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。     

内皮祖细胞培养上清对高氧暴露下Ⅱ型肺泡上皮细胞增殖和分化的影响

 目的: 研究高氧暴露对内皮祖细胞(EPCs)旁分泌功能的影响,并探索EPCs旁分泌因子对高氧暴露下Ⅱ型肺泡上皮细胞(AECⅡ)功能的影响。方法: 分离大鼠骨髓来源的单个核细胞,EGM-2MV培养基培养7~10 d获取EPCs,并对其进行鉴定。分别在空气条件下及60%的氧浓度条件下培养EPCs,收集空气组EPCs培养上清(E-CM-RA)和高氧组EPCs培养上清(E-CM-O₂),ELISA法检测2组上清中VEGF、FGF10、EGF及PDGF-BB的表达水平。分离纯化培养成年大鼠的AECⅡ,常规培养至第2 天,分为4组:(1)RA组:空气条件下培养;(2) O₂组:60%的氧浓度条件下培养;(3) O₂+E-CM-RA组:在O₂组的基础上,在培养基中加入E-CM-RA;(4) O₂+E-CM-O₂组:在O₂组的基础上,在培养基中加入E-CM-O₂。分别在干预12 h、24 h、2 d和3 d,用MTT法及细胞计数法检测4组AECⅡ的增殖情况;在24 h、2 d和3 d用real-time PCR法检测4组AECⅡ中SP-C和AQP5的mRNA表达量。结果: E-CM-RA中VEGF及FGF10的表达水平明显高于E-CM-O₂(P<0.05)。干预不同时间,各组间AECⅡ的活力及数量均有明显差异(P<0.01)。与RA组相比,O₂组AECⅡ各时点的活力明显下降,数量明显减少(P<0.05);与O₂组相比,各时点O₂+E-CM-RA组AECⅡ的活力明显增高,数量明显增加(P<0.05);但O₂+E-CM-O₂组与O₂组AECⅡ的活力及数量均无显著差异。干预24 h、2 d及3 d,4组间AECⅡ的SP-C与AQP5 mRNA的表达量均具有明显差异(P<0.01)。与RA组相比,O₂组AECⅡ中SP-C的mRNA表达量显著降低(P<0.01),AQP5的mRNA表达量显著升高(P<0.01)。与O₂组相比,在24 h、2 d及3 d时, O₂+E-CM-RA组和O₂+E-CM-O₂组AECⅡ中SP-C的mRNA表达量显著增高(P<0.05),而在2 d及3 d时,AQP5的mRNA表达量显著降低(P<0.01)。结论: EPCs可分泌肺发育相关生长因子VEGF和FGF10,高氧暴露抑制EPCs表达 VEGF和FGF10。高氧暴露抑制AECⅡ的增殖,降低AECⅡ表达SP-C mRNA,促进AECⅡ表达AQP5 mRNA。EPCs培养上清能够改善高氧暴露对AECⅡ增殖的抑制作用,维持AECⅡ本身特性,抑制AECⅡ向AECⅠ分化。而高氧暴露部分削弱了EPCs培养上清的这种作用,这可能与高氧暴露抑制EPCs表达肺发育相关因子VEGF和FGF10有关。     

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。     

STAT1 ASON对肺纤维化大鼠BALF细胞因子和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响

目的:观察信号转导子与转录活化子1(STAT1)反义寡核苷酸(ASON)雾化吸入对博莱霉素(BLM)致肺纤维化大鼠肺泡灌洗液(BALF)PDGF-BB(血小板衍生生长因子-BB)、 TNF-α(肿瘤坏死因子-α )、 TGF-β1(转化生长因子-β1)、 IFN-γ(干扰素-γ)表达及肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响.方法:取45只健康雌性Wistar大鼠随机分成ASON组、 BLM组和生理盐水(NS)组各15只, 气管内分别灌注BLM(ASON组、 BLM组)和NS(NS组)复制肺纤维化模型和空白对照, ASON组于第0、 2、 4、 6天雾化吸入STAT1 ASON, BLM组和NS组雾化吸入NS, 各组分别于第7、 14、 28天均处死动物5只.观察肺泡炎和肺纤维化改变;收集BALF测定PDGF-BB、 TNF-α、 IFN-γ、 TGF-β1的水平;测定肺组织Ⅰ、Ⅲ型胶原mRNA表达.结果:ASON组第7、 14、 28天肺泡炎和肺纤维化程度明显低于同时间点BLM组( P <0.05).BLM组各时间点BALF中PDGF-BB、 TNF-α、 TGF-β1表达水平较NS组升高( P <0.05), ASON组较BLM组各时间点均降低( P <0.05);BLM组BALF中IFN-γ表达水平各时间点均低于NS组( P <0.05) , 但ASON组各时间点均较BLM组高( P <0.05).ASON组和BLM组第7、 14、 28天Ⅰ、Ⅲ型胶原mRNA表达高于NS组( P <0.05);ASON组第7、 14、 28天Ⅰ、Ⅲ型胶原mRNA的表达均较同时间点BLM组降低( P <0.05).结论:STAT1 ASON雾化吸入能够减轻BLM诱导的肺泡炎和肺纤维化程度, 其机制可能与抑制BALF中TGF-β1、 PDGF-BB、 TNF-α的表达, 抑制IFN-γ下降而减少肺组织胶原的沉积有关.     

穿心莲内酯对肺纤维化大鼠BALF中细胞因子和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响

目的:探讨穿心莲内酯灌胃对博来霉素(BLM)致肺纤维化大鼠支气管肺泡灌洗液(BALF)中TNF-α、TGF-β1浓度和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响。方法:取健康雄性SD大鼠90只,随机分为生理盐水(NS)组、BLM组、泼尼松(Pred)组、不同剂量穿心莲内酯组(即穿A组62.5mg/kg、穿B组125 mg/kg、穿C组250 mg/kg),各组大鼠15只,分别气管内灌注BLM(BLM组、Pred组、穿A组、穿B组、穿C组)或NS(NS组)后,每天给予NS(NS组、BLM组)、Pred(Pred组)或穿心莲内酯(穿A组、穿B组、穿C组)灌胃,各组分别于气管内灌注药物后第7、14、28天处死大鼠5只。用HE、Masson染色观察肺泡炎症和纤维化改变;实时荧光定量逆转录-聚合酶链反应检测肺组织Ⅰ、Ⅲ型胶原mRNA表达;酶联免疫吸附试验测定BALF中TNF-α、TGF-β1浓度;同时监测肾功能指标血尿素氮(BUN)、肌酐(Cr)及肝功能指标血丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)。结果:肝肾功能监测显示:不同剂量穿心莲内酯组、NS组、BLM组、Pred组所监测的AST、ALT、BUN、Cr比较,差异无统计学意义(P>0.05)。NS组大鼠肺组织未发现肺泡间隔水肿、炎性细胞浸润和纤维化形成。BLM组第7天时肺泡腔内可见大量炎性细胞浸润,第14天时肺泡炎仍存在,但炎症细胞明显减少,肺泡间隔内成纤维细胞明显增多,肺泡结构破坏,肺泡隔增宽,第28天时炎症较前减轻,肺纤维化程度加重,部分肺泡腔消失,形成严重纤维化。穿A组病理形态改变与BLM组相似。Pred组、穿B组、穿C组大鼠第7天有较多的炎症细胞浸润及局部聚积,第14天和第28天的纤维化病理改变均较BLM组、穿A组明显减轻。NS组各个时间点BALF中TGF-β1、TNF-α含量均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组BALF中的浓度(P<0.05)。BLM组3个时间点BALF中TGF-β1、TNF-α含量较Pred组、穿B、穿C组高(P<0.05)。与BLM组比较,穿A组BALF中TGF-β1、TNF-α含量无统计学意义。NS组各个时间点肺组织Ⅰ、Ⅲ型胶原mRNA表达均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组肺组织中的表达(P<0.05)。BLM组3个时间点Ⅰ、Ⅲ型胶原mRNA表达较Pred组、穿B组、穿C组高(P<0.05)。与BLM组比较,穿A组肺组织Ⅰ、Ⅲ型胶原mRNA表达无统计学差异。结论:穿心莲内酯灌胃可减轻BLM致肺纤维化大鼠肺泡炎和肺纤维化程度,降低肺组织Ⅰ、Ⅲ型胶原mRNA表达,降低BALF中TNF-α、TGF-β1浓度,且对肝肾无明显毒副作用。     

骨桥蛋白单抗抑制大鼠肺纤维化的研究

探讨骨桥蛋白(osteopontin,OPN)对博莱霉素(bleomycin,BLM)诱导的大鼠肺纤维化肺组织基质金属蛋白酶-9(MMP-9)、金属基质蛋白酶抑制剂-1(TIMP-1)和Ⅰ、Ⅲ型胶原mRNA表达的影响。取72只健康雌性Wistar大鼠随机分成生理盐水对照组(NS组)、博莱霉素模型组(BLM组)和骨桥蛋白抗体干预组(OPN-Ab组)各24只,气管内灌注BLM复制肺纤维化模型(BLM组、OPN-Ab干预组),NS组气管内灌注生理盐水,OPN-Ab组于0d、2d、4d、6d尾静脉注射骨桥蛋白抗体(1:32),BLM组和NS组尾静脉注射生理盐水,各组分别于7d、14d、28d处死动物8只。收集肺组织作切片行HE、MASSON染色测定肺泡炎症和纤维化改变,免疫组织化学PAP法测定肺组织中OPN、MMP-9、TIMP-1的表达;RT-PCR测定肺组织Ⅰ/Ⅲ型胶原mRNA的表达。结果与BLM组比较,在第7天和第14天OPN-Ab组肺泡炎明显减轻(P0.01);而肺纤维化程度在各时间点均明显减轻(P0.05);与NS组比较,BLM组、OPN-Ab组肺组织中OPN、MMP-9、TIMP-1表达水平均显著升高(P0.01)。BLM组第7天OPN、MMP-9、TIMP-1在肺组织中表达水平显著升高,随后下降,第28天时仍高于NS组(P0.01)。与BLM组比较,OPN-Ab组各时间点OPN、MMP-9、TIMP-1表达水平降低(P0.01)。OPN-Ab组第7、14、28天肺组织Ⅰ、Ⅲ型胶原mRNA表达均较同时间点BLM组明显降低(P0.05)。OPN可能是肺纤维化形成机制中的重要因素,骨桥蛋白抗体可能通过抑制细胞因子MMP-9、TIMP-1表达,减少肺组织中胶原的生成,最终抑制肺纤维化。     

穿心莲内酯对肺纤维化大鼠BALF中细胞因子和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响

目的:探讨穿心莲内酯灌胃对博来霉素(BLM)致肺纤维化大鼠支气管肺泡灌洗液(BALF)中TNF-α、TGF-β1浓度和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响.方法:取健康雄性SD大鼠90只,随机分为生理盐水(NS)组、BLM组、泼尼松(Pred)组、不同剂量穿心莲内酯组(即穿A组62.5mg/kg、穿B组125 mg/kg、穿C组250 mg/kg),各组大鼠15只,分别气管内灌注BLM(BLM组、Pred组、穿A组、穿B组、穿C组)或NS(NS组)后,每天给予NS(NS组、BLM组)、Pred(Pred组)或穿心莲内酯(穿A组、穿B组、穿C组)灌胃,各组分别于气管内灌注药物后第7、14、28天处死大鼠5只.用HE、Masson染色观察肺泡炎症和纤维化改变;实时荧光定量逆转录-聚合酶链反应检测肺组织Ⅰ、Ⅲ型胶原mRNA表达;酶联免疫吸附试验测定BALF中TNF-α、TGF-β1浓度;同时监测肾功能指标血尿素氮(BUN)、肌酐(Cr)及肝功能指标血丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST).结果:肝肾功能监测显示:不同剂量穿心莲内酯组、NS组、BLM组、Pred组所监测的AST、ALT、BUN、Cr比较,差异无统计学意义(P＞0.05).NS组大鼠肺组织未发现肺泡间隔水肿、炎性细胞浸润和纤维化形成.BLM组第7天时肺泡腔内可见大量炎性细胞浸润,第14天时肺泡炎仍存在,但炎症细胞明显减少,肺泡间隔内成纤维细胞明显增多,肺泡结构破坏,肺泡隔增宽,第28天时炎症较前减轻,肺纤维化程度加重,部分肺泡腔消失,形成严重纤维化.穿A组病理形态改变与BLM组相似.Pred组、穿B组、穿C组大鼠第7天有较多的炎症细胞浸润及局部聚积,第14天和第28天的纤维化病理改变均较BLM组、穿A组明显减轻.NS组各个时间点BALF中TGF-β1、TNF-α含量均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组BALF中的浓度(P＜0.05).BLM组3个时间点BALF中TGF-β1、TNF-α含量较Pred组、穿B、穿C组高(P＜0.05).与BLM组比较,穿A组BALF中TGF-β1、TNF-α含量无统计学意义.NS组各个时间点肺组织Ⅰ、Ⅲ型胶原mRNA表达均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组肺组织中的表达(P＜0.05).BLM组3个时间点Ⅰ、Ⅲ型胶原mRNA表达较Pred组、穿B组、穿C组高(P＜O.05).与BLM组比较,穿A组肺组织Ⅰ、Ⅲ型胶原mRNA表达无统计学差异.结论:穿心莲内酯灌胃可减轻BuM致肺纤维化大鼠肺泡炎和肺纤维化程度,降低肺组织Ⅰ、Ⅲ型胶原mRNA表达,降低BALF中TNF-α、TGF-β1浓度,且对肝肾无明显毒副作用.     

维甲酸下调高氧暴露下胎鼠肺成纤维细胞及Ⅱ型肺泡上皮细胞MMP-2表达

目的探讨维甲酸(RA)对高氧暴露下胎鼠肺成纤维细胞(LFs)和肺泡Ⅱ型上皮细胞(AECⅡ)基质金属蛋白酶-2 (MMP -2 )表达的影响及调控。方法原代培养胎鼠肺LFs及AECⅡ,待其生长至亚汇合状态时,随机分为:空气组、空气+RA组、高氧组和高氧+RA组。于培养2、6、12、2 4和4 8(AECⅡ)或72h(LFs)时,采用半定量RT- PCR方法检测MMP 2和TIMP 2mRNA表达,应用Westernblot检测p38和磷酸化p38(p p38)蛋白表达水平。结果(1)与空气组比较,高氧可促进胎鼠LFs、AECⅡMMP 2mRNA和胎鼠LFsp p38表达(P <0 . 0 1或P <0. 0 5 ) ;(2 )RA能部分下调高氧诱导的胎鼠LFs、AECⅡMMP 2mRNA高表达和明显降低胎鼠LFsp p38表达;(3)高氧、RA对胎鼠LFs、AECⅡTIMP -2mRNA和胎鼠LFsp38蛋白表达无明显影响;(4)胎鼠LFsp p38与MMP 2mRNA表达呈显著性正相关(r =0. 76 7,P <0. 0 1,n =18)。结论高氧可促进胎鼠LFs、AECⅡMMP 2mRNA表达;p38通路参与高氧状态下胎鼠LFsMMP -2表达调控;RA可在转录后水平调节p38的活性状态从而实现对MMP-2的表达调控。     

角化生长因子在慢性肺疾病早产大鼠的表达

目的探讨高氧致CLD早产大鼠KGF表达的变化规律.方法 60只新生早产大鼠随机分为2组,每组30只:A组-对照组;B组-高氧组(吸入95%O2).分别采用Western Blotting及RT-PCR检测肺组织KGF及其mRNA表达.结果高氧组肺内KGF及其mRNA表达高于对照组.结论高氧引起新生早产大鼠肺内KGF及其mRNA的表达随时间发生变化.     

布地奈德对肺纤维化大鼠Smad4、PDGF-A及PAI-1表达的影响

目的:探讨布地奈德(BUD)对肺纤维化大鼠肺组织转化生长因子-β1(TGF-β1)、血小板衍化生长因子-A(PDGF-A)、抗生物皮肤生长因子同源物4母体(Smad4)、纤溶酶原激活物抑制物-1(PAI-1)表达的影响。方法:取健康雌性Wistar大鼠随机分成博莱霉素(BLM)组、生理盐水(NS)组和BUD组,每组15只。NS组气管内灌注NS,BUD组、BLM组气管内灌注BLM,继之0～6 d每天雾化1次,BLM组和NS组雾化吸入NS,BUD组雾化吸入BUD,分别于第7、14、28天处死3组大鼠各5只。用HE、Masson染色观察肺泡炎症和纤维化改变;免疫组化法测定肺组织中TGF-β1、PDGF-A、Smad4、PAI-1的表达。结果:第7、14天,BUD组肺泡炎程度较BLM组减轻(P<0.05);第7、14、28天,BUD组肺纤维化程度均较BLM组减轻(P<0.05)。第7、14、28天,BUD组肺组织中TGF-β1、PDGF-A、Smad4、PAI-1表达较BLM组明显降低(P<0.05)。结论:BUD雾化吸入可降低肺纤维化大鼠肺组织TGF-β1、PDGF-A、Smad4、PAI-1的表达,同时亦可减轻其肺泡炎及肺纤维化程度。     

Expression of keratinocyte growth factor/fibroblast growth factor-7 and its receptor in human lung cancer: correlation with tumour proliferative activity and patient prognosis

Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7), a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation, has been implicated in the repair of lung tissue. The present study was designed to determine the expression and role of KGF and its receptor (KGFR) in human lung cancer tissues, particularly in relation to cancer cell kinetics and prognosis. Thirty-one adenocarcinomas and 30 squamous cell carcinomas, and ten normal lung tissues as a control, were examined. The expression of KGF and KGFR proteins was examined using rabbit polyclonal anti-human KGF and anti-human KGFR antisera raised in the authors' laboratories against synthetic peptides corresponding to parts of human KGF KGFR, respectively. Their specificity was confirmed in lung tumour tissues by western blotting various controls. Proliferative activity was assessed by determining the labelling index (LI) for Ki-67 antigen. Immunohistochemistry revealed that tissue co-expression of KGF KGFR correlated significantly with higher differentiation grades in squamous cell carcinoma. Conversely, in adenocarcinoma, co-expression correlated with lower differentiation grades high Ki-67 LI, was significantly associated with lymph node metastasis shorter 5-year survival. Therefore, the results indicate that co-expression of KGF KGFR correlates significantly with poor prognosis in adenocarcinoma, but not in squamous cell carcinoma, of the lung.     

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.     

Enhanced expression of keratinocyte growth factor and its receptor correlates with venous invasion in pancreatic cancer

Keratinocyte growth factor (KGF) and KGF receptor (KGFR) have been implicated in cancer growth as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-2 cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-2 cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-2 cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC.     

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.     

胰岛素样生长因子-Ⅰ对高氧致新生鼠慢性肺疾病Ⅰ型胶原表达的影响

目的研究collagen Ⅰ在高氧致新生鼠慢性肺疾病中的表达、作用及IGF-Ⅰ对collagenⅠ表达的影响。方法将足月新生大鼠144只随机分为高氧组和空气组，分别于实验1d，3d，7d，10d，14d和21d应用免疫组化染色分别检测collagenⅠ和IGF～Ⅰ蛋白表达，并将IGF—Ⅰ和collagenⅠ蛋白表达进行相关分析。结果高氧组在3d～10d IGF—Ⅰ和collagenⅠ蛋白表达与空气组相比较明显降低（P〈0．05）；14d和21d IGF—Ⅰ和collagenⅠ蛋白表达与空气组相比较明显增加（P〈0．05），IGF—Ⅰ和collagenⅠ表达呈正相关。结论collagenⅠ与CLD时肺泡发育受阻和肺纤维化发生有关，IGF—Ⅰ是影响collagenⅠ合成的重要生长因子之一。     

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。     

活性维生素D3在大鼠肺纤维化发生发展中的作用及对miR-29a表达的影响

目的探讨活性维生素D3[1,25(OH)2D3]在大鼠肺纤维化发生发展中的作用及对miR-29a的影响。方法 150只SD雄性大鼠随机分为纤维化发生干预组(90只)和纤维化后干预组(60只),纤维化发生干预组分为模型组Ⅰ、给药组Ⅰ和对照组Ⅰ(n=30),纤维化后干预组分为模型组Ⅱ、给药组Ⅱ和对照组Ⅱ(n=20)。模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ经气管注入博来霉素(5mg/kg)建立肺纤维化模型,对照组Ⅰ/Ⅱ经气管注入等体积生理盐水。给药组Ⅰ/Ⅱ分别于手术后第2天和第14天腹腔注射活性维生素D3,模型组Ⅰ/Ⅱ分别于手术后第2天和第14天腹腔注射等量的活性维生素D3溶剂(0.1%乙醇和99.9%的丙二醇),对照组Ⅰ/Ⅱ分别在术后第2天和第14天腹腔注射等量的生理盐水。各种处理均为两天1次。纤维化发生干预组分别于手术后第14天、第21天和第28天处死大鼠取材,纤维化后干预组分别于手术后第21天和第28天处死大鼠取材,各小组每个时间点10只大鼠。用Masson染色法观察各组实验大鼠肺中胶原纤维的差异,用碱水解法检测羟脯氨酸含量的变化,用实时定量PCR检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(Col)ⅠmRNA及miR-29a的相对表达量,用免疫组织化学方法检测α-SMA和ColⅠ蛋白质表达水平,并经图像分析进行量化。结果博来霉素处理后,第14天大鼠肺部已经出现纤维化,随着时间推移,纤维化进一步加重。模型组Ⅰ/Ⅱ和给药组Ⅰ/Ⅱ中的羟脯氨酸含量、α-SMA和ColⅠ的mRNA和蛋白质表达都明显高于对照组Ⅰ/Ⅱ,而其miR-29a表达与对照组Ⅰ/Ⅱ相比则明显减少。给药组Ⅰ/Ⅱ中miR-29a的表达与模型组Ⅰ/Ⅱ相比有所增加(P0.05)。在纤维化发生干预组中,与模型组Ⅰ相比,给药组Ⅰ在3个时间点羟脯氨酸含量、α-SMA、ColⅠ的mRNA和蛋白质表达都显著降低(P0.05),但在纤维化后干预组中的给药组Ⅱ中羟脯氨酸含量、α-SMA、ColⅠ的mRNA和蛋白质表达比模型组Ⅱ虽有所降低,但差异无显著性(P0.05)。结论活性维生素D3对大鼠肺纤维化的发生发展具有一定的抑制作用,其预防效果更好一些,并且能促进miR-29a的表达,活性维生素D3可能是通过促进miR-29a的表达来抑制肺纤维化的发生发展。