维甲酸对高氧暴露新生大鼠肺组织角化细胞生长因子及其受体表达的影响

目的: 探讨高氧暴露新生大鼠肺组织结构的变化和角化细胞生长因子(KGF)及其受体(KGFR)的表达情况及维甲酸(RA)对其的影响。方法: 将出生24 h内SD大鼠90只随机分为3组,Ⅰ组:空气+生理盐水(NS);Ⅱ组:高氧+NS;Ⅲ组:高氧+RA。Ⅱ、Ⅲ组持续暴露于85% O₂中,Ⅰ组置于空气中;Ⅲ组每天腹腔注射RA,Ⅰ、Ⅱ组每天腹腔注射NS。分别于生后3、7、14 d取肺标本,用HE染色法观察肺组织结构变化及辐射状肺泡计数(RAC);RT-PCR检测KGF和KGFR mRNA表达强度和免疫组织化学法检测KGF蛋白表达水平。结果: (1)生后第14 d,Ⅱ、Ⅲ组较Ⅰ组RAC值显著减少(P<0.05),但Ⅲ组较Ⅱ组明显增高(P<0.05)。(2)与Ⅰ组相比,Ⅱ组3 d时,KGF mRNA表达明显增强(P<0.05);其后开始下降,7 d仍高于Ⅰ组(P<0.05);14 d时较Ⅰ组有所降低(P<0.05);Ⅲ组各时点表达量均高于同期Ⅱ组(P<0.05)。3 d时,各组KGFR mRNA表达量无明显差异;7 d、14 d时,Ⅱ、Ⅲ组表达量明显低于Ⅰ组(均P<0.05),且Ⅱ组和Ⅲ组之间无显著差异(P>0.05)。(3)KGF阳性细胞主要分布在部分肺泡壁及肺泡周围血管内皮细胞和间质细胞。KGF蛋白表达强度与其mRNA表达变化相似。结论: RA可促进肺组织KGF表达,改善高氧所致肺发育受阻。对未成熟肺高氧损伤有一定的保护作用。     

内皮祖细胞培养上清对高氧暴露新生大鼠肺结构的改善作用

目的:研究内皮祖细胞培养上清(endothelial progenitor cell-conditioned medium,EPC-CM)对高氧暴露新生大鼠肺损伤时肺泡结构的改善作用及其机制。方法:从新生SD大鼠骨髓中获取内皮祖细胞并鉴定,收集第3代细胞的培养上清备用。另取新生SD大鼠40只随机分为4组,即空气组:仔鼠在空气(21%O_2)中喂养21天;高氧组:仔鼠在85% O_2中喂养21天;内皮细胞基础培养基(endothelial cell basal medium,EBM)干预组:仔鼠在85%O_2中喂养至第14天时,经气道给予100μL EBM,然后喂养至第21天;EPC-CM干预组:仔鼠在85% O_2中喂养至第14天,经气道给予100μL EPC-CM,喂养至第21天。第21天处死小鼠,左肺用4%多聚甲醛固定,留作石蜡切片,随后HE染色进行肺组织病理形态学观察,并做辐射状肺泡计数(radical alveolar count,RAC)及肺泡平均线性截距(mean linear intercept,MLI)测量;免疫组织化学方法对血管内皮细胞FVIII染色,计数肺组织微血管密度;右肺留作实时荧光定量PCR检测肺组织KGF、VEGF、SP-A和SP-C的mRNA表达。结果:培养所得细胞具有典型的EPCs形态改变,能结合异硫氰酸荧光素标记的荆豆凝集素1并摄取Di I荧光标记的乙酰化低密度脂蛋白。高氧组及EBM干预组的仔鼠体重、RAC、MLI和微血管密度较空气组显著降低(P0.05),EPC-CM干预组的RAC和微血管密度较高氧组和EBM干预组明显增加(P0.05),而体重和MLI的变化无明显差异,但有增高的趋势。高氧组和EBM干预组肺组织KGF、VEGF、SP-A和SP-C的mRNA表达较空气组显著降低(P0.05),EPC-CM干预组的表达显著高于高氧组和EBM干预组(P0.05)。结论:EPC-CM可改善高氧暴露新生大鼠的肺泡化和肺血管发育,可能与促进肺内KGF和VEGF mRNA的表达相关。     

高氧下调早产大鼠肺组织AQP5的表达

目的探讨水通道蛋白5(AQP5)在早产大鼠及吸高氧过程中的肺动态表达。方法剖宫术取出孕21d SD早产鼠,于12～24h内随机分为对照组和高氧组。分别在1、4、7、10和14d时提取肺组织,RT-PCR测定AQP5 mRNA表达,免疫组化和Western blot检测AQP5蛋白的表达。结果早产大鼠生后肺组织AQP5表达不断增强,其阳性染色主要定位于Ⅰ型肺泡上皮细胞。高氧暴露1d时,仅肺组织AQP5 mRNA表达显著高于对照组(P<0·05),而高氧暴露4、7、10及14d时,其mRNA及蛋白表达均明显减少(P<0·05或P<0·01)。结论AQP5通过调节肺水平衡,在早产大鼠肺泡化形成的关键时期发挥重要作用;高氧暴露导致AQP5表达下调是促使肺损伤发生、发展的重要因素。     

表皮生长因子对新生大鼠高氧损伤肺组织EGFR及EGF mRNA表达的影响

目的： 探讨外源性表皮生长因子（EGF）对新生大鼠高氧损伤肺组织EGFR及EGF mRNA表达的影响。方法： 取胎龄21 d剖宫产出生的新生Sprague dawley （SD）大鼠持续吸入95％的O2制作未成熟肺高氧损伤模型，随机分为高氧表皮生长因子（EGF）组和高氧生理盐水（NS）组，另设空气NS对照组；所有组按给药（或NS）时间分为3个亚组，即：a亚组(1-3 d)、b亚组(4-6 d)、c亚组(1-6 d)；各亚组均于生后3、7、14 d分批处死取肺组织。应用免疫组化观察各组肺组织EGF-R的表达，RT-PCR方法检测EGF-mRNA的表达。结果： EGF mRNA的表达随着日龄增加而递增，生后7、14 d高氧组EGF-R及EGF mRNA的表达高于空气对照组，14 d EGFa和c亚组EGF-R的表达均明显高于相应的高氧NS组(P＜0.05)，14 d时EGF组内源性EGF mRNA的表达较NS组明显增加(P＜0.01)。结论： 早期应用EGF可促进肺泡上皮细胞EGF-R的表达，改善高氧所致肺发育受阻，对未成熟肺高氧损伤有一定的保护作用。     

高氧致新生鼠肺PECAM-1mRNA及蛋白质表达动态改变的研究

目的研究高浓度氧持续吸入后新生鼠肺部组织PECAM-1mRNA及蛋白质的表达规律,探讨高氧对发育中鼠肺微血管发育的影响.方法新生SD大鼠生后12h内放入氧箱,持续吸入高氧制作高氧CLD模型,采用RT-PCR和免疫组织化学技术测定实验组和对照组在生后1d、7d、7d、14d、21d肺组织内PECAM-1mRNA及蛋白质表达并半定量.结果新生鼠肺部PECAM-1mRNA及蛋白质表达随生后日龄增长而增加,高氧持续吸入后PECAM-1表达减少.结论高氧导致肺组织PECAM-1mRNA和蛋白质表达减少,提示肺微血管生成减少,肺微循环发育异常.推测,高氧可能直接损伤内皮细胞干扰肺血管发育过程,进而影响肺泡分化导致CLD的病理改变.     

高氧、维甲酸对早产大鼠肺成纤维细胞MMP-2及TIMP-2表达的调控

目的:探讨维甲酸（RA）对高氧暴露下早产大鼠肺成纤维细胞（LFs）基质金属蛋白酶-2（MMP-2）及其特异性组织抑制物-2（TIMP-2）表达的影响。方法:建立原代培养的早产大鼠LFs高氧暴露模型,采用半定量RT-PCR方法检测MMP-2和TIMP-2 mRNA表达,明胶酶谱法检测MMP-2酶原和活酶表达, Western blotting检测其磷酸化和总的ERK1/2、JNK1/2、p38和c-Jun表达。结果:（1）与对照组比较,高氧可促进早产大鼠LFs MMP-2 mRNA及其酶原和活酶表达（P<0.05,P<0.01）,同时使其p-ERK1/2、p-JNK1/2、p-p38和p-c-Jun表达水平显著提高（P<0.01,P<0.05）;（2）RA能不同程度下调高氧诱导的早产大鼠LFs MMP-2 mRNA高表达和明显降低其p-JNK1/2、 p-p38和p-c-Jun表达（P<0.01,P<0.05）,但进一步提高p-ERK1/2表达;（3）高氧、RA对TIMP-2 mRNA和总ERK1/2、JNK1/2 、p38及c-Jun表达无明显影响。结论:高氧暴露通过激活MAPKs信号转导通路（主要是JNK和p38）使c-Jun磷酸化水平提高,促进MMP-2表达和激活;RA通过抑制JNK和p38磷酸化,下调MMP-2表达与活化,从而拮抗高氧诱导的肺损伤。     

组蛋白H2AX在高氧致肺损伤新生大鼠肺组织的表达

目的 研究组蛋白H2AX在高氧致肺损伤新生大鼠肺组织中的表达,并探讨其在高氧肺损伤中的作用.方法 将80例新生大鼠随机分为实验组(高氧组)和对照组(空气组),建立高氧致新生大鼠肺损伤的模型,在实验开始后1、2、3、5、7天取肺组织标本,运用免疫荧光技术和Western-blot方法检测高氧肺损伤过程中H2AX的表达规律...     

高浓度氧增加未成年大鼠肺血红素加氧酶-1表达

目的 探讨高浓度氧对未成年大鼠肺血红素加氧酶-1(HO-1)表达的影响.方法 将生后21 d SD大鼠40只随机分为空气组和12、24、48及72 h高氧组,分别置于空气和常压高氧箱(92%~94% O<,2>)中.检测左肺湿/干重比及肺组织病理学改变,用RT-PCR和Western blot法分别检测肺HO-1 mRNA及HO-1蛋白表达情况.结果 高氧12 h组肺HO-1 mRNA吸光度积分相对值和高氧24 h组HO-1蛋白表达水平分别为0.350±0.043和0.455±0.046,较对照组0.263±0.037和0.280±0.044明显升高,且均随高氧暴露时间延长而表达进一步增加(P<0.05,P<0.01).与空气组比较,高氧48 h组和高氧72 h组左肺湿重/干重及肺损伤评分显著增高(P<0.05,P<0.01).结论 高浓度氧可引起未成年大鼠肺组织HO-1表达增多.     

高氧对新生大鼠肺血管内皮生长因子及其受体表达的影响

目的研究高浓度氧持续吸入后新生大鼠肺部VEGF及其受体VEGFR1和VEGFR2的表达规律,探讨高氧性BPD肺血管发育异常的损伤机制;方法采用新生Wister大鼠制作BPD模型,免疫组化方法观察实验组和对照组在生后1d、4d、7d、14d、21d肺组织内VEGF、VEGFR1、VEGFR2的合成和分布情况;结果新生鼠肺部VEGF及其受体VEGFR1和VEGFR2表达生后随日龄增长而增加,高氧造成肺泡简化和肺纤维化的病理改变,导致上述血管因子表达减少;结论肺血管的生长是正常肺泡发育重要环节,推测高氧诱导的VEGF信号通路的改变可能是BPD病理改变的重要成因.     

高氧致新生鼠肺VEGF、PECAM-1、HIF-1α mRNA的动态改变

目的研究高氧吸入致新生鼠肺VEGF、HIF—1α，PECAM-1mRNA的动态改变，探讨高氧对发育中鼠肺微血管损伤的调节机制。方法采用新生SD大鼠制作CLD模型，RT—PCR技术检测实验组和对照组在生后1、4、7、14、21天肺组织VEGF、PECAM-1、HIF-1αmRNA表达。结果新生鼠肺VEGF、PECAM-1mRNA表达量随生后日龄增长而增加，高氧导致VEGF、PECAM-1及HIF—1α表达减少。结论高氧导致肺组织VEGF、PECAM—1及HIF-1αmRNA表达减少。推测，高氧可能通过抑制HIF-1-VEGF信号通路或者直接损伤内皮细胞干扰肺血管发育过程。致CLD的病理改变。     

Keratinocyte growth factor is a growth factor for mammary epithelium in vivo. The mammary epithelium of lactating rats is resistant to the proliferative action of keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF is secreted by stromal cells and affects epithelial but not mesenchymal cell proliferation. KGF injected intravenously was found to cause dramatic proliferation of mammary epithelium in the mammary glands of rats. KGF causes ductal neogenesis and intraductal epithelial hyperplasia but not lobular differentiation in nulliparous female rats. KGF causes ductal and lobular epithelial hyperplasia in male rats. KGF causes proliferation of ductal and acinar cells in the mammary glands of pregnant rats. On the other hand, the ductal epithelium of lactating postpartum rats is resistant to the proliferative action of KGF. The mammary glands of lactating rats did not express less KGF receptor mRNA than the glands of pregnant rats, suggesting that the resistance of the ductal epithelium to KGF during lactation is not related to KGF receptor mRNA down-regulation. The mammary glands of both pregnant and postpartum lactating rats express KGF mRNA with more KGF present in the glands of lactating rats. In conclusion, the KGF and KGF receptor genes are expressed in rat mammary glands and recombinant KGF is a potent growth factor for mammary epithelium.     

补阳还五汤的抗氧化作用

目的：探讨复方中药制剂补阳还五汤的抗氧化机制。方法：用水煎醇沉法制备补阳还五汤提取液,从新生鼠坐骨神经分离纯化雪旺细胞,建立过氧化氢（H2O2）损伤模型。将培养细胞分为补阳还五汤处理组、氧化损伤组及正常组和空白对照组,培养12h时,检测丙二醛（MDA）的含量,用免疫组化方法检测Caspase-3的表达,用RT-PCR技术检测胶质细胞源性神经营养因子（GDNF）的表达。结果：与氧化损伤组相比,补阳还五汤处理组的MDA含量明显减弱,Caspase-3呈弱阳性表达,GDNF mRNA的表达量减少不明显。结论：补阳还五汤具有良好的抗过氧化损伤效果,对H2O2引起雪旺细胞的损伤具有保护作用。     

人非小细胞肺癌中KGF mRNA的表达及意义

目的研究人非小细胞肺癌(non-small cell lung cancer,NSCLC)角化细胞生长因子(keratinocyte growth factor,KGF)mR-NA的表达,及其在NSCLC发生过程中肿瘤细胞与间质细胞间的相互作用。方法采用原位杂交和免疫组化法检测KGF mR-NA与Ki-67在50例NSCLC的表达,并与正常组织对照。结果KGF mRNA的表达除在NSCLC某些实质细胞内观察到外,主要见于NSCLC的纤维母细胞和血管平滑肌细胞胞质。肿瘤组织KGF mRNA表达的阳性率86%明显高于正常肺组织的24%(P<0·05)。有淋巴结转移者比无淋巴结转移者的表达更强,且与肺癌的分化相关,分化程度越低,KGF mRNA表达越强。在50例肺癌中Ki-67表达的分布与KGF mRNA相似。结论NSCLC存在KGF mRNA高表达。KGF可通过旁分泌、自分泌两种方式发挥作用。KGF可能与NSCLC的发生有一定的相关性。     

Possible involvement of keratinocyte growth factor and its receptor in enhanced epithelial-cell proliferation and acquired recurrence of middle-ear cholesteatoma

Middle-ear cholesteatoma is characterized by enhanced proliferation of epithelial cells and granular tissue formation. However, the molecular mechanism underlying these pathological changes is largely unknown. Keratinocyte growth factor (KGF) is a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation. In the present study, we investigated the possible involvement of KGF and its receptor, KGFR, in the pathogenesis of cholesteatoma using in situ hybridization and immunohistochemistry, respectively. We examined 56 cholesteatoma specimens, and 8 normal skin areas as control. KGF and KGFR expression was examined by immunohistochemistry using rabbit anti-human KGF and anti-human KGFR polyclonal antisera raised in our laboratories against synthetic peptides corresponding to parts of human KGF and KGFR, respectively. KGF protein and mRNA were detected exclusively in stromal fibroblasts and infiltrating T lymphocytes in 80% of cholesteatoma cases, whereas KGFR protein and mRNA were localized in the epithelium in 72% of cases. Assessment of the proliferative activity of cholesteatoma using the labeling index for Ki-67 showed a significantly higher Ki-67 labeling index (66%) in KGF+/KGFR+ cases than other cases. There was a significant correlation between KGF+/KGFR+ expression and recurrence. Our results indicate the possible involvement of both KGF and KGFR in enhanced epithelial cell proliferative activity and recurrence of cholesteatoma.     

复方SZ滴眼液对晶状体上皮细胞凋亡的抑制作用及其信号转导机制

目的： 探讨复方SZ滴眼液（Co-SZ）抑制H₂O₂诱导的晶状体上皮细胞（LEC）凋亡的作用及信号转导机制。为将Co-SZ作为防治白内障的有效药物提供实验依据。 方法： (1)将H₂O₂与Co-SZ和SD大鼠晶状体共同孵育后：TUNEL法检测LEC凋亡率。透射电镜观察LEC超微结构改变及凋亡形成。(2)H₂O₂与Co-SZ和体外培养的牛LEC共同孵育后,四甲基偶氮唑蓝法（MTT）检测不同浓度的Co-SZ抑制LEC凋亡率。流式细胞仪（FCM）检测LEC细胞核内DNA含量。荧光分光光度法检测LEC内游离Ca2+浓度。放射免疫分析法检测LEC内环化腺苷酸（cAMP）和环化鸟苷酸（cGMP）浓度。 结果： TUNEL法检测Co-SZ组LEC凋亡率显著低于H₂O₂组。Co-SZ组LEC超微结构变化也显著轻于H₂O₂组。MTT检测Co-SZ组细胞活性明显高于H₂O₂并具有剂量依赖关系。Co-SZ组LEC核内DNA含量增加。Co-SZ使LEC内游离Ca2+、cAMP降低、cGMP升高。结论：Co-SZ能有效抑制H₂O₂诱导的LEC发生的凋亡 。Co-SZ抑制LEC凋亡的机制可能是通过抑制LEC核内DNA降解，并通过抑制细胞内游离钙离子浓度升高、阻断Ca2+-钙调蛋白依赖性蛋白激酶途径和Ca2+-蛋白激酶C信号转导途径。     

Growth factor expression in the obstructed developing and mature rat kidney

The purpose of this study is to evaluate the role of keratinocyte growth factor (KGF), transforming growth factor-alpha (TGF-alpha), and their receptors in altered renal growth caused by complete ureteral obstruction in the developing kidney. Neonatal and adult rats underwent complete unilateral ureteral ligation or sham operation. The kidneys were harvested at 1, 5, 10, 20, and 30 days after obstruction. Renal growth and development was assessed by histology and immunohistocytochemical localization of vimentin, cytokeratin and smooth muscle-alpha actin. Cellular proliferation was measured by [3H]thymidine labeling index of all cells. RNase protection assays were used to quantify mRNA encoding for KGF, KGF receptor, TGF-alpha, and epidermal growth factor (EGF) receptor. Ureteral obstruction in the developing kidneys resulted in decreased DNA synthesis, rapid parenchymal loss, myofibroblast proliferation in the interstitium, decreased tubular epithelial cells formation, and development of cystic dysplasia. In comparison, obstruction in the mature kidneys resulted in transient growth in the medullary ductal cells, parenchymal loss, and myofibroblast proliferation at a later time, lymphocytic infiltration in the interstitium but not cystic dysplasia. KGF and KGF receptor mRNA levels were increased in obstructed neonatal kidneys. Similarly, TGF-alpha and EGF receptor mRNA levels were increased. Delayed and more moderate increases in KGF, KGF receptor, and TGF-alpha expression were also seen in the obstructed mature kidneys. Of importance, the amount of EGF receptor mRNA was not increased in the obstructed compared with the contralateral or sham-operated adult kidneys. This study suggests that obstruction alters the normal expression pattern of KGF, TGF-alpha, and their receptors in renal development. These changes may be responsible for the impaired renal growth and altered development seen in ureteral obstruction of the kidneys. Although some changes are similar to those seen in the adult kidney, the increased expression of TGF-alpha and cystic dysplasia are unique to neonatal obstruction.     

Keratinocyte growth factor (KGF) decreases ICAM-1 and VCAM-1 cell expression on bronchial epithelial cells

Activation of leucocytes during airway inflammatory reaction involves adhesion to bronchial epithelial cells (BEC), a process implicating specific interactions between glycoproteins with epithelial cell surface proteins, mainly intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In this study, the effect of keratinocyte growth factor (KGF), a growth factor involved in pulmonary epithelium repair, was evaluated on adhesion molecule expression with BEAS-2B cells and BEC and granulocyte adherence to BEAS-2B. The modulation by KGF of membrane and mRNA expression of ICAM-1 and VCAM-1 was studied on confluent cells stimulated or not with tumour necrosis factor-alpha (TNF) (200 UI/ml) or TNF and interleukin (IL)-4 (50 UI/ml and 10 ng/ml). Levels of soluble-(s)ICAM-1 and sVCAM-1 were measured by ELISA. Although moderately, KGF significantly decreased membrane ICAM-1 expression in unstimulated BEAS-2B cells (24% inhibition at 100 ng/ml) or in TNF- or TNF + IL-4-stimulated cells (22.5 and 18.7% inhibition, respectively). Treatment with KGF tended to decrease VCAM-1 expression in TNF- and TNF + IL-4-stimulated BEAS-2B (P = n.s. and P < 0.05, 14 and 15% inhibition, respectively). In primary culture of BEC, adhesion molecule expression was also reduced. ICAM-1 and VCAM-1 mRNA expression were also inhibited by KGF. Levels of sICAM-1 and sVCAM-1 were not significantly increased in supernatants from KGF-treated cells (30% and 24% increase at 100 ng/ml, respectively) compared to controls. Moreover, KGF decreased by 31% the adherence of neutrophils to TNF-activated BEAS-2B. In conclusion, KGF decreases ICAM-1 and VCAM-1 expression and neutrophil adherence in BEC. These suggest its involvement in the resolution of the inflammatory reaction.