The results showed that the strongest expression of IGF-Ⅰ in group A and D occurred at ages of 5—7 days (alveolar stage). The stronger their expressions, the better the alveolar develop. The peak stage of expression in group B occurred earlier, on the day 3 after birth. Compared with group A, the expression of IGF-Ⅰ during gestation age of 18 days to age of 3 days in group B were significantly higher (P＜0.01), but significantly lower at other time points (P＜0.01). The expression of IGF-Ⅰ was lower in group C all the time and always higher in group D than those in group A (P＜0.01). The peak expression of IGF-Ⅱ took place at the gestation age of 18 days, then gradually dropped to trace. During 18 days of gestation to age of 3 days, the expression of IGF-Ⅱ in group B was significantly higher than that in group A (P＜0.01). No difference was found among all other groups. The change in the expression of IGF-Ⅰ, -Ⅱ mRNA in all 4 groups was similar to that of their polypeptides. The results suggested that there is a close linking between IGF- Ⅰ, -Ⅱ and lung development in newborns. The IGF-Ⅱ works at early stage and the that of IGF-Ⅰ works at the stage of new septa formation and alveoli maturation. The stronger their expressions, the more mature the lung development.     

412例早产儿临床分析

目的 探讨如何提高早产儿存活率和生存质量。方法 回顾分析本院新生儿病房收治的412例早产儿临床资料。结果 早产最常见的因素是多胎妊娠、妊娠合并高血压、前置胎盘或胎盘早剥、母妊娠合并其他疾病、胎膜早破、脐带因素等。母亲妊娠年龄以20～29岁为主,分娩方式以剖宫产为主。早产儿常见疾病的顺位为：新生儿缺氧缺血性脑病、新生儿窒息、血糖紊乱等。早产儿死亡42例,病死率10．2％,体重、胎龄越小、病死率越高,死亡率最高的疾病是肺透明膜病(47．1％)。结论 避免多胎妊娠;立即进行复苏,机械通气治疗十分重要,且要严格掌握上呼吸机指征。     

高氧、维甲酸对早产鼠肺组织基质金属蛋白酶-2、-9 及特异性组织抑制物-1、-2表达的影响

目的探讨基质金属原蛋白-2(MMP-2)、MMP-9、基质金属蛋白酶特异性组织抑制物-1(TIMP-1)和TIMP-2在高氧肺损伤中的作用及维甲酸(RA)的保护作用机制。方法建立高氧(FiO285%)暴露早产SD大鼠肺损伤模型,应用RT-PCR法检测MMP-2、MMP-9、TIMP-1和TIMP-2mRNA表达,采用明胶酶谱检测MMP-2和MMP-9酶原及活酶表达,采用Western blot技术检测TIMP-1和TIMP-2蛋白表达。结果与空气组比较,高氧暴露4、7、14d,MMP-2、MMP-9和TIMP-1 mRNA的表达均显著升高(P均<0.01),MMP-2活酶、MMP-9酶原及活酶和TIMP-1蛋白的表达明显上调(P<0.05);RA对空气暴露下它们的表达均无明显影响(P均>0.05),但不同程度下调高氧暴露后MMP-2、MMP-9、TIMP-1mRNA的表达和MMP-2活酶、MMP-9酶原及活酶的表达,进一步提高TIMP-1蛋白表达;高氧、RA对TIMP-2 mRNA和蛋白的表达均无明显影响(P均>0.05)。结论高氧暴露明显改变MMPs/TIMPs的表达,在肺泡形成关键时期,MMPs/TIMPs之间平衡关系的破坏是造成肺发育受阻和纤维化的重要因素;通过协调MMPs/TIMPs之间的表达,改善肺泡结构,降低肺纤维化程度,从而逆转高氧所致肺损伤,是RA发挥保护作用的重要机制之一。     

高氧、维甲酸对胎鼠肺成纤维细胞c-Jun、c-Fos表达的影响

目的 探讨维甲酸对高氧暴露下胎鼠肺成纤维细胞c—Jun、c—Fos表达的影响。方法 取第2代胎鼠肺成纤维细胞作体外培养，待生长至接近融合状态时，随机分为：空气组，空气 维甲酸组，高氧组，高氧 维甲酸组。于培养30min和1、2、6、12、24、48、72h时，行细胞固定后，应用免疫组化方法捡测c—Jun、c—Fos表达强度并结合计算机图像处理系统进行分析。结果 与空气组相比，高氧1h时c—Jun表达明显增强(P＜0．01)，6—12h时达高峰，以后开始下降，但仍高于正常水平(P＜0．01)；c—Fos的表达在高氧30min即增强(P＜0．01)，2—6h达高峰，以后开始下降，24h后恢复到正常水平。维甲酸能明显下调高氧所诱导的c—Jun、c—Fos高表达，但并不能完全阻止c—Jun、c—Fos的过度表达。结论 c—Jun、c—Fos可能参与了高氧暴露下胎鼠肺成纤维细胞的信号转导过程；维甲酸可部分抑制由高氧所诱导的c—Jun、c—Fos的过度表达。